Hepatobiliary Transport in Health and Disease 9783110279344, 9783110278996

Table of contents :
Preface
List of Contributors
Abbreviations
1 Physiology of bile formation: Hepatocellular bile salt transporters
1.1 Introduction
1.2 Sodium-dependent bile salt uptake into hepatocytes
1.3 Sodium-independent bile salt uptake into hepatocytes
1.4 Bile salt export across the canalicular membrane
1.5 Bile salt salvage systems
1.6 Concluding remarks
1.7 References
2 Structure and function of hepatic ABC transporters
2.1 Introduction to human ABC transporters expressed in the liver
2.2 Structure and function of the bile salt export pump (ABCB11; BSEP)
2.3 Structure and function of the multidrug resistance protein 3 (ABCB4; MDR3)
2.4 Structure and function of the breast cancer resistance protein (ABCG2; BCRP)
2.5 Concluding remarks
2.6 References
3 Short- and long-term regulation of hepatobiliary transport
3.1 Introduction
3.2 Short-term regulation of sinusoidal transport systems
3.3 Long-term regulation of sinusoidal transport systems
3.4 Short-term regulation of canalicular secretion
3.5 Long-term regulation of canalicular transport systems
3.6 Methods of studying subcellular transporter distribution
3.7 Summary
3.8 References
4 Nuclear bile acid receptor FXR and hepatobiliary transport systems
4.1 Introduction
4.2 Nuclear receptors
4.3 Bile acids and the enterohepatic circulation
4.4 Bile acid homeostasis, enterohepatic circulation, and FXR
4.5 The role of FXR in the pathogenesis of biliary diseases
4.6 Concluding remarks
4.7 References
5 Bile acid signaling in the liver and the biliary tree
5.1 Introduction
5.2 Bile acid signaling in liver parenchymal cells (hepatocytes)
5.3 Bile acid signaling in sinusoidal endothelial cells
5.4 Bile acid signaling in Kupffer cells
5.5 Bile acid signaling in hepatic stellate cells
5.6 Bile acid signaling in the biliary tree
5.7 References
6 Modulation of innate immunity and inflammation by bile acids and their receptors
6.1 Introduction
6.2 Impact of FXR deletion on immunity and inflammation - lessons from FXR knockout mice
6.3 Role of TGR5 in the modulation of immune function
6.4 Effects of bile acids on immunological function independently of bile acid receptors
6.5 Obstructive jaundice and its impact on immune function
6.6 Role of bile acids and FXR in viral infections
6.7 Concluding remarks
6.8 References
7 Bile acids as extrahepatic and interorgan signaling molecules
7.1 Introduction
7.2 Bile acid-dependent modulation of glucose homeostasis
7.3 Impact of bile acids on energy expenditure
7.4 Bile acid receptors and immune response
7.5 Role of bile acid receptors in the cardiovascular system
7.6 Role of bile acid receptors in the kidney
7.7 Bile acid receptors in the central and peripheral nervous system
7.8 Summary and future perspectives
7.9 References
8 Disorders of bile duct development
8.1 Introduction
8.2 Morphogenesis of the intrahepatic bile duct epithelium: molecular players involved and their relationship with arterial morphogenesis
8.3 Ductal plate malformation (DPM): definition, clinical heterogeneity, and classification based on animal models
8.4 Cilia in cholangiocytes: a multifunctional transducing system
8.5 DPM-related cholangiopathies
8.6 Alagille's syndrome (AGS)
8.7 References
9 Mutations of the bile salt export pump (BSEP) and multidrug-resistance protein 3 (MDR3)
9.1 Introduction
9.2 BSEP-related liver diseases
9.3 MDR3-related liver diseases
9.4 Treatment of BSEP- and MDR3-associated liver diseases
9.5 Concluding remarks
9.6 References
10 MRP2 (ABCC2) and disorders of bilirubin handling in liver
10.1 Introduction
10.2 The conjugate efflux pump MRP2 in the hepatocyte canalicular membrane
10.3 Formation of unconjugated bilirubin and its uptake into hepatocytes
10.4 Formation of bilirubin glucuronides and their transport into bile by MRP2
10.5 Uptake of bilirubin glucuronides into hepatocytes
10.6 MRP3, a basolateral efflux pump, contributes to conjugated hyperbilirubinemia
10.7 Genetic disorders and drug-induced inhibition of bilirubin uptake into hepatocytes
10.8 Genetic variants of the MRP2 (ABCC2) gene and MRP2 deficiency in Dubin-Johnson syndrome
10.9 Impairment of MRP2 localization in the hepatocyte canalicular membrane
10.10 References
11 Hepatobiliary transport during pregnancy: Cross talk between transporters and hormones
11.1 Introduction
11.2 Estrogens as cholestatic agents
11.3 Cholestatic activity of progesterone
11.4 Biochemical observations in symptom-free pregnant women
11.5 Genetic lessons from congenital cholestasis
11.6 Transporter variants and cholestasis of pregnancy
11.7 Nuclear receptors and cholestasis of pregnancy
11.8 Gallstones and pregnancy
11.9 Concluding remarks
11.10 References
12 Hepatobiliary transport and gallstone formation
12.1 Introduction
12.2 Epidemiology and risk factors of cholelithiasis
12.3 Hepatobiliary transporters and the pathophysiology of gallstone formation
12.4 Genetic variation in hepatobiliary transporter genes and gallstone susceptibility
12.5 Concluding remarks
12.6 References
13 Molecular basis of primary biliary cirrhosis
13.1 Introduction
13.2 Pathogenesis of PBC
13.3 Concluding remarks
13.4 References
14 The molecular basis of primary sclerosing cholangitis
14.1 Introduction
14.2 Immune-mediated bile duct injury
14.3 Toxic bile duct injury
14.4 Fibrosis and cirrhosis
14.5 Cancer development
14.6 Concluding remarks
14.7 References
15 Drug-induced cholestatic liver injury
15.1 Introduction
15.2 Diagnostic criteria of drug-induced cholestasis
15.3 Hepatocellular drug concentration
15.4 Hepatic bile salt accumulation
15.5 Susceptibility to drug-induced cholestasis
15.6 Concluding remarks
15.7 References
16 Bile acids and receptors: Therapeutic relevance
16.1 Introduction
16.2 Nuclear and membrane BA receptors: general concepts
16.3 Therapeutic potential of BAs
16.4 Role of BA receptors in BA homeostasis and bile production: therapeutic implications in cholestasis
16.5 Role of BA receptors for targeting hepatic inflammation and fibrosis
16.6 Role of BA receptors in the pathogenesis and treatment of gallstone disease
16.7 BA receptors in intestine: therapeutic implications for the gut-liver axis and inflammatory bowel disease
16.8 Role of BAs in lipid metabolism: therapeutic implications for atherosclerosis and nonalcoholic fatty liver disease (NAFLD)
16.9 Role of BAs in hepatic glucose metabolism and beyond
16.10 BA receptors in hepatobiliary and colorectal cancer
16.11 BA receptors beyond the liver and gastrointestinal tract
16.12 Concluding remarks
16.13 References
17 Analysis of bile acids by tandem mass spectrometry
17.1 Introduction
17.2 Experimental procedures
17.3 Applications
17.4 Summary
17.5 References
Index

Citation preview

Hepatobiliary Transport in Health and Disease Edited by Häussinger, Keitel and Kubitz

Hepatobiliary Transport in Health and Disease Edited by Dieter Häussinger, Verena Keitel and Ralf Kubitz

DE GRUYTER

Editors Prof. Dr. med. Dieter Häussinger Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany

Prof. Dr. med. Ralf Kubitz Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany

Dr. med. Verena Keitel Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany

This book has 48 figures and 10 tables. ISBN 978-3-11-027899-6 e-ISBN 978-3-11-027934-4

A CIP catalogue record for this book is available from the Library of Congress. Bibliografic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. © Copyright 2012 by Walter de Gruyter GmbH & Co. KG, Berlin/Boston Printing and Binding: Hubert & Co. GmbH & Co. KG, Göttingen Front cover image: MedicalRF.com/Getty Images Printed in Germany www.degruyter.com The publisher, together with the authors and editors, has taken great pains to ensure that all information presented in this work (programs, applications, amounts, dosages, etc.) reflects the standard of knowledge at the time of publication. Despite careful manuscript preparation and proof correction, errors can nevertheless occur. Authors, editors and publisher disclaim all responsibility and for any errors or omissions or liability for the results obtained from use of the information, or parts thereof, contained in this work. The citation of registered names, trade names, trade marks, etc. in this work does not imply, even in the absence of a specific statement, that such names are exempt from laws and regulations protecting trade marks etc. and therefore free for general use.

Preface

One major function of the liver is the uptake of endo- and xenobiotics from the bloodstream and their excretion into bile. Examples are the excretion of bile acids, bilirubin glucuronides, and glutathione conjugates into bile. The underlying transcellular vectorial transport processes involve specific transport systems in both the sinusoidal (“blood-side”) and the canalicular membrane (bile-side“) of the hepatocyte. Uptake and excretion mechanisms are remarkably coordinated in order to prevent a harmful overload of the hepatocytes with toxic bile acids or other toxic agents. The transport systems involved in hepatobiliary transport have been cloned and characterized at the molecular level and it became recently clear that mutations and polymorphisms of individual transporter molecules underlie a variety of liver diseases. This not only includes rare forms of severe pediatric liver diseases but also liver disorders of the adult, which are caused or modified by disturbances in hepatobiliary transport systems. Furthermore, recently it became clear that bile acids, whose function in digestion is well known, also behave as signal molecules in a variety of organs, including the intestinal and biliary epithelia, sinusoidal endothelial and immune cells. Nuclear and membrane-bound bile acid receptors have been identified that mediate bile acid effects on gene expression, metabolism, and cell viability in liver and other organs. Interestingly, many bile acids are potent inducers of hepatocyte apoptosis, whereas other bile acids are hepatoprotective and are used therapeutically in several liver diseases. This book gives an in-depth survey on the structure and function of transport molecules involved in hepatobiliary transport, on the role of different bile acid receptors in various organs and their function in health and disease, the mechanisms of bile salt-induced apoptosis and hepatocyte protection, and the role of transporter mutations as causes and modifiers of liver diseases. The book will be of interest not only for scientists in biochemisty, structural chemistry, molecular biology, medicine, and cell biology but also for physicians involved in patient care. The chapters were written by renowned experts and active researchers in the field of hepatobiliary transport with some of them members of the Collaborative Research Center 575 “Experimental Hepatology”, the Collaborative Research Center 974 "Communication and System Relevance in Liver Damage and Regeneration" and the Clinical Research Group 217 “Hepatobiliary Transport.” We would like to express our sincere thanks not only to the authors for their excellent contributions, but also to Mrs. Julia Lauterbach, Editor for Science,Technology and Medicine, from DeGruyter Publishers for her excellent collaboration and professional help in preparing and producing this book project, and to Mr. Martin Lay for the artwork and beautiful illustrations. Dieter Häussinger Verena Keitel Ralf Kubitz Düsseldorf, February 2012

List of Contributors

Anna Baghdasaryan Hans Popper Laboratory of Molecular Hepatology Division of Gastroenterology and Hepatology Department of Internal Medicine III Medical University of Vienna, Austria And Laboratory of Experimental and Molecular Hepatology Division of Gastroenterology and Hepatology Department of Internal Medicine Medical University of Graz, Austria Prof. Dr. Ulrich Beuers Department of Gastroenterology and Hepatology Academic Medical Center University of Amsterdam 1100 DE Amsterdam, Netherlands Prof. Dr. Johannes Bode Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Dr. Thierry Claudel Hans Popper Laboratory of Molecular Hepatology Division of Gastroenterology and Hepatology Department of Internal Medicine III Medical University of Vienna, Austria Philipp Ellinger Institute of Biochemistry Heinrich-Heine-University, Geb. 26.23 Universitätsstrasse 1 40225 Düsseldorf, Germany

Dr. Luca Fabris Department of Clinical Medicine, University of Milan-Bicocca, Milan, Italy and Department of Surgical and Gastroenterological Sciences University of Padova, Italy Dr. Raffaella M. Gadaleta Laboratory of Lipid Metabolism and Cancer Department of Translational Pharmacology Mario Negri Sud Institute via Nazionale n.8/A 66030 Santa Maria Imbaro, Italy PD Dr. Dirk Graf Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Prof. Dr. Dieter Häussinger Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Dr. Diran Herebian Clinic for Pediatrics and Neonatology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Dr. Tom Hemming Karlsen Medical Department, Rikshospitalet University Hospital Oslo 27 Oslo, Norway Dr. Simon Hohenester Department of Gastroenterology, Metabolic Disorders

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List of contributors

and Intensive Care Medicine University Hospital Aachen Pauwelsstraße 30 52074 Aachen, Germany Dr. Johannes Roksund Hov Medical Department Rikshospitalet University Hospital Oslo 27 Oslo, Norway PD Dr. Verena Keitel Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Prof. Dr. Dietrich Keppler German Cancer Research Center Im Neuenheimer Feld 280 69120 Heidelberg, Germany Marianne Kluth Institute of Biochemistry Heinrich-Heine-University, Geb. 26.23 Universitätsstrasse 1 40225 Düsseldorf, Germany Prof. Dr. Ralf Kubitz Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Prof. Dr. Frank Lammert Clinic for Gastroenterology and Endocrinology University Hospital Homburg Kirrberger Strasse 100 66421 Homburg, Germany Prof. Dr. Ertan Mayatepek Clinic for Pediatrics and Neonatology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany

Prof. Dr. Peter J. Meier Division of Clinical Pharmacology & Toxicology University Hospital 8091 Zürich, Switzerland Prof. Dr. Antonio Moschetta Laboratory of Lipid Metabolism and Cancer Department of Translational Pharmacology Consorzio Mario Negri Sud Via Nazionale 8/A 66030 Santa Maria Imbaro, Italy Dr. Roman Müllenbach Clinic for Gastroenterology and Endocrinology University Hospital Homburg Kirrberger Strasse 100 66421 Homburg, Germany Prof. Dr. Christiane Pauli-Magnus Clinical Trial Unit Universitätsspital Basel Schanzenstrasse 55 4031 Basel, Switzerland Susanne Pryzbylla Institute of Biochemistry Heinrich-Heine-University, Geb. 26.23 Universitätsstrasse 1 40225 Düsseldorf, Germany Dr. Aileen Raizner Pediatric Gastroenterology and Hepatology Department of Medicine 300 Cedar Street, Room S241 New Haven, CT 06520 USA Prof. Dr. Roland Reinehr Clinic for Gastroenterology, Hepatology and Infectiology University Hospital Düsseldorf Moorenstrasse 5 40225 Düsseldorf, Germany Prof. Dr. Lutz Schmitt Institute of Biochemistry Heinrich-Heine-University, Geb. 26.32 Universitätstrasse 1 40225 Düsseldorf, Germany

List of Contributors Prof. Dr. Erik Schrumpf Medical Department Rikshospitalet University Hospital Oslo 27 Oslo, Norway Dr. Sander H.J. Smits Institute of Biochemistry Heinrich-Heine-University, Geb. 26.23 Universitätsstrasse 1 40225 Düsseldorf, Germany Prof. Dr. Bruno Stieger Clinical Pharmacology and Toxicology University Hospital Zürich Schönleinstr. 2 8032 Zurich, Switzerland Prof. Dr. Mario Strazzabosco Transplant Hepatology, Yale Liver Center and Digestive Disease Section

Department of Medicine 300 Cedar Street, Room S241 New Haven, CT06520, USA Prof. Dr. Michael Trauner Hans Popper Laboratory of Molecular Hepatology Division of Gastroenterology and Hepatology Department of Internal Medicine III Medical University of Vienna, Austria Prof. Dr. Henning Wittenburg Clinic for Gastroenterology and Rheumatology University Hospital Leipzig Liebigstrasse 20 04103 Leipzig, Germany

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Contents

Preface ........................................................................................................................v List of Contributors ....................................................................................................vii Abbreviations ..........................................................................................................xvii 1 Physiology of bile formation: Hepatocellular bile salt transporters ............................................................................................. 1 1.1 Introduction................................................................................................ 1 1.2 Sodium-dependent bile salt uptake into hepatocytes................................... 3 1.3 Sodium-independent bile salt uptake into hepatocytes................................ 7 1.4 Bile salt export across the canalicular membrane ....................................... 8 1.5 Bile salt salvage systems ........................................................................... 12 1.6 Concluding remarks ................................................................................. 12 1.7 References ................................................................................................ 13 2 Structure and function of hepatic ABC transporters ........................................... 23 2.1 Introduction to human ABC transporters expressed in the liver ................. 23 2.2 Structure and function of the bile salt export pump (ABCB11; BSEP).............................................................................. 25 2.3 Structure and function of the multidrug resistance protein 3 (ABCB4; MDR3) ........................................................................ 32 2.4 Structure and function of the breast cancer resistance protein (ABCG2; BCRP) ............................................................ 37 2.5 Concluding remarks ................................................................................. 40 2.6 References ................................................................................................ 41 3 Short- and long-term regulation of hepatobiliary transport ................................................................................... 49 3.1 Introduction.............................................................................................. 49 3.2 Short-term regulation of sinusoidal transport systems ............................... 49 3.3 Long-term regulation of sinusoidal transport systems ................................ 51 3.4 Short-term regulation of canalicular secretion........................................... 54 3.5 Long-term regulation of canalicular transport systems ............................... 56 3.6 Methods of studying subcellular transporter distribution ........................... 58 3.7 Summary .................................................................................................. 59 3.8 References ................................................................................................ 60 4 Nuclear bile acid receptor FXR and hepatobiliary transport systems ................................................................... 71 4.1 Introduction.............................................................................................. 71 4.2 Nuclear receptors ..................................................................................... 71 4.3 Bile acids and the enterohepatic circulation ............................................. 74 4.4 Bile acid homeostasis, enterohepatic circulation, and FXR ....................... 75 4.5 The role of FXR in the pathogenesis of biliary diseases .............................. 79

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Contents

Concluding remarks ................................................................................. 80 References ................................................................................................ 81

5 Bile acid signaling in the liver and the biliary tree .............................................. 85 5.1 Introduction.............................................................................................. 85 5.2 Bile acid signaling in liver parenchymal cells (hepatocytes) ...................... 85 5.3 Bile acid signaling in sinusoidal endothelial cells ..................................... 89 5.4 Bile acid signaling in Kupffer cells ............................................................ 90 5.5 Bile acid signaling in hepatic stellate cells ................................................ 91 5.6 Bile acid signaling in the biliary tree ......................................................... 93 5.7 References ................................................................................................ 96 6 Modulation of innate immunity and inflammation by bile acids and their receptors....................................................................... 103 6.1 Introduction............................................................................................ 103 6.2 Impact of FXR deletion on immunity and inflammation – lessons from FXR knockout mice ............................................................ 106 6.3 Role of TGR5 in the modulation of immune function .............................. 108 6.4 Effects of bile acids on immunological function independently of bile acid receptors ....................................................... 109 6.5 Obstructive jaundice and its impact on immune function ....................... 110 6.6 Role of bile acids and FXR in viral infections .......................................... 111 6.7 Concluding remarks ............................................................................... 111 6.8 References .............................................................................................. 112 7 Bile acids as extrahepatic and interorgan signaling molecules .......................................................................................... 117 7.1 Introduction............................................................................................ 117 7.2 Bile acid–dependent modulation of glucose homeostasis ....................... 118 7.3 Impact of bile acids on energy expenditure ............................................ 120 7.4 Bile acid receptors and immune response............................................... 120 7.5 Role of bile acid receptors in the cardiovascular system ......................... 121 7.6 Role of bile acid receptors in the kidney ................................................. 122 7.7 Bile acid receptors in the central and peripheral nervous system ............ 124 7.8 Summary and future perspectives ........................................................... 124 7.9 References .............................................................................................. 125 8 Disorders of bile duct development.................................................................. 131 8.1 Introduction............................................................................................ 131 8.2 Morphogenesis of the intrahepatic bile duct epithelium: molecular players involved and their relationship with arterial morphogenesis........ 131 8.3 Ductal plate malformation (DPM): definition, clinical heterogeneity, and classification based on animal models ...................... 136 8.4 Cilia in cholangiocytes: a multifunctional transducing system................. 136 8.5 DPM-related cholangiopathies .............................................................. 138 8.6 Alagille’s syndrome (AGS) ...................................................................... 145 8.7 References .............................................................................................. 146

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9 Mutations of the bile salt export pump (BSEP) and multidrug-resistance protein 3 (MDR3) .................................................... 151 9.1 Introduction............................................................................................ 151 9.2 BSEP-related liver diseases ...................................................................... 151 9.3 MDR3-related liver diseases ................................................................... 158 9.4 Treatment of BSEP- and MDR3-associated liver diseases ......................... 162 9.5 Concluding remarks ............................................................................... 162 9.6 References .............................................................................................. 163 10 MRP2 (ABCC2) and disorders of bilirubin handling in liver ............................................................................................... 171 10.1 Introduction ........................................................................................... 171 10.2 The conjugate efflux pump MRP2 in the hepatocyte canalicular membrane ............................................................................ 171 10.3 Formation of unconjugated bilirubin and its uptake into hepatocytes .............................................................. 173 10.4 Formation of bilirubin glucuronides and their transport into bile by MRP2 ..................................................... 174 10.5 Uptake of bilirubin glucuronides into hepatocytes .................................. 174 10.6 MRP3, a basolateral efflux pump, contributes to conjugated hyperbilirubinemia ........................................................... 175 10.7 Genetic disorders and drug-induced inhibition of bilirubin uptake into hepatocytes ....................................................... 175 10.8 Genetic variants of the MRP2 (ABCC2) gene and MRP2 deficiency in Dubin-Johnson syndrome........................................ 176 10.9 Impairment of MRP2 localization in the hepatocyte canalicular membrane .......................................................... 177 10.10 References ............................................................................................. 177 11 Hepatobiliary transport during pregnancy: Cross talk between transporters and hormones ......................................................... 183 11.1 Introduction ........................................................................................... 183 11.2 Estrogens as cholestatic agents ............................................................... 183 11.3 Cholestatic activity of progesterone ........................................................ 185 11.4 Biochemical observations in symptom-free pregnant women ................ 186 11.5 Genetic lessons from congenital cholestasis ........................................... 186 11.6 Transporter variants and cholestasis of pregnancy ................................... 187 11.7 Nuclear receptors and cholestasis of pregnancy ..................................... 189 11.8 Gallstones and pregnancy ...................................................................... 189 11.9 Concluding remarks ............................................................................... 190 11.10 References ............................................................................................. 190 12 Hepatobiliary transport and gallstone formation.............................................. 195 12.1 Introduction ........................................................................................... 195 12.2 Epidemiology and risk factors of cholelithiasis ........................................ 195 12.3 Hepatobiliary transporters and the pathophysiology of gallstone formation............................................................................. 196

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Contents

Genetic variation in hepatobiliary transporter genes and gallstone susceptibility ........................................................... 199 Concluding remarks ............................................................................... 202 References.............................................................................................. 203

13 Molecular basis of primary biliary cirrhosis ..................................................... 207 13.1 Introduction ........................................................................................... 207 13.2 Pathogenesis of PBC ............................................................................... 210 13.3 Concluding remarks ............................................................................... 214 13.4 References.............................................................................................. 215 14 The molecular basis of primary sclerosing cholangitis ...................................... 223 14.1 Introduction ........................................................................................... 223 14.2 Immune-mediated bile duct injury ......................................................... 227 14.3 Toxic bile duct injury ............................................................................. 231 14.4 Fibrosis and cirrhosis .............................................................................. 232 14.5 Cancer development .............................................................................. 233 14.6 Concluding remarks ............................................................................... 233 14.7 References.............................................................................................. 235 15 Drug-induced cholestatic liver injury ............................................................... 241 15.1 Introduction ........................................................................................... 241 15.2 Diagnostic criteria of drug-induced cholestasis ....................................... 241 15.3 Hepatocellular drug concentration ......................................................... 242 15.4 Hepatic bile salt accumulation .............................................................. 244 15.5 Susceptibility to drug-induced cholestasis .............................................. 245 15.6 Concluding remarks ............................................................................... 247 15.7 References.............................................................................................. 247 16 Bile acids and receptors: Therapeutic relevance ............................................... 253 16.1 Introduction ........................................................................................... 253 16.2 Nuclear and membrane BA receptors: general concepts ......................... 253 16.3 Therapeutic potential of BAs ................................................................... 254 16.4 Role of BA receptors in BA homeostasis and bile production: therapeutic implications in cholestasis.................................................... 254 16.5 Role of BA receptors for targeting hepatic inflammation and fibrosis ....................................................................... 257 16.6 Role of BA receptors in the pathogenesis and treatment of gallstone disease ................................................................................ 258 16.7 BA receptors in intestine: therapeutic implications for the gut-liver axis and inflammatory bowel disease........................................ 259 16.8 Role of BAs in lipid metabolism: therapeutic implications for atherosclerosis and nonalcoholic fatty liver disease (NAFLD) ................. 260 16.9 Role of BAs in hepatic glucose metabolism and beyond ........................ 262 16.10 BA receptors in hepatobiliary and colorectal cancer............................... 264 16.11 BA receptors beyond the liver and gastrointestinal tract .......................... 265

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16.12 Concluding remarks ............................................................................... 265 16.13 References ............................................................................................. 266 17 Analysis of bile acids by tandem mass spectrometry......................................... 277 17.1 Introduction ........................................................................................... 277 17.2 Experimental procedures ........................................................................ 278 17.3 Applications ........................................................................................... 281 17.4 Summary ................................................................................................ 286 17.5 References.............................................................................................. 287 Index ...................................................................................................................... 289

Abbreviations

μM

μmol/l

ABC ADPKD AE2 AGS AMA ApoE ARPKD ASBT

ATP-binding cassette Autosomal Dominant Polycystic Kidney Disease (ADPKD) anion exchanger 2 (SLC4A2) Alagille syndrome antimitochondrial autoantibodies apolipoprotein E Autosomal Recessive Polycystic Kidney Disease apical sodium-dependent bile acid transporter (SLC10A2)

BA BDL BSEP BRIC

bile acids bile duct ligation bile salt export pump (ABCB11) benign recurrent intrahepatic cholestasis

CA CBDL CD CDCA CE CFTR CHX CREB CV CYP7A

cholic acid common bile duct ligation Caroli’s disease chenodeoxycholic acid collision energy cystic fibrosis transmembrane conductance regulator (ABCC7) cycloheximide cAMP-responsive element binding protein cone voltage 7 alpha-hydroxylase

DBD DCA DHCA DILI DISC DPM

DNA-binding domain deoxycholic acid dehydrocholic acid drug-induced liver injury death-inducing signaling complex ductal plate malformation

ECM EGFR ER ESE ESI

extracellular matrix epidermal growth factor receptor estrogen receptor exonic splicing enhancer electrospray ionisation

FAK FGF

focal adhesion kinase fibroblast growth factor

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Abbreviations

FOXA2 FXR

forkhead box A2 farnesoid X receptor/bile acid receptor (NR1H4)

G/T G-BAs γGT GLP-1

ratio of glycine / taurine glycine conjugated bile acids gamma-glutamyltranspeptidase glucagon-like peptide-1

HDCA HLA HNF1D HNF3β HNF4α HPLC HSC

hyodeoxycholic acid human leukocyte antigen complex hepatocyte nuclear factor 1α hepatocyte nuclear factor 3β hepatocyte nuclear factor 4α high performance liquid chromatography hepatic stellate cells

IBD ICP IFN IL IS

inflammatory bowel disease intrahepatic cholestasis of pregnancy interferon interleukin internal standard

JNK

c-Jun-N-terminal kinase

KC

Kupffer cells

LCA LCA-3S LC-MS LC-MS/MS LDB LLE LOD LOQ LPAC LRH-1 LXR

lithocholic acid lithocholic acid-3-sulfate liquid chromatography-mass spectrometry liquid chromatography-tandem mass spectrometry ligand-binding domain liquid-liquid extraction limit of detection limit of quantification low phospholipid associated cholelithiasis syndrome liver receptor homolog liver X receptor

m/z MAPK MCA MDR MHC MRP

mass-to-charge mitogen-activated protein kinase muricholic acid multidrug resistance gene major histocompatibility complex multidrug resistance-associated protein

NBD NF-κB NKT

nucleotide binding domain nuclear factor-κB natural killer T cells

Abbreviations

NOX NR NRF2 NTCP

NADPH oxidase nuclear receptor nuclear factor erythroid 2–related factor 2 Na+-taurocholate cotransporting polypeptide (SLC10A1)

OAT OATP OCT OSTα/β

organic anion transporter organic anion-transporting protein organic cation transporter organic solute transporter α/β

PBC PC PEPCK PFIC PI3K PKC PL PNAC PPARα PSC PXR

primary biliary cirrhosis phosphatidylcholine phosphoenolpyrovate carboxykinase progressive familial intrahepatic cholestasis phosphatidylinositol 3-kinase protein kinase C phospholipids parenteral nutrition associated cholestasis peroxisome proliferator-activated receptor alpha primary sclerosing cholangitis pregnane X receptor

RARα ROS RXRα

heterodimer of retinoic acid receptor α reactive oxygen species retinoid X receptor α

SEC Shp1 SNP SOCS SPE SREBP STAT

sinusoidal endothelial cells small heterodimer partner 1 (NROB2) single nucleotide polymorphism suppressor of cytokine signaling solid phase extraction sterol-regulatory element binding protein signal transducer and activator of transcription

T-BAs TLCA TLCS TMD TNF TUDCA

taurine conjugated bile acids taurolithocholic acid taurolithocholylsulfate transmembrane spanning domain tumor necrosis factor tauroursodeoxycholic acid

UDCA UGT1A1 UHPLC ULC UPLC

ursodeoxycholic acid uridine diphosphate glucuronosyltransferase ultra/high performance liquid chromatography ultra liquid chromatography ultra performance liquid chromatography

VEGF VMC

vascular endothelial growth factor Von Meyenburg complex

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1 Physiology of bile formation: Hepatocellular bile salt transporters Bruno Stieger and Peter J. Meier

1.1 Introduction Among the many functions of the liver, bile formation is at center stage. Bile, which enters the upper part of the small intestine from the liver, is essential for the digestion of fat and absorption of lipids. Lipids from the diet contribute significantly to the energy needs of the body. In addition, bile promotes the absorption of fat-soluble vitamins in the small intestine. Disturbed bile formation may therefore lead to a shortage in the energy supply; in small children, for example, this can lead to a failure to thrive. Also, a critical shortage of vitamin K disturbs the blood clotting system and may lead to microhemorrhage. Quantitatively the most relevant parts of bile are bile salts, lipids (owing to their high biliary concentration) organic anions, and small ions such as bicarbonate (1), but proteins are also present. Bile salts (owing to their high biliary concentration) are amphipathic molecules that, together with biliary phospholipids form mixed micelles (2,3). These mixed micelles act as acceptors for poorly water soluble biliary constituents such as cholesterol; importantly, they also reduce the detergent action of bile salts and thereby protect the biliary tree from the toxic action of bile salts (4). High bile salt concentrations within hepatocytes impair the function of mitochondria and are cytotoxic (5,6). In this chapter, bile salt transporters and their role in hepatocellular bile formation are described. Aspects of the transcriptional regulation of the expression of the respective genes, the ontogenesis of the transporters, the sensing of bile salt concentrations in serum and within hepatocytes, as well as the impact of mutations in the genes coding for the involved transporters are covered in their respective chapters. In addition, more detailed information on the sodium-taurocholate cotransporting polypeptide (Ntcp in rodents, NTCP in humans, gene symbol Slc10a1 in rodents, SLC10A1 in humans) and the bile salt export pump (Bsep in rodents, BSEP in humans, gene symbol Abcb11 in rodents, ABCB11 in humans) may be found in the following review (7). Bile salt synthesis occurs in hepatocytes and starts from cholesterol. It requires a multitude of steps catalyzed by a large variety of enzymes (8). The primary bile acids in rats and humans are cholic acid and chenodeoxycholic acid, whereas in mice cholic acid and β-muricholic acid are predominant (9). The conjugation of bile acids with glycine or taurine leads to the bile salts, which have a lower pKa than the bile acids. Enzyme defects in the biosynthesis of bile salts may lead to liver disease in humans, which in some instances is severe (9,10). After their synthesis, newly synthesized bile salts are mixed with the preexisting pool of hepatocytes, followed by their export across the canalicular membrane into the canaliculi. Canaliculi are the starting points of the biliary tree, which ultimately drains via the gallbladder (unless the latter is absent, as in some species or after cholecystectomy in humans) into the duodenum, where they assist in the further processing of the chyme (2,3,10). Bile salts are absorbed to more

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than 90% along the small intestine and transported back to the liver by the portal blood. In the liver, bile salts undergo high extraction and are to a large extent taken up into hepatocytes, from which they are again secreted into bile (2,11–18). In humans, the total bile salt pool constitutes 3–4 g and cycles 6–10 times between the liver and the small intestine, a route called the enterohepatic circulation. This process is very efficient, as humans lose only about 0.5 g/day of bile salts via the feces (19). The size of the bile salt pool is supervised and regulated in the ileum, where the expression of the nuclear receptor farnesoid-X-receptor (FXR) parallels the bile salt load of ileocytes. FXR regulates the transcription of fibroblast growth factor (FGF)15 in rodents or FGF19 in man. FGF15/19 is released into the portal blood and transported to the liver. In the liver, FGF15/19 suppresses, via a plasma membrane receptor−mediated mechanism, the biosynthesis of bile salts (20–22). Low serum levels of FGF19 lead to diarrhea in humans, illustrating the importance of a tight control of the bile salt pool (23,24). Hepatocytes are highly polarized cells with a distinct protein pattern in the blood plasma-facing basolateral and the canalicular membranes (25). This distinct protein expression is also reflected in a specific expression of different bile salt transport proteins in the basolateral and canalicular membranes of hepatocytes (11,12,26). Uptake of bile salts across the basolateral membrane occurs predominantly in a sodium-dependent manner and to a smaller degree in a sodium-independent manner. The sodium-dependent uptake is mediated by NTCP, while the sodium-independent uptake is mediated by members of the organic anion transporting polypeptides (Oatps in rodents, OATPs in humans, gene symbol Slco in rodents, SLCO in humans) (11). Canalicular bile salt secretion is an ATP-dependent process (27–30) mediated by BSEP (11). The transporters in the basolateral and the canalicular membranes need a high degree of coordination in order to keep the intracellular concentration of the potentially cytotoxic bile salts low. Transport of bile salts across hepatocytes is a very rapid process. For example, after intravenous injection, a taurocholate bolus can already be detected in bile after 1 minute; the bolus is almost completely secreted into bile after 10 minutes (31). The mechanistic details of the transcellular movement of bile salts are not known in detail, but it is known that the process involves binding proteins such as, for example, 3α-hydroxysteroid dehydrogenase, glutathione-S-transferase, rat liver fatty acid-binding protein, or human bile salt-binding protein (32,33). These proteins help to keep the intracellular concentration of free bile salts low (it is likely below 1 μM [34]) and to facilitate diffusion of the bile salts after their release from the basolateral uptake transporters to the canalicular membrane. In hepatocytes challenged with a high bile salt load, bile salts, as a consequence of their hydrophobic properties, partly partition into intracellular membranes and vesicles (35–38). Under such conditions, transcellular vesicular transport becomes a relevant component of bile salt flux across hepatocytes (31). This route seems not to be relevant under a normal physiologic bile salt load of hepatocytes (32,39). Vesicular mechanisms are also important for the regulation of the carrier density in the canalicular membrane. As a result, microtubules provide the tracks for vesicles and are functionally regulated by bile salts (40,41). Consequently the transcellular transport of bile salts by vesicles is not easily distinguished from the vesicular insertion of transporters into the cananlicular membrane. Any situation where the flow of bile salts is disturbed is called cholestasis, which can lead to the intracellular accumulation of bile salts in hepatocytes. Increased hepatocellular concentrations of bile salts can impair the functioning of the mitochondria and, in

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turn, become cytotoxic to hepatocytes (5,6). If bile flow is not restored to normal, either spontaneously or by therapeutic intervention, hepatocytes will be severely impaired and liver disease will ensue. In turn, serum bile salts and liver enzymes in the plasma will increase.

1.2 Sodium-dependent bile salt uptake into hepatocytes 1.2.1 Molecular properties Rat Ntcp was the first sodium-dependent bile salt transporter to be identified at the molecular level; it was cloned from rat liver by expression cloning in Xenopus laevis oocytes (42). Subsequently, orthologues from other species such as humans (43), mice (44), and rabbits (45) were cloned and additional orthologues from more species are predicted (46). These orthologues are all members of the gene family 10 (SLC10A) of the solute carrier superfamily (47). NTCP is the first member (SLC10A1), of the SLC10A family, which in addition hosts the ileal apical sodium bile salt transporter (ASBT, SLC10A2) and the steroid sulfate transporter (SOAT, SLC10A6) as well as four orphan transporters. The genes coding for rat and human NTCP vary considerably in size, including 21.4 and 13.6 kb. Rat Slc10a1 localizes to chromosome 6q24 (48) and human SLC10A1 to chromosome 12q24 (43). Mouse Slc10a1 extends to 12.5 kb and is found on chromosome 12qD1 (49). This gene is transcribed into two alternatively spliced variants (44). Rat Ntcp is predicted to have a molecular weight (MW) of around 39 kDa and its deglycosylated form in X. laevis oocytes has an MW around 33 kDa with no cleavable signal sequence (42). In vitro site-directed mutagenesis showed the putative N-linked glycosylation sites near the N-terminus to be glycosylated. Western blotting of isolated liver plasma membrane vesicles revealed a MW of 51 kDa, and immunofluorescence of isolated rat hepatocytes pointed to an intracellular localization of the C-terminus (50,51). In addition to glycosylation, phosphorylation and ubiquitination have been found as posttranslational modifications of rat Ntcp (52–54). The crystal structure of NTCP has not yet been determined. The extracellular localization of the N-terminus and the intracellular localization of the C-terminus suggest an odd number of transmembrane-spanning domains. Site-directed mutagenesis, including translation/insertion scanning and alanine insertion scanning of NTCP, provided evidence for seven transmembrane spanning domains and helices seven and eight assembled in an extracellular loop close to the plasma membrane, totaling nine helices (55). Later studies found helices seven and eight to be essential for the function of NTCP (56). Different methods, including coimmunoprecipitation experiments and fluorescence resonance energy transfer, strongly suggest that NTCP forms a homodimer and may also form heterodimers with SLC10A4 and SLC10A6 (57). Earlier studies with radiation inactivation found a functional MW of 170 kDa for sodium-dependent bile salt uptake into basolateral rat liver plasma membrane vesicles (58), which is in line with these new findings. Recently the crystal structure of a bacterial homologue of ASBT with 2.2 Å resolution identified a core domain of six helices with two sodium ions and an additional four helices forming a panel-like flat domain (59). It should be noted that this bacterial homologue of ASBT has the N- and the C-termini at the intracellular side of the plasma membrane. Nevertheless, this structure can certainly help to refine the structure of mammalian NTCP and ASBT and to obtain a crystal structure of NTCP.

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K쎵 Na쎵 NTCP

2 Na쎵 cBS앥

~ 앥40 mV

cBS앥

BSEP

cBS앥

uBA앥 OATP1B1 X앥 OATP1B3

uBA앥 X앥

OSTa /OSTb MRP3

cBS 앥

MRP4

uBA: unconjugated bile acid cBS: conjugated bile salt X: anion

Fig. 1.1: Bile salts transporters in hepatocytes. Uptake of conjugated bile salts (cBS) from the portal venous blood across the basolateral membrane into the hepatocyte is predominately mediated in a sodium-dependent manner by NTCP. Sodium-independent transport of unconjugated bile acids (uBA) is facilitated by members of the OATP family. Bile salts are excreted into bile across the canalicular membrane by the bile salt export pump (BSEP). The basolateral hepatocyte membrane also contains bile salt export transporters, such as MRP4, MRP3 and OSTα /β. These effl ux pumps are upregulated under cholestatic conditions and may protect hepatocytes from a toxic bile salt load (see text for details). uBA = unconjugated bile acid; cBS = conjugated bile salt; X = anion.

1.2.2 Cellular expression and tissue distribution Rat Ntcp and human NTCP, as assessed by immunofluorescence studies, are expressed specifically in hepatocytes in the basolateral membrane (fFig. 1.1)(50,51,60). NTCP is expressed throughout the liver acini. This complements an earlier finding demonstrating efficient taurocholate uptake in the periportal and pericentral areas of the rat liver acini (61). Tissue distribution analysis by Northern blotting demonstrated expression of Ntcp in the kidney as well as the liver (42). This finding has not yet been verified at the protein level. Expression of Ntcp has also been reported in the luminal membrane of pancreatic acinar cells (62). NTCP expression in the placenta, where a fetal-to-maternal bile salt gradient prevails, is species-specific. NTCP expression is absent in humans (62–64), while low levels of Ntcp mRNA have been reported in rat placenta (62,65,66). Tissue distribution of NTCP expression is subject to regulation by DNA-methylation of the SLC10A1 gene (67).

1.2.3 Transport properties of NTCP Coexpression of total rat liver mRNA with antisense oligonucleotides against Ntcp leads to a complete knockdown of sodium-dependent bile salt uptake into X. laevis oocytes (68). This suggests that Ntcp is the major or likely the only sodium-dependent uptake system for bile salts. Comparison of Km values for Ntcp and sodium-dependent bile salt

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transport activity obtained from studies with perfused rat liver, primary cultured hepatocytes and isolated membrane vesicles, reveals affinities from 12 to 61 μM (7). This compares favorably with Km values of Ntcp as determined in heterologous expression systems such as X. laevis oocytes, COS-7 HPTC, McArdle RH-7777, CHO, HepG2, or MDCK cells ranging from 8 to 30 μM (7). Comparable Km values for Ntcp and sodiumdependent transport activity can be taken as evidence for a single transporter mediating hepatocellular sodium-dependent bile salt uptake. However, this view has been challenged and microsomal epoxide hydrolase has been postulated to constitute an additional sodium-dependent bile salt transporter (69). Other studies could not confirm this additional transport activity (70,71). In support of a role of microsomal epoxide hydrolase in bile salt uptake, a patient with hypercholanemia was reported to have normal NTCP expression but an altered promoter for micrososmal epoxide hydrolase (72). Analysis of a liver biopsy taken from this patient demonstrated a marked reduction in the activity of the promoter and a massive reduction in micrososmal epoxide hydrolase protein. Mice with a knocked out gene for microsomal epoxide hydrolase display a normal phenotype (73), providing evidence for a minor role of this enzyme in the bile salt homeostasis of mice. Generating mice with a disrupted Slc10a1 gene might help to resolve these discrepancies. Ntcp is strictly dependent on sodium and transports two sodium ions with every bile salt molecule transported (74). Consequently Ntcp has been reported as an electrogenic transporter (75). Mechanistically, these are hallmarks of a secondary active transporter, which requires an out-to-in sodium gradient. This sodium gradient is maintained by the Na+, K+-ATPase, which is expressed in the basolateral plasma membrane of rat hepatocytes (76–78). As a consequence of this transport mechanism, Ntcp is able to accumulate bile salts in hepatocytes against a concentration gradient. The kinetic properties of Ntcp are affected by the presence of albumin, which lowers the Km of Ntcp and stimulates taurocholate transport activity (79,80). Expression of total rat liver mRNA followed by comparison of sodium-dependent transport activity of Ntcp in the presence and absence of albumin in X. laevis oocytes (81) and characterization of human NTCP in CHO cells (82) confirmed this observation at the molecular level. The substrate specificity of Ntcps has recently been summarized (7). Ntcps have a rather narrow substrate specificity with a preference for conjugated over unconjugated bile salts. Mouse Ntcp is a very poor transporter of unconjugated bile salts, since mice with disrupted Slco1 genes have massively elevated plasma levels of unconjugated bile salts (84). In many cases, trihydroxy bile salts have a higher affinity than dihydroxy bile salts for Ntcp (7). Next to bile salts, steroid metabolites such as estrone-3-sulfate (7) or the 3-O-sulfate conjugate of 17α-ethinylestradiol (85) and bromosulphophthalein (7) are substrates. Ntcp is capable of transporting experimental drugs (7) and, importantly, human NTCP but not rat Ntcp mediates transport of rosuvastatin (7). Lately, human NTCP has also been demonstrated to transport atorvastatin (87). High-throughput screening is also gaining more and more relevance for transporter studies during drug development. To this end, fluorescent bile salts present an option for assaying bile salt transporters. Several fluorescently labeled substrates have been developed and tested on Ntcp, whereby cholylgycylfluorescein and chenodeoxycholyllysylNBD have been demonstrated to be a substrate of rat Ntcp (88), NBD-cholyltaurine of rat Ntcp (89), and cholylglycylamidofluorescein as well as chenodeoxycholylglylamidofluorescein of human NTCP (90,91). In contrast, cholyl-L-lysyl-fluorescein (CLF) is

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not transported by human NTCP (92), which demonstrates the relevance of the bile salt side chain for substrate recognition by Ntcps (93). Transport proteins are important for the hepatocellular uptake of markers used for monitoring liver function (94). NTCP has been found to transport with high affinity the magnetic resonance imaging (MRI) contrast agent Gd-EOB-DTPA (95) and the classic liver function marker indocyanine green (96). In addition to transporting a variety of substrates, Ntcp is inhibited by several drugs and compounds. While cyclosporine is a potent inhibitor of rat Ntcp (97) and human NTCP (90) with an IC50 value of 1 μM (82), it is not transported by rat Ntcp (97). This is one of many examples where an inhibitor of a transport system is not also a substrate of the same transporter. An additional inhibitor of rat Ntcp is bumetanide (89,97). Additional drugs reported to inhibit NTCP include, among others β-blockers, antihypertensives, antibiotics, and antivirals (7). Importantly, the fluorescent bile salt CLF is also a potent inhibitor of NTCP (92). In principle, inhibition of NTCP should lead to an elevation of bile salt concentrations in plasma. However, bile salts can also enter hepatocytes via OATPs expressed in the basolateral plasma membrane of hepatocytes. This concept is complicated by the fact that many inhibitors of NTCP are also substrates and/or inhibitors of the hepatocellular OATPs (7). Experimental verification of this scenario would require pharmacokinetic studies with model drugs and determination of bile salt patterns and concentrations in the plasma.

1.2.4 Pathophysiology To date, no severe human disease related to a mutation in the SCL10A1 gene is known. However, primary hypercholanemia has been described in patients with a normal SLC10A1 gene (98) and in those with mutations in the tight junction protein 2 (99). Information on NTCP in patients with liver disease is available on only a small number of patients and consequently limited. Progressive familial intrahepatic cholestasis (PFIC) leads to a downregulation of NTCP at the protein but not the mRNA level (100). In these patients, therefore, posttranslational regulation is the cause of lowered NTCP expression. The SLC10A1 gene is indirectly regulated by the bile salt sensor farnesoid X receptor (FXR) (101), activation of which leads to repression. Patients with biliary atresia leading to progressive obstructive choleastasis indeed have low NTCP mRNA levels (98,102). The mRNA level shows a negative correlation with serum bile salt levels (98,103) and increases after restoration of bile flow (98). In patients with early stages of primary biliary cirrhosis (PBC) or with chronic viral hepatitis C, NTCP mRNA is unchanged (103–105); in late stages of PBC, mRNA coding for NTCP is downregulated (104). Inflammatory liver disease (103) and treatment of human liver slices with LPS (106) both repress NTCP expression. In the tumor tissue of patients with hepatocellular carcinoma, NTCP expression is lower than in the surrounding normal tissue (60,107). In patients having low serum bilirubin levels after surgery, NTCP mRNA is lower than in those with high postoperative bilirubin (108). Liver transplantation is a major stress for the transplanted organ. In this situation, expression of SLC10A1 at the mRNA level increases considerably posttransplant (109). Animal models of liver disease have made it possible to investigate the expression of Ntcp in more detail. In essence, these experiments have reproduced the findings observed in biopsies from patients (7). Liver disease, in particular cholestatic liver disease,

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leads generally to a downregulation of NTCP. This reflects a protective mechanism of hepatocytes against the toxic action of intracellular bile salts, since downregulation of NTCP lowers the hepatocellular bile salt load. Finally, experiments have provided evidence for a regulation of Ntcp by phosphorylation/dephosphorylation, leading to a cycling between the plasma membrane and an endosomal compartment (110). In a model cell line, translocation of NTCP to the basolateral membrane involves Rab4 (111).

1.2.5 Pharmacogenomics So far, only limited information is available on the impact of variants of SLC10A1 on the function of NTCP. In a small Japanese cohort, several single-nucleotide polymorphisms (SNPs) were identified without functional characterization (112). In a cohort of 50 European Americans, 50 African Americans, and 50 Chinese Americans, several SNPs including four nonsynonymous SNPs were identified: p.I223T, p.S267F, p.I279T, and p.K314E (113). These variants were functionally compared with the NTCP*1 variant in HeLa cells. All except the p.I223T variant were targeted to the plasma membrane. The p.S267F variant practically lacked cholate and taurocholate transport, while all the other variants displayed normal transport for cholate, taurocholate, and estronhe-3sulfate. In a later study, all variants were found to transport rosuvastatin (114), whereas the p.S267F variant had a much lower Km (3 μM) than the NTCP*1 variant (65 μM) for rosuvastatin. Later, NTCP, in particular NTCP*2, was also found to transport atorvastatin (87). Currently no genotype-phenotype correlations have been published regarding the physiologic or clinical consequences of NTCP variants.

1.3 Sodium-independent bile salt uptake into hepatocytes 1.3.1 Role in physiology The molecular properties of OATPs are beyond the scope of this chapter; they are reviewed in great detail elsewhere (115–119). Human hepatocytes express OATP1B1, OATP1B3, and OATP2B1, which are all localized in the basolateral plasma membrane (11,12). Among these three transporters, OATP2B1 has so far a more narrow substrate specificity and is not involved in bile salt transport (11,12). A hallmark of these and other OATPs is their broad substrate specificity, including many drugs. The substrate specificity of the various OATPs has been detailed by several investigators (115–118). With respect to bile salt transport, OATPs have a preference for unconjugated over conjugated bile salts (71). This was recently demonstrated in genetically modified mice, where the Slco1a and Slco1b genes were inactivated (84). These mice have elevated total bile salts. Analysis of the bile salt pattern revealed that conjugated bile salts are normal in these animals, whereas the unconjugated bile salts are 13-fold greater as compared with the wild-type mice (84). Another research group reported that mice lacking a functional Slco1b2 gene have up to 45-fold increased serum levels of bile salts (120). Mouse Oatp1b2 is the murine orthologue of human OATP1B1 and OATP1B3 (115) and is transcribed into splice variants (121). Hence the data from the Oatp1b2 knockout mouse support the concept that among the OATPs, OATP1B1 is the major sodium-independent bile salt uptake system (12). The transport mechanism of OATPs

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is not known in detail. It is assumed that they act as organic anion exchangers (116). Many OATPs exchange their substrates for bicarbonate and are activated by a low extracellular pH (122). Indirect evidence suggests that some OATPs may work against a concentration gradient (94).

1.3.2 Pathophysiology OATP1B1 was found to be downregulated in patients with inflammatory cholestasis (103) as well as in those with advanced stages of PBC (105) and with primary sclerosing cholangitis (PSC) (105,123). Also, PFIC2 and PFIC3 lead to a downregulation of OATP1B1 and OATP1B3 (100). In patients with hepatocellular carcinoma, expression of OATP1B1 (107,124), OATP1B3 (125,126), and OATP2B1 (127) is reduced. However, the extent of this reduction may vary between patients (125,128). In animal models of inflammatory cholestasis and in ethinylestradiol-induced cholestasis as a model of estrogen-induced cholestasis, the expression of hepatic Oatps (namely Oatp1a1, Oatp1a4, and Oatp2b1) parallels the expression pattern of Ntcp – that is, they are downregulated (129). However, in the model of obstructive cholestasis, only Oatp1a1 is downregulated while Oatp1a4 and Oatp2b1 remain unaltered (129).

1.3.3 Pharmacogenomics Owing to the role of OATP variants in drug disposition and the pharmacodynamic effects of drugs, a large number of pharmacogenomic studies on OATPs have been published. Such studies are reviewed in in various publications (118,130–133). Pharmacogenetic studies of the effects of OATP variants on serum bile salt levels and bile salt secretion in humans have to our knowledge so far not been published. However, variants of hepatic SCLO genes have been associated with increased serum bilirubin levels and are summarized in a recent overview (133).

1.4 Bile salt export across the canalicular membrane 1.4.1 Molecular properties Bsep was first cloned from rat liver (134) and is highly similar to a fragment isolated from pig liver called sister of p-glycoprotein (135). Bsep is a member of the B family of the ATP-binding cassette (ABC), namely ABCB11 (86). With the use of a positional cloning approach in families with PFIC2, the genetic defect of this disease was localized to the ABCB11 gene, situated on the human chromosome 2q24–31. ABCB11 includes 28 exons and encodes 1321 amino acids. Mouse and rat Bseps have the same length and show an identity of more than 80% with human BSEP, with the highest identities in the two nucleotide binding domains (7,136). Rat Bsep has four N-linked potential glycosylation sites, all of which have linked sugar chains (137). In addition, BSEP is phosphorylated (136) and ubiquitinylated (138). The detailed molecular structure of BSEP is not yet known. BSEP is predicted to consist of two transmembrane units each with six transmembrane helices (134,139). The first transmembrane unit is linked by a large cytoplasmic loop harboring the first ATP-binding site to the second transmembrane unit followed by an additional large

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cytoplasmic domain harboring the second ATP binding site (134). This basic assembly of modules is typical for ABC transporters (140).

1.4.2 Cellular expression and tissue distribution Bsep is specifically localized to the canalicular domain of hepatocytes and displays homogenous expression throughout the acinus (fFig. 1.1)(134). Immunogold labeling has revealed microvillar expression of Bsep, but it is absent from the intermicrovillar connecting membranes, indicating the presence of microdomains in the canalicular membrane. Bsep is found in detergent-inducible (141) and bile salt−inducible microdomains (142). In addition, subcanalicular vesicles stain positive on immunohistochemistry (134,143). Northern blot analysis has identified a predominant if not exclusive Bsep mRNA expression in liver (7,86). Using PCR-based methodology, mRNA for BSEP has also been described in brain, small and large intestine, kidney, and testis. However, these findings were to some extent conflicting and species-dependent (86). In summary, a consistently high BSEP expression has been reported for the liver.

1.4.3 Transport properties Studies from several laboratories using isolated rat canalicular plasma membrane vesicles have identified an ATP-dependent transport activity for bile salts (27–30). This transport activity requires hydrolysis of ATP and is electrogenic (7). The bile salt transport properties of cloned rat Bsep characterized in the Sf9 cell expression system has revealed comparable transport properties. Rat, mouse, and human BSEPs transport mainly bile salts (7). With respect to unconjugated bile acids, cholate has been studied as a BSEP substrate. Rat Bsep shows a low transport activity for cholate (134), whereas the data on human BSEP are conflicting and depend on the expression system. A polar cell expression system using LLC-PK1 cells showed that BSEP mediates cholate transport (144); however, the Sf9 cell expression system shows no BSEP-mediated cholate transport (144,145). The latter fact is supported by findings in patients with a defect in bile salt conjugation who have practically no bile acids in their bile (99). The fluorescently labeled bile salts cholylglycylamidofluorescein and chenodeoxycholylglylamidofluorescein are substrates of BSEP (144), whereas CLF is not a BSEP substrate but an inhibitor of BSEP (92). Hence not only for NTCP but also for BSEP the side chain of bile salts is a structural determinant of transport. CLF, in contrast to bile salts, carries two negative charges on the side chain and is a substrate for the canalicular multidrug resistance−associated protein 2 (Mrp2) from rat and mouse (92). Taurolithocholate-3 sulfate also carries two negative charges on the side chain and is not transported by rat Bsep in the Sf9 cell expression system (146,147) but very weakly in the HEK cell expression system (148). However, this bile salt is a good substrate for human BSEP (148). A comparison of selected substrates with rat Bsep and Mrp2 revealed no overlap (146), suggesting that Mrp2 cannot compensate for a lacking function of Bsep. This is indeed the case, as mutations in the ABCB11 gene rendering BSEP nonfunctional lead to PFIC2, which is a severe liver disease (149). The intrinsic clearances of mouse, rat, and human BSEPs are comparable (145), supporting a high conservation of BSEP between different mammalian species also at the functional level.

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Impairment of BSEP function can also occur from its inhibition. The consequence is again an impairment of bile flow and consequent cholestasis. This has been demonstrated, for example, for cyclosporine, which is associated with cholestasis in humans (150), inhibits bile flow in rats (151), and is a competitive inhibitor of rat and human BSEP (146,152). Many drugs have the potential of BSEP inhibition (83,153,154) and can lead to drug-induced cholestasis, an acquired form of liver disease. A different form of acquired liver disease is cholestasis of pregnancy, which is associated with steroid (estrogen or progesterone) metabolites (155). In animal experiments, the estrogen metabolite estradiol-17β-glucuronide (E17βG) has been demonstrated to be strongly cholestatic and was found to be dependent on Mrp2 expression in rats (156). E17βG alone does not inhibit rat Bsep as expressed in Sf9 cells; this occurs only if Mrp2 is coexpressed in the same vesicles (146). Hence E17βG is an indirect inhibitor of Bsep, potentially acting from the trans side of the membrane after its secretion (83,155). Indirect Bsep inhibition was also reported for sulfated progesterone metabolites (157). Bosentan can lead to cholestasis and has also been demonstrated in the Sf9 cell expression system to be a BSEP inhibitor (145,158). In studies with rats, bosentan stimulated rather than inhibited bile flow (159). This stimulation of bile flow was dependent on the expression of Mrp2 and was mainly due to an increase of the bile salt−independent bile flow. Bosentan stimulation of the bile flow leads to reduced biliary lipid secretion. This, in turn, may affect the composition of the canalicular membrane and hence also the functioning of Bsep. In summary, BSEP is subject to inhibition by various compounds with different and sometimes complex mechanisms.

1.4.4 Pathophysiology Functional impairment of BSEP leads to liver disease, which can be either acquired or inherited. Acquired forms of liver disease due to BSEP malfunction can be caused, among others, by inflammatory liver diseases (such as sepsis of acute and chronic viral hepatitis), inhibition of BSEP by drugs, xenobiotics, or endogenous metabolites and obstructive cholestasis. PBC and PSC cholangitis can also be considered as acquired BSEP impairment, since the primary cause of these diseases is not related to BSEP. Examples of BSEP expression in specimens from patients with various forms of liver disease have been published, as, for example, from cholestatic patients with percutaneous biliary drainage, where mRNA for BSEP was significantly downregulated in poorly drained patients in comparison with well-drained patients or control biopsies (160). Immunohistochemical staining of BSEP displayed fuzzy extensions of BSEP into the subcananlicular cytoplasm in addition to the canalicular membrane. Subcanalicular staining was more pronounced in poorly drained patients. In children with biliary atresia, mRNA of BSEP was downregulated at early stages but normal at late stages (102). Immunohistochemical analysis of biopsies from the same patients displayed comparable staining at all stages. In patients with inflammatory cholestasis and in early stages of PBC, BSEP expression was reported to be downregulated (103–105). However, another report published an upregulation of BSEP and conserved immunostaining in PBC (161). Use of the LPS model for inflammatory liver disease in human liver slices (106) or treatment of primary human hepatocytes with interleukin 1β (162) also leads to BSEP downregulation. In patients with cholesterol calculi, BSEP expression at the mRNA and protein levels was reported to be downregulated (163). In patients treated with

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ursodeoxycholic acid for gallstone disease, BSEP was upregulated at the protein but not at the mRNA level (164). Biopsies from hepatocellular carcinomas have revealed a highly variant and inconsistent pattern of BSEP expression. However, there seems to be a general trend of BSEP downregulation (107,165). After liver transplantation, bile salt secretion increases, as does mRNA for BSEP (109). In patients who have undergone liver resection and had low postoperative levels of serum bilirubin, BSEP mRNA is lower than in patients with high postoperative bilirubin (108). Studies in animal models of liver disease such as obstructive cholestasis or ethinylestradiol-induced cholestasis showed practically unchanged Bsep expression in total liver (7,86). Functionally, ethinylestradiol treatment leads to a downregulation of ATP-dependent taurocholate transport activity in canalicular vesicles (166) and, after acute treatment with E17βG, Bsep is retrieved into a subapical vesicular system (167). The rate-limiting step of hepatocellular bile salt transport is localized to the canalicular membrane (168). Consequently Bsep expression in the canalicular membrane is extensively regulated by insertion/retrieval in addition to posttranslational modification. Bsep internalization can be induced in animals by treatment with lithocholate or taurolithocholate, but it can be prevented by prior treatment with cAMP or tauroursodeoxycholate (7). Bile salt secretion can be stimulated in normal physiologic conditions – that is, independent of the biosynthesis of Bsep but involving an intact microtubular system (7). Bsep is recruited from a preexisting intracellular pool, which is at the crossroads of the transport of newly synthesized Bsep to the cananlicular membrane and of constitutive cycling of Bsep between subapical compartment(s) and the canaliculus (169). It should be noted that in obstructive cholestasis also, canalicular proteins other than transporters are reversibly internalized into a subapical compartment (170). Other models of liver disease, such as sepsis or toxic liver injury, tend to reproduce findings in human liver. All together, the expression of BSEP in liver disease is much less affected than Ntcp. Keeping Bsep at a high expression is an additional protective mechanism of hepatocytes in liver diseases to lower the intracellular concentration of bile salts (7).

1.4.5 Pharmacogenetics of ABCB11 Because BSEP is a bile salt rather than a drug transporter, only limited information about variants of the ABCB11 gene has been published so far and information on their functional consequences is even more scarce. A study including 48 Japanese individuals listed 82 single-nucleotide polymorphisms of the ABCB11 gene without further functional characterization (171). Many of the reported SNPs are rather rare, while only the c.1331T>C and the c.2029A>G nonsynonymous SNPs have consistently been reported with frequencies higher than 0.5% in different unrelated cohorts (171–175). The frequencies of the SNPs differ between ethnicities. The c.1331T>C SNP is the most common variant of ABCB11 and was found to have no functional impact on BSEP (176). The p.I206V, p.Q558H, p.N591S, and p.E1186K variants have reduced transport activity, and the p.E1186K variant displays reduced cell-surface expression in a heterologous expression system (175). In a human liver tissue bank of 110 individuals, the p.444A (c.1331C) variant tended toward lower expression levels. The BSEP protein expression level varied about 20-fold in this cohort (177). In a different cohort, a 19-fold variation of BSEP mRNA and 31-fold variation of BSEP protein was seen (175). The c.1331C variant was found to be a susceptibility factor for drug-induced cholestasis (83,176) and

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intrahepatic cholestasis of pregnancy (155,178,179) in independent cohorts. In a large cohort, a polymorphism in the vicinity the ABCB11 gene was linked to fasting glucose levels, and two additional SNPs were found in intron 19 of ABCB11. These new SNPs were also strongly associated with fasting glucose levels. Two additional studies found an association of an SNP in the ABCB11 gene with serum cholesterol, triglycerides, and apoA levels (180). In two Asian populations, an SNP in the chromosome region comprising the genes of both glucose-6-phosphate catalytic subunit-related protein and BSEP (181) was found to be associated with fasting glucose levels. These association studies support the role of BSEP in body energy homeostasis.

1.5 Bile salt salvage systems As outlined in sections 1.2.4 and 1.3.2, hepatocellular uptake systems for bile salts are downregulated and BSEP is maintained to protect hepatocytes from the intracellular accumulation of potentially toxic bile salts. In addition, three transport systems capable of mediating basolateral bile salt efflux back into the portal blood may contribute to the protection of hepatocytes from a potentially toxic bile salt load. The basolateral rat Mrp3 mediates bile salt transport in heterologous expression systems with very low affinity (182) and human MRP3 with higher affinity (183,184). Biopsies from patients with cholestatic liver disease showed no change in MRP3 mRNA (103), while in another study with cholestatic patients undergoing biliary drainage MRP3 was found to be upregulated (160,185). Similarly, MRP3 expression is increased in patients with PBC (105), which was not confirmed in patients with early stages of PBC (104). In PFIC patients, MRP3 showed no upregulation (100). In rodents, cholestasis leads to a massive upregulation of rat Mrp3 (186,187) and mouse Mrp3 (188). Hence, MRP3 could act as bile salt salvage system in liver diseases. Human MRP4 mediates bile salt transport in cotransport with reduced glutathione (189). In PFIC and cholestatic patients, MRP4 is upregulated (100,190,191). In patients with PBC, MRP4 protein but not mRNA is upregulated (192). These findings are reproduced in cholestatic rats (193). Ostα /Ostβ is a heterodimeric transporter and the major basolateral efflux transporter in ileal enterocytes, which is also expressed in the basolateral membrane of hepatocytes (17,194). Ostα /Ostβ is upregulated in cholestatic rats and in humans with PBC (185,195). However, the functional role of this upregulation is not clear, since Ostα /Ostβ functions as a heterodimer. But in liver, the mRNA for Ostβ is predominantly upregulated under cholestatic conditions (194).

1.6 Concluding remarks NTCP and BSEP are the most important hepatocellular bile salt transporters. Based on their strategic location in hepatocytes, they are the key transporters in liver for maintaining the enterohepatic circulation. As a consequence, both transporters are extensively regulated in health and disease. These two transporters are in addition complemented by the basolateral OATPs, which are able to take up unconjugated bile acids. These bile acids are produced by the bacterial gut flora, which deconjugate bile salts. In addition, there is an export system in the basolateral membrane of hepatocytes that can mediate bile salt export from hepatocytes in patients with liver disease. This intricate network of

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transporters primarily maintains the enterohepatic circulation of bile salts and, in liver disease, protects hepatocytes from potentially toxic bile salts.

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121. Meyer zu Schwabedissen HE, Ware JA, Tirona RG, et al. Identification, expression, and functional characterization of full-length and splice variants of murine organic anion transporting polypeptide 1b2. Mol. Pharm. 2009;6:1790–7. 122. Leuthold S, Hagenbuch B, Mohebbi N, et al. Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters. Am. J. Physiol. Cell Physiol. 2009;296:C570–82. 123. Oswald M, Kullak-Ublick GA, Paumgartner G, et al. Expression of hepatic transporters OATP-C and MRP2 in primary sclerosing cholangitis. Liver 2001;21:247–53. 124. Kinoshita M, Miyata M. Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci. Hepatology 2002;36:433–8. 125. Cui Y, Konig J, Nies AT, et al. Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma. Lab. Invest. 2003;83:527–38. 126. Vavricka SR, Jung D, Fried M, et al. The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma. J. Hepatol. 2004;40:212–18. 127. Wlcek K, Svoboda M, Riha J, et al. The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer. Cancer Biol. Ther. 2011;11:801–11. 128. Vasuri F, Golfieri R, Fiorentino M, et al. OATP 1B1/1B3 expression in hepatocellular carcinomas treated with orthotopic liver transplantation. Virchows Arch. 2011;459:141–6. 129. Geier A, Wagner M, Dietrich CG, et al. Principles of hepatic organic anion transporter regulation during cholestasis, inflammation and liver regeneration. Biochim. Biophys. Acta 2007;1773:283–308. 130. Zair ZM, Eloranta JJ, Stieger B, et al. Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney. Pharmacogenomics 2008;9:597–624. 131. Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. 2009;158:693–705. 132. Fahrmayr C, Fromm MF, Konig J. Hepatic OATP and OCT uptake transporters: their role for drug-drug interactions and pharmacogenetic aspects. Drug Metab. Rev. 2010;42:380–401. 133. Stieger B, Meier PJ. Pharmacogenetics of drug transporters in the enterohepatic circulation. Pharmacogenomics 2011;12:611–31. 134. Gerloff T, Stieger B, Hagenbuch B, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J. Biol. Chem. 1998;273:10046–50. 135. Childs S, Yeh RL, Georges E, et al. Identifi cation of a sister gene to P-glycoprotein. Cancer Res. 1995;55:2029–34. 136. Noe J, Hagenbuch B, Meier PJ, et al. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system. Hepatology 2001;33:1223–31. 137. Mochizuki K, Kagawa T, Numari A, et al. Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2007;292:G818–28. 138. Hayashi H, Sugiyama Y. Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11). Mol. Pharmacol. 2009;75:143–50. 139. Thompson R, Strautnieks S. Inherited disorders of transport in the liver. Curr. Opin. Genet. Dev. 2000;10:310–3. 140. Borst P, Elferink RO. Mammalian ABC transporters in health and disease. Annu. Rev. Biochem. 2002;71:537–92.

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1 Physiology of bile formation: Hepatocellular bile salt transporters

141. Ismair MG, Hausler S, Stuermer CA, et al. ABC-transporters are localized in caveolin-1positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes. Hepatology 2009;49:1673–82. 142. Guyot C, Stieger B. Interaction of bile salts with rat canalicular membrane vesicles: evidence for bile salt resistant microdomains. J. Hepatol. 2011;55:1368–76. 143. Dombrowski F, Stieger B, Beuers U. Tauroursodeoxycholic acid inserts the bile salt export pump into canalicular membranes of cholestatic rat liver. Lab. Invest. 2006;86:166–74. 144. Mita S, Suzuki H, Akita H, et al. Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep. Am. J. Physiol. Gastrointest. Liver Physiol. 2006;290:G550–6. 145. Noe J, Stieger B, Meier PJ. Functional expression of the canalicular bile salt export pump of human liver. Gastroenterology 2002;123:1659–66. 146. Stieger B, Fattinger K, Madon J, et al. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology 2000;118:422–30. 147. Akita H, Suzuki H, Ito K, et al. Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump. Biochim. Biophys. Acta 2001;1511:7–16. 148. Hayashi H, Takada T, Suzuki H, et al. Transport by vesicles of glycine- and taurineconjugated bile salts and taurolithocholate 3-sulfate: a comparison of human BSEP with rat Bsep. Biochim. Biophys. Acta 2005;1738:54–62. 149. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat. Genet. 1998;20:233–8. 150. Arias IM. Cyclosporin, the biology of the bile canaliculus, and cholestasis. Gastroenterology 1993;104:1558–60. 151. Bohme M, Muller M, Leier I, et al. Cholestasis caused by inhibition of the adenosine triphosphate-dependent bile salt transport in rat liver. Gastroenterology 1994;107: 255–65. 152. Byrne JA, Strautnieks SS, Mieli-Vergani G, et al. The human bile salt export pump: characterization of substrate specifi city and identification of inhibitors. Gastroenterology 2002;123:1649–58. 153. Morgan RE, Trauner M, van Staden CJ, et al. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 2010;118:485–500. 154. Dawson S, Stahl S, Paul N, et al. In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab. Dispos. 2012;40:130–8. 155. Pauli-Magnus C, Meier PJ, Stieger B. Genetic determinants of drug-induced cholestasis and intrahepatic cholestasis of pregnancy. Semin. Liver Dis. 2010;30:147–59. 156. Huang L, Smit JW, Meijer DK, et al. Mrp2 is essential for estradiol-17beta(beta-Dglucuronide)-induced cholestasis in rats. Hepatology 2000;32:66–72. 157. Vallejo M, Briz O, Serrano MA, et al. Potential role of trans-inhibition of the bile salt export pump by progesterone metabolites in the etiopathogenesis of intrahepatic cholestasis of pregnancy. J. Hepatol. 2006;44:1150–7. 158. Fattinger K, Funk C, Pantze M, et al. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 2001;69:223–31. 159. Fouassier L, Kinnman N, Lefevre G, et al. Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan. J. Hepatol. 2002;37:184–91. 160. Shoda J, Kano M, Oda K, et al. The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their

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association with the impairment of biliary secretory function. Am. J. Gastroenterol. 2001;96:3368–78. Ros JE, Libbrecht L, Geuken M, et al. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease. J. Pathol. 2003;200:553–60. Le VM, Gripon P, Stieger B, et al. Down-regulation of organic anion transporter expression in human hepatocytes exposed to the proinflammatory cytokine interleukin 1beta. Drug Metab. Dispos. 2008;36:217–22. Kong FM, Sui CY, Li YJ, et al. Hepatobiliary membrane transporters involving in the formation of cholesterol calculus. Hepatobiliary Pancreat. Dis. Int. 2006;5:286–9. Marschall HU, Wagner M, Zollner G, et al. Complementary stimulation of hepatobiliary transport and detoxifi cation systems by rifampicin and ursodeoxycholic acid in humans. Gastroenterology 2005;129:476–85. Van der Borght S, Libbrecht L, Katoonizadeh A, et al. Nuclear beta-catenin staining and absence of steatosis are indicators of hepatocellular adenomas with an increased risk of malignancy. Histopathology 2007;51:855–6. Bossard R, Stieger B, O'Neill B, et al. Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver. J. Clin. Invest. 1993;91:2714–20. Crocenzi FA, Mottino AD, Cao J, et al. Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 2003;285:G449–59. Reichen J, Paumgartner G. Uptake of bile acids by perfused rat liver. Am. J. Physiol. 1976;231:734–42. Wakabayashi Y, Kipp H, Arias IM. Transporters on demand: intracellular reservoirs and cycling of bile canalicular ABC transporters. J. Biol. Chem. 2006;281:27669–73. Stieger B, Meier PJ, Landmann L. Effect of obstructive cholestasis on membrane traffic and domain-specific expression of plasma membrane proteins in rat liver parenchymal cells. Hepatology 1994;20:201–12. Saito S, Iida A, Sekine A, et al. Three hundred twenty-six genetic variations in genes encoding nine members of ATP-binding cassette, subfamily B (ABCB/MDR/TAP), in the Japanese population. J. Hum. Genet. 2002;47:38–50. Pauli-Magnus C, Kerb R, Fattinger K, et al. BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 2004;39:779–91. Lang T, Haberl M, Jung D, et al. Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11). Drug Metab. Dispos. 2006;34:1582–99. Kim SR, Saito Y, Itoda M, et al. Genetic variations of the ABC transporter gene ABCB11 encoding the human bile salt export pump (BSEP) in a Japanese population. Drug Metab. Pharmacokinet. 2009;24:277–81. Ho RH, Leake BF, Kilkenny DM, et al. Polymorphic variants in the human bile salt export pump (BSEP; ABCB11): functional characterization and interindividual variability. Pharmacogenet. Genomics 2010;20:45–57. Lang C, Meier Y, Stieger B, et al. Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet. Genomics 2007;17:47–60. Meier Y, Pauli-Magnus C, Zanger UM, et al. Interindividual variability of canalicular ATP-binding-cassette (ABC)-transporter expression in human liver. Hepatology 2006;44:62–74. Meier Y, Zodan T, Lang C, et al. Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the

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1 Physiology of bile formation: Hepatocellular bile salt transporters 1331T>C polymorphism in the bile salt export pump. World J. Gastroenterol. 2008;14:38–45. Dixon PH, van Mil SW, Chambers J, et al. Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy. Gut 2009;58:537–44. Andreotti G, Menashe I, Chen J, et al. Genetic determinants of serum lipid levels in Chinese subjects: a population-based study in Shanghai, China. Eur. J. Epidemiol. 2009;24:763–74. Takeuchi F, Katsuya T, Chakrewarthy S, et al. Common variants at the GCK, GCKR, G6PC2-ABCB11 and MTNR1B loci are associated with fasting glucose in two Asian populations. Diabetologia 2010;53:299–308. Hirohashi T, Suzuki H, Takikawa H, et al. ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3). J. Biol. Chem. 2000;275:2905–10. Zeng H, Liu G, Rea PA, et al. Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res. 2000;60:4779–84. Akita H, Suzuki H, Hirohashi T, et al. Transport activity of human MRP3 expressed in Sf9 cells: comparative studies with rat MRP3. Pharm. Res. 2002;19:34–41. Schaap FG, van der Gaag NA, Gouma DJ, et al. High expression of the bile salthomeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis. Hepatology 2009;49:1228–35. Soroka CJ, Lee JM, Azzaroli F, et al. Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology 2001;33:783–91. Donner MG, Keppler D. Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 2001;34:351–9. Slitt AL, Allen K, Morrone J, et al. Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis. Biochim. Biophys. Acta 2007;1768:637–47. Rius M, Nies AT, Hummel-Eisenbeiss J, et al. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 2003;38:374–84. Gradhand U, Lang T, Schaeffeler E, et al. Variability in human hepatic MRP4 expression: influence of cholestasis and genotype. Pharmacogenomics J. 2008;8:42–52. Chai J, Luo D, Wu X, et al. Changes of organic anion transporter MRP4 and related nuclear receptors in human obstructive cholestasis. J. Gastrointest. Surg. 2011;15:996–1004. Zollner G, Wagner M, Fickert P, et al. Expression of bile acid synthesis and detoxification enzymes and the alternative bile acid efflux pump MRP4 in patients with primary biliary cirrhosis. Liver Int. 2007;27:920–9. Denk GU, Soroka CJ, Takeyama Y, et al. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J. Hepatol. 2004;40:585–91. Dawson PA, Hubbert ML, Rao A. Getting the mOST from OST: role of organic solute transporter, OSTalpha-OSTbeta, in bile acid and steroid metabolism. Biochim. Biophys. Acta 2010;1801:994–1004. Boyer JL, Trauner M, Mennone A, et al. Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents. Am. J. Physiol. Gastrointest. Liver Physiol. 2006;290:G1124–30.

2 Structure and function of hepatic ABC transporters Philipp Ellinger, Marianne Kluth, Susanne Przybylla, Sander H. J. Smits, and Lutz Schmitt

2.1 Introduction to human ABC transporters expressed in the liver Several membrane transporters that belong to a group of ATP-dependent primary transporters, the so-called ABC (ATP binding cassette) transporters, are found in the human genome. In general, ABC transporters contain two transmembrane-spanning domains (TMDs) and two characteristic nucleotide-binding domains (NBDs) localized in the cytosol. In the membrane the two TMDs form a pore-like structure, which facilitates substrate transport against a chemical gradient. One TMD is predicted to have six α-helices, whereas the soluble NBDs are essential for the supply of energy by hydrolysis of ATP. Compared with the TMD, the NBD harbors highly conserved sequence motifs: the Walker A (GXXGXGKS/T, where X can be for any amino acid), Walker B (ΦΦΦΦD, where Φ can be any hydrophobic residue) motifs, and the C-loop (ABC–signature motif, LSGGQ) (1). The C loop, which is located roughly 90 amino acids downstream of the Walker A motif and roughly 30 amino acids upstream of the Walker B motif, is actually the characteristic sequence motif of this family; together with the Walker A and B motifs, it serves as a diagnostic clue to the identification of new family members. Additional sequence motifs present in ABC transporters are the Q loop, the D loop (SALD), and a highly conserved histidine residue essential for ATP hydrolysis, which is positioned 30 amino acid downstream of the D loop (2). To achieve a thermodynamic uphill transport of the substrate, transport has to be coupled to the cycle of ATP hydrolysis. Several high-resolution structures of full-length ABC transporters and isolated NBDs, in combination with biochemical analysis, have provided important contributions to a molecular understanding of substrate binding, ATP hydrolysis, and substrate transport. For example, the highly conserved NBD has an L-shaped structure consisting of a catalytic domain and a helical domain. The catalytic domain contains the Walker A and B motifs while the helical domain harbors the C loop. These two domains are connected by the Q and Pro loops (3). Further analysis of, for example, the isolated haemolysin B-NBD demonstrated that in the presence of ATP, the two NBDs form a homodimer (2). The Walker A and B motifs of one NBD and the C loop of the opposing NBD bind one ATP, so that the two NBDs are set in a head-totail arrangement. This ATP-induced dimerization generates mechanical work, which in principle can be transmitted to the TMDs and might serve as another source of energy (see section 2.2.4). The dimeric NBDs cooperate in hydrolysing ATP and provide the free energy to drive the directional transport of the substrate against a concentration gradient. After ATP hydrolysis, ADP and Pi dissociate from the NBD, the dimer falls apart, and the ground state of the NBDs is restored. Furthermore, different models for the transport mechanism have been proposed. The simplest model, the alternating access model, describes two basic conformations. One conformation is open to the cytosolic side (inward-facing), with a substrate-binding

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2 Structure and function of hepatic ABC transporters

site accessible for the substrate only from the cytosol, and the second conformation is open to the extracellular side, containing a binding site, which has a low affinity to the substrate and is accessible only from the extracellular space (4). A continuous model is the ATP-switch model. In the ground state the transporter is in the inwardfacing conformation with a high-affinity substrate-binding site and the NBDs exist as monomers, with low affinity to ATP. The ATP-induced dimerization of the NBDs leads to a conformational change in the TMDs such that the substrate-binding site is exposed to the extracellular space, the substrate affinity is reduced, and the bound substrate is finally released (5). However, the exact molecular coupling of the ATP–hydrolysis cycle and substrate transport is still not entirely clear. In the human hepatocyte, several ABC transporter are expressed: for example, the bile salt export pump (BSEP, ABCB11), responsible for bile salt transport; ABCG5/ABCG8, involved in sterol transport; multidrug resistance protein 3 (MDR3, ABCB4), flopping phosphatidylcholine from the inner to the outer membrane leaflet; and ABCG2, transporting a variety of hydrophobic substances (fFig. 2.1). Mutations in one of these transporters are associated with different kinds of liver diseases of varying severity. For example, Dubin-Johnson disease is related to mutations in MRP2 (ABCC2), and progressive familial intrahepatic cholestasis type 2 (PFIC2) is associated with a mutations with the bile salt export pump BSEP. This chapter summarizes experimental insights and focuses on the canalicular ABC transporters BSEP, MDR3, and ABCG2, highlighting their discovery and evolution and the in vitro assays from which a mechanistic understanding may be derived.

Thight junction Hepatocyte

Ca na l

u ic

la r m e m

BS

p P– g

EP

Bile canaliculus OSTa/b

Cholesterol ABCG5/G8

MRP2

Bile salts

OATPs

Bile salts, bilirubin, estrone–3–sulfate

Phosphatidylcholine

MD

R3

AB CG 2

Hydrophobic Glucoronides, substances sulfated bile salts Organic anions, divalent bile salts

MRP3

Cyclic MRP4 nucleotides, bile salts, folates Cyclic nucleotides

MRP5

Hydrophobic substances MRP6 BQ123, GSH conjugates

Sinusoidal membrane

Bile salts

Monovalent bile salts

e an br

NTCP

Glucoronides, sulfated and GSH conjuMRP1 gates, folates

Fig. 2.1: Localization of transporters in the hepatocytes. Bile salts are taken up at the sinusoidal (basolateral) membrane through the sodium–taurocholate cotransporting peptide (NTCP) in a sodium–dependent manner and to a lesser extent through a sodium– independent transport by organic anion transporting proteins (OATPs). They are then further shuttled to the canalicular membrane and transported via the bile salt export pump (BSEP) into the canaliculus. Multidrug resistance protein 3 (MDR3) and Sterolin 1 (Continued )

2.2 Structure and function of the bile salt export pump

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25

Fig. 2.1: (Continued ) (ABCG5/G8) complete the bile formation by flopping phosphatidylcholine (PC) from the inner to the outer leaflet as well as transporting cholsterol. Bile salts, PC and cholesterol form mixed micelles which constitute the basis of bile. P–glycoprotein (P–gp) and the breast cancer resistance protein (ABCG2) transport a variety of hydrophobic substances into the bile and confer multidrug resistance (MDR). Furthermore, a substanial number of multidrug–related proteins (MRPs) are localized in the sinusoidal membrane except MRP2. They transport a broad range of organic anions and conjugated substances. They also participate in MDR and some of them, e.g. MRP4 as well as the organic solute transporter (OST) act as salvage system for too high bile salt concentrations within the cell to prevent toxicity.

2.2 Structure and function of the bile salt export pump (ABCB11; BSEP) 2.2.1 Liver transport of bile salts Bile salts are essential for the absorption of lipids and fat-soluble vitamins, originated from food intake, by the enterocytes of the small intestine and also for the excretion of endo- and xenobiotics with the bile. They are synthesized by multiple enzymatic reactions in the liver, more precisely in the hepatocytes from cholesterol as educt; this constitutes one of the key function of the liver (6). From there bile salts enter the biliary tree and are stored in the gallbladder upon food intake (7). After they have fulfilled “their mode of action”, bile salts pass through the enterohepatic circulation, meaning that they are reabsorbed to ~90% in the small intestine and then transported back to the liver via the portal blood. There, they are transported again into the hepatocyte and the cycle starts anew with their secretion into the canaliculi (8,9). A single bile salt molecule traverses the cycle approximately up to 10 times a day until it is excreted via the intestine, which makes this circulation an extremely efficient recycling system (10). Because bile salts are amphipathic molecules, they display a detergent character. Hence a high concentration within the cell is deleterious, leading to damaged mitochondria and apoptosis or necrosis of the hepatocytes owing to the salts’ ability to solubilize or create defects within biological membranes. To prevent this and keep bile salts circulating, a specialized set of bile salt transporters in the hepatocyte is required (9,11,12). In the basolateral membrane (also called the sinusoidal membrane), bile salts are taken up from the portal blood. This is accomplished by the sodium taurocholate cotransporting peptide (NTCP, SLC10A1) in a sodium-dependent transport process (13). In addition, there is the less frequently used sodium-independent transport by the organic anion-transporting polypeptides (OATPs) (14). After entering the cell, bile salts reach the apical membrane (also called the canalicular membrane); the exact mechanism of this is not yet completely understood. For example, one mechanism involves bile salt−binding proteins (15). At the canalicular membrane, bile salts are transported into the canalicular lumen by the ATP-binding cassette transporter (ABC transporter) bile salt export pump (ABCB11;

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BSEP) (16,17). BSEP is the main driving force for the bile salt−dependent part of bile flow and a bottleneck in the enterohepatic circulation. It must transport bile salts against a steep concentration gradient to maintain circulation, since the concentration of bile salts in the canaliculus is 1000 fold higher than in the cell, 1 mM and 1 μM, respectively (10).

2.2.2 Discovery of the bile salt export pump The electrochemical gradient across the canalicular membrane is ~ ⫺35 mV and its discovery marked the first explanation for bile salt transport across this membrane (18,19). However, this electrochemical gradient alone could not be the entire explanation. Finally, in 1991, an ATP-dependent system for the transport of taurocholate in isolated canalicular membranes of rat liver was described. Other laboratories subsequently confirmed this finding (20,21). Evidence that an ABC transporter was responsible for bile salt secretion into the canaliculus appeared in 1995. It involved an increased level of mRNAs, detected by Northern blotting, in combination with the overexpression of an ABC transporter found via the Western blot technique with a P-glycoprotein antibody (ABCB1, MDR1, P-gp); these were demonstrated in a bile salt−resistant rat hepatoma−derived cell line (22). This suggested that an ABC transporter closely related to P-gp became upregulated in this system. In the same year, Childs et al. screened a pig cDNA library with a probe consisting of a P-gp sequence and identified a gene exclusively expressed in the liver that had a sequence identity of 61% to human P-gp on the amino acid level (23). This gene was named “sister of P-gp” (sP-gp), but its function remained unknown. Gerloff et al. were the first to demonstrate that oocytes exhibited a stimulated taurocholate efflux when liver sP-gp cRNA was injected into Xenopus laevis oocytes and the first to express sP-gp in Sf 9 (Spodoptera frugiperda) cells (24). Furthermore, membrane vesicles derived from these Sf 9 cells demonstrated an ATPdependent taurocholate uptake, much as in previous studies with isolated canalicular membranes. Because of these findings the “sister of P-gp” was renamed “bile salt export pump (BSEP)” and was considered to be the predominant bile salt transporter in the apical membranes of hepatocytes (24). Further strong support for this consideration was obtained by positional cloning of the human BSEP gene and mapping it to chromosome 2q24, a locus linked to progressive familial intrahepatic cholestasis type 2 (PFIC2), a severe liver disease (25).

2.2.3 Evolution of the bile salt export pump The production of bile salts and their subsequent transport into the canaliculi is highly conserved among the livers of vertebrates. Over the years, BSEP has been detected and studied in the pig (23), rat (26), mouse (27,28), rabbit (29), dog (30) and human (31,32). Interestingly, full-length BSEP cDNAs has been identified in a variant of the small skate (Raja erinacea), a 200-million-year-old marine vertebrate with an amino acid sequence identity of 68.5% to the human orthologue (33). Here, bile salts are transported in large amounts by BSEP. Furthermore, it was demonstrated that mutations leading to PFIC2 in humans had the same effect on substrate transport in skate BSEP (33,34). The bile of this elasmobranch normally consists of bile alcohols (scymnol sulfate) rather than bile salts, which cannot be found in its bile. Probably bile alcohols were the original

2.2 Structure and function of the bile salt export pump

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27

substrates for BSEP, and mammalian evolution led to different substrates owing to a selective pressure – for example, more fat in the diet. Interestingly, skate bile does not contain any phospholipids and no MDR3 protein (a phospholipid floppase) is found in the hepatocyte. The function of BSEP and its tight correlation with MDR3 is described in section 2.3. Importantly, however, this finding suggests that BSEP evolved much earlier than the highly identical MDR3 protein (sequence identity between P-gp and MDR3 of ~80%) and probably also by gene duplication (35). The occurrence of lipids in bile was potentially the result of the more deleterious bile acids than of the bile alcohols that arose during evolution. All of these indications demonstrate that BSEP diverged very early from P-gp and that it is highly conserved in vertebrate evolution.

2.2.4 The bile salt export pump – a member of the ABC transporter family BSEP belongs to the group of ABC transporters. They can be found in all the taxonomic kingdoms (from bacteria to humans), and all possess the same modular architecture and act either as importers or exporters (36). In humans, 48 ABC transporter genes have been identified in addition to a small number of pseudogenes, which are not expressed (37). All known eukaryotic ABC transporters are exporters, whereas ABC importers can be found only in Archaea and Bacteria. In humans, ABC transporters are expressed throughout the body, but some highly tissue-specific and ABC transporters are restricted to the liver (37). Phylogenetic analysis of the entire human ABC transporter sequences has led to the classification of seven subfamilies (A to G) (38). Because of their important roles in human physiology, dysfunction is the cause of very severe diseases, such as cystic fibrosis (39). In terms of mutations of liver ABC transporters BSEP and MDR3, for example, PFIC2 (25) and PFIC3 (40) may develop (see chapter 9). BSEP belongs to the group B (MDR/TAP) subfamily of human ABC transporters because of its high sequence identity to P-gp. The gene is located on chromosomes 2q24 (25) and the 28 exons code for a 1321 amino acid glycosylated ABC transporter with a molecular mass of ~160 kDa (31,32). ABC transporters have a core architecture consisting of two NBDs and two TMDs. In eukaryotes these modules are encoded on a single gene, but one must distinguish between the full-size transporters (two TMDs and two NBDs) and half-size transporters (only one of each domain). The latter homo- or heterodimerize to form a functional transporter. BSEP is a full-size ABC transporter with a core molecular weight of 146 kDa. Interestingly, the N-terminal NBD (NBD1) of BSEP contains a methionine instead of a glutamate within the Walker B motif. The glutamate normally interacts with ATP through a catalytic water molecule that catalyzes the nucleophilic attack onto the γ–phosphate. ATP binding sites in ABC transporters are composed of the Walker A and B motifs of one NBD and the C-loop of the other NBD. Therefore the ATP-binding site that contains NBD1 (site 1) is a degenerated site. Degeneration of this conserved residue is also seen in other human ABC transporters like TAP1/2 or CFTR (41). Functional studies of other degenerated ABC transporters have demonstrated that this mutation leads to an ATPdeficient site within the NBD dimer. This, of course, implies an asymmetric function of the two NBDs and further suggests that ATP binding site 1 of BSEP is catalytically inactive or active only at drastically reduced levels compared with the other ATP binding site in the composite dimer. This phenomenon has not been investigated for BSEP so far but is of high concern for a molecular understanding of ATP hydrolysis coupled to bile salt transport. The TMDs are located within the membrane and provide the translocation

28

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2 Structure and function of hepatic ABC transporters

Models of

(A) BSEP

(B) MDR3

(C) ABCG2

Fig. 2.2: Models of BSEP, MDR3 and ABCG2. (A) The model of BSEP based on the known structure of Sav1866 from Staphylococcus aureus. The transmembrane domain is highlighted in blue and the nucleotide binding domain in cyan. The used template is deposited under protein data bank (PDB) code 2HYD (B) The model of MDR3 based on the known structure of P-gp from Mus musculus. The transmembrane domain is highlighted in blue and the nucleotide binding domain in cyan. The used template is deposited under PDB code 3G61. (C) The model of ABCG2 based on the known Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP. The used template is deposited under PDB code 2ONJ. Since ABCG2 is a halfsize transporter the two monomers are colour coded differently. Monomer I is highlighted in blue and monomer II in purple. Due to the bound AMP-PNP in the template structure the conformation of ABCG2 represents the potential nucleotide bound state. It is important to clarify that these models are based on the known X-ray structure and the structures obtained from the actual protein might look differently.

pathway for the substrate. In contrast to the NBDs, the TMDs are highly variable in their sequence and thus determine the substrate specificity. It is assumed that many human ABC transporters show the 6 ⫻ 6 topology, meaning that they contain six TM helices (TMH) traversing the membrane followed by a cytoplasmically located NBD and again six TMHs and an NBD. This assumption was originally proposed based on cysteine scanning mutagenesis of P-gp and the recently developed x-ray structures of mouse P-gp and bacterial homologues (42); it may be true for BSEP as well. BSEP is also a full-size ABC transporter containing 12 TMHs and two NBDs (fFig. 2.2). This number of helices is derived from hydrophobicity calculations because structural information on BSEP is lacking. To date only one eukaryotic ABC transporter structure (of mouse P-gp) has been published (42). The second available structure (ABCB10) is deposited only in the Protein Data Bank (PDB database). The P-gp structure shows the typical bundle of six helices crossing the membrane. However, as first observed for Sav1866, a domain swap is present in P-gp, suggesting that such a swapping is a conserved feature of ABC drug pumps. Here, four helices of one bundle and two helices of the other bundle build up one TMD. The TMDs provide a large cavity for substrate binding for mouse P-gp, which may also be true for BSEP. According to the “cholesterol fill-in mechanism,” cholesterol also participates in substrate recognition and fills the volume of the cavity that is not occupied by the substrate, as postulated for P-gp. Besides the amino acids, which constitute the substrate-binding site, this could also e an additional explanation for the fact that those two closely homologous transporters have different substrate spectra and

2.2 Structure and function of the bile salt export pump

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BSEP is restricted to bile salts. It has been demonstrated that the activity of BSEP critically depends on cholesterol. This might be because BSEP is targeted to detergent-resistant microdomains (DRMs) in the canalicular membrane, which exhibit a high amount of cholesterol and sphingomyelin, or because of the “cholesterol fill-in model.” Whatever the molecular reason(s) for the differences between P-gp and BSEP may be, all the models proposed must be verified experimentally for BSEP in the future. So far most if not all functional information on BSEP results from disease-linked mutations found in patients with, for example, PFIC2 (see chapter 9) and offers insights into the way single amino acids influence the trafficking, stability, and transport capabilities of BSEP.

2.2.5 Cloning and expression systems for BSEP To study a protein biochemically in vitro, it is often necessary to obtain sufficient amounts of pure, homogeneous protein. Therefore an expression system must be chosen and recombinant expression constructs must be cloned. All this is true for BSEP, but it has one big drawback. The human cDNA that codes for BSEP has been found to be unstable in Escherichia coli (31,32,43). This phenomenon has been observed for several other mammalian membrane proteins as well (44). Because E. coli is the most widely used cloning and expression host for standard molecular biology techniques, other strategies must be applied in utilizing the favored expression system. In the case of human BSEP, after several years of struggle, efforts to clone the cDNA into an expression vector were eventually successful. However this led, even after a bacterial promoter in the cDNA was silenced, to a construct with several point mutations within the coding sequence (six missense mutations) and the loss of specific parts of the coding sequence during expression construct propagation (32). All these findings led to the notion that the cDNA of BSEP is “toxic” or “unstable” for cloning and/or the expression host since colonies would no longer grow. One way of circumventing this laborious work is to use homologous recombination (HR) in the yeast Saccharomyces cerevisiae (45,46). We therefore established a workflow for human BSEP that can also be applied to any other target (47). Here the expression vector of interest was modified by the introduction of an origin of replication (ori) and a selection marker for S. cerevisiae into the backbone of the plasmid. The linearized expression vector and the PCR-amplified BSEP cDNA, which has overlapping ends to the expression vector, are then transformed into yeast. S. cerevisiae is capable of recombining those overlapping ends to a circular vector, and only clones that do this correctly are able to grow under selection. The expression vector can be recovered from yeast and transformed in E. coli for amplification. We have found that E. coli is capable of handling the BSEP cDNA if it is in a closed, circular plasmid form (no nicks, etc.) and grown strictly at or below 30°C. If required, our expression construct can be designed to remove the origin of replication or selection marker to prevent a potential influence of these additional sequences on balanced expression systems. Furthermore, we developed a mutagenesis strategy relying only on yeast. A changed primer design (primers carrying the mutation are not completely complementary to each other) results in a PCR product with overlapping ends (5’ and 3’ ends) that can be recombined by yeast, resulting in a plasmid containing the desired mutation. With the directed recombination–assisted mutagenesis (DREAM) method, mutations can be introduced more easily and quickly than with commercially available strategies. Therefore this method is seen as a DREAM (47).

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The major bottleneck in studying membrane proteins in vitro (e.g. structural and functional studies), is their homo– or heterologous overexpression, making it difficult to purify the protein in adequate amounts. Therefore one must choose between prokaryotic and eukaryotic expression systems. As a prokaryotic expression system, E. coli is the most widely used host (48), although there are others like Lactococcus lactis, which is also successfully used for the overexpression of membrane proteins (49). Mammalian transporter can be expressed in E. coli, but sometimes in an inactive manner. Therefore we also tried to overexpress human BSEP in E. coli using the T7-RNA polymerase/promotor system in combination with a synthetic gene, which sequence was optimized for use in E. coli. Unfortunately cells stopped growing upon induction of BSEP expression and we were not able to detect BSEP in cell lysates via Western blotting. BSEP is a plasma membrane protein and E. coli does not possess the eukaryotic posttranslational modification system, and no cholesterol is present in the inner membrane. Therefore, eukaryotic expression systems are likely the methods of choice for BSEP. Three different expression systems, which are also commercially available, are used the most: yeast, insect, and mammalian cell lines. Mammalian cell lines have the great advantage that they present the native environment of BSEP; these cells contain the native lipid environment, the native secretory/posttranslational pathways, and a known functional expression. Human BSEP could be expressed in different mammalian cell lines (e.g. HEK293 cells (50), HepG2 cells (51), MDCK cells (52) and LLC PK1 (53) cells) and characterized functionally without purification. The most widely used system for the heterologous expression of BSEP is the insect cell system. Human BSEP was expressed in Sf 9 (32,54) as well as HighFive cells (31). Insect cells exhibit a nonnative lipid environment with low levels of cholesterol as well as nonnative glycosylation (generally of the high-mannose type), but they resemble the native conditions more than yeast does. Expression in this system is used to investigate the transport properties of BSEP in vesicular-based transport assays. Cell culture−based systems may generally be suitable for addressing questions of a cellular phenotype, protein trafficking, and the modification of protein interactions. But mammalian and insect cell systems are also costly and maybe not be producible in large the amounts required for purification and structural studies of BSEP. From this point of view, a better choice might be yeast. Two yeast-based systems are used to overexpress mammalian membrane proteins: the previously mentioned S. cerevisiae and Pichia pastoris. The advantages are obvious: yeast is inexpensive, requires simple culture media, and exhibits well-studied genetics. Furthermore, yeasts have the eukaryotic modification machinery. Of course there are disadvantages, since, for example, the lipid composition of the membrane is different from that of mammalian cells (they contain ergosterol instead of cholesterol) and S. cerevisiae often hyperglycosylates proteins (highly branched and extended high-mannose structures), which is not observed in P. pastoris. It has been shown that BSEP requires glycosylation for transport activity in MDCK cells (55), although the type of glycosylation seems not to be important, as shown by functional expression in insect cells. S. cerevisiae was also used to express human P-gp (56) and MRP1 (57), and we were also able to express BSEP in this host (unpublished data), yielding only low amounts of fully translated protein. Therefore we switched to P. pastoris. This yeast was shown to overexpress 25 human ABC transporters (BSEP was not among them) and was also the expression host for mouse P-gp, which in the end and after a long endeavor resulted in the three dimensional x-ray structure (42,58). Recently we demonstrated the heterologous overexpression of human BSEP in

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2.2 Structure and function of the bile salt export pump

31

this yeast (47). Another advantage of P. pastoris is that this methylotrophic yeast strain can be fermented to high cell densities generating large amounts of biomass, which can be used for subsequent purification.

2.2.6 In vitro assays to study BSEP Although BSEP has not been purified to homogeneity yet, assays have been described to study the function of BSEP in vivo. A vesicular transport assay is the most important one. It consists of three steps: (a) preparation of membrane vesicles, (b) addition of substrate and an energy source, and (c) readout of substrate uptake into the vesicles. In general there are two ways to prepare membrane vesicles from cells, right-side-out (RSO) and inside-out (IO) vesicles. In RSO vesicles, the cytosolic side of the transporter is localized in the lumen of the vesicles, whereas in IO vesicles it is vice versa. The latter ones are commonly used for primary transporters such as BSEP. With the addition of ATP and substrate, transport is initiated and the substrate begins to accumulate in the lumen of the vesicles. After a defined amount of time, the reaction is stopped by, for example, a rapid-filtration method; then the transported amount of substrate, which is retained in the vesicle on the filter, is quantified (via radioactivity, fluorescence, or LC/MS). With this assay, the substrate spectrum of BSEP was elucidated (see fTab. 2.1 for human BSEP). These assays were mainly performed with BSEP derived from insect cell vesicles (31,32) but also with vesicles originated from HEK293 cells or isolated canalicular membranes (52). Human BSEP transports monovalent conjugated bile salts in the order of taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate (it has to be

Tab. 2.1: Substrate spectra and Michaelis-Menten constant for human BSEP from different expression systems. For an excellent overview, see reference 60. Substrate Taurocholate

KM / μM

Source

8 (32), 20 (30, 61), 15(54)

Sf9

4 (31)

HighFive

6 (50) Taurochenodeoxycholate

4 (54), 5 (32), 13 (61)

Sf9

7 (50)

HEK293

Tauroursodeoxycholate

12 (32)

Sf9

Taurodeoxycholate

34 (61)

Sf9

4 (61)

Sf9

Taurolithocholate Taurolithocholate 3 sulfate

10 (50)

HEK293

Glycocholate

11 (32), 36 (54)

Sf9

22 (50)

HEK293

Glycochenodeoxycholate Pravastatin

2 (54)

Sf9

8 (50)

HEK293

124 (62)

HEK293

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2 Structure and function of hepatic ABC transporters

mentioned, that the KM values vary slightly between different expression systems but not unconjugated ones) (see fTab. 2.1). In most of these studies bile acids are tritium-labeled for readout, but fluorescent bile acid derivatives, such as cholylglycylamidofluorescein and chenodeoxycholylglycylamidofluorescein, have also been investigated (59). In addition, inhibitors were analyzed for their potential impact on BSEP. Inhibition of BSEP by different drugs causes drug-induced cholestasis, leading to severe liver injury (63). Examples of inhibitors that were determined for human BSEP by a vesicular uptake assay in competition experiments with bile salts include cyclosporine, rifampicin, and bosentan. These assays and the recommendation of the European Medicines Agency (64) emphasize the importance of BSEP for drug development. With a vesicular uptake assay for BSEP commercially available, the screening of drug libraries is in principle straightforward. One disadvantage of this kind of assay, however, is that besides the target transporter, the vesicles contain many irrelevant membrane proteins that probably affect the uptake assay. This can be excluded if proper controls are performed, but it complicates the assay. Mutations in the BSEP gene can lead to an impairment of bile salt transport due to the protein’s dysfunction. This can lead to PFIC2 or BRIC2, a severe liver disease, which at present can be cured only by liver transplantation (65). Currently, according to the Human Gene Mutation Database (http://www.hgmd.org/), 179 disease-related BSEP mutations are known. Thus an understanding of the effect of such mutations could, in the future, lead to therapeutic innovations that might cure this disease without transplantation. If, for example, mutated BSEP is still able to transport and the disease is caused by a trafficking defect, it could also be investigated by the vesicular transport assay, with mutation and localization studies in cell culture systems involving immunostaining or with a fluorescent tag like eGFP or YFP. Trafficking and the regulation of BSEP in the apical membrane of hepatocytes also requires adaptor proteins. HCLS1-associated protein X-1 (Hax1), for example, was identified using yeast two-hybrid screens as well as pull-down assays with glutathioneS-transferase (GST) tag fusion proteins (soluble parts of BSEP with GST tag) and coimmunoprecipitation (66). Other adaptor proteins are still not known and would be of high interest, especially for the short-term regulation of BSEP or for their potential involvement in trafficking mutants.

2.3 Structure and function of the multidrug resistance protein 3 (ABCB4; MDR3) As described in section 2.2.1, BSEP is essential for the circulation of bile salts. However, bile salts are harsh detergents and possess the power to solubilize any biological membrane. The outer leaflet of the canalicular membrane is destabilized by bile salts, which are translocated in the canaliculus by BSEP (ABCB11). To dampen this effect, bile salts and phosphatidylcholine (PC) form mixed micelles with cholesterol translocated by ABCG5/G8. These mixed micelles have a lower capacity to extract lipids from the membrane. A second function of PC is the solubilization of cholesterol, which prevents the crystallization of cholesterol in the biliary duct and the formation of cholesterol gallstones. The bulk of PC is reabsorbed in the intestine and returns to the hepatocyte within the enterohepatic cycle. However, the half-time of PC to flip spontaneously from

2.3 Structure and function of the multidrug resistance protein 3

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the inner to the outer leaflet of a lipid bilayer is very low; therefore PC must be translocated across the membrane of the hepatocyte by an active transporter. The multidrug resistance protein 3 (MDR3), also called ABCB4, is localized only in the canalicular membrane of the hepatocyte (fFig. 2.1) and is indispensable for the primary active transport of PC from the inner to the outer leaflet of the canalicular membrane against a concentration gradient. The mouse homologue is called Mdr2 and fulfills the same function as MDR3 to flop PC across the apical membrane of hepatocytes. Mutations in the MDR3 gene caused different types of liver diseases, such as progressive familial intrahepatic cholestasis type 3 (PFIC3), intrahepatic cholestasis of pregnancy (ICP), and low phospholipid−associated cholestasis (LPAC).

2.3.1 A brief history of MDR3 During an analysis of cDNAs from human liver in 1987, van der Bliek et al. identified a gene that is highly homologous to the human P–gp and designated it MDR3. One year later the complete cDNA sequence was published (67). This sequence is composed of two similar halves. One half consists like BSEP of six putative TMHs and one NBD. The NBDs are identical to those of the human MDR1. Furthermore, the TMDs showed up to 80% identity. Divergence between MDR1 and MDR3 is greatest at the N-terminus and in the 60−amino acid linker connecting the two halves (67). While MDR1 transports a wide variety of structural unrelated substances and is involved in multidrug resistance (MDR), no drug-pumping activity has been demonstrated for MDR3 (68). Smit and coworkers characterized mice with a disruption of mdr2 in 1993. They ascertained that the homozygous disruption of the murine homologous mdr2 gene leads to a complete absence of PC and cholesterol from bile (69). Furthermore, mice heterozygous for Mdr2 (Mdr2⫺/+) have normal amounts of cholesterol and only 40% of PC in bile. Human MDR3 can functionally replace mdr2 in knockout mice (70). This demonstrates that the closely related Mdr2 and MDR3 carry out the same function. Direct evidence that MDR3 can translocate endogenous PC has been obtained in enhanced transport of newly synthesized [3H]choline-labeled PC to the surface of transgenic fibroblast (71). This suggested that MDR3 translocates specifically PC from the inner to the outer leaflet of the canicular membrane. Van Helvoort and coworkers (72) were the first to demonstrated specific transport of a short-chain PC in polarized pig kidney epithelial cells transfected with MDR3. In this study they measured lipid translocation across the plasma membrane by extracting fluorescently labeled short-chain lipids from the cell surface into the basolateral and apical media. MDR3 translocated fluorescently labeled PC but not the other lipid analogues (72). However van Helvoort et al. showed that radiolabeled short-chain PC lacking the fluorescence moiety was not translocated into the apical medium by MDR3.

2.3.2 MDR3 – an ATP-binding cassette (ABC) transporter The 141-kDa lipid translocase is postranslationally modified by glycosylation at two predicted asparagine residues (N91 and N97) and is allocated to the group of P glycoproteins based on amino acid sequence homology. Like BSEP, MDR3 is a so called full-size transporter and is encoded on one structural gene (NBD-TMD)2 (see fFig. 2.2).

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2.3.2.1 Transport machinery – the flippase model Two models, the “vacuum cleaner model” and the “flippase model”, are postulated for the transport of hydrophobic substrates by ABC transporters. The vacuum cleaner model proposes that the molecule in the cytosol interacts with the transporter, enters a hydrophobic cavity of the ABC-transporter, and is pumped into the extracellular space. In contrast, Higgins and Gottesman proposed a flippase model for mammalian P-glycoproteins (73). P-gp binds an amphipathic molecule located in the inner leaflet of the plasma membrane and flips the molecule to the exoplasmic leaflet. Therefore a substrate-binding site must be accessible from the lipid phase. The substrate accumulates in the outer leaflet, forming a concentration gradient between the cytosolic and exoplasmic leaflet of the plasma membrane. From the leaflet the substrate can freely diffuse into the extracellular medium. On the basis of the flippase model, it is feasible to explain the observation that PC secretion depends on the expression of Mdr2, the mouse homolog of MDR3, and the bile salt concentration (74). Elferink and coworkers showed if either PC or bile salts were lacking, PC would not be detectable in bile, concluding that bile salts translocation is the main driving force for the secretion of phospholipids (75). It is assumed that P-gp, which is over 76% identical to MDR3, can bind substrates within the inner leaflet of the membrane as well as from the cytosol. How ABC transporters recognize and translocate substrates is still unclear and the subject of intensive investigation.

2.3.2.2 MDR3 – a drug ABC transporter? MDR3 shares 78% amino acid sequence identity with the well-characterized drugpumping ABC transporter P-gp. Because of the high amino acid sequence homology between MDR3 and MDR1 (over 85%) it was assumed that MDR3 also translocates drugs. However, initial experiments with MDR3 cDNA or its mouse homolog Mdr2 transfected cells showed no drug resistance (67,76–78) and MDR3 was not detected in MDR cell lines (67,79). The first indication that MDR3 translocates drugs was obtained by Kino et al. (80). They observed that MDR3 transfected yeast cells showed low-level resistance against the antifungal agent aureobasidin A. Another study of MDR3 was performed by Smith et al. (81), who investigated vectorial substrate transport by polarized pig kidney monolayers transfected with MDR3 cDNA of several MDR1 substrates. They observed that the transport of digoxin, paclitaxel, vinblastine, and ivermectine into the apical medium was significantly increased in the MDR3-transfected cells compared with the control cells. Digoxin transport by MDR3 was efficiently inhibited by the MDR1-specific inhibitor verapamil, cyclosporine, and PSC833, which also inhibited the transport of short-chain PC. Verapamil had also previously been shown to inhibit the translocation of short-chain C6-NBD-PC (72,82). No significant transport of some other MDR1 substrates, such as cyclosporine or dexamethasone, was determined. These results suggest that MDR3 is not specific for PC and is able to translocate various typical MDR1 substrates as well. But why is drug transport observed only in polarized monolayers transfected with MDR3 cDNA? Currently there is no satisfactory explanation. Further studies on the translocation of long-chain PC and drugs by MDR3 are required.

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2.3.3 Analysis of the substrate specificity of the PC translocator The analysis of lipid transporters is very complex by reason of the difficulty of developing a reliable assay for the molecular mechanism of lipid transporters. Following are described two different ways of analyzing the function of P-glycoproteins and especially lipid translocases. On the one hand, MDR3 translocates PC across the membrane; three different approaches to this have been reported. On the other hand, MDR3 hydrolyzes ATP. The resultant ATPase activity correlates indirectly with the substrate transport.

2.3.3.1 Transport of lipids and lipid analogues by the ABC transporter Currently no sensitive assay for measuring naturally occurring long-chain proteinmediated lipid translocation from one leaflet to the other leaflet of the membrane exists. Nevertheless Sleight and Pagano used the lower hydrophobicity of short-chain lipids (C5-C6 acyl chain) to determine lipid transport of lipid translocases, which allows their free exchange as monomers via the aqueous phase (83). At first short-chain lipids are easily integrated into the surface of the membrane of interest and can be detected by a spin-, fluorescent-, or radiolabel on the short-chain. The transport can be measured by chemically quenching of the spin-labeled or fluorescent analogue in the outer leaflet (84) or by “back-exchange.” To date two different systems to determine short-chain PC transport by MDR3 or the mouse-homologous Mdr2 have been described. Ruetz and Gros expressed Mdr2 in the membrane of secretory vesicles obtained from a yeast secretion mutant (82). These vesicles can be easily isolated and consist of a pure population of inside-out vesicles, meaning that the cytoplasmic NBDs of the ABC transporter are located on the outside of the vesicle. To determine Mdr2-driven transport of PC from the outer leaflet into the inner leaflet of the vesicular membrane they used fluorescent-labeled short-chain PC – C6-NBD-PC: (N-6[7-nitro-2,1,3-benzoxadiazol-4yl]-amino-hexanoyl-phosphatidylcholine) – which is chemically reduced to the nonfluorescent compound by a membrane-impermeable reducing agent such as sodium dithionite. Dithionite reduces only the C6-NBD-PC located in the outer leaflet, whereas the translocated C6-NBD-PC in the inner leaflet remains unaffected. Detergent disruption of the vesicles lead to a decrease of fluorescence emission because of the release of translocated C6-NBD-PC. With this system Ruetz and Gros proved indeed a very small but specific transport of short-chain PC analogue by Mdr2. Second, they showed that transport was ATP-dependent and inhibited by verapamil, a specific inhibitor for MDR1. In the “back-exchange” method, short-chain lipids are extracted from the outer leaflet by bovine serum albumin (BSA). BSA has the ability to selectively bind short-chain lipids from the outer leaflet. The lipids are analyzed by two-dimensional thin layer chromatography (TLC) and the transport activity is calculated by the ratio between translocated and total amount of short-chain PC (85). The floppase activity of MDR3 was confirmed by van Helvoort et al. using LLC-PK1 pig cells transfected with an MDR3 cDNA construct (72). LLC-PK1 cells are able to grow as monolayers on filters and MDR3 is found only in the apical membrane. Cells are cultured in the presence of a short-chain lipid precursor, which is taken up and converted into the corresponding short-chain lipid analogue. The intracellularly synthesized C6-NBD-PC was specifically transported by MDR3 but not C6-NBD-phosphatidylethanolamine, C6-NBD-sphingomyelin, or C6-NBD-glucosylceramide. Remarkably, radiolabeled short-chain PC with two C8 fatty acids lacking the fluorescence moiety

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2 Structure and function of hepatic ABC transporters

(C8C8-[3H]PC) were slightly translocated into the apical medium. Van Helvoort and colleagues confirmed that the high specificity of MDR3 is determined by the choline head group. To date, only Smith and coworkers have been able to generate a system for the translocation of long-chain PC through the membrane of fibroblasts from transgenic mice by MDR3 (71). Intracellular synthesized radioactively labeled PC is inserted into the inner leaflet and translocated to the outer leaflet in the presence of MDR3. PC-TP, a PCspecific transfer protein, carries out the exchange of labeled PC from the outer leaflet to acceptor liposomes in the medium. In this study Smith et al. determined an increased translocation of long-chain PC in the presence of MDR3. One main drawback of this system is the high background in the absence of MDR3 by vesicular transport. This makes usage of this assay extremely complicated. Thus far no in vitro system for the translocation of PC by MDR3 is established because of the challenge of cloning, expressing, and purifying functional MDR3 in sufficient amounts and the technical difficulty of measuring the translocation of natural PC.

2.3.3.2 Substrate-stimulated ATPase activity ABC transporters hydrolyze ATP to energize the transport across the membrane. Since ATP hydrolysis is linked by substrate translocation, the transport activity can be visualized indirectly. Most ABC transporters offer a basal ATPase activity. This ATPase activity is stimulated or inhibited by adding the substrate or inhibitor. There are two assays for measuring the ATPase activity by the determination of released inorganic phosphate: the malachite green assay (86) and the NADH-coupled assay (87,88). Both assays measure the release of free orthophosphate. The highly sensitive malachite green assay is based on the complex formation of free phosphate with molybdate. The reaction of phosphomolybdate and the dye malachite green results in a green complex, whose absorbance can be easily determined at a wavelength of 620 to 650 nm. Nevertheless, a disadvantage of this method is its inability to observe the hydrolytic reaction continuously. The NADHcoupled assay enables one to follow the rate of ATP hydrolysis in real time by coupling the release of Pi and the oxidation of NADH to NAD+. The ATPase hydrolyzes ATP to ADP and Pi. ADP is converted to ATP and phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase. The lactate dehydrogenase reduces pyruvate to lactate, while NADH is oxidized to NAD+. The decrease of NADH is then determined at a wavelength of 340 nm. The precondition to measuring ATPase activity is simple: sufficient expression of MDR3. To date it has not been possible to clone and express functional MDR3 in bacterial systems such as E. coli or L. lactis because of the “toxic” or “unstable” DNA sequence (as described in section 2.2.5). The expression of MDR3 in mammalian cell lines such as LLC PK1 and insect cells has been demonstrated by different groups (72,82). However, the obtained protein amounts are not sufficient to purify MDR3. Thus, up to now, it has not been possible to measure the PC-stimulated ATPase activity of membrane vesicles containing MDR3 and/or of isolated MDR3 in detergent solution or reconstituted into liposomes. To overcome this major obstacle it is crucial to study MDR3 in vitro and obtain a more detailed knowledge of this interesting ABC transporter as expressed inside the liver.

2.4 Structure and function of the breast cancer resistance protein

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37

2.4 Structure and function of the breast cancer resistance protein (ABCG2; BCRP) 2.4.1 History of ABCG2 ABCG2 was first identified in human (BCRP, ABCG2) carcinoma cells. Despite the absence of overexpression of known multidrug transporters, like P-gp or MRP1, these cells displayed a remarkable resistance to multiple chemotherapeutic drugs such as doxorubicin and mitoxantrone. The gene conferring this resistance was isolated and subsequently used to transfect carcinoma cells, which then displayed a diminished accumulation of daunorubicin in flow cytometry assays. Additionally, this transport function appeared to depend on the presence of ATP, and this transport protein was termed breast cancer resistance protein (BCRP; ABCG2) (89). Independently, ABCG2 was discovered as the determinant responsible for the resistance of human colon carcinoma cells selected in mitoxantrone. Isolated cDNA clones displayed high levels of resistance to mitoxantrone. The gene showed relation to the Drosphila melanogaster white gene and homology to ABC transporters; it was named MXR for “mitoxantrone resistance” (90). Furthermore, ABCG2 was identified among a group of new human ABC transporters that were found to be highly expressed in the placenta. The isolated cDNA contained an open reading frame of 655 amino acids consisting an ABC halfsize transporter with an N-terminal NBD and a C-terminal TMD (91). Although it was discovered three times in different contexts, the gene involved always encoded ABCG2.

2.4.2 Structure and function of ABCG2 ABCG2 is a 72-kDa 655−amino acid glycoprotein. Among the members of the ABC transporter family, ABCG2 has, like other members of the ABCG subfamily, a reverse topology, meaning that the NBD is located N-terminal to the TMD. With only one NBD and one TMD encoded on a single gene, ABCG2 is considered to be a half-size transporter and thought to dimerize to become a functional ABC transporter (see fFig. 2.2). Several studies have focused on this oligomerization behavior. Interestingly, intermolecular disulfide bonds are required to obtain a dimeric protein. Cysteine scanning mutagenesis revealed that residue C603 of ABCG2 is involved in intermolecular crosslinking via disulfide bonds (92). Additionally, no mutation of any other cysteine residue had an effect on the dimerization of ABCG2 or its activity. In agreement with these results, Henriksen et al. showed that the oligomeric species of ABCG2 was observed with the use of a nonreducing SDS-PAGE can be gradually disrupted by the addition of a reducing agent (93). Here, mutational analysis of the three cysteine residues located in the third extracellular loop showed that only the C603A mutant impaired dimerization. However, a cell survival assay with mitoxantrone showed that this mutation was still as resistant as the wild type, indicating that the disulfide bond is not essential for the transport function. A biotinylation assay supported the idea that the other two cysteine residues in this loop, C592 and C608, form an intramolecular disulfide bond. However, this disulfide bond is important for protein degradation (94). An important feature of ABCG2 is the GXXXG motif, which has been identified as a recurring transmembrane sequence and is proposed to be an interaction site between the transmembrane α-helices of different monomers. Polgar et al. investigated the only putative GXXXG motif in transmembrane helix 1 of ABCG2. Mutation of one or both of

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the glycine residues resulted in lower ATP hydrolysis and a reduced substrate transport rate, although the protein was still expressed at similar levels on the cell surface. These findings support the hypothesis that the GXXXG motif plays a role for the correct orientation of the transmembrane segments toward each other in the functional transporter. Mutational studies of G553 indicate an involvement of this residue in the dimerization of ABCG2 (95). Another important characteristic of ABCG2 is its hyperglycosylation, deduced from the apparent molecular weight of the protein in SDS PAGE gels and susceptibility to PNGaseF treatment. The glycosylation, however, appears to have no influence on the trafficking of ABCG2 to the plasma membrane. Surface expression was investigated by immunostaining of human ovarian carcinoma cells and hamster ovary cells. Although three glycosylation sites are predicted to be potentially located in the third extracellular loop, only the N569Q mutant showed impaired glycosylation. As mentioned in section 2.2.5, this impaired glycosylation does not result in misstrafficking, in contrast to, for example, the N557 alanine mutation, which results in a ER localization of ABCG2 (96). Nonglycosylated ABCG2 still showed reduced accumulation of the substrate rhodamine 123 in flow-cytometric assays and normal ATPase activity, which can be stimulated by prazosin. The results were comparable to levels found for glycosylated ABCG2 in crude membrane preparations, indicating that glycosylation is not essential for the function of ABCG2. Many studies investigating the function of ABCG2 have employed mutagenesis to clarify the role of different residues in the protein. Residues C592, C603, and C608 are involved in intra- or intermolecular disulfide bonds and N596 is glycosylated. Furthermore, residue R482 has been extensively characterized. Early isolates of ABCG2 from carcinoma cell lines showed a mutation at this position. By testing the accumulation of rhodamine 123 in cells expressing the variants R482G and R482T, broader substrate specificity was observed (97). Whereas the wild-type protein conferred no resistance to compounds like rhodamine 123, doxorubicin, or daunorubicin, expression of ABCG2 and the mutants R482G and R482T reduced the accumulation of the drugs and prolonged cell survival in cytotoxicity assays. Other compounds – like mitoxantrone, prazosin, and Hoechst 33342 – are substrates for both mutant and wild-type transporters (98,99) . A later study confirmed previous results and additionally observed binding of substrates, which are not transported to the wild-type transporter (100). A common single-nucleotide polymorphism encoding the mutation Q141K is linked with the occurrence of gout. ABCG2 was shown to be located in the brush-border membrane of kidney proximal tubule cells. Functional assays with X. laevis oocytes expressing wild-type ABCG2 or Q141K mutant showed that the latter exhibited urate efflux, thereby linking ABCG2 to this genetic disease (101). A recent study revealed that this mutant is exhibiting increased susceptibility for lysosomal and proteasomal degradation (102).

2.4.3 Analysis of the substrate specificity of ABCG2 Owing to the discovery of ABCG2 in drug-resistant cells, the first reported substrates for it were predominantly chemotherapeutic drugs. These included mitoxantrone, flavopiridol, metothrexate, irinotecan and its active metabolite SN-38, porphyrines, and tyrosinkinase inhibitors such as imatinib and gefitinib (103). Other substrates are antibiotics (104,105), flavonoids, antivirals (106,107), folic acid (108), and fluorescent dyes such

2.4 Structure and function of the breast cancer resistance protein

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as Hoechst 33342. Mutation of the arginine residue at position 482 conveys a broader substrate spectrum including rhodamine 123 and anthracyclines such as doxorubicin. Because of its broad substrate spectrum and its expression in several tissues apart from the liver – such as the small intestine, colon, central nervous system, testis, ovary, and placental syncytiotrophoblasts – the transporter is thought to have a protective role (103,109,110). The number of ABCG2 inhibitors identified is equally large. Fumitremorgin C was the first inhibitor described (111). Its analog, Ko143, was found to be one of the most effective ABCG2 inhibitors (112). Some inhibitors were also inhibitors of P-gp or MRP – among them cyclosporine (113) and elacridar (GF120918)(114). Many compounds are both inhibitors and transported substrates, such as dihydropyridines (115). Despite the great number of substrates and inhibitors described to date, no clear structural requirements for a binding compound could be identified.

2.4.4 Expression, purification, and biochemical studies of ABCG2 To date, ABCG2 has been successfully expressed in a number of different vector systems and host organisms. Early studies have been done with drug-selected mammalian cell lines. Finally, the isolation of the cDNA offered the opportunity to move the expression to some heterologous hosts, such as Xenopus oocytes, insect cells, yeast, or bacteria. Baculovirus-infected insect ovary cells (Sf 9) and High Five cells offer an alternative to mammalian cell lines and have been successfully used to overexpress ABCG2, although in both cases hypoglycosylation, transport, and ATPase activity were observed (116,117). Other expression systems include yeasts like P. pastoris and S. cerevisiae. Mao et al. expressed ABCG2 in P. pastoris, obtaining active protein comprising about 3% of the total protein in microsome preparations (118). Similar expression levels could be observed in baker’s yeast, yielding protein with ATPase activity, which could be stimulated by substrate (119). Additionally, a prokaryotic expression system has been reported employing the gram-positive bacterium L. lactis (120). Expression in another bacterial system, E. coli, did not yield functional protein (121). Especially for the purification of ABCG2 from the membrane fraction of the expression host, a high yield is needed. Protein expression of the systems mentioned previously in this section was tested on the ability to obtain high yields of ABCG2 after purification. Solubilization of ABCG2 using different detergents showed the best results with the use of lysophosphatidylcholine (LPC) and n-dodecyl-β-D-maltoside (β-DDM) for P. pastoris membranes and FosCholine-14 and -16 for ABCG2 expressed in insect cells (122,123). Also used for solubilization of protein from insect cell membranes was CHAPS (117). Purification steps of the amino-terminal-histidine-tagged protein in all cases yielded sufficiently pure protein after immobilized metal-ion affinity chromatography (IMAC). Because of weak binding to the affinity resin, further purification steps were necessary when the insect cell expression system was used. These included ion exchange and size exclusion chromatography (117). ABCG2 retained ATPase activity and substrate binding after its purification. Because of its ability to efflux a broad variety of substrates, multiple drug binding sites have been proposed for ABCG2. Clark et al. investigated this with heterologous displacement assays. [3H]daunomycin binding constants were measured in the presence of other known substrates of the ABCG2 gain-of-function mutant R482G. Three distinct binding sites were proposed, which are interlinked by allosteric communication (124). Several

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studies of the substrate specificity and drug binding could be obtained by employing fluorescent substrates of ABCG2. For example 1,4-dihydropyridines could be identified as ABCG2 substrates by photoaffinity labeling with [125I]Iodoarylazidoprazosin (IAAP) and [3H]azidopine (125). Since the translocation process is ATP-dependent, the ATPase activity of ABCG2 has been measured to confirm its physiologic activity. The majority of kinetic parameters were obtained on membrane preparations containing other ATPases. The wild type and the R482G isoform are capable of hydrolyzing ATP in the absence of any substrate (98). Since the influence of substrates and inhibitors on hydrolytic activity is an indicator of interaction with the protein, the measurement of ATPase activity is the focus of several studies. ATPase activity has been used as a readout in order to identify cholesterol content of the membrane as a major factor in ABCG2 activity (126). Cholesterol loading and depletion experiments showed stimulation of ATPase activity by substrates and improved drug transport in cholesterol-loaded membranes. In contrast, ATPase activity could not be stimulated in cholesterol-depleted membranes, indicating an essential role of membrane cholesterol. Another tool to gain further knowledge about the topology of ABCG2 is epitope insertion mutagenesis. One study employed hemagglutinin (HA) tags to probe the predicted hydrophilic regions of ABCG2 via immunofluorescence (127). The results supported a model of six transmembrane helices with the amino and carboxy termini located intracellularly. A later study investigated a current homology model of ABCG2 by epitope insertion and found significant differences in the location of the predicted transmembrane segments (122). To date there are no high-resolution structural data on ABCG2, although some attempts to obtain such data have been undertaken. These include negative-stain electron cryomicroscopy of purified protein. ABCG2 overexpressed in insect cells was solubilized and retained its stimulated ATPase activity. Analysis of the electron microscopy data revealed large particles (~170 Å in diameter) with a noticeable fourfold symmetry, in agreement with a higher oligomer as postulated by biochemical analysis. The final three-dimensional structure with an estimated resolution of ~18 Å could be accurately fitted with homology models of ABCG2, forming a tetramer. Data from sizeexclusion chromatography and blue native PAGE supported the idea that ABCG2 forms a higher-order oligomeric species under the tested conditions (122). Rosenberg et al. used purified ABCG2 expressed in P. pastoris to obtain two-dimensional crystals. The substrate mitoxantrone had a noticeable effect on the crystal shape. Analysis showed a significant change in unit cell dimensions, indicating a conformational change upon drug binding. A new homology model verified by epitope insertion mutagenesis supported the structural data by showing rigid body motion of two transmembrane helices, leading to a more compact conformation of the transporter in the drugbound state. However, a three-dimensional structure is still a long way off. Eventually that will provide a detailed look at the function of ABCG2 at the molecular level.

2.5 Concluding remarks Several interesting transporters are expressed within the hepatocytes, contributing to the function of these cells. This chapter focused on three of them, BSEP, MDR3, and

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ABCG2. A wealth of information is to be derived from studies of their expression and mutation in different mammalian cell lines – studies focusing on their localization, trafficking, and activity. Although such studies have revealed extremely valuable and often essential information, the next step must be to achieve a molecular understanding of these transport mechanisms. Here, the first prerequisite is to elucidate the overexpression of these transporters, which will lead to their characterization directly in isolated membranes and/or after subsequent solubilization and purification in detergent solution. As described and summarized, the expression of membrane proteins is by no means trivial and often hampered by a too low expression in homo- or heterologous expression systems. However, if overexpression can be achieved for BSEP, MDR3, or ABCG2, the gain in knowledge derived from localization studies as well as mutational analysis will shed much light on the molecular mechanism of transport of a large variety of substrates with ATP only as an energizing molecule. Truly it will be a long way to go, but the information obtained will be worth the effort.

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115. Zhou S, et al. Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels. Blood 2005;105(6):2571–6. 116. Ozvegy C, et al. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochemical and biophysical research communications 2001;285(1):111–17. 117. Pozza A, et al. Purification of breast cancer resistance protein ABCG2 and role of arginine-482. Cellular and molecular life sciences: CMLS 2006;63(16):1912–22. 118. Mao Q, et al. Functional expression of the human breast cancer resistance protein in Pichia pastoris. Biochemical and biophysical research communications 2004;320(3):730–7. 119. Jacobs A, et al. Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study. The protein journal 2011;30(3):201–11. 120. Janvilisri T, et al. Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis. The Journal of biological chemistry 2003;278(23):20645–51. 121. Pozza A, JM Perez-Victoria, A Di Pietro. Insect cell versus bacterial overexpressed membrane proteins: an example, the human ABCG2 transporter. Methods in molecular biology 2010;654:47–75. 122. Rosenberg MF, et al. The human breast cancer resistance protein (BCRP/ABCG2) shows conformational changes with mitoxantrone. Structure 2010;18(4):482–93. 123. McDevitt CA, et al. Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2. Structure 2006;14(11):1623–32. 124. Clark R, ID Kerr, R Callaghan. Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. British journal of pharmacology 2006;149(5):506–15. 125. Shukla S, et al. The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2. Biochemistry 2006;45(29):8940–51. 126. Pal A, et al. Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2. The Journal of pharmacology and experimental therapeutics 2007;321(3):1085–94. 127. Wang H, et al. Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence. Biochemistry 2008;47(52):13778–87.

3 Short- and long-term regulation of hepatobiliary transport Dieter Häussinger and Ralf Kubitz

3.1 Introduction Bile formation is a complex process that involves sinusoidal (basolateral) and canalicular (apical) transporter proteins. Regulation of these transporters occurs at the level of gene expression (1), transporter degradation (2–4), covalent modifications of transporters (5,6), and their regulated exocytic insertion into the membrane or endocytic retrieval from it (7–13). Short-term regulation also involves substrate availability (14) and competition between different substrates (15,16). Because the load of cholephilic compounds to the liver can vary significantly within short time periods – for example, in response to food intake – short-term regulation of basolateral uptake and canalicular secretion is mandatory. Within the enterohepatic circulation bile acids (BAs) recirculate several times per day to the liver (17). Intracellular bile salt homeostasis is required, because bile salts per se influence several other liver functions such as cholesterol biosynthesis (18,19), immune function (20–22), apoptosis (23,24), stellate cell proliferation (25), and glucose homeostasis (26–29) (also see Chapter 7). On the other hand, secretion of GSH or glutathione conjugates changes the intracellular glutathione disulfide/glutathione (GSSG/GSH) ratio and thereby influences the antioxidative potential. Bilirubin is a metabolic end product and also acts as an antioxidant (30). Gene polymorphisms of the multidrug-resistance protein 2 (MRP2), which transports conjugated bilirubin into bile, therefore can augment antioxidant defense, especially under conditions of limited antioxidant supply.

3.2 Short-term regulation of sinusoidal transport systems Transport of endogenous or exogenous substrates or waste products from the sinusoidal blood into the cells is mediated by various mechanisms and transport systems in the sinusoidal membrane of hepatocytes (14,17,31,32). Lipophilic substrates may diffuse across the plasma membrane along the chemical gradients. This process depends on substrate gradients across the membrane, which are determined not only by substrate delivery to the liver but also metabolic and excretory sinks. Several transport systems allow facilitated transport. These systems include the organic anion-transporting polypeptide (Oatp/OATP) family (33). Oatps/OATPs (for the rodent and human orthologues) transport anions such as conjugates of estrogens, leukotrienes, ajmalin, or ouabain (33). These transporters do not build up high substrate concentrations and their transport efficacy similarly depends on downstream metabolism and excretion. In addition to facilitated transport, secondarily active transport systems exist, which use the electrochemical Na+ gradient built up by the constitutively expressed Na+K+-ATPase in the sinusoidal membrane. Regarding bile salt transport, the Na+-taurocholate cotransporting polypeptide (Ntcp/ Slc10a1) (34,35) is the most important transport system, which uses the Na+ gradient as the driving force in a stoichiometry of Na+ to bile salt at 2:1.

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The organic anion transporters OAT/Oat, including the human OAT2 (Slc22A7) and OAT5 (Slc22A10) (36,37), mediate Na+-independent uptake of xenobiotics such as methotrexate, p-aminohippurate, and salicylate (36). Organic cations are transported by the organic cation transporters OCT/Oct (38), including the liver-specific human OCT1 (SLC22A1) (39) and OCT3 (SLC22A3) (40). Their genes are located in a cluster together with SLC22A2 on chromosome 6q25.3. OCT1 transports antidiabetic drugs such as metformin (41) and antivirals such as lamivudine (42). Under certain conditions resecretion of substrates from hepatocytes back into the blood is favorable in order to avoid intracellular accumulation of potentially toxic substrates. For this purpose, primary active transporters are expressed at low levels in the lateral membrane of hepatocytes, but their expression may be considerably induced. These transporters include the multidrug resistance–associated protein 3 (MRP3/ABCC3) (43), with a substrate specificity overlapping with that of MRP2 (44). Bile salts may be secreted together with reduced glutathione (GSH) back into blood by Mrp4 (ABCC4) in the sinusoidal membrane (45). Likewise, Mrp5 (ABCC5) functions as an export pump for cyclic nucleotides (46), while the physiological substrate for MRP6 (ABCC6) is still unclear (47). Canalicular secretion is widely assumed to be the rate-limiting step for transhepatocellular transport. However, rate control may also be exerted by uptake at the sinusoidal membrane, especially at physiologically low BA concentrations or under pathophysiological conditions, such as sepsis or estrogen treatment, in which the expression of Ntcp and other transporters is downregulated (48,49), which may largely occur at a posttranscriptional level (50). Ntcp underlies short-term control by cAMP, Ca2+/calmodulin, and okadaic acid-sensitive protein phosphatases (51–54). Vmax of Ntcp is increased via cAMP within minutes by a microfilament-dependent translocation of intracellularly stored Ntcp molecules to the plasma membrane (52,55). Downstream of cAMP, PI3 kinase (PI3K) (56) and the Ca+-independent PKC isoform delta (PKCdelta) (57) may in part mediate this effect. Furthermore, PI3K activates PKCzeta. PKCzeta in large amounts localizes to the canalicular membrane (58) and enhances the motility of Ntcp-containing intracellular vesicles (59). Similar signaling events including PI3K (60) and the protein kinase B/Akt (61) were identified in the swelling-induced translocation of Ntcp. On the other hand, glyco- and taurochenodeoxycholic acid (GCDC, TCDC) (but not taurocholic acid (TC) or the secondary BA taurodeoxycholic acid (TDC)) induce rapid endocytosis of Ntcp at concentrations reached in portal blood after food intake. This response may protect hepatocytes from high intracellular BA concentrations. Whereas TCDC (but not TC) induces endocytosis of Ntcp, TC was shown to induce the insertion of the concentrative nucleoside cotransporter (CNT2) into the sinusoidal membrane in a PI3K-, ras/raf-, MEK-, and Erk-dependent manner (62), suggesting a transporter and BA-specific network of regulation. TCDC-induced endocytosis of Ntcp involves protein kinase C as well as the protein phosphatase 2B (63). PKC activation induces the internalization of about 40% of membranebound Ntcp within 1 hour (64) in transfected HepG2 cells, which is followed by lysosomal degradation of Ntcp (65), whereas proteasomal degradation is involved in the protein maturation of Ntcp (66). PKC activation induced short-term downregulation of Mrp3 via endocytosis, whereas exocytosis is triggered in response to glucagon/cAMP (67). Apart from interference with the subcellular localization of transporter proteins, PKC may directly inactivate transporters such as rat Oatp1 (Slc21a1) or Oatp2 (Slc21a5). PKC, but not PKA, inhibits Oatp1/2-mediated transport when expressed in Xenopus laevis oocytes (68).

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Taken together, several mechanisms for the modulation of Ntcp transport activity in the sinusoidal membrane exist and may indirectly control transcellular BA secretion.

3.3 Long-term regulation of sinusoidal transport systems Ntcp is an important target of long-term regulation. Downregulation of Ntcp represents an adaptive response to limit BA uptake into hepatocytes and adjust sinusoidal BA uptake in relation to canalicular secretion (14,69). In general, our knowledge about hepatobiliary transporter regulation results from different animal and cell models. For some aspects of bile formation, there are significant differences between species (70–72).

3.3.1 Long-term regulation of Ntcp/NTCP Downregulation of Ntcp by BAs involves the farnesoid X receptor (FXR) together with the small heterodimer partner 1 (Shp1/NROB2) (73) as well as the hepatocyte nuclear factor (HNF)1α (74), the heterodimer of retinoic acid receptor α, and retinoid X receptor α (RARα:RXRα), HNF4α, liver receptor homologue (LRH)-1 (75), and forkhead box A2 (FOXA2), also known as hepatocyte nuclear factor 3-beta (HNF3β) (71,76). Under near-physiological conditions, nutrition-dependent variations of transhepatic bile salt flux have only a minor influence on the expression of Ntcp (and Bsep) in mice (77). Nevertheless, FXR can indirectly repress Ntcp expression via the induction of Shp1 (73). However, a classic BA response element was not found in the human or rodent NTCP/Ntcp promoters (71). It was suggested that BA-dependent NTCP repression occurs via Shp-mediated inhibition of RXRα:RARα or BA-mediated repression of HNF1α or HNF4α (71). Interestingly, in Shp knockout mice, cholic acid still reduced Ntcp mRNA expression, indicating additional pathways of repression (78). A potential pathway represents the interaction of c-Jun N-terminal kinase ( JNK) (79) with RXRα. JNK may phosphorylate RXRα, thereby disrupting its binding to the Ntcp promoter (80). During sepsis-associated cholestasis, interleukin-1β (IL-1β) represses rat Ntcp expression by an IL-1β-induced reduction in the concentration of nuclear RXRα:RARα heterodimers (81). Furthermore, tumor necrosis factor α (TNF-α) was suggested to contribute to the downregulation of Ntcp (82), whereas IL-6 may have a minor impact (83). The signaling pathways seem to be redundant, because knockout of TNFαR, IL-1βR, and IL-6 still allowed Ntcp repression in LPS- or BDL-induced cholestasis (84). Downregulation involves disruption of Ntcp-transactivation by RXRα and may occur at the level of reduced expression (85), altered JNK-dependent phosphorylation of RXR (80), reduced nuclear availability of RXR (86,87) or by competition with coactivators of RXRα (88), such as the steroid receptor coactivator-3 (SRC-3). Furthermore, downregulation of HNF1α may reduce Ntcp expression (89), which may be the consequence of IL-1β-mediated HNF4α downregulation (90). Taurine supplementation can counteract the LPS-induced downregulation of Bsep and Mrp2, probably by ameliorating LPS-induced TNF-α and interleukin 1ß secretion (91). fFig. 3.1 summarizes important aspects on the regulation of Ntcp expression In the bile duct ligation (BDL) model, the process of Ntcp downregulation may involve two phases. Initially, when BAs accumulate inside the hepatocyte, increased BA

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LPS

Ntcp

IL-1b Lrh1 RXR

Shp1

FXR

JNK

Shp

RXR RAR

Ntcp

RXR RAR HNF1a

HNF4a HNF4a

HNF1a

Fig. 3.1: Long-term regulation of Ntcp. Schematic representation of the regulation of the Na+-taurocholate cotransporting polypeptide (Ntcp) by bile salts and interleukin 1β (Il-1β), which involves inhibition of the RXRα:RARα heterodimer. Bile salts activate the expression of small heterodimer partner 1 (shp1) through binding to the farnesoid X receptor (FXR), which in turn interferes with RXRα:RARα binding to the Ntcp promoter. Lipopolysaccharides induce IL-1β secretion, which inhibits RXRα:RARα through JNK-dependent phosphorylation and downregulation of RXRα:RARα expression on the one side and increased degradation of HNF4α and subsequent reduction of HNF1α-expression on the other. (HNF1α: hepatocyte nuclear factor 1α; HNF4α: hepatocyte nuclear factor 4α; JNK:C-Jun N-terminal kinase LRH-1: liver receptor homologue 1; RARα: heterodimer of retinoic acid receptor α; RXRα: retinoid X receptor α.)

concentrations may activate Fxr (92) and increased Shp expression may trigger decreased Ntcp expression (93). Seven days after BDL Shp levels are restored (92), however, repression of Ntcp continues, suggesting an Shp-independent mechanism for perpetuated Ntcp downregulation. Interestingly, Fxr knockout mice had markedly reduced HNF1α and RXRα:RARα levels, whereas Ntcp repression was abrogated, excluding a major role of these nuclear factors in BDL-induced Ntcp regulation (92). Factors responsible for the sustained phase have not yet been identified and apparently do not involve cytokines (85). On the other hand, at least upregulation of Mrp3, an ABC transporter from the ABC-C subfamily expressed at the basolateral membrane of hepatocytes, was mediated by the TNF receptor (TNFR) and the liver receptor homologue 1, an effect that was abrogated in TNFR⫺/⫺ mice (94). In the regenerating liver, Ntcp is downregulated. After partial hepatectomy, Na+dependent TC uptake is rapidly decreased and Ntcp protein expression is reduced by 90%, whereas expression levels of Na+K+-ATPase and ectoATPase are preserved (95). Also, Oatp1 and 2 (rat) are reduced at the mRNA and protein levels by 50% to 60%, whereas canalicular transporter expression remains largely unaffected (96), which may augment removal of potentially toxic substrates from the hepatocyte. However, although uptake transporters are downregulated and serum BA levels are increased about

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10-fold, bile flow and BA excretion into bile per gram of body weight remain unaltered (97) or even increase after partial hepatectomy (98), suggesting that a new steady state of BA homeostasis is achieved during liver regeneration. Although increased bile salt levels are considered to be toxic, elevated BA concentrations and downstream signaling are required for proper liver regeneration (99). In this context it is important to note that BAs can trigger the proliferation (25) and differentiation (100) of hepatic stellate cells, which were identified recently as stem/progenitor cells in the liver (101). Apart from sinusoidal uptake transporters, Mrp3 is also involved in liver regeneration, because regeneration is delayed in Mrp3 knockout mice (102). Hormones can also suppress Ntcp expression. Ethinyl estradiol application strongly reduces transporter expression within days (49). Consistent with an effect of female hormones, male rats have a twofold higher basal expression of Ntcp (103), and administration of estrogens (17β-ethinylestradiol) to male rats decreases Ntcp. This effect is growth hormone-dependent (104). In the chronic cholestasis of humans, Ntcp as well as OATPs and canalicular transport systems are downregulated (105,106), partially because of posttranscriptional mechanisms. As shown in the rat model of bile duct ligation, which is associated with portal inflammation, downregulation of Ntcp and Bsep occurs predominantly in the periportal area of the hepatocyte, whereas the perivenous area remains largely unaffected (107). Evidence has been presented that TNF-α and IL-1ß are involved in triggering Ntcp and Bsep downregulation. However, in this animal model, bile duct ligation was accompanied by an upregulation of Oatps, predominantly in periportal hepatocytes (107). Apart from pathological situations such as sepsis-associated cholestasis, bile duct ligation, or liver regeneration, in which Ntcp expression is downregulated, several inducers of Ntcp expression have been identified. Like canalicular transporter genes, the NTCP gene is transactivated by glucocorticoids via a glucocorticoid response element (GRE), as shown for the human NTCP promoter (108). Furthermore, thyroid hormones were shown to induce Ntcp (104). Finally, fasting mediates the upregulation of Ntcp via HNF4α and PPAR γ coactivator-1α (109).

3.3.2 Long-term regulation of other sinusoidal transporters Like Ntcp, Mrp4 is probably under the negative control of Fxr, because Fxr knockout mice show an increased expression of Mrp4 as compared with normal mice, and further induction by cholic acid feeding of Mrp4 occurred independent of Fxr (110). In line with this, in bile duct-ligated mouse livers, upregulation of Mrp4 was further increased in Fxr knockout mice as compared with wild-type animals (111,112). Chronic cholestasis is a strong stimulus for MRP4 expression in humans (105), which is also increased by UDCA treatment in a not yet identified mode of action (113). There is a zonal heterogeneity in the long-term regulation of multidrug-resistance proteins. In LPS-treated rats, downregulation of canalicular Mrp2 predominantly occurs in the perivenous region, with minor downregulation in the periportal area. Conversely, basolateral Mrp3 is upregulated under these conditions in perivenous hepatocytes (114). Also, Mrp5 mRNA was found to be upregulated under these conditions. This suggests that upregulation of perivenous Mrp3 and mRNA induction of Mrp5 may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides during sepsis (114).

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3.4 Short-term regulation of canalicular secretion Rapid Bsep and Mrp2 insertion and retrieval into and from the canalicular membrane is a major mechanism for the short-term regulation of bile formation (8,14). This mechanism allows the number of transporter molecules in the canalicular membrane to change within minutes, and thus also the secretory capacity (Vmax). Major regulator of this process are changes in hepatocellular hydration, which occur in response to hormones, nutrients, oxidative stress, and changes in ambient osmolarity (for review, see references 115 and 116). In perfused rat liver, cell shrinkage inhibits whereas cell swelling stimulates taurocholate (TC) excretion into bile regardless of whether cell volume is modified by anisotonic exposure, insulin, cumulative amino acid uptake, or ethanol (7,14,117). A 10% increase of the hepatocyte water content is sufficient to double within minutes the Vmax of TC excretion into bile (7). This swelling-induced increase in transport requires intact microtubules (118) and is, as shown by immunohistochemistry, due to a rapid insertion of intracellularly stored Bsep into the canalicular membrane, whereas hyperosmotic hepatocyte shrinkage is cholestatic by triggering a rapid retrieval of Bsep from the canalicular membrane and its transfer into an intracellular vesicular compartment (11). A similar osmoregulation is also found for Mrp2 localization (8,119), which is also reflected at the functional level by corresponding changes in the canalicular secretion of the glutathione conjugates (119,120). Interestingly, Bsep and Mrp2 are retrieved following hyperosmotic hepatocyte shrinkage into different intracellular vesicular compartments. As shown by immunohistochemistry, only 15% of the retrieved vesicles contained both, Bsep and Mrp2, whereas the remainder of vesicles contained either Mrp2 or Bsep (10). This may indicate different retrieval mechanisms for the two transporters in response to hyperosmolarity or the existence of canalicular membrane domains being enriched in either Bsep or Mrp2. Because taurocholate induces hepatocyte swelling and cell swelling triggers Bsep insertion into the canalicular membrane, a feedforward regulation of canalicular secretion with increasing taurocholate loads to the liver has been proposed (7). Such a response would also accelerate enterohepatic circulation of BAs after ingestion of a meal, which by itself also triggers nutrient-driven hepatocyte swelling – for example, by the concentrative uptake of amino acids into hepatocytes. Bsep and Mrp2 insertion into the canalicular membrane is also triggered by tauroursodeoxycholate (TUDC) (121–123), whereas oxidative stress (10) and LPS treatment (119,124) were reported to induce Mrp2 retrieval from the canalicular membrane, possibly due to a dissociation of radixin from Mrp2 (125). Oxidative stress also triggers Bsep internalization (126). Taurolithocholic acid and estradiol-17ß-glucuronide induce Bsep retrieval (12,127), which is counteracted by silibinin (128). Apart from endo- and exocytosis, transporter phosphorylation was also suggested to regulate activity of Bsep. Coexpression of mouse Bsep (mBsep) and mouse PKC alpha in Sf9 insect cells led to strongly increased phosphorylation of mBsep (129). BAs such as TUDC (130) and taurolithocholic acid (131) activate different PKC isoforms. In the intact organ, activation of PKC isoforms by phorbolesters or thymeleatoxin, a selective agonist for Ca2+-dependent PKC isoforms, induces cholestasis (132). Taken together, PKC-dependent Bsep phosphorylation may represent a positive or negative feedback mechanism of canalicular BA secretion. The mechanisms underlying the cell hydration-dependent Mrp2 and Bsep insertion/ retrieval into/from the canalicular membrane are depicted schematically in fFig. 3.2 (see also Chapter 7) (for review, see also reference 133). In brief, hepatocyte swelling

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Hepatocyte shrinkage Oxidative stress

TUDC integrins FAK EGFR

[Cl앥]cyt앖

Src pHves앗

Endosome

ASM PKCz p47phox Erk

p38

NOX

ROS

Bsep and Mrp2 insertion Choleresis

Fyn Cortactin

Yes EGFR CD95

JNK

Apoptosis

Bsep and Mrp2 retrieval Cholestasis

Fig. 3.2: Osmoregulation of canalicular transporter insertion/retrieval. Hepatocyte swelling is sensed by integrins and triggers an activation of focal adhesion kinase, c-Src, EGFR, and MAP kinases. Dual MAP-kinase activation is required for the microtubule-dependent insertion of Bsep, which is located in vesicles underneath the canalicular membrane. Cell shrinkage is sensed by chloride-dependent acidification of endosomes, which activates acidic sphingomyelinase and triggers ceramide formation. Ceramide activates protein kinase Cζ, which phosphorylates p47phox, which activates NADPH oxidase(s). The resulting generation of reactive oxygen species activates a pathway not only to CD95 activation but also to Fyn, which mediates retrieval of Bsep and Mrp2 from the canalicular membrane and triggers cholestasis.

is mechanosensed by α5ß1 integrins and swelling-induced integrin activation triggers the activation of focal adhesion kinase, c-Src, and downstream activation of mitogen activated protein kinases Erks and p38MAPK, which trigger insertion of Bsep into the canalicular membrane (121,122,134,135). Dual activation of both Erks and p38MAPK is required for the choleretic effect of cell swelling, and choleresis induced by hypoosmotic cell swelling is abolished after inhibition of either the integrin system, Src, or one of the two MAP-kinases (11,121,122,136). Interestingly, tauroursodeoxycholate (TUDC) nonosmotically activates the same pathway (122) through a directly activating interaction with the ß1-integrin molecule (137). Ntcp is required for TUDC-induced integrin activation, which largely occurs inside the hepatocyte and explains why the effects of TUDC on integrin signaling are hepatocyte-specific. The signaling mechanisms underlying hyperosmotic Mrp2 and Bsep retrieval have been unraveled recently (see

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fFig. 3.2; see also Chapter 7) and involve endosomal osmosensing with activation of acidic sphingomyelinase, subsequent ceramide formation, protein kinase Cζ-activation, p47phox phosphorylation, and NADPH oxidase activation. The resulting oxidative stress signal induces an activation of the Src family kinase Fyn, which triggers, probably via cortactin phosphorylation, the retrieval of Mrp2 and Bsep (13). A similar mechanism may underlie the transporter retrieval induced by externally added hydroperoxides or toxic hydrophobic BAs, because the latter were also shown to activate NADPH oxidase and to induce oxidative stress (138–140).

3.5 Long-term regulation of canalicular transport systems The structure and biochemistry of the canalicular transport systems are described in Chapter 1. The bile salt export pump BSEP (ABCB11) belongs to the family of the ATP-binding cassette transporter (ABC transporter). At least in humans, BSEP has a major role in maintaining BA homeostasis (see also Chapter 9). Major regulators of BSEP expression at the level of transcription are the farnesoid X receptor (FXR), the liver receptor homologue 1 (Lrh1) (141), and the nuclear factor erythroid 2–related factor 2 (Nrf2) (see fFig. 3.3). In contrast to Ntcp, the farnesoid X receptor (FXR) directly transactivates the BSEP promoter after BA binding to FXR and heterodimer formation with the obligate partner RXRα as shown for the human (142,143) and rat BSEP/Bsep promoter (144). In Fxr⫺/⫺ mice, inducibility of Bsep expression is almost completely abolished and basal expression strongly reduced (145). In mice four different Fxr isoforms have been described with organ-specific expression patterns and different affinities for DNA binding (146). Whether splice variants exist in humans is not yet known; however, there are some

Ntcp

ROS

Nrf2

Lrh1

GC

RXR FXR

Bsep

BS EP

Fig. 3.3: Long-term regulation of BSEP. Schematic representation of the regulation of BSEP expression. Bile acids enter the hepatocytes – for example, by Ntcp-mediated uptake, and bind to FXR, which transactivates the BSEP promoter. Liver receptor homologue (Lrh1) and the ROS-sensor nuclear factor erythroid 2–related factor 2 (Nrf2) probably bind to response elements more distal from the first exon of BSEP. Furthermore, glucocorticoids (GCs) may induce BSEP through glucocorticoid response elements. Increased expression of BSEP eventually lowers intracellular bile acid concentrations (ROS: reactive oxygen species).

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genetic variants of FXR (single nucleotide polymorphisms with prevalences of 2.5%– 12%) and altered function and expression of target genes (147). The most potent natural agonist is chenodeoxycholate (CDCA) (148,149), which is the most abundant BA in humans. Apart from CDCA, the synthetic FXR agonist GW 4064 activates FXR, whereas lithocholic acid was identified as a “natural” antagonist (150). BAs activate the BSEP promoter (via FXR) in the potency rank order CDCA>DCA>CA>UDCA with a ratio of 25:20:18:8 in vitro, similar to the ratios found in HepG2 cells in vivo (∼50:17:5:1) (151). Interestingly, repression of Cyp7A1, another target of FXR action, was almost equal for all BAs (151), and a target-specific efficacy of BA-binding to FXR was suggested. The therapeutically used BA UDCA can induce alternative BA transporters such as Mrp2 and Mrp3 independent of Fxr in mice (152). As mentioned previously, FXR is involved in the adaptive response in obstructive cholestasis. In this setting, maintenance or upregulation of Bsep expression prevents liver damage. Bsep is not expressed after BDL in Fxr⫺/⫺ mice. This is associated with a decreased bile duct pressure on the one hand but with bile infarcts on the other hand (111). However, it has been suggested that disruption of the FXR pathway may even be beneficial in obstructive cholestasis owing to a stronger upregulation of Mrp4 expression, which is otherwise repressed by Fxr (112). MRP4 probably represents an overflow mechanism in humans, and an almost 10-fold induction of MRP4 was seen in patients with progressive familial intrahepatic cholestasis (105). Whether stimulation or inhibition of Fxr is more beneficial in cholestasis is under debate, because a protective effect of the FXR agonist GW4064 has been observed in rats with obstructive cholestasis (153). In line with a protective role of FXR is the observation that stigmasterol acetate, a phytosterol component of intravenous nutrition, acts as an FXR antagonist and may account for total parenteral nutrition-associated cholestasis (154). Furthermore, liver injury was more evident in Fxr⫺/⫺ than in pregnane X receptor knockout mice (Pxr⫺/⫺) after alpha-naphthylisothiocyanate (ANIT) treatment (155), underlining the role of Fxr. On the other hand, lack of adaptation in Fxr⫺/⫺ mice is partially compensated by increased hydroxylation by Cyp3a11 and alternative excretion of hydroxylated bile salts via the kidneys (156). In contrast to FXR, knockout of the pregnane X receptor (PXR) consistently leads to increased liver injury in (obstructive) cholestasis (157,158). It has been suggested that this is due to deficient detoxifying mechanisms rather than elevated BA levels (159). Low PXR expression in children with chronic cholestasis due to biliary atresia has been associated with a poorer prognosis (106). The liver receptor homologue 1 (Lrh1, NR5A2; synonyms: fetoprotein transcription factor, cholesterol-7 alpha-hydroxylase promoter factor) is a key regulator of cholesterol 7 alpha-hydroxylase (CYP7A1), a rate-controlling enzyme for BA synthesis from cholesterol. Lrh1 probably is another regulator of Bsep and Mrp3. In hepatocyte-specific Lrh1 knockout mice, reduced Bsep expression has been observed (141) along with reduced cholic acid synthesis (160). Likewise, Mrp3, Mrp2, and Mrp4 are positively regulated by the nuclear factor erythroid 2–related factor 2 (Nrf2) (161,162) in addition to Bsep (163). Nrf2 is a sensor for oxidative stress (164,165), which may be relevant for counteracting the oxidative stress induced by toxic BAs (166). A single application of perfluorodecanoic acid, a known agonist of peroxisome proliferator-activated receptor α (PPARα), induces Mrp3 up to 4-fold and Mrp4 more than 30-fold in a Nrf2-dependent manner (167), underscoring the hepatoprotective role of Nrf2. It has even been suggested that some beneficial effects of UDCA are mediated by Nrf2 (162).

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Lipopolysaccharide triggers the downregulation of Bsep and Mrp2 (89,124). On the other hand, glucocorticoids are strong inducers of Mrp2 (9,124) and Bsep (168). Furthermore, the expression of Bsep and Mrp2 is subject to osmoregulation. Upregulation of these transporters in response to hypoosmotic hepatocyte swelling and downregulation in response to hyperosmotic cell shrinkage complements the short-term effects of hepatocyte volume changes on biliary secretion (see fFig. 3.2).

3.6 Methods of studying subcellular transporter distribution Various methods have been developed to study the subcellular localization of transporter proteins in epithelial cells such as hepatocytes. Immunostaining of transporters in isolated cells or tissues sections in connection with confocal laser scanning microscopy (8,40) or electron microscopy (119,123) have widely been used. This allows for colocalization with subcellular compartments by the use of marker proteins. Otherwise assignment to subcellular compartments may be achieved by differential (169) or sucrose gradient centrifugation (53). Transport capacity of cells or the intact organ may be used as a surrogate marker of transporter availability (7,118,132). Biotinylation of membrane proteins at the cell surface (60) or flow cytometry (using extracellular epitopes and antibodies) may be used for the quantification of transporter molecules at the plasma membrane (64,170) or the analysis of endocytosis by confocal microscopy (an example is given in fFig. 3.4). Rapid and reversible transporter retrieval and insertion from and into the canalicular membrane of liver parenchymal cells in response to anisoosmotic challenge was shown for Mrp2 (8) and Bsep (11), in response to LPS (124), oxidative stress (10) and tauroursodeoxycholate (121) and hyperosmolarity (119). This technique was also employed for the investigation of estrogen-induced retrieval of Mrp2 (172) and Bsep (12) and phalloidin-induced bulk retrieval of canalicular proteins (173). An important step in the determination of canalicular transporter localization was the covisualization of the transporter proteins together with the tight junction associated protein zonula occludens protein 1 (ZO-1), which delineates the canalicular domain from the basolateral membrane. Canalicular transporter proteins within the canalicular membrane are located between these two lines, whereas retrieved transporter proteins are situated in co-localization or aside from ZO-1 immunofluorescence (8,13). Hyperosmotic exposure has induced retrieval of Bsep and Mrp2, but only 15% of endocytic vesicles were shared by both transporters, indicating that endocytosis is transporter-specific (11). More recently automated methods have been developed for observer-independent rapid determinations of transporter distribution in liver tissue (174). The bile canalicular membrane has a dense architecture and contains many filamentous proteins (175); therefore bile canaliculi are characterized by a high mass density. Centrifugation with low centrifugal forces allows the enrichment of canalicular membranes (169), which may also be achieved by a simplified protocol to separate canalicular membrane from plasma membranes and endomembranes (170). Several independent techniques can be used to demonstrate transporter insertion or retrieval in hepatocytes; all of them support the concept that bile formation is controlled at the short-term time scale by a regulated insertion/retrieval of transporter molecules. Future developments may aim at the visualization of such transporter movements in the living cell and the disclosure of protein/protein interactions involved in this dynamic

3.7 Summary Control

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Fig. 3.4: Visualization of transporter endocytosis. An example of induced endocytosis is given by double-tagged Na+-taurocholate cotransporting polypeptide (FLAG-Ntcp-EGFP), expressed in HepG2 cells. Overall expression of Ntcp was visualized by the intracellular EGFP-tag (green), whereas membrane-bound Ntcp was detected by the extracellular FLAG tag and the use of anti-FLAG antibodies in nonpermeabilized cells. HepG2 cells were preincubated with anti-FLAG-antibodies at 4°C for 45 minutes, followed by extensive washing. Thereafter, cells were incubated for 1 hour at 37°C in the absence or presence of the PKC activator phorbol-12-myristate-13-acetate. In PKC-activated cells, numerous Ntcp-bearing vesicles (green) contained FLAG-antibodies (red), which were endocytosed together with Ntcp (resulting in yellow), whereas most Ntcp-bearing vesicles in control cells remained green, indicating that these vesicles were not recycled from the cell membrane (171).

process; fluorescence resonance energy transfer techniques may have some potential here. However, such studies must be complemented by functional assessments such as the measurement of transporter function in order to obtain information on structural-functional relationships.

3.7 Summary Excretion of lipophilic and hydrophilic compounds into bile is essential for the function of the liver and other organs. In liver parenchymal cells, several transporter proteins are involved in bile formation, which determines substrate concentrations in the blood and bile as well as at intracellular levels. Coordinated action of these transport proteins in the basolateral and canalicular membrane is mandatory and subject to short- and longterm regulation. Apart from mRNA or protein expression, transporter activity is also determined by covalent modifications (e.g., phosphorylation), substrate competition, and subcellular transporter localization. The latter is very dynamic and major target of

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the short-term regulation of bile secretion and involves rapid endo- and exocytosis of transporter-bearing vesicles from and into the respective cell membrane. In liver parenchymal cells, several signaling pathways that regulate these processes have been identified. On a long-term scale, expression is largely controlled by signaling pathways that target nuclear receptors and gene transcription. Most effectors in biliary secretion trigger both short- and long-term regulatory mechanisms.

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116. Lang F, Busch GL, Ritter M, et al. Functional significance of cell volume regulatory mechanisms. Physiol. Rev. 1998;78(1):247–306. 117. Hallbrucker C, Lang F, Gerok W, et al. Cell swelling increases bile flow and taurocholate excretion into bile in isolated perfused rat liver. Biochem. J. 1992;281 (Pt 3):593–5. 118. Häussinger D, Saha N, Hallbrucker C, et al. Involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport in perfused rat liver. Biochem. J. 1993;291 (Pt 2):355–60. 119. Dombrowski F, Kubitz R, Chittattu A, et al. Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide. Biochem. J. 2000;348 (Pt 1):183–8. 120. Wettstein M, Noe B, Häussinger D. Metabolism of cysteinyl leukotrienes in the perfused rat liver: the influence of endotoxin pretreatment and the cellular hydration state. Hepatology 1995;22(1):235–40. 121. Kurz AK, Graf D, Schmitt M, et al. Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 2001;121(2):407–19. 122. Häussinger D, Kurz AK, Wettstein M, et al. Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis. Gastroenterology 2003;124(5):1476–87. 123. Beuers U, Bilzer M, Chittattu A, et al. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 2001;33(5):1206–16. 124. Kubitz R, Wettstein M, Warskulat U, et al. Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone. Gastroenterology 1999b;116(2):401–10. 125. Saeki J, Sekine S, Horie T. LPS-induced dissociation of multidrug resistance-associated protein 2 (Mrp2) and radixin is associated with Mrp2 selective internalization in rats. Biochem. Pharmacol. 2011;81(1):178–84. 126. Perez LM, Milkiewicz P, Elias E, et al. Oxidative stress induces internalization of the bile salt export pump, Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: a role for protein kinase C and prevention by protein kinase A. Toxicol. Sci. 2006;91(1):150–8. 127. Crocenzi FA, Mottino AD, Sanchez Pozzi EJ, et al. Impaired localisation and transport function of canalicular Bsep in taurolithocholate induced cholestasis in the rat. Gut 2003b;52(8):1170–7. 128. Crocenzi FA, Basiglio CL, Perez LM, et al. Silibinin prevents cholestasis-associated retrieval of the bile salt export pump, Bsep, in isolated rat hepatocyte couplets: possible involvement of cAMP. Biochem. Pharmacol. 2005;69(7):1113–20. 129. Noé J, Hagenbuch B, Meier PJ, et al. Characterization of the mouse bile salt export pump overexpressed in the baculovirus system. Hepatology 2001;33(5):1223–31. 130. Beuers U, Throckmorton DC, Anderson MS, et al. Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996;110 (5):1553–63. 131. Beuers U, Probst I, Soroka C, et al. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology 1999;29(2):477–82. 132. Kubitz R, Saha N, Kühlkamp T, et al. Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver. J. Biol. Chem. 2004;279(11):10323–30. 133. Häussinger D, Reinehr R. Osmotic regulation of bile acid transport, apoptosis and proliferation in rat liver. Cell. Physiol. Biochem. 2011;28(6):1089–98.

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155. Cui YJ, Aleksunes LM, Tanaka Y, et al. Compensatory induction of liver efflux transporters in response to ANIT-induced liver injury is impaired in FXR-null mice. Toxicol. Sci. 2009;110(1):47–60. 156. Marschall HU, Wagner M, Bodin K, et al. Fxr⫺/⫺ mice adapt to biliary obstruction by enhanced phase I detoxifi cation and renal elimination of bile acids. J. Lipid Res. 2006;47(3):582–92. 157. Xie W, Radominska-Pandya A, Shi Y, et al. An essential role for nuclear receptors SXR/PXR in detoxifi cation of cholestatic bile acids. Proc. Natl. Acad. Sci. USA 2001;98(6):3375–80. 158. Stedman CA, Liddle C, Coulter SA, et al. Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury. Proc. Natl. Acad. Sci. USA 2005;102(6):2063–8. 159. Stedman C, Robertson G, Coulter S, et al. Feed-forward regulation of bile acid detoxifi cation by CYP3A4: studies in humanized transgenic mice. J. Biol. Chem. 2004;279(12):11336–43. 160. Mataki C, Magnier BC, Houten SM, et al. Compromised intestinal lipid absorption in mice with a liver-specific defi ciency of liver receptor homolog 1. Mol. Cell. Biol. 2007;27(23):8330–9. 161. Maher JM, Dieter MZ, Aleksunes LM, et al. Oxidative and electrophilic stress induces multidrug resistance-associated protein transporters via the nuclear factor-E2-related factor-2 transcriptional pathway. Hepatology 2007;46(5):1597–610. 162. Okada K, Shoda J, Taguchi K, et al. Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 2008;295(4):G735–G47. 163. Weerachayaphorn J, Cai SY, Soroka CJ, et al. Nuclear factor erythroid 2-related factor 2 is a positive regulator of human bile salt export pump expression. Hepatology 2009;50(5):1588–96. 164. Klaassen CD, Reisman SA. Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver. Toxicol. Appl. Pharmacol. 2010;244(1):57–65. 165. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011;16(2):123–40. 166. Tan KP, Yang M, Ito S. Activation of nuclear factor (erythroid-2 like) factor 2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress. Mol. Pharmacol. 2007;72(5):1380–90. 167. Maher JM, Aleksunes LM, Dieter MZ, et al. Nrf2- and PPAR alpha-mediated regulation of hepatic Mrp transporters after exposure to perfluorooctanoic acid and perfluorodecanoic acid. Toxicol. Sci. 2008;106(2):319–28. 168. Warskulat U, Kubitz R, Wettstein M, et al. Regulation of bile salt export pump mRNA levels by dexamethasone and osmolarity in cultured rat hepatocytes. Biol. Chem. 1999;380(11):1273–9. 169. Kipp H, Arias IM. Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver. J. Biol. Chem. 2000;275(21):15917–25. 170. Kubitz R, Helmer A, Häussinger D. Biliary transport systems: short-term regulation. Methods Enzymol. 2005;400:542–57. 171. Stross, Kubitz, Häussinger, unpublished results. 172. Mottino AD, Cao J, Veggi LM, et al. Altered localization and activity of canalicular Mrp2 in estradiol-17beta-D-glucuronide-induced cholestasis. Hepatology 2002;35(6): 1409–19.

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4 Nuclear bile acid receptor FXR and hepatobiliary transport systems Raffaella Gadaleta and Antonio Moschetta

4.1 Introduction Bile acids (BAs) are synthesized from cholesterol in the liver and released in the small intestine after food intake to facilitate intestinal absorption of lipids and lipid-soluble molecules. Then, BAs travel back to the liver, where they are reabsorbed and subjected to a cycle called BA enterohepatic circulation. The nuclear farnesoid X receptor (FXR) is a transcription factor known as the master regulator of BAs homeostasis. In fact, FXR critically regulates BA synthesis and enterohepatic circulation. This chapter focuses on the role of FXR in the transcriptional regulation of genes encoding proteins directly involved in hepatobiliary transport.

4.2 Nuclear receptors Nuclear receptors (NRs) are ligand-activated transcription factors regulating vital aspects of mammalian physiology such as development, metabolism, and reproduction (1). NRs transactivate target genes through the binding of consensus NR-response elements (NR-RE), which are located in the regulatory 5’-flanking region of the target genes. NRs are unique transcription factors because their activity is regulated by specific ligands that easily pass biological membranes, making them ideal drug targets. Small lipophilic molecules such as retinoids, steroids, prostanoids, oxysterols, bile acids, vitamin D, and thyroid hormone as well as semisynthetic and synthetic compounds have been shown to activate NRs (2). The NR superfamily of transcription factors consists of three subfamilies: endocrine, orphan, and adopted NRs. The endocrine subfamily encompasses those NRs having known ligands even before cloning. In 1985, the glucocorticoid receptor (GR) was the first cloned NR (3). The orphan NRs have no identified ligands yet. The members of the adopted NR subfamily were first cloned on the basis of sequence homology with members of the endocrine subfamily. Then their natural ligands were identified with the process of “reverse endocrinology” (4,5). This deorphanization process has been pivotal for NRs such as liver X receptor (LXR), farnesoid X receptor (FXR), peroxisome proliferator− activated receptors (PPARs), and the consequent discovery of cholesterol-, bile acid-, fatty acid − and eicosanoid-related metabolic signaling pathway. All of the NRs are thought to be evolutionary derived from a common ancestor. It has been suggested that the ancestral receptors were constitutive homodimeric transcription factors that acquired the ability to bind a ligand and heterodimerize. Like other transcription factors, NRs have a similar modular structure characterized by different regions corresponding to different functional domains. A typical NR presents a variable N-terminal domain, a highly conserved DNA-binding domain (DBD), a moderately conserved

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ligand-binding domain (LBD), and a hinge region between the DBD and LBD (6). The N-terminal domain is the most variable both in size and sequence and frequently contains a ligand-independent activation domain. The N-terminal domain contributes to the specificity of NR isoforms because it can interact with cell type−specific factors. Furthermore, this modulatory domain is a phosphorylation site. This modification is mediated by different signaling pathways and can significantly affect the NR transcriptional activity. The DBD gives NRs the ability to recognize specific DNA consensus sequences on their target genes – e.g., nuclear responsive elements (NREs). NREs are palindromic sequences organized as inverted, everted, or direct repeats separated by n nucleotides (IRn, ERn, and IRn, respectively). In general NRs of the adopted subfamily constitutively bind the NRE of their target genes as heterodimers with a common partner, a NR called retinoid X receptor (RXR). However, some of them can bind the DNA with high affinity as monomers. On the contrary, members of the endocrine NR subfamily are commonly located in the cytoplasm. After ligand binding, they move to the nucleus and regulate the expression of their target genes almost exclusively as homodimers. Two steroid hormone receptor monomers bind cooperatively to their response elements, and some dimerization interfaces have been identified both in the LBD and in the DBD (7). The hinge domain is not well conserved among the different receptors and connects the DBD to the LBD, allowing rotation of the DBD. Frequently the hinge domain bears nuclear localization signals and contains residues whose mutation abolishes interaction with NR corepressor proteins. The LBD is a multifunctional domain that mediates ligand-dependent transcriptional activity, homo- and heterodimerization, and in some circumstances hormone-reversible transcriptional repression. Generally NRs become active after ligand binding. Through a conformational change they release corepressors and interact with coactivator proteins (8,9). However, there are NRs that are constitutively active even without ligand activation, such as the nuclear receptor related 1 (NURR1) and neuron-derived receptor 1 (NOR1). Furthermore, in order to transactivate genes, NRs interact not only with coactivators and corepressors but also with other proteins of the basal transcriptional machinery. Although the functionality of (direct) protein-protein interactions between NRs and proteins of the basal transcriptional machinery is yet to be determined, it is likely that these interactions cause the recruitment of the basal transcriptional components to the promoter as well as the enhancement of transcription. In promoters, NREs are located close to recognition sequences for other transcription factors, and the interaction between NRs and these factors can play an important role in determining transcriptional rates, resulting in functional synergism or repression. The current model of gene regulation by these receptors assumes that the unliganded receptors bind the NRE together with corepressors, silencing the transcriptional activity. Upon ligand binding, the conformational changes of the receptors cause the dissociation of corepressors and the recruitment of coactivator complexes, resulting in transcriptional activation. NRs also work independently of direct DNA-binding, as in the transrepression, in which NRs interfere with other transcription factors (e.g., AP-1, NF-κB). NRs are also regulated by several posttranslational modifications (e.g., phosphorylation, acetylation, sumoylation). Nevertheless, these molecular pathways are largely elusive, and how these modifications and interactions contribute to transcriptional rate changes is largely unknown. The NR superfamily has a high potential as drug targets

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because their lipophilic ligands can easily pass biological membranes. Currently, at least 13% of all FDA-approved drug targets are NRs (10), which are used for the management of several pathological conditions such as breast and prostate cancer, inflammation, and metabolic diseases.

4.2.1 Farnesoid X receptor (FXR) In 1995 the first report about FXR claimed the discovery of a new protein able to interact with RXR, which was termed RXR-interacting protein 14 (RIP14) (11). In the same year Forman et al. (12) cloned the rat homologue of the mouse RIP14 and showed that it could be activated by superphysiological levels of an intermediate of the mevalonate pathway, e.g., the farnesol. For this reason, the rat RIP14 was termed farnesoid X receptor (FXR). Its expression was detected in the liver, kidney, intestine, and adrenal cortex. However, only superphysiological concentrations of farnesol were able to induce FXR activity, and this was true for the rat protein only. In 1999, it was demonstrated that primary bile acids (BAs) were the endogenous ligands of FXR, ending up the “deorphanization” process (13−15). As a consequence, FXR was also named BAR (bile acid receptor). Given their intrinsic high cytotoxicity, the concentration of BAs in the body must be tightly regulated. In fact, FXR is the master regulator of bile acid synthesis, transport, and metabolism. This regulation is exploited through the integration of dietary and hormonal signals. There are two genes encoding for FXR – e.g., FXRα and FXRβ. The FXRβ gene is functional in rabbits, rodents, and dogs only, whereas it is a pseudogene in humans and primates. In this chapter we will further refer to the FXRα form only. The FXRα gene maps to human chromosome 12q23.1 and mouse chromosome 10c.2; it consists of 11 exons and 10 introns. The FXR gene has two functional promoters and undergoes alternative splicing, resulting in four possible isoforms (16). These isoforms are expressed in a tissue-specific way and few FXR target genes are regulated in an isoform-dependent manner (17−19). In humans and mice, FXR is mainly expressed along the entire gastrointestinal tract (with a particularly high peak in the liver and ileum) and in the kidney and adrenal gland, while low expression profiles have been detected in the heart and adipose tissue (19). Consistent with its most predominant expression in the liver and ileum, FXR has an established role in the regulation of the enterohepatic circulation of BAs and in the negative feedback regulation of BA biosynthesis. In the absence of BAs, FXR binds to the FXR-responsive element (FXRE) on the promoter of its own target genes as a heterodimer FXR:RXR, and in association with corepressor proteins. The FXRE consists of an inverted repeat of the canonical hexanucleotides (AGGTCA) spaced by one nucleotide (IR-1). Besides IR-1, FXR might bind other FXREs such as DR-1 and ER-8. After ligand binding, FXR undergoes a conformational change, which determines the release of corepressors such as the nuclear corepressors (NCor), and the recruitment of coactivators such as the steroid receptor coactivator 1 (SRC-1) or peroxisome-proliferator-receptorγ coactivator-1α (PGC1α) (20,21). The generation of FXR knockout mice was a breakthrough for understanding the role of FXR in many BA-related physiological and pathophysiological conditions. In 2000, FXR knockout mice confirmed the role of FXR as master regulator of BA homeostasis in vivo (22). In fact, loss of FXR was responsible for the enlargement of the BA pool size owing to the increased expression of CYP7A1, which is the rate-limiting enzyme in the BA biosynthetic

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pathway. Furthermore, the impairment of BA transport and clearance makes FXR knockout mice highly susceptible to hepatic tumors (23,24). These observations underline the crucial physiological need of a tight control of BA levels in the body. In this context, FXR plays the key role of BA sensor.

4.3 Bile acids and the enterohepatic circulation BAs are amphipathic molecules synthesized from cholesterol in the liver, stored in the gallbladder, and released in the small intestine after food intake. They have several fundamental roles, ranging from bile formation to biliary cholesterol solubilization and intestinal absorption of lipids and lipid-soluble molecules. Currently, BAs are also increasingly recognized as signaling molecules in a variety of activities, including lipid and glucose metabolism and energy homeostasis, which are exploited via their major underlying pathway, the BA-mediated activation of FXR (25). BAs are detergent-like molecules and BA accumulation has highly detrimental effects, especially in organs exposed to a high flux of these cytotoxic molecules. Therefore BA levels must be tightly regulated. BAs are synthesized in the liver from cholesterol through a pathway of multiple enzymatic steps. Primary human BAs, chenodeoxycholate (CDCA) and cholate (CA), are the end products of cholesterol catabolism. In order to increase their water solubility and consequently decrease their cytotoxicity (26), after synthesis CA and CDCA are conjugated in the liver to taurine or glycine (27). Conjugated BAs cannot exit the liver by passive diffusion, and a process of active secretion is required. Various transporters for BAs and other major bile lipids (phosphatidylcholine and cholesterol) have been identified in the liver, where they are tightly regulated by FXR and LXR. Bile formation takes place at the level of the hepatocyte apical canalicular membrane and is driven by active secretion of BAs by the bile salt export pump (BSEP/ABCB11) (28). Together with BAs, two other major lipids, cholesterol and phosphatidylcholine, are secreted into the human bile by the canalicular membrane transporters ABCG5/ ABCG8 (29) and the multidrug-resistance protein 3 MDR3/ABCB4, respectively (30). The secreted bile is subsequently modified in the biliary tract. During fasting, human bile is temporarily stored in the gallbladder, where absorption of water and bile concentration (up to 10-fold) occur. Upon food ingestion, the hormone cholecystokinin is released from the proximal intestinal tract, causing gallbladder contraction and delivery of the bile to the intestine. Then BAs travel along the intestine and in the distal ileum, the majority (95%) is actively reabsorbed by the apical sodium-dependent bile acid transporter (ASBT, SLC10A2) (31). After their reabsorption, BAs are secreted from the enterocyte into the portal blood by the basolateral heterodimeric organic solute transporters OSTα-OSTβ (32) and transported back to the liver, where their great majority is reabsorbed by the sodium (Na)-taurocholate cotransporter protein (NTCP, SLC10A1). Finally, BAs are resecreted into the bile (33) via a route known as the enterohepatic circulation (fFig. 4.1). The enterohepatic circulation of BAs is extremely important in view of the high energy expenditure required for BAs biosynthesis (33). In fact, only 0.5 g /day of BAs are lost through the feces. This is then made up by de novo hepatic synthesis.

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Liver

BA

Bile acids

CH

Cholesterol

PC

Phosphatidylcoline

PS

Phosphatidylserine

Gallbladder

Portal vein

Small intestine

Fig. 4.1: The enterohepatic circulation. At the hepatocytic canalicular membrane BSEP/ ABCB11 transports BAs, MDR3/ABCB4, and ABCG5/G8 transport phosphatidylcholine and cholesterol, respectively, into bile. After their biliary secretion, BAs are stored in the gallbladder. After feeding, the gallbladder contracts and pushes the bile into the intestine, where it helps the absorption of lipids and liposoluble molecules. In the ileum BAs are taken up at the apical membrane via ASBT and secreted into the portal vein from the basolateral membrane via the OSTα /β transporters. In the ileocytes, BA-dependent FXR activation results in production/secretion of FGF15/19. This hormone exerts negative feedback on bile salt de novo synthesis in the liver and induces gallbladder dilatation. The BAs secreted into the portal vein are transported back to the liver, where they are taken up by NTCP.

4.4 Bile acid homeostasis, enterohepatic circulation, and FXR Proper bile formation and transport is based on the coordinate activity of the liver and intestine and is modulated by dietary and hormonal signals. In order to maintain a functional BA pool and avoid an overexpenditure of energy for their de novo synthesis, BAs are extensively recycled in the body by a complex transport system, which mainly involves the liver and intestine. The synthesis, transport, and metabolism of BAs are interconnected processes that undergo extensive feedback and feedforward regulation.

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Cyp7A1 Bile acids Cyp8B1 neosynthesis FGF15/19 SHP Bile acids transport

NTCP ABCB11 ABCB4

BA

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Liver

FXR RXR

Intestine

Repression of bile acids de novo synthesis and gallbladder dilation

FGF15/19

FXRE Bile acids reabsorption Induced

ASBT IBABP OST OST

Repressed

Fig. 4.2: Simplified gene regulation network controlled by FXR. FXR binds on the DNA to FXREs, as heterodimer with RXR. Upon binding of BAs, FXR transcriptional activity turns on, resulting in liver-intestine cross-talk to coordinately regulate genes involved in BAs synthesis and transport.

An increase of intracellular BA levels results in their increased binding to FXR and transcriptional activation of FXR target genes. Therefore BAs synthesis and transport, as well as proteins involved in these processes, are transcriptionally regulated by FXR (fFig. 4.2). This process ensures a very efficient enterohepatic circulation of BAs, limiting their fecal and urinary loss. The coordinated action of BA transport proteins keeps ongoing hepatic extraction from the portal blood, biliary secretion, and absorption of BAs in the intestine.

4.4.1 Bile acid de novo synthesis and its regulation: CYP7A1, CYP8B1 and FXR Every day approximately 500 mg of cholesterol is converted into BAs in the liver of a healthy man. Cholesterol 7α-hydroxylase, encoded by the gene CYP7A1, is the first and rate-limiting enzyme of the classic neutral pathway that accounts for the majority of total BA synthesis (34). CYP7A1 is a member of the cytochrome P450 superfamily. This key enzyme catalyzes the conversion of cholesterol in 7α-hydroxycholesterol, which is crucial for the following enzymatic steps ultimately leading to BA synthesis. Mice deficient in the CYP7A1 have a high incidence of postnatal lethality due to liver failure, vitamin deficiencies, and lipid malabsorption. The BA pool size of these animals is reduced by 75%, and the reduction in BA synthesis is not compensated by the increased expression of other BA biosynthetic enzymes. In humans, the “classic” neutral pathway leads to an equal amount of CDCA and CA. An “alternative” acidic pathway is responsible for the production of oxidized cholesterol molecules, which are then converted predominantly to CDCA in the liver (35). CYP7A1 expression is induced by the orphan nuclear liver receptor homolog-1 (LRH-1) and an oxysterols sensor, LXR, in rodents (36−39). In humans, LXR is not involved in the regulation of CYP7A1 expression, since no LXREs have been found on the human CYP7A1 promoter (40). BAs are able to repress their own synthesis in a feedforward way through FXR, which acts synergistically in the intestine and liver. During fasting, BAs are synthesized from cholesterol in the liver, stored in the gallbladder, and ready to be delivered to the small intestine. After a postprandial stimulus, BAs are delivered from the gallbladder

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to the duodenum, pass through the enterohepatic circulation, and ultimately end up into the liver. The first pathway governing this feedback inhibitory circuit starts in the liver with the BA-mediated activation of FXR, which in turn induces the hepatic expression of the atypical NR small heterodimer partner (SHP). SHP lacks the DBD (thus it is not able to bind to the DNA), and inhibits the activity of several NRs. In rodents, SHP represses the activity of LRH-1 (36,37,41) and LXR, thereby indirectly repressing the expression of CYP7A1 and, consequently BAs synthesis (36,41). FXR not only downregulates CYP7A1 in the liver but also suppresses the expression of CYP8B1, the rate-limiting enzyme of the alternative acidic pathway for BA synthesis. CYP8B1 is also repressed by FXR via SHP (42). In this case, SHP represses the ability of the hepatocyte nuclear factor 4α (HNF-4α) to induce CYP7B1 expression. Importantly, hepatic CYP7A1 mRNA levels are repressed after administration of BAs to SHP knockout mice (43). This stresses the existence of a redundant SHP-independent pathway, which represses the BA synthesis. Indeed, there is a second pathway through which BAs regulate their own production through FXR activation in the ileum. In the enterocytes, FXR mediates the induction of the hormone-like molecule fibroblast growth factor 15/19 (FGF15/19 in mouse and human, respectively), which is immediately secreted into the portal circulation and reaches the liver. There, it binds the fibroblast growth factor receptor 4 (FGFR4), which in turn activates a phosphorylation cascade via a c-jun N-terminal kinase-dependent pathway (44) and this strongly suppresses the CYP7A1 expression in a SHP-independent manner (44). Additionally, FGF15/19 controls gallbladder filling and opposes the action of cholecystokinin, which triggers gallbladder emptying (45).

4.4.2 Hepatic export of bile acids: BSEP, MRP2, MDR3, and FXR Newly synthesized BAs are conjugated to taurine or glycine and then actively secreted into the gallbladder. Newly synthesized BAs activate FXR in the liver, which in turn induces the expression of the bile salt export pump (BSEP/ABCB11) and promotes BA hepatic export. Under physiological conditions, monoanionic conjugated BAs are excreted into the bile via BSEP. BSEP is an adenosine triphosphate (ATP)-binding cassette (ABC) transporter localized in the canalicular or apical domain of the hepatocyte plasma membrane. ABC transporters are membrane proteins that use the energy of ATP hydrolysis to transport solutes against their gradient of concentration. BSEP was cloned in 1998 and identified as the gene mutated in the progressive familial intrahepatic cholestasis (PFIC) type 2 (46), which is a pathological condition characterized by impaired bile secretion and flow resulting in the accumulation of toxic BAs in the liver. In mammalian species, the canalicular BA concentration is about 1000-fold higher than that of the portal blood, underscoring the need of an active ATP-dependent efflux system at the canalicular pole of the hepatocytes. Mice with homozygous disruption of BSEP show radical impairment of bile flow with a reduction of the total BA output by about 30%. In the human liver, BSEP is a 160-KDa protein. Orthologous gene products for BSEP exist in a variety of animal species, including the mouse, rat, rabbit, and pig. In addition to BSEP, a second canalicular efflux pump for conjugated BAs is the multidrugresistance protein 2 (MRP2, ABCC2). MRP2 mediates the export of multiple organic anions from the hepatocytes into the bile. Its enterohepatic expression is confined to the canalicular domain of the hepatocytes and the apical domain of the enterocytes in the

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proximal small intestine. MRP2 is positively regulated by another NR, which is called pregnane X receptor (PXR). Notably, FXR also directly induces the expression of the canalicular phospholipid transporter ABCB4 to avoid the damage of the biliary epithelia by increased biliary concentration of BAs. The ABCB4 protein was previously termed multidrug-resistance P-glycoprotein 2/3 (MDR2/3 in mice and human, respectively), since it has been found highly expressed in neoplastic cells characterized by a notable resistance against chemotherapeutics. Homozygous ABCB4 knockout mice develop inflammatory liver disease, leading to fibrosis and end-stage cirrhosis. Likewise, mutations of ABCB4 in humans result in progressive familial intrahepatic cholestasis (PFIC) type 3 (47). Phenotypic analysis of homozygous ABCB4 knockout mice has revealed the complete absence of biliary phospholipids, and about half the levels in heterozygous mice compared with control animals. The FXR-dependent concomitant activation of BSEP and MDR3 has an extensive and protective physiological value because the BAs cytotoxicity is avoided by their incorporation into phospholipid micelles. As a result, the FXR-mediated regulation of these transporters prevents the cytotoxicity of detergent-like BA molecules in the liver and biliary tract.

4.4.3 Intestinal import/export of bile acids After a postprandial stimulus, BAs are released from the gallbladder and travel along the small intestine to facilitate the absorption of lipids and lipid-soluble molecules. In the terminal ileum, BA reabsorption from the intestinal lumen occurs, which is a critical step in BA homeostasis and the major determinant of the BA pool size. The first step involves BA uptake into the enterocytes via the apical sodium-dependent bile salt transporter (ASBT, SLC10A2). The ASBT gene maps to chromosome 13q33.55, and the human ASBT protein consists of 348 amino acids. After their uptake at the apical side of the enterocytes, BAs reach the basolateral side and undergo efflux into the portal circulation. Intracellular transport is mediated by the 14-kDa ileal bile acid−binding protein (IBABP), which is cytoplasmatically attached to ASBT (48−50). Finally, BAs are secreted into the portal blood by the heterodimeric organic solute transporter α /β (OSTα /β) (51). This complex process of active BA intestinal absorption is entirely headed by FXR. Activation of FXR in the distal ileum downregulates the expression of ASBT while also inducing the expression of IBABP (52,53) and OSTα /β (54,55). Notably, FXREs have been found in the promoter of IBABP and OSTα /β, indicating a direct regulation of these genes by FXR. However, FXR-mediated negative regulation of the intestinal apical BA uptake by ASBT is more complex and still under investigation, with considerable species differences (56−61). Nevertheless, FXR likely inhibits ASBT expression in most instances. In mice it has been shown that BA-activated FXR induces SHP, which in turn regulates LRH1. LRH1 ultimately represses the ASBT expression. On the contrary, in the rat, no LRH1-binding site has been found in the promoter region of ASBT. In humans it has been proposed that BA-activated FXR can repress the ASBT expression via the SHP-mediated activation of RXRα-RARα heterodimer.

4.4.4 Hepatic import of bile acids The final step in the BAs enterohepatic circulation is their extraction from the portal blood by the hepatocytes. More than 80% of conjugated BAs are extracted by the liver, mainly via the Na-taurocholate cotransporting polypeptide (NTCP). NTCP

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belongs to the same transporter family as ASBT. However, its substrate specificity is not limited to BAs but also includes sulfated sex steroids, bromosulfophthalein, thyroid hormones, and the drug conjugate chlorambucil-taurocholate. The human protein consists of 349 amino acids and shows a high affinity for taurocholate (62). In the rat and humans, NTCP has been localized on the basolateral hepatocyte membrane. The role of FXR-driven and NTCP-mediated regulation of hepatic sinusoidal apical BA uptake is complex and subject to species differences. In the rat, it has been shown that the FXR-induced activation of SHP negatively modulates the RXRα-RARα-mediated activation of NTCP. However, further research about SHP-independent mechanisms is currently ongoing, since the bile acid-induced downregulation of NTCP expression also occurs in SHP knockout mice. Also, the human NTCP promoter does not contain RXRα-RARα consensus elements as in the rat (60). Therefore, it is likely that there are RXRα-RARα−independent mechanisms for the BA-dependent downregulation of NTCP expression.

4.5 The role of FXR in the pathogenesis of biliary diseases Since the BA-FXR axis is involved in the regulation of bile production and transport, FXR is of crucial importance in the pathophysiology of liver- and biliary tract-related diseases such as cholesterol gallstone disease and cholestasis. Following, we briefly focus on the role of FXR in these two pathological conditions.

4.5.1 Cholesterol gallstones disease (CGD) Cholesterol is insoluble in an aqueous environment. Thus it is incorporated in the bile in micelles with BAs and phosphatidylcholine and/or in phosphatidylcholine vesicles. The correct ratio among biliary BAs, phospholipids, and cholesterol is of crucial importance to maintain cholesterol in solution in the bile and prevent the precipitation of cholesterol crystals, which is the first step in cholesterol gallstone formation (63). FXR knockout mice on a lithogenic diet show biliary cholesterol supersaturation and are more susceptible to cholesterol gallstone formation than wild-type mice. The explanation is that the amount of BAs and phosphatidylcholine in the bile is decreased as the transport proteins BSEP and MDR3 are less expressed in the absence of FXR, whereas ABCG5/8 shows no change. In wild-type mice on a lithogenic diet, gallstone formation can be prevented by the synthetic FXR agonist GW4064. In fact, in these conditions increased amounts of solubilizing BAs and phospholipids prevent cholesterol supersaturation and nucleation of cholesterol crystals (64). For these reasons, FXR agonists may be useful for the prevention or management of CGD. However, caution is needed because FXR activation also inhibits BA synthesis by reducing CYP7A1 expression. This results in a decreased BA pool size, which subsequently leads to decreased BA content and increased cholesterol levels in the bile and thus susceptibility to gallstone formation.

4.5.2 Intrahepatic cholestatic conditions Cholestasis consists of impaired bile secretion and flow. As a result, intrahepatic accumulation of BAs may occur and potentially cause fibrosis, inflammation, and cirrhosis. In cholestatic conditions, changes in the expression of biliary transporters

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represent a compensatory response that limits the hepatocellular accumulation of potentially toxic biliary constituents and provides alternative excretory routes. These compensatory mechanisms depend on the BA-induced FXR activation. Besides the activation of BSEP and MRP2, BA-activated FXR also drives the expression of the hepatic basolateral OSTα /β. As a result, BAs are secreted into the systemic circulation and eliminated via the urine. Under physiological conditions, no BAs are excreted into the urine. The minimal amounts of BAs reaching the kidney are promptly reabsorbed by the ASBT transporter (which is localized at the apical membrane of proximal renal tubular cells) and then secreted into the systemic circulation by the basolateral membrane OSTα /β (65−67). During cholestasis, passive glomerular filtration accounts for the loss of BAs through the urine owing to high serum BA levels and repression of ASBT expression in the kidney (68). It is still controversial whether cholestasis leads to decreased or increased ileal BA reabsorption in vivo (69), since the quantities of BAs within the intestinal lumen are often decreased. It has been suggested that decreased BA absorption and insufficient FXR-dependent FGF15/19 secretion by the enterocytes can lead to a vicious cycle where inappropriately high hepatic BAs are de novo synthesized and the liver is progressively damaged. Animal models have also been used to investigate the role of FXR in the pathogenesis and treatment of cholestasis. For instance, in the α-naphthylisothiocyanate−induced acute model of intrahepatic cholestasis of short duration (70), GW4064-dependent activation of FXR markedly reduces liver injury. Also, in the 17α-ethinylestradiolmediated chronic rat model of cholestasis the FXR semi-synthetic agonist 6-ethyl chenodeoxycholic acid (6-ECDCA) protects from cholestasis by increasing the expression of SHP, BSEP/ABCB11 and MDR3/ABCB4 while reducing the expression of CYP7A1 and CYP8B (which are involved in BA neosynthesis) or NTCP (which accounts for basolateral BA uptake) (71).

4.6 Concluding remarks In this chapter we have discussed the physiological role of the BA-FXR axis in the BA synthesis and transport and in two pathological conditions, cholesterol gallstone disease and cholestasis. Although further research is needed, there is extensive experimental evidence suggesting that the modulation of FXR expression and activity may be transferred from the bench to bedside using this NR as a drug target for the treatment of hepatobiliary diseases. Several FXR agonists, such as 6-ECDCA, are currently being tested in phase II/III clinical trials for metabolic and chronic liver diseases with the goal of demonstrating their safety and patients’ tolerance to these drugs (72). Nevertheless, it is important to underline that indiscriminate use of NR ligands such as FXR ligands would definitely have undesirable side effects along with the beneficial ones. Several experimental models and clinical trials support the need for further research in order to identify selective FXR modulators. Clinical data are necessary to prove the long list of positive and negative results observed so far in different animal models. The dissection of FXR transcriptional functions as well as elucidation of its tissue-specific transcriptional activity are necessary to unravel whether and how a certain ligand acts as an agonist or antagonist for a specific gene. This will improve the drug design for FXR and lead to the development of a new generation of target-specific drugs.

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23. Kim I, Morimura K, Shah Y, et al. Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice. Carcinogenesis 2007;28:940–6. 24. Yang F, Huang X, Yi T, et al. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Cancer Res. 2007;67:863–7. 25. Thomas C, Pellicciari R, Pruzanski M, et al. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 2008;7:678–93. 26. Moschetta A, vanBerge-Henegouwen GP, Portincasa P, et al. Hydrophilic bile salts enhance differential distribution of sphingomyelin and phosphatidylcholine between micellar and vesicular phases: potential implications for their effects in vivo. J. Hepatol. 2001;34:492–9. 27. Falany CN, Johnson MR, Barnes S, et al. Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and expression of human liver bile acid CoA:amino acid N-acyltransferase. J. Biol. Chem. 1994;269:19375–9. 28. Gerloff T, Stieger B, Hagenbuch B, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J. Biol. Chem. 1998;273: 10046–50. 29. Berge KE, Tian H, Graf GA, et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 2000;290:1771–5. 30. Smit JJ, Schinkel AH, Oude Elferink RP, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993;75:451–62. 31. Wong MH, Oelkers P, Dawson PA. Identifi cation of a mutation in the ileal sodiumdependent bile acid transporter gene that abolishes transport activity. J. Biol. Chem. 1995;270:27228–34. 32. Dawson PA, Hubbert M, Haywood J, et al. The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter. J. Biol. Chem. 2005;280:6960–8. 33. Love MW, Dawson PA. New insights into bile acid transport. Curr. Opin. Lipidol. 1998;9:225–9. 34. Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 2003;72:137–74. 35. Chiang JY. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr. Rev. 2002;23:443–63. 36. Goodwin B, Jones SA, Price RR, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 2000;6:517–26. 37. Lee YK, Moore DD. Liver receptor homolog-1, an emerging metabolic modulator. Front. Biosci. 2008;13:5950–8. 38. Lehmann JM, Kliewer SA, Moore LB, et al. Activation of the nuclear receptor LXR by oxysterols defi nes a new hormone response pathway. J. Biol. Chem. 1997;272: 3137–40. 39. Peet DJ, Turley SD, Ma W, et al. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 1998;93:693–704. 40. Goodwin B, Watson MA, Kim H, et al. Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Mol. Endocrinol. 2003;17: 386–94. 41. Lu TT, Makishima M, Repa JJ, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell 2000;6:507–15. 42. Zhang M, Chiang JY. Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression. J. Biol. Chem. 2001;276:41690–9.

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43. Kerr TA, Saeki S, Schneider M, et al. Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis. Dev. Cell 2002;2: 713–20. 44. Inagaki T, Choi M, Moschetta A, et al. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell Metab. 2005;2:217–25. 45. Choi M, Moschetta A, Bookout AL, et al. Identification of a hormonal basis for gallbladder filling. Nat. Med. 2006;12:1253–5. 46. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat. Genet. 1998;20: 233–8. 47. de Vree JM, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc. Natl. Acad. Sci. USA 1998;95:282–7. 48. Gong YZ, Everett ET, Schwartz DA, et al. Molecular cloning, tissue distribution, and expression of a 14-kDa bile acid-binding protein from rat ileal cytosol. Proc. Natl. Acad. Sci. USA 1994;91:4741–5. 49. Tochtrop GP, DeKoster GT, Covey DF, et al. A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein. J. Am. Chem. Soc. 2004;126: 11024–9. 50. Toke O, Monsey JD, DeKoster GT, et al. Determinants of cooperativity and site selectivity in human ileal bile acid binding protein. Biochemistry 2006;45:727–37. 51. Dawson PA, Hubbert M, Haywood J, et al. The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter. J. Biol. Chem. 2005;280:6960–8. 52. Grober J, Zaghini I, Fujii H, et al. Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9cis-retinoic acid receptor heterodimer. J. Biol. Chem. 1999;274:29749–54. 53. Kok T, Hulzebos CV, Wolters H, et al. Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein. J. Biol. Chem. 2003;278:41930–7. 54. Landrier JF, Eloranta JJ, Vavricka SR, et al. The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes. Am. J. Physiol. Gastrointest. Liver Physiol. 2006;290:G476–G85. 55. Rao A, Haywood J, Craddock AL, et al. The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis. Proc. Natl. Acad. Sci. USA 2008;105:3891–6. 56. Chen F, Ma L, Dawson PA, et al. Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J. Biol. Chem. 2003;278:19909–16. 57. Denson LA, Auld KL, Schiek DS, et al. Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J. Biol. Chem. 2000;275:8835–43. 58. Denson LA, Sturm E, Echevarria W, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–7. 59. Gartung C, Ananthanarayanan M, Rahman MA, et al. Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 1996;110:199–209. 60. Jung D, Hagenbuch B, Fried M, et al. Role of liver-enriched transcription factors and nuclear receptors in regulating the human, mouse, and rat NTCP gene. Am. J. Physiol. Gastrointest. Liver Physiol. 2004;286:G752–G61.

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61. Neimark E, Chen F, Li X, et al. Bile acid-induced negative feedback regulation of the human ileal bile acid transporter. Hepatology 2004;40:149–56. 62. Hagenbuch B, Meier PJ. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter. J. Clin. Invest. 1994;93:1326–31. 63. Lo SG, Petruzzelli M, Moschetta A. A translational view on the biliary lipid secretory network. Biochim. Biophys. Acta 2008;1781:79–96. 64. Moschetta A, Bookout AL, Mangelsdorf DJ. Prevention of cholesterol gallstone disease by FXR agonists in a mouse model. Nat. Med. 2004;10:1352–8. 65. Ballatori N, Christian WV, Lee JY, et al. OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia. Hepatology 2005;42:1270–9. 66. Christie DM, Dawson PA, Thevananther S, et al. Comparative analysis of the ontogeny of a sodium-dependent bile acid transporter in rat kidney and ileum. Am. J. Physiol. 1996;271:G377–G85. 67. Craddock AL, Love MW, Daniel RW, et al. Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter. Am. J. Physiol. 1998;274:G157–G69. 68. Lee J, Azzaroli F, Wang L, et al. Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology 2001;121:1473–84. 69. Zollner G, Marschall HU, Wagner M, et al. Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Mol. Pharm. 2006;3:231–51. 70. Liu Y, Binz J, Numerick MJ, et al. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J. Clin. Invest. 2003;112:1678–87. 71. Fiorucci S, Clerici C, Antonelli E, et al. Protective effects of 6-ethyl chenodeoxycholic acid, a farnesoid X receptor ligand, in estrogen-induced cholestasis. J. Pharmacol. Exp. Ther. 2005;313:604–12. 72. Thomas C, Pellicciari R, Pruzanski M, et al. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 2008;7:678–93.

5 Bile acid signaling in the liver and the biliary tree Dieter Häussinger, Roland Reinehr, and Verena Keitel

5.1 Introduction Bile acids are involved not only in nutrient absorption in the intestine but also act as signal molecules in the liver and can activate intracellular signal transduction pathways. Bile acid signaling in the liver is cell type – and bile acid-specific. Whereas some bile acids, such as glycochenodeoxycholate (GCDC), can trigger hepatocyte apoptosis and cholestasis, others, such as tauroursodeoxycholate (TUDC) are hepatoprotective and choleretic. Several molecular structures that can sense the different bile acids and initiate bile acid signaling toward effector sites have now been identified. These include not only nuclear bile acid receptors such as FXR (see Chapters 3 and 4) or the membranebound bile acid receptor TGR5 (see Chapter 7) but also putative chloride channels and integrins (fFig. 5.1). This chapter focuses on the bile acid sensors and bile salt-activated signal transduction pathways in the different hepatic cell types. For bile acid signaling in extrahepatic organs, see Chapter 7.

5.2 Bile acid signaling in liver parenchymal cells (hepatocytes) Ursodeoxycholic acid is widely used for the treatment of cholestatic liver disease, such as primary biliary cirrhosis (1–3). In vivo ursodeoxycholic acid is rapidly conjugated with taurine or glycine and the taurine conjugate of ursodeoxycholic acid (TUDC) prevents bile acid-induced cholestasis, stimulates the excretion of other bile acids, and protects against hepatocellular injury induced by various forms of stress (4–6). The choleretic action of TUDC is largely due to a rapid insertion of intracellularly stored bile salt export pump (Bsep) into the canalicular membrane of the hepatocyte (7), which increases the bile salt excretory capacity in a microtubule-dependent way (6,8). The rapid insertion of Bsep-containing vesicles under the influence of TUDC is in line with early observations that TUDC stimulates exocytosis and triggers an intracellular Ca2+-signal (9,10). Recent studies identified the integrin system as one major TUDC-sensor in hepatocytes (11,12). Integrins are a family of extracellular matrix adhesion molecules involved in “mechanotransduction” and growth factor signaling; the most important ones in liver are the α1β1, α 5β1, and α 9β1 integrins (13,14). As shown by immunohistochemistry, TUDC induces the rapid appearance of the active conformation of the ß1 subunit in the cytosol of hepatocytes, indicating integrin activation in response to TUDC. TUDC-induced ß1 integrin activation requires the presence of the Na+/taurocholate cotransporting peptide (Ntcp), which points to a liver-specificity of TUDC-induced integrin activation and is abolished in the presence of an integrin-antagonistic RGD-motif containing hexapeptide. Other bile acids – such as taurocholate (TC), glycochenodeoxycholate (GCDC), taurochenodeoxycholate (TCDC), and taurolithocholylsulfate (TLCS) – were without effect on ß1 integrins, indicating specificity of the TUDC effect. Molecular dynamics simulations of a 3D model of the ectodomain of α5β1 integrin have suggested that TUDC directly

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5 Bile acid signaling in the liver and the biliary tree • Choleresis • Cytoprotection • Proliferation • Immunomodulation • Apoptosis

Chloride channels

• ERK1/2 signalling • AKT signalling

Integrins

GCDC TLCS

TUDC

Bile acid sensing

TC M2R

S1P2 TC

CDCA FXR

TLC LCA

(Neonatal cardiomyocytes) • Arrhythmia

• • • • •

TGR5

• • • • •

Gene expression Bile acid homeostasis Lipid metabolism Glucose metabolism Antiinflammation

Biliary secretion Antiinflammation Anti-apoptosis Glucose homeostasis Energy expenditure

Fig. 5.1: Bile acid sensing. Several molecular structures that sense the different bile acids and thereby initiate bile acid-induced signaling toward effector sites have been identified in the liver and other organs. These include not only nuclear bile acid receptors such as FXR, the membrane-bound bile acid receptor TGR5, the muscarinergic receptor M2R, or the sphingosin-1-phosphate receptor 2 (S1P2) but also putative chloride channels and integrins.

interacts with the integrin to trigger its activation (12). Downstream consequences of TUDC-induced integrin activation include the activation of focal adhesion kinase (FAK), c-Src-kinase, mitogen-activated protein (MAP) kinases Erk-1/2 and p38MAPK (7,11,15), and the epidermal growth factor receptor (EGFR) (12). This is in line with previous findings of a swelling-mediated EGFR activation by either hypoosmolarity or insulin, which is also triggered by α5β1 integrin activation (16). TUDC also activates PI3-kinase and Ras, with Ras being a downstream signaling event toward Erk (17), but it has not yet been determined whether this effect is also triggered via the integrin system. However, the p85α subunit of PI3-kinase can directly bind to FAK and activate PIs-kinase (18). Such FAK-mediated PI3-kinase activation by TUDC may explain why Src inhibition has no effect on TUDC-induced Erk activation, whereas TUDC-induced p38MAPK activation is sensitive to inhibition of Src but not to PI3-kinase inhibition. Dual activation of both, Erks and p38MAPK is required for the TUDC-induced choleretic effect, which is abolished in presence of inhibitors of either the integrin system, c-Src, or one of the two MAP-kinases. The role of integrins as TUDC-sensors is underlined by the fact that integrin-inhibitory peptides containing an RGD motif abolish TUDC signaling toward c-Src and MAP kinases as well as the stimulation of bile formation, which is otherwise

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triggered by TUDC (11). The TUDC-signal transduction pathway strongly resembles the one triggered by hypoosmotic hepatocyte swelling, which uses ß1 integrins as osmosensors and the FAK/c-Src/Erks and p38MAPK pathway for osmosignaling toward choleresis (for review see reference 19). The similarities between TUDC and hypoosmotic signaling in hepatocytes are also reflected by the fact that both, hepatocyte swelling and TUDC trigger an inhibition of autophagic proteolysis via p38MAPK (11,20). Activation of protein kinase C (PKC) by TUDC has also been reported (10), which is apparently involved in Mrp2 targeting to the canalicular membrane in cholestatic livers (21). This finding may reflect a feature of cholestatic livers, because PKC activation triggers cholestasis in normal hepatocytes (22). fFig. 5.2 summarizes the TUDC-signaling events toward choleresis. Other bile acids – such as taurolithocholylsulfate (TLCS), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC) – are proapoptotic and can trigger cholestasis. These bile acids can induce apoptosis by activation of CD95 (23) and CD95 targeting to the plasma membrane (24). These bile acids trigger within seconds an increase of the cytosolic chloride concentration, probably by interfering with anion channels in the plasma membrane. The rise of intracellular chloride activates vacuolar-type proton-ATPase and induces endosomal acidification with subsequent activation of acidic sphingomyelinase (25). The resulting increase in ceramide leads to an activation of protein kinase C (PKC)ζ, which triggers p47phox phosphorylation, activation of NADPH oxidase (Nox) and generation of an oxidative stress signal (26). This bile salt-induced formation of reactive oxygen species (ROS) on the one hand promotes

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Fig. 5.2: TUDC-induced choleresis. TUDC leads to an activation of α5β1 integrins, which triggers FAK and c-Src activation. c-Src then mediates EGFR phosphorylation and subsequent Erk-dependent proliferation. In addition, c-Src activates p38MAPK. Dual MAP kinase activation, i.e., activation of Erks and p38MAPK, triggers microtubule-dependent Bsep and Mrp2 insertion into the canalicular membrane thereby promoting choleresis.

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hepatocyte shrinkage (27) and on the other activation of the Src family kinase Yes and of c-Jun-N-terminal kinase ( JNK) (28). Yes associates with the epidermal growth factor receptor (EGFR), which becomes activated by a Yes-catalyzed tyrosine phosphorylation at Y845, followed by an activating autophosphorylation of Y1173 (28). These events occur within 1 minute of GCDC, TLCS or TCDC addition and are not observed upon addition of taurocholate (TC) or TUDC (26–29). The activated EGFR then associates with CD95 and catalyzes tyrosine phosphorylation of CD95 (29). CD95/EGFR association is strongly dependent on the JNK signal, which is generated in response to bile acidinduced ROS formation and EGFR/CD95 complex is fairly stable, because it persists at least until apoptotic bleb formation. CD95-tyrosine phosphorylation is the signal for CD95 oligomerization (30) and translocation of the CD95-oligomer/EGFR complex to the plasma membrane, where formation of the death-inducing signaling (i.e., recruitment of FADD and caspase 8) occurs. Apoptosis is then executed within a few hours in response to TLCS and GCDC, whereas in response to TCDC the CD95 becomes activated without execution of cell death. This was explained by activation of a PI3-kinasedependent survival pathway, because TCDC induced apoptosis only after inhibition of PI3-kinase (31). TUDC, which also activates PI3-kinase (17), also protects hepatocytes from TLCS-induced apoptosis; however, this protective effect was insensitive to PI3kinase inhibition (32). The signaling pathways underlying this protective effect of TUDC do not involve TUDC-induced MAP kinase, PI3-kinase, or protein kinase B activation (32), indicating that different signaling pathways are engaged in mediating the antiapoptotic and choleretic effects of TUDC, respectively. TUDC prevents TLCS-induced JNK activation and CD95 trafficking to the plasma membrane and its site of action is upstream of caspase 8 activation (32). Also, cyclic AMP (cAMP) inhibits bile acid-induced apoptosis in isolated rat hepatocytes (33–35) and modulates the apoptotic program in other cell types (36). Inhibition of hepatocyte apoptosis by cAMP involves activation of PKA and PI3-kinase (35,37–40), modulation of the bile acid-induced activation pattern of Erks, p38MAPK and JNK, and prevents CD95 membrane trafficking to the plasma membrane (33) and DISC formation (34). The mechanisms involved in cAMP’s inhibition of CD95 membrane targeting and activation in response to proapoptotic bile acids (29,32) have been investigated in detail. Its action resides in a protein kinase A-dependent serine/ threonine phosphorylation of CD95, which induces CD95 internalization and simultaneous inhibition of bile acid-induced CD95 tyrosine phosphorylation. The latter is due to a PKA-dependent inhibition of bile salt-induced Yes/EGFR association and EGFR activation and is accompanied by a serine/threonine phosphorylation of Yes (41). Cyclic AMP does not affect the JNK signal induced by proapoptotic bile acids and therefore does not prevent EGFR/CD95 association. Interestingly, proapoptotic bile acids can trigger by themselves as a late response formation of cAMP followed by CD95 internalization and inhibition of bile salt-induced apoptosis (34). Bile acid-induced apoptosis is also counteracted by betaine, which does not affect CD95 targeting to the plasma membrane but inhibits the downstream proapoptotic mitochondrial pathway (42). fFig. 5.3 schematically depicts the bile acid-signaling pathway toward CD95 activation and apoptosis. Bile acid-induced activation of the CD95 system can also cross talk with other signal transduction pathways. For example, hydrophobic bile acids inhibit interleukin-6 signaling through a caspase-mediated cleavage of signal transducing gp130 and a p38MAPK-dependent downregulation of STAT3 phosphorylation (43). TLCS also inhibits

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Fig. 5.3: Proapoptotic bile acid-induced CD95 activation. Proapoptotic bile acids, such as GCDC or TLCS trigger an almost instantaneous elevation of the intracellular chloride concentration which further leads to an acidic sphingomyelinase (ASM)-driven Nox activation and subsequent ROS formation. This further promotes Yes-mediated EGFR activation and a JNK-mediated CD95/EGFR association. Activated EGFR phosphorylates CD95 on tyrosine residues, which is required for CD95 oligomerization, translocation to the plasma membrane, DISC formation and apoptosis induction.

interferon-α signaling through inhibition of Jak1 and Tyk2 phosphorylation, STAT1/2 phosphorylation and inhibits by such means the expression of interferon-stimulated genes, such as dsRNA-activated protein kinase (PKR) (44). In addition, TLCS can induce insulin resistance by a PKC-dependent suppression of insulin-induced phosphorylation of the insulin receptor ß-subunit and of the PI3-kinase pathway (45). Interestingly, TUDC can counteract the TLCS-induced suppression of insulin signaling. Other bile acids such as TC were shown to stimulate hepatocyte polarization through a cAMP-Epac-AMPkinase pathway (46). For regulation of the Na+-taurocholate cotransporting protein (Ntcp) by bile acids, see Chapter 3.

5.3 Bile acid signaling in sinusoidal endothelial cells The endothelial cells lining the liver sinusoids are a very distinct population of endothelial cells characterized by fenestrations and the lack of a basement membrane (47,48). These sinusoidal endothelial cells (SECs) mediate receptor-induced clearance of endotoxins and bacteria, leukocyte adhesion and transmigration, orchestration of the immune response (49), and, with their fenestrae, form an adjustable barrier between the portal blood and hepatocytes. Depending on food intake, SECs are exposed to highly variable concentrations of nutrients, including bile acids. Bile acid levels rise in portal

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venous blood postprandially and concentrations between 14 and 43 μM have been measured in humans and rats (50–52). While the nuclear bile acid receptor farnesoid X receptor (FXR) has not been detected in SECs (53), the membrane-bound bile acid receptor TGR5 (Gpbar-1) is highly expressed in SECs (54). TGR5 is coupled to a stimulatory G protein and is activated by bile acids, with taurolithocholate (TLC) and taurodeoxycholate (TDC) being the most potent agonists (TLC EC50 = 0.29 μM; TDC EC50 = 0.79 μM) (55–57). Activation of TGR5 by bile acids in isolated SECs leads to a significant rise in intracellular cyclic AMP (cAMP), activation of protein kinase A and subsequent phosphorylation of the endothelial NO synthase (eNOS) at serine residue 1177. Phosphorylation of serine 1177 of eNOS enhances enzyme activity, leading to an increased NO production as measured by DAF-FM fluorescence on rat liver slices (54). Furthermore, stimulation of SEC with TLC induced a significant upregulation of eNOS mRNA, while the mRNA expression of intercellular adhesion molecule-1 (ICAM-1) was unaffected (54). Activation of TGR5 also increased serine phosphorylation of the CD95 receptor, which facilitates CD95 internalization, thereby preventing apoptosis induction (34,54). TGR5-mediated serine phosphorylation of the CD95 may protect SEC from bile acid-induced apoptosis, which is highly relevant since these cells are exposed to high bile acid concentrations following food intake. The TGR5-cAMP-PKA-eNOS-NO pathway may allow adaptation of blood flow through the liver sinusoids to nutrient intake, thus promoting hepatic metabolism. SECs are also an important donor of NO in the hepatic vasculature. Decreased NO production in SECs is one hallmark of portal hypertension (48,58–60). Thus activation of the TGR5-cAMP-PKA-eNOS-NO pathway may be beneficial in portal hypertension.

5.4 Bile acid signaling in Kupffer cells Macrophage function is impaired during cholestasis and infection- and endotoxinrelated complications are frequent in patients with obstructive cholestasis (61–65). Common bile duct ligation (CBDL) serves as a model for obstructive jaundice and is associated with bacterial overgrowth in the intestine, bacterial translocation, and portal and systemic bacteremia (66). The increased bacteremia and endotoxemia in CBDL rats has been attributed to impaired phagocytic activity of hepatic Kupffer cells (KCs) and is reversible after biliary decompression (67–71). Whereas hydrophobic bile acids, such as deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) and their taurine conjugates, dose-dependently inhibit KC phagocytosis, the hydrophilic bile acid ursodeoxycholic acid (UDCA) even enhances KC phagocytic activity (67,70,72). The molecular mechanisms underlying the bile acid effects on phagocytosis and bacterial killing are unclear. Bile acids, such as chenodeoxycholic acid (CDCA), have also been shown to inhibit macrophage cytokine production, whereas UDCA had only minimal effects (73). Although CDCA and DCA are potent agonists for the membrane-bound bile acid receptor TGR5 (Gpbar-1) (CDCA EC50 = 6.7 μM; DCA EC50 = 1.25 μM), UDCA is only a weak TGR5 agonist (UDCA EC50 = 36.4 μM) (57). TGR5 is expressed in KCs, and stimulation with taurocholate and taurolithocholate (TLC) led to a significant increase of intracellular cAMP levels in isolated rat KCs (74). Activation of TGR5 in isolated KC with TLC or a synthetic TGR5 agonist reduced lipopolysaccharide (LPS)-induced expression of

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the cytokines interleukin-1D, interleukin-1β, interleukin-6, and tumor necrosis factor-α (74). KCs from TGR5 knockout mice express higher proinflammatory cytokine mRNA levels than their wild-type litter mates, both under control conditions and after LPS injection. Administration of a TGR5 agonist to wild-type mice suppressed LPS-induced serum cytokine levels (75). Activation of TGR5 reduced nuclear factor-κB (NF-κB) transcriptional activity, thus inhibiting cytokine production (76). In the liver of CBDL rats, TGR5 immunofluorescence staining was enhanced in KCs but not in SECs (74). This upregulation of TGR5 in obstructive cholestasis could enhance the bile acid-mediated suppression of cytokine production. Whether TGR5 is also responsible for the reduced phagocytic activity is as yet unknown.

5.5 Bile acid signaling in hepatic stellate cells Hepatic stellate cells (HSCs) have been widely looked upon as “key players” in the initiation and progression of hepatic fibrosis (77,78), which is characterized by quantitative and qualitative changes in the composition and deposition of the extracellular matrix (ECM), resulting from an imbalance between ECM deposition and degradation by activated HSCs (79,80). Because fibrosis is a dynamic process and depends upon the extent and duration of parenchymal and nonparenchymal liver cell injury, the transformation of quiescent HSCs into a myofibroblast-like activated phenotype after liver injury is induced by cytokines, reactive oxygen species, and changes in the composition of the perisinusoidal matrix (80,81); it results in the migration of HSCs to sites of tissue damage (80,82,83). Thereafter, activated HSCs are replaced by the genuine tissue, most likely after selective apoptosis of activated HSCs (79,80). In contrast, sustained HSC transformation induced by chronic liver injury is thought to lead to progressive accumulation of persistently activated HSCs with accumulation of ECM and the development of liver fibrosis and cirrhosis (79,80). However, more recent studies have revealed that HSC represent a hepatic stem/progenitor cell compartment (84) and identified the space of Dissé as their stem cell niche (85). Therefore it is attractive to speculate that the transformation of HSCs into myofibroblasts following liver injury is an aberrant differentiation process or a stress response of HSCs rather than a regular wound-healing reaction. Thus the signaling pathways induced by bile acids in HSCs are of interest with respect not only to liver injury but also to liver regeneration. Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95-death receptor in liver parenchymal cells (see section 5.2). In hepatocytes the underlying molecular mechanisms involve a Yes-dependent but ligandindependent activation of the epidermal growth factor-receptor (EGFR), which catalyzes CD95-tyrosine-phosphorylation as a prerequisite for CD95-oligomerization, formation of the death-inducing signaling complex (DISC) and apoptosis induction (26,29,41). Bile acids also activate EGFR in cholangiocytes (see below and reference 86) and in activated HSCs (87). Surprisingly, bile acid–induced EGFR-activation in HSC does not trigger apoptosis but results in the stimulation of cell proliferation. The behavior of shortterm cultured quiescent HSCs toward CD95-ligand (CD95L) is also unusual. CD95L, a potent inducer of hepatocyte apoptosis, triggers activation of the EGFR in quiescent HSCs, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death

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signaling through CD95-tyrosine nitration (88). Similar observations were made with other death receptor ligands – i.e., TNF-α and TRAIL (88). The mitogenic action of CD95L in quiescent HSC is due to a c-Src-dependent shedding of EGF and subsequent auto-/paracrine activation of the EGFR. This unusual behavior of quiescent HSCs toward death receptor ligands may relate to the recent findings that quiescent HSCs are stem/ progenitor cells in the liver with a capacity to differentiate not only into myofibroblasts but also toward hepatocyte- and endothelium-like cells (84,89). Thus apoptosis inhibition and stimulation of HSC proliferation in the hostile cytokine milieu, which accompanies liver injury, may help HSCs to fulfill their role as stem cells during liver regeneration. During cholestatic liver injury HSCs may be exposed to increased concentrations of circulating bile acids. As shown recently, cholestatic bile acids, such as TLCS or GCDC, induce a rapid NADPH oxidase activation in quiescent HSCs, which triggers a Yesmediated EGFR activation and HSC proliferation (90). In contrast to hepatocytes, hydrophobic bile acids induce only a weak JNK signal in HSCs. However, when a stronger JNK signal is induced by co-administration of either cycloheximide (CHX) or hydrogen peroxide (H2O2), the bile acid–induced mitogenic signal is shifted to an apoptotic one (90). Thus in quiescent HSCs hydrophobic bile acids activate the EGFR, and this can couple to both cell proliferation and apoptosis, depending on the accompanying JNKsignaling context. Therefore JNK act as a switch between bile acid–induced proliferation and apoptosis in these cells. This may offer new perspectives for the understanding of cholestatic liver disease, especially in view of the proposed role of quiescent HSCs as stem/progenitor cells. In line with this, bile acids such as TLCS or cholic acid can induce the expression of hepatocellular markers in quiescent HSCs in vitro (Kordes and Häussinger, unpublished result), indicating a modulatory effect of bile acids during HSC differentiation. These findings are further corroborated by the observation that elevated bile acid levels accelerate liver regeneration, whereas decreased bile acid levels inhibit liver regeneration in a mouse model of partial hepatectomy via a farnesoid X receptor (FXR) –dependent pathway (91). Whereas an inactivating CD95-tyrosine nitration occurs in response to CD95 ligand in quiescent but not activated HSCs no CD95-tyrosine nitration is induced by hydrophobic bile acids in HSCs. Like quiescent HSCs, activated (cultured for 7–10 days) HSCs respond to hydrophobic bile acids with an antioxidant-sensitive EGFR activation and proliferation (87,90). Again, only a weak JNK signal is induced and proliferation can be switched toward apoptosis when a JNK signal is coinduced by cycloheximide or hydrogen peroxide in activated HSCs (90). Thus hydrophobic bile acid signaling toward proliferation/apoptosis is similar in quiescent and activated HSCs and also resembles the signaling events initiated in liver parenchymal cells except for the JNK signal. The explanation for why hydrophobic bile acids induce a strong JNK signal in hepatocytes but not in HSCs may reside at least in part in the high expression of DNA damage-inducible gene Gadd45ß in HSCs but not in hepatocytes (90). Gadd45ß is a negative regulator of MKK7-mediated JNK activation (92,93), which also promotes hepatocyte survival during liver regeneration (94). fFig. 5.4 summarizes bile acid-induced signaling in quiescent and activated HSCs with respect to proliferation and apoptosis

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5.6 Bile acid signaling in the biliary tree

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Fig. 5.4: Bile acid-induced signaling towards proliferation and apoptosis in hepatic stellate cells. In quiescent hepatic stellate cells (HSC), hydrophobic bile acids, such as GCDC or TLCS, lead to NADPH oxidase-driven ROS formation and subsequent Yes-mediated EGFR activation whereas, in contrast to hepatocytes, no JNK activation occurs. Also in activated HSC an antioxidant-sensitive bile acid-induced EGFR activation has been shown. Therefore, bile acid-induced EGFR activation triggers HSC proliferation unless a second stimulus comes into play leading to a JNK activation which couples EGFR to CD95-mediated apoptosis. Apart from bile acids also CD95L leads to an EGFR-mediated proliferation in quiescent HSC. Here, CD95L induces a c-Src- and matrix metalloproteinase (MMP)9-mediated EGF shedding which further leads to EGFR activation. In addition, a CD95 tyrosine nitration is induced which leads to apoptosis resistance. In activated HSC, upon addition of a second stimulus leading to JNK activation CD95L-induced EGFR activation can be coupled to CD95-mediated apoptosis because no CD95 tyrosine nitration occurs in these transformed HSC. *Note: A role of Yes in triggering bile salt-induced EGFR activation was shown for quiescent HSC (90) but has not yet been investigated in activated HSC.

5.6 Bile acid signaling in the biliary tree The epithelial cells lining the bile ducts (cholangiocytes) and gallbladder are exposed to millimolar bile acid concentrations, necessitating cytoprotective mechanisms to prevent bile acid-induced cell damage. Bile acids regulate cholangiocyte proliferation and survival as well as secretion (95). Taurocholate (TC) feeding of rats ameliorates biliary damage and cholangiocyte apoptosis induced by carbon tetrachloride (CCl4) administration (96), vagotomy (97), adrenergic denervation (98), hepatic artery ligation (99), or the

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injection of tumor necrosis factor-α (100). The cytoprotective effect of TC was mediated through activation of protein kinase A (PKA) and subsequent stimulation of phosphatidylinositol 3-kinase (PI3K) activity, which in turn inhibited caspase activity (96–98). Furthermore, TC can induce expression and secretion of vascular endothelial growth factor-A in cholangiocytes, thereby promoting cholangiocyte protective mechanisms and proliferation (99,101). Feeding of TC in these studies not only prevented apoptosis but also triggered cholangiocyte proliferation (96,97,101). It has been suggested that bile acids, such as TC and taurolithocholate (TLC), must be transported into cholangiocytes by the apical sodium-dependent bile acid transporter (ASBT, SCL10A2) (102,103) in order to stimulate the cAMP-PKA-PI3K-survival/proliferation pathways, since ASBT expression is also downregulated by vagotomy and restored upon TC feeding (97). However, it has recently been shown that cholangiocytes also express the membranebound bile acid receptor TGR5 (Gpbar-1), and activation of TGR5 in biliary epithelial cells increased intracellular cAMP levels and may therefore also activate PKA and PI3K pathways (104–106). Since ASBT inhibitors have recently become available (107), the role of bile acid uptake for TC-mediated antiapoptotic and proliferative effects can be clarified. In contrast to TC and TLC, which promote cholangiocyte proliferation (108), ursodeoxycholic acid (UDCA) and tauroursodeoxycholate (TUDC) inhibited cholangiocyte proliferation (without increasing apoptosis) through activation of protein kinase C α (PKCα) (109). ASBT expression was also downregulated by UDCA treatment (109). Bile acids have also been shown to affect cholangiocyte secretion (95). TC and TLC enhanced the secretin-dependent rise in intracellular cAMP levels and Cl⫺/HCO3- exchanger activity, thereby promoting fluid secretion and bile flow (110,111). These effects were dependent upon the activation of PI3K and subsequent elevation of cAMP levels (110,112). UDCA, in turn, promotes ATP release into bile through activation of the cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7). ATP is a ligand for purinergic P2Y receptors and thus stimulates chloride efflux and fluid secretion by cholangiocytes (113). Norursodeoxycholic acid (norUDCA) is a C23 homologue of UDCA. Both UDCA and norUDCA induce cholangiocyte bicarbonate secretion, however, the effect of norUDCA was superior to the effect of UDCA and was largely independent of CFTR function (114). It has been suggested that the beneficial effect of norUDCA depends on increased cholehepatic shunting of bile acids, which are reabsorbed from the bile duct lumen by cholangiocytes, returned to hepatocytes and subsequently resecreted into bile by hepatocytes (114). Further studies using ASBT inhibitors are required to clarify the role of cholehepatic shunting for bile secretion. Recently the bile acid receptor TGR5 has been localized to the primary cilium as well as in the apical membrane of cholangiocytes and gallbladder epithelial cells (104– 106) (fFig. 5.5). Stimulation of TGR5 with a specific agonist in gallbladder epithelial cells increased intracellular cAMP levels and activated the cAMP-regulated chloride channel CFTR, resulting in increased chloride secretion (104). This effect was not observed in gallbladder epithelial cells derived from TGR5 knockout mice (104). Interestingly, elevation of intracellular cAMP leads to the insertion of CFTR and ASBT from an intracellular vesicular pool into the apical membrane, thereby enhancing transport activity (115–117). In gallbladder epithelial cells, TGR5 was detected in colocalization with CFTR and ASBT, both in an intracellular, rab-11 positive, vesicular compartment as well as in the apical membrane domain (104). Therefore TGR5 may function as a

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Fig. 5.5: Localization of TGR5 in biliary epithelial cells. A. TGR5 (Gpbar-1; shown in red) was detected in the apical membrane of human gallbladder epithelial cells in colocalization with the organic anion transporter MRP2 (ABCC2; shown in purple). The multidrug resistance-associated protein 3 (MRP3, ABCC3; shown in green) was localized in the basolateral membrane domain of gallbladder epithelial cells. Nuclei were stained with Hoechst (104). Bars = 10 μm. B. Isolated mouse cholangiocytes were stained for TGR5 (in red) and for acetylated α-tubulin (in green), which is a marker protein of primary cilia. Vertical sections in the xz-plane demonstrate the localization of TGR5 in the primary cilium, which protrudes from the apical membrane (105).

bile acid sensor coupling biliary bile acid concentration to cholangiocyte bile acid absorption and fluid secretion. Through elevation of cAMP, TGR5 may also mediate bile acid-induced antiapoptotic and proliferative effects (106). In the gallbladder, TGR5 is also localized in smooth muscle cells and activation of the receptor results in an opening of ATP-sensitive potassium channels leading to smooth muscle cell relaxation and gallbladder filling (118,119). Through decreased contractile activity of gallbladder smooth muscle cells, TGR5 agonists may promote cholesterol gallstone formation. This is underscored by the finding that TGR5 knockout mice are protected from cholesterol gallstone development even when fed a lithogenic diet (120). In summary, cholangiocyte fluid secretion is promoted by both hydrophobic and hydrophilic bile acids through various signaling pathways including the TGR5-cAMP-PKApathway, the activation of PI3-kinase, and increased cholehepatic shunting (fFig. 5.6). Furthermore, taurocholate and taurolithocholate protect cholangiocytes from apoptosis and promote cell proliferation most likely via TGR5-cAMP and PI3-kinase pathways. UDCA also has antiapoptotic effects, however, in contrast to TC, and TLC inhibits cholangiocyte proliferation through elevation of calcium and activation of protein kinase C. This antiproliferative effect was also observed in patients on long-time UDCA treatment for primary sclerosing cholangitis (PSC) who developed less cholangiocarcinomas over time, as predicted (121).

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Fig. 5.6: Bile acid-induced modulation of cholangiocyte secretion, proliferation and survival pathways. The bile acid receptor TGR5 (Gpbar-1) is localized in the apical membrane and the primary cilium of biliary epithelial cells. Activation of TGR5 leads to a rise in intracellular cAMP and subsequent activation of the cAMP-regulated chloride channel CFTR. The elevation of cAMP also promotes cell proliferation and activates anti-apoptotic mechanisms. Furthermore, cAMP may induce the insertion of CFTR and ASBT from intracellular vesicular structures into the apical membrane domain. Taurocholate (TC) and taurolithocholate (TLC) can increase intracellular cAMP concentrations and activate PI3-kinase, which in turn facilitates cell proliferation and inhibits caspase activity, thereby preventing apoptosis. Whether ASBT-dependent uptake is required for the secretory, proliferative, and antiapoptotic effects of TC and TLC is unclear. Dotted lines represent indirect effects (106).

5.7 References 1. Poupon RE, Balkau B, Eschwege E, et al. A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. UDCA-PBC Study Group. N. Engl. J. Med. 1991;324:1548–54. 2. Poupon R. Primary biliary cirrhosis: a 2010 update. J. Hepatol. 2010;52:745–58. 3. Leuschner U, Fischer H, Kurtz W, et al. Ursodeoxycholic acid in primary biliary cirrhosis: results of a controlled double-blind trial. Gastroenterology 1989;97:1268–74. 4. Heuman DM, Mills AS, McCall J, et al. Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts. In vivo studies in the rat. Gastroenterology 1991;100:203–11. 5. Kitani K, Kanai S. Tauroursodeoxycholate prevents taurocholate induced cholestasis. Life Sci. 1982;30:515–23. 6. Häussinger D, Hallbrucker C, Saha N, et al. Cell volume and bile acid excretion. Biochem. J. 1992;288 (Pt 2):681–9. 7. Kurz AK, Graf D, Schmitt M, et al. Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 2001;121:407–19. 8. Häussinger D, Saha N, Hallbrucker C, et al. Involvement of microtubules in the swellinginduced stimulation of transcellular taurocholate transport in perfused rat liver. Biochem. J. 1993;291 (Pt 2):355–60.

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9. Beuers U, Nathanson MH, Isales CM, et al. Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca++ mechanisms defective in cholestasis. J. Clin. Invest. 1993;92:2984–93. 10. Beuers U, Throckmorton DC, Anderson MS, et al. Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. Gastroenterology 1996;110:1553–63. 11. Häussinger D, Kurz AK, Wettstein M, et al. Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis. Gastroenterology 2003;124:1476–87. 12. Gohlke H, Reinehr R, Schmitz B, et al. α5β1-integrins act as sensors for tuaroursodeoxycholate in rat liver. Manuscript in preparation 2012. 13. Torimura T, Ueno T, Kin M, et al. Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of beta1 integrins. Hepatology 2001;34:62–71. 14. Carloni V, Mazzocca A, Pantaleo P, et al. The integrin, alpha6beta1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the migration of human hepatocarcinoma cells. Hepatology 2001;34:42–9. 15. Schliess F, Kurz AK, vom Dahl S, et al. Mitogen-activated protein kinases mediate the stimulation of bile acid secretion by tauroursodeoxycholate in rat liver. Gastroenterology 1997;113:1306–14. 16. Reinehr R, Sommerfeld A, Häussinger D. Insulin induces swelling-dependent activation of the epidermal growth factor receptor in rat liver. J. Biol. Chem. 2010;285:25904–12. 17. Kurz AK, Block C, Graf D, et al. Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver. Biochem. J. 2000;350 (Pt 1):207–13. 18. Chen HC, Appeddu PA, Isoda H, et al. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J. Biol. Chem. 1996;271:26329–34. 19. Häussinger D, Reinehr R. Osmotic regulation of bile acid transport, apoptosis and proliferation in rat liver. Cell. Physiol. Biochem. 2011;28:1089–98. 20. vom Dahl S, Schliess F, Reissmann R, et al. Involvement of integrins in osmosensing and signaling toward autophagic proteolysis in rat liver. J. Biol. Chem. 2003;278:27088–95. 21. Beuers U, Bilzer M, Chittattu A, et al. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 2001;33:1206–16. 22. Kubitz R, Saha N, Kuhlkamp T, et al. Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver. J. Biol. Chem. 2004;279:10323–30. 23. Faubion WA, Guicciardi ME, Miyoshi H, et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J. Clin. Invest. 1999;103:137–45. 24. Sodeman T, Bronk SF, Roberts PJ, et al. Bile salts mediate hepatocyte apoptosis by increasing cell surface traffi cking of Fas. Am. J. Physiol. Gastrointest. Liver Physiol. 2000;278:G992–G9. 25. Becker S, Reinehr R, Grether-Beck S, et al. Hydrophobic bile salts trigger ceramide formation through endosomal acidification. Biol. Chem. 2007;388:185–96. 26. Reinehr R, Becker S, Keitel V, et al. Bile salt-induced apoptosis involves NADPH oxidase isoform activation. Gastroenterology 2005;129:2009–31. 27. Becker S, Reinehr R, Graf D, vom Dahl S, Häussinger D. Hydrophobic bile salts induce hepatocyte shrinkage via NADPH oxidase activation. Cell. Physiol. Biochem. 2007;19:89–98. 28. Reinehr R, Becker S, Höngen A, et al. The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95. J. Biol. Chem. 2004;279:23977–87.

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29. Reinehr R, Graf D, Häussinger D. Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation. Gastroenterology 2003;125:839–53. 30. Eberle A, Reinehr R, Becker S, et al. CD95 tyrosine phosphorylation is required for CD95 oligomerization. Apoptosis 2007;12:719–29. 31. Rust C, Karnitz LM, Paya CV, et al. The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J. Biol. Chem. 2000;275:20210–16. 32. Graf D, Kurz AK, Fischer R, et al. Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner. Gastroenterology 2002;122:1411–27. 33. Graf D, Reinehr R, Kurz AK, et al. Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms. Arch. Biochem. Biophys. 2003;415:34–42. 34. Reinehr R, Häussinger D. Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95. Gastroenterology 2004;126:249–62. 35. Webster CR, Anwer MS. Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes. Hepatology 1998;27:1324–31. 36. Martin MC, Dransfi eld I, Haslett C, et al. Cyclic AMP regulation of neutrophil apoptosis occurs via a novel protein kinase A-independent signaling pathway. J. Biol. Chem. 2001;276:45041–50. 37. Webster CR, Anwer MS. Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp. Am. J. Physiol. 1999;277:G1165–G72. 38. Webster CR, Blanch CJ, Phillips J, et al. Cell swelling-induced translocation of rat liver Na(+)/taurocholate cotransport polypeptide is mediated via the phosphoinositide 3-kinase signaling pathway. J. Biol. Chem. 2000;275:29754–60. 39. Webster CR, Usechak P, Anwer MS. cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release. Am. J. Physiol. Gastrointest. Liver Physiol. 2002;283:G727–G38. 40. Webster CR, Blanch C, Anwer MS. Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP. Am. J. Physiol. Gastrointest. Liver Physiol. 2002;283:G44–G50. 41. Reinehr R, Becker S, Wettstein M, et al. Involvement of the Src family kinase yes in bile salt-induced apoptosis. Gastroenterology 2004;127:1540–57. 42. Graf D, Kurz AK, Reinehr R, et al. Prevention of bile acid-induced apoptosis by betaine in rat liver. Hepatology 2002;36:829–39. 43. Graf D, Kohlmann C, Haselow K, et al. Bile acids inhibit interleukin-6 signaling via gp130 receptor-dependent and -independent pathways in rat liver. Hepatology 2006;44:1206–17. 44. Graf D, Haselow K, Munks I, et al. Inhibition of interferon-alpha-induced signaling by hyperosmolarity and hydrophobic bile acids. Biol. Chem. 2010;391:1175–87. 45. Mannack G, Graf D, Donner MM, et al. Taurolithocholic acid-3 sulfate impairs insulin signaling in cultured rat hepatocytes and perfused rat liver. Cell. Physiol. Biochem. 2008;21:137–50. 46. Fu D, Wakabayashi Y, Lippincott-Schwartz J, et al. Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc. Natl. Acad. Sci. USA 2011;108:1403–8. 47. Wisse E, Braet F, Luo D, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol. Pathol. 1996;24:100–11. 48. Iwakiri Y, Groszmann RJ. Vascular endothelial dysfunction in cirrhosis. J. Hepatol. 2007;46:927–34.

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70. Van Bossuyt H, Desmaretz C, Gaeta GB, et al. The role of bile acids in the development of endotoxemia during obstructive jaundice in the rat. J. Hepatol. 1990;10:274–9. 71. Clements WD, McCaigue M, Erwin P, et al. Biliary decompression promotes Kupffer cell recovery in obstructive jaundice. Gut 1996;38:925–31. 72. Funaoka M, Komatsu M, Toyoshima I, et al. Tauroursodeoxycholic acid enhances phagocytosis of the cultured rat Kupffer cell. J. Gastroenterol. Hepatol. 1999;14:652–8. 73. Calmus Y, Guechot J, Podevin P, et al. Differential effects of chenodeoxycholic and ursodeoxycholic acids on interleukin 1, interleukin 6 and tumor necrosis factor-alpha production by monocytes. Hepatology 1992;16:719–23. 74. Keitel V, Donner M, Winandy S, et al. Expression and function of the bile acid receptor TGR5 in Kupffer cells. Biochem. Biophys. Res. Commun. 2008;372:78–84. 75. Wang YD, Chen WD, Yu D, et al. The G-Protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-kappaB) in mice. Hepatology 2011;54:1421–32. 76. Pols TW, Nomura M, Harach T, et al. TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell Metab. 2011;14:747–57. 77. Gressner AM. The cell biology of liver fibrogenesis - an imbalance of proliferation, growth arrest and apoptosis of myofibroblasts. Cell Tissue Res. 1998;292:447–52. 78. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fi brosis. Mechanisms and treatment strategies. N. Engl. J. Med. 1993;328:1828–35. 79. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 2000;275:2247–50. 80. Friedman SL. Evolving challenges in hepatic fibrosis. Nat. Rev. Gastroenterol. Hepatol. 2010;7:425–36. 81. Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 2000;279:G245–G9. 82. Ikeda K, Wakahara T, Wang YQ, et al. In vitro migratory potential of rat quiescent hepatic stellate cells and its augmentation by cell activation. Hepatology 1999;29:1760–7. 83. Marra F, DeFranco R, Grappone C, et al. Expression of the thrombin receptor in human liver: up-regulation during acute and chronic injury. Hepatology 1998;27:462–71. 84. Kordes C, Sawitza I, Muller-Marbach A, et al. CD133+ hepatic stellate cells are progenitor cells. Biochem. Biophys. Res. Commun. 2007;352:410–17. 85. Sawitza I, Kordes C, Reister S, et al. The niche of stellate cells within rat liver. Hepatology 2009;50:1617–24. 86. Werneburg NW, Yoon JH, Higuchi H, et al. Bile acids activate EGF receptor via a TGF-alpha-dependent mechanism in human cholangiocyte cell lines. Am. J. Physiol. Gastrointest. Liver Physiol. 2003;285:G31–G6. 87. Svegliati-Baroni G, Ridolfi F, Hannivoort R, et al. Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor. Gastroenterology 2005;128:1042–55. 88. Reinehr R, Sommerfeld A, Häussinger D. CD95 ligand is a proliferative and antiapoptotic signal in quiescent hepatic stellate cells. Gastroenterology 2008;134:1494–506. 89. Kordes C, Sawitza I, Häussinger D. Stellate cells in the regenerating liver. In: Häussinger D, ed. Liver Regeneration: Walter de Gruyter; 2011:85–98. 90. Sommerfeld A, Reinehr R, Häussinger D. Bile acid-induced epidermal growth factor receptor activation in quiescent rat hepatic stellate cells can trigger both proliferation and apoptosis. J. Biol. Chem. 2009;284:22173–83. 91. Huang W, Ma K, Zhang J, et al. Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration. Science 2006;312:233–6.

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92. Papa S, Zazzeroni F, Bubici C, et al. Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat. Cell Biol. 2004;6:146–53. 93. Papa S, Monti SM, Vitale RM, et al. Insights into the structural basis of the GADD45betamediated inactivation of the JNK kinase, MKK7/JNKK2. J. Biol. Chem. 2007;282:19029–41. 94. Papa S, Zazzeroni F, Fu YX, et al. Gadd45beta promotes hepatocyte survival during liver regeneration in mice by modulating JNK signaling. J. Clin. Invest. 2008;118:1911–23. 95. Xia X, Francis H, Glaser S, et al. Bile acid interactions with cholangiocytes. World J. Gastroenterol. 2006;12:3553–63. 96. Marucci L, Alpini G, Glaser SS, et al. Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism. Am. J. Physiol. Gastrointest. Liver Physiol. 2003;284:G290–G301. 97. Marzioni M, LeSage GD, Glaser S, et al. Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct-ligated rats. Am. J. Physiol. Gastrointest. Liver Physiol. 2003;284:G837–G52. 98. Marzioni M, Ueno Y, Glaser S, et al. Cytoprotective effects of taurocholic acid feeding on the biliary tree after adrenergic denervation of the liver. Liver Int. 2007;27:558–68. 99. Glaser S, Onori P, Gaudio E, et al. Taurocholic acid prevents biliary damage induced by hepatic artery ligation in cholestatic rats. Dig. Liver Dis. 2010;42:709–17. 100. Ueno Y, Francis H, Glaser S, et al. Taurocholic acid feeding prevents tumor necrosis factor-alpha-induced damage of cholangiocytes by a PI3K-mediated pathway. Exp. Biol. Med. (Maywood) 2007;232:942–9. 101. Mancinelli R, Onori P, Gaudio E, et al. Taurocholate feeding to bile duct ligated rats prevents caffeic acid-induced bile duct damage by changes in cholangiocyte VEGF expression. Exp. Biol. Med. (Maywood) 2009;234:462–74. 102. Alpini G, Glaser SS, Rodgers R, et al. Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes. Gastroenterology 1997;113:1734–40. 103. Lazaridis KN, Pham L, Tietz P, et al. Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. J. Clin. Invest. 1997;100:2714–21. 104. Keitel V, Cupisti K, Ullmer C, et al. The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders. Hepatology 2009;50:861–70. 105. Keitel V, Ullmer C, Häussinger D. The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes. Biol. Chem. 2010;391:785–9. 106. Keitel V, Häussinger D. TGR5 in the biliary tree. Dig. Dis. 2011;29:45–7. 107. Chen L, Yao X, Young A, et al. Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes. Am. J. Physiol. Endocrinol. Metab. 2012;302:E68–E76. 108. Alpini G, Glaser SS, Ueno Y, et al. Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion. Gastroenterology 1999;116:179–86. 109. Alpini G, Baiocchi L, Glaser S, et al. Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha. Hepatology 2002;35:1041–52. 110. Alpini G, Glaser S, Robertson W, et al. Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. Am. J. Physiol. 1997;273:G518–G29. 111. Alpini G, Glaser SS, Ueno Y, et al. Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion. Gastroenterology 1999;116:179–86. 112. Alpini G, Glaser S, Alvaro D, et al. Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats. Gastroenterology 2002;123:1226–37.

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113. Fiorotto R, Spirli C, Fabris L, et al. Ursodeoxycholic acid stimulates cholangiocyte fl uid secretion in mice via CFTR-dependent ATP secretion. Gastroenterology 2007;133:1603–13. 114. Halilbasic E, Fiorotto R, Fickert P, et al. Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice. Hepatology 2009;49:1972–81. 115. Alpini G, Glaser S, Baiocchi L, et al. Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats. Hepatology 2005;41:1037–45. 116. Cheng SH, Rich DP, Marshall J, et al. Phosphorylation of the R domain by cAMPdependent protein kinase regulates the CFTR chloride channel. Cell 1991;66:1027–36. 117. Howard M, Jiang X, Stolz DB, et al. Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells. Am. J. Physiol. Cell Physiol. 2000;279:C375–C82. 118. Lavoie B, Balemba OB, Godfrey C, et al. Hydrophobic bile salts inhibit gallbladder smooth muscle function via stimulation of GPBAR1 receptors and activation of KATP channels. J. Physiol. 2010;588:3295–305. 119. Li T, Holmstrom SR, Kir S, et al. The G protein-coupled bile acid receptor, TGR5, stimulates gallbladder filling. Mol. Endocrinol. 2011;25:1066–71. 120. Vassileva G, Golovko A, Markowitz L, et al. Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation. Biochem. J. 2006;398:423–30. 121. Rudolph G, Kloeters-Plachky P, Rost D, et al. The incidence of cholangiocarcinoma in primary sclerosing cholangitis after long-time treatment with ursodeoxycholic acid. Eur. J. Gastroenterol. Hepatol. 2007;19:487–91.

6 Modulation of innate immunity and inflammation by bile acids and their receptors Dirk Graf and Johannes Bode

6.1 Introduction Bile acids are synthesized from cholesterol in the liver and are normally secreted into bile, thereby reaching the small intestine. In the terminal ileum they are reabsorbed, transported through enterocytes into the portal blood, and recirculated to the liver. In cholestatic syndromes, bile acids accumulate and reach levels up to 300 μmol/L (1). In the past it became increasingly evident that bile acids also function as signaling molecules involved in regulation of diverse cellular processes and that patients with cholestasis and high serum concentrations of bile acids show impaired cell-mediated innate and adaptive immunity (2,3). That bile acids mediate anti-inflammatory effects was further supported by studies on cholestatic animal models and patients with obstructive jaundice, suggesting that increased levels of bile acids correlate with reduced LPS-induced complications after surgery (4,5) and that oral application of bile acids leads to an impaired intestinal bacterial translocation (6). However, patients with obstructive jaundice were also shown to have higher perioperative complications after surgeries, with increased morbidity and mortality compared with noncholestatic patients (7–10). This was mainly due to the increased susceptibility of these patients to bacterial infections and sepsis, bacterial translocation, impaired wound healing, gastrointestinal bleeding, and blood coagulation disorders (10,11). These complications have at least in part been attributed to a compromised immune system, as observed in cholestatic patients, involving impaired function of immune-competent cells of the innate and adaptive immune system. In support of the in vivo data, the results of a variety of previous studies have provided strong evidence that bile acids mediate inhibitory effects on the function not only of peripheral blood–derived monocytic cells (PBMCs), monocytes, and macrophages but also on B cells in vitro. Hence, it is well documented by a variety of previous reports that bile acids inhibit the production of lipopolysaccharide (LPS)-induced inflammatory cytokines such as IL-1, IL-6, and tumor necrosis factor α (TNF-α) in PBMCs, monocytes, and macrophages (fFig. 6.1) (12,13). Consistently, bile acids have also been reported to affect the phagocytic activity of macrophages or Kupffer cells (13–17) and to repress the humoral response in human PBMC cultures by inhibiting the cell proliferation and exocytosis of immunoglobulins (18). Thereby the suppressive potential of bile acids on the inflammatory response was demonstrated to depend on the bile acid used. For example, chenodeoxycholic acid (CDCA) exhibited strong inhibitory properties in vitro, whereas ursodeoxycholic acid (UDCA) had only weak effects on inflammatory cytokine production by monocytes (13). Meanwhile, strong evidence exists that these anti-inflammatory or immune-modulatory effects of bile acids are largely due to the bile acid–induced activation of two different receptor systems: the group of bile acid sensing nuclear receptors and the more recently

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6 Modulation of innate immunity and inflammation PAMP e.g. LPS PRR

O ATP TGR5

NH

cAMP PKA FXR

Macrophage

Expression of inflammatory genes

Expression of e.g. TNFa; IL-1b; IL-6; IL-12; IFNg

FXR Expression of bile salt transporters

FXR Expression of bile salt transporters

Hepatocyte

Fig. 6.1: Schematic summary of the crosstalk between bile acids and pattern recognition receptors (PRR)-dependent signaling. In general, macrophages and other mammalian cells sense the presence of pathogens through a group of receptor complexes, also termed as PRR, which are specialized to detect conserved molecular structures. These pathogen-related molecular structures are also termed as pathogen associated molecular patterns (PAMP). The family of PRRs comprises so called toll like receptor (TLR), which are membrane-bound PRRs expressed by a variety of different cell types either located at the cellular surface or in the endosome. The prototype of the TLR-family is TLR4, a plasma membrane located receptor, which recognizes lipopolysaccharide (LPS) of gram-negative bacteria in presence of CD14 and MD2. In macrophages LPS induces an inflammatory- and antiviral response by expression of different cytokines like TNF-α, IL-1ß, IL-6, IL-12, IFNß and IFN-γ. The pro-infl ammatory cytokines mediate a suppression of bile salt transporter mRNA and protein expression in hepatocytes probably via inhibition of the nuclear bile acid receptor farnesoid X receptor (FXR), thereby impairing bile acid secretion with a consecutive accumulation of bile acids in hepatocytes and blood stream. On the other hand bile acids repress LPS-induced expression of proinflammatory cytokines mainly in a TGR5 and /or FXR dependent pathway, so that a tight interplay between bile acids and TLRsignaling can be postulated.

studied G protein–coupled bile acid receptor TGR5 (also called Gpbar-1 or M-bar) (fFig. 6.1). Both of these receptor systems are activated by bile acids under physiological conditions (15). The group of nuclear receptors, that are able to recognize bile acids consists of the farnesoid X receptor (19–21), pregnane X receptor (PXR) (22), constitutive androstane receptor (CAR) (23), and vitamin D receptor (VDR) (24). Among these, the farnesoid X receptor

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105

(FXR) is the best studied nuclear bile acid receptor, which is potently activated by conjugated and unconjugated chenodeoxycholic acid with an EC50 of about 5–10 μM (19–21). Other bile acids with lower potency to activate FXR are deoxycholic acid and lithocholic acid. Further on, lithocholic acid leads to the activation of PXR and VDR at higher concentrations of about 30–300 μmol/L (22,24). Stimulation of these nuclear receptors results in transcriptional regulation of target genes involved in the regulation of bile acid, glucose, and lipid homeostasis (25). Whereas nuclear receptors sense intracellular bile acids, the G-coupled receptor TGR5 is located at the outer cell membrane and activated by different primary and secondary bile acids. Thereby, taurine-conjugated LCA is the most potent agonist of TGR5, with an EC50 of about 290 nM, followed by deoxycholic acid, chenodeoxycholic acid, and cholic acid (15). The pattern of nuclear receptors (FXR, PXR, CAR and VDR) and TGR5 expressed in immune-competent cells, and both parenchymal and nonparenchymal liver cells is only partially characterized (fTab. 6.1). Expression of FXR is described for human monocytes/ macrophages and Kupffer cells (27,28), CD4+, CD19+, CD8+ cells (27), hepatocytes (19,20), biliary epithelial cells (29), and liver-derived murine natural killer T cells (NKTs) (30), whereas its expression in regulatory T cells, natural killer cells, and dendritic cells has not yet been evaluated. In sinusoidal endothelial cells (31) and human stellate cells (32), protein expression of FXR is not detected. TGR5 expression has been described in CD14+ human monocytes (15), rat alveolar macrophages (15), murine macrophages (33), rat Kupffer cells (34), sinusoidal liver endothelial cells (SECs) (35), biliary epithelial cells (36), and human macrophages (authors’ unpublished data) as well as in neurons, astrocytes, and microglia (37) (Görg, Keitel and Häussinger; unpublished data). In contrast to FXR, TGR5 is not expressed in CD4+, CD19+, or CD8+ cells, hepatocytes (15), or quiescent hepatic stellate cells (25), indicating that this receptor is restricted mainly to cells of the innate immune system (fTab. 6.1).

Tab. 6.1: Expression of bile acid receptors in immunocompetent cells and in parenchymal and non-parenchymal liver cells (adapted from reference 26). Receptor MO/MC/ KC

CD4⫹ CD8⫹ CD19⫹ DC

HC SEC

HSC

BEC

FXR

⫹

⫹

⫹

⫹

not ⫹ known

⫺

human ⫺ ⫹

PXR

⫹

⫹

⫹

⫹

not ⫹ known

not ⫺ known

not known

CAR

⫹

⫹

⫹

⫹

not ⫹ known

not ⫺ known

not known

VDR

⫹

⫹

⫹

⫹

⫹

⫹

⫹

⫹

⫹

TGR5

⫹

⫺

⫺

⫺

not ⫺ known

⫹

⫺ in ⫹ quescient

Abreviations: MO: monocytes; MC: macrophages; KC: Kupffer cells; DC: dendritic cells; HC: hepatocytes; SEC: liver sinusoidal endothelial cells; HSC: hepatic stellate cells; BEC: biliary epithelial cells.

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6 Modulation of innate immunity and inflammation

The present review focuses on the relevance of bile acids and their receptors FXR and TGR5 to the regulation of innate and adaptive immunity and inflammation.

6.2 Impact of FXR deletion on immunity and inflammation – lessons from FXR knockout mice Nuclear hormone receptors are known to modulate innate immunity and inflammation through different pathways. In case of the glucocorticoid receptor (GR) as a prototype of nuclear receptors, its activation results in the repression of proinflammatory genes such as TNF-α, IL-6, and IL-1 in macrophages and T lymphocytes. GR signaling interferes with the activation of PRR (pathogen recognition receptors) activated transcription factors such as NF-κB and activator protein-1 (AP-1), by different pathways. On the one hand, direct interaction of GR–dependent signal elements with components of NF-κB pathways is described and, on the other hand, other mechanisms like induction of negative feedback mechanisms that target signaling molecules involved in activating NF-κB and AP-1, for example, IL-10, MKP-1, and IκBα or disruption of activator/coactivator complexes, are identified (38). Moreover, recent evidence suggests that the glucocorticoid receptor modulates the IL-6–induced hepatic acute-phase response by inhibition of suppressor of cytokine signaling (SOCS)3 expression, an endogenous inhibitor of STAT3-dependent cytokine signaling, resulting in enhanced STAT3 activation and STAT3-mediated expression of acute-phase proteins (39). The liver acts as an “immune organ” comprising the largest pool of resident tissue macrophages (Kupffer cells) in the body. Apart from this, other immune-competent cells such as dendritic cells, T lymphocytes, NK cells, and NKTs are also enriched in the liver. Pathogens and antigens from gut and spleen reach the liver via portal blood and are sensed by different PRRs including Toll-like receptors, NOD-like receptors and RIG-I-like helicases differentially expressed in hepatocytes, endothelial cells, and immune-competent cells (40,41). FXR knockout mice suffer from a variety of defects in immunity and inflammation, especially in liver and intestine, where the highest density of FXR-expressing cells exists. The consequences of FXR deletion for immune and inflammatory processes is outlined further on; however, the impact on metabolic processes such as glucose, lipid, and bile acid homeostasis is not within the scope of this chapter. Likewise the effects of other nuclear receptors including pregnane X receptor, vitamin D receptor, and constitutive androstane receptors, which can also modulate immune function and inflammation (for review see reference 26), are reviewed elsewhere in detail and are not addressed here.

6.2.1 “Liver phenotype” FXR depletion results in an increase of blood and hepatic bile acid levels, age-dependent elevated expression of the proinflammatory cytokines (e.g. IL-1ß, IL-6, IFN-γ, and TNF-α), raised beta-catenin and c-myc expression, and displayed pronounced liver injury (42,43). Furthermore FXR⫺/⫺ mice suffer from the spontaneous development of liver tumors (42,43). Lipopolysaccharide (LPS) administration lead to massive necrosis and hepatic infiltration of inflammatory cells, accompanied by higher levels of inflammatory cytokines such as iNOS, COX-2, IFN-γ, and IP-10 in FXR⫺/⫺ mice as compared with wild-type mice (43,44). Moreover, transfection of a constitutively active FXR expression construct

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repressed iNOS, COX-2, interferon-inducible protein 10, and IFN-γ mRNA levels induced by LPS administration (44). Also in vitro, TLR-4 and TNF-α–induced expression of proinflammatory cytokines like IL-6 and IL-1 and molecules such as iNOS was increased in the hepatocytes of FXR⫺/⫺ mice as compared with wild-type hepatocytes (44). FXR⫺/⫺ mice show hepatic steatosis with inflammation, which mimics all the features that occur in nonalcoholic steatohepatitis (45,46). Zhang et al. demonstrated that a synthetic FXR agonist inhibits IL-6-mediated CRP expression in a hepatocellular cell line and blocks LPS-induced expression of the acute-phase protein amyloid P component and serum amyloid A3 (47). All together, these data provide substantial evidence for the direct anti-inflammatory actions of bile acids, which at least in part are FXR-dependent (47–49). In this context it should be noted that inflammatory endotoxinemia and carbon tetrachloride can induce cholestatic conditions through membrane retrieval and inflammatory cytokine–mediated suppression of the bile salt transporter mRNA and protein expression in hepatocytes (50–55) (fFig. 6.1). Considering that the liver plays key roles in innate and adaptive immunity, harboring the body’s largest pool of macrophages and a variety of other immune-competent cells, one may conclude that this inflammationinduced cholestasis represents a kind of bile acid–mediated feedback loop to safeguard liver tissue from an overwhelming local activation of macrophages in particular. If this is the case, FXR and, as discussed in section 6.3, also TGR5 is a key molecule within this bile acid–mediated autoprotective feedback loop (fFig. 6.1). Increased expression of osteopontin (OPN) has been implicated in various inflammatory diseases including rheumatoid arthritis, Crohn’s disease, and fulminant hepatitis. In the case of Concanavalin A (Con A)–induced hepatitis as model for T-cell immunemediated hepatitis, OPN expression by NKTs is involved in self-activation and triggers neutrophil infiltration (56). Con A–treated FXR⫺/⫺ mice develop pronounced liver damage as compared with wild-type mice; on the other hand, supplementation with the synthetic FXR agonist ameliorates Con A–induced hepatic injury. It has been suggested that osteopontin as an NKT-derived extracellular matrix protein and immune regulatory cytokine is important for the observed phenotype in FXR⫺/⫺ mice after Con A treatment. Consistently, FXR⫺/⫺ mice show an increased OPN expression, and synthetic ligands of FXR attenuated Con A–induced liver injury and hepatic expression of the OPN gene. Apart from this, it has been demonstrated that FXR is expressed in liver-derived NKTs and blocks Con A–mediated expression of OPN, thereby probably ameliorating liver damage (30). These data support the assumption that upon bile acid–induced activation, the bile acid receptor FXR modulates activation of liver-derived NKTs and thereby mediates hepatoprotective effects.

6.2.2 “Gut phenotype” Obstructive cholestasis is often accompanied by bacterial proliferation and mucosal injury in the small intestine, which promotes the translocation of bacteria and other pathogens across the epithelial barrier so that systemic infection occurs. Bile acids induce FXR-dependent genes involved in enteroprotection and inhibit bacterial overgrowth and mucosal injury in ileum caused by bile duct ligation. FXR⫺/⫺ mice have raised ileal levels of bacteria and show epithelial deterioration (57). FXR deletion results in a proinflammatory state with development of moderate intestinal inflammation and infiltration of CD11b+ cells as well as the development of fibrosis under naive conditions.

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In models of acute and chronic colitis, FXR⫺/⫺ mice have an increased susceptibility to inflammation and experimentally induced colitis is aggravated in them. These models show similiarities to inflammatory bowel disease, the pathogenesis of which involves a dysregulation of innate immunity (28). Treatment of FXR wild-type mice with the synthetic FXR ligand 6-ethyl chenodeoxycholic acid prevents chemically induced intestinal inflammation, and results in an amelioration of the symptoms of colitis and inhibition of epithelial permeability (28,58). Furthermore, in murine intestinal macrophages, FXR downregulates LPS-induced target genes such as iNOS, TNF-α, IL-1ß, IL-6, COX-1, and COX-2 (28). On the other hand, FXR expression in the ileum and colon is decreased in patients with Crohn’s colitis (28,59). This may be secondary to the altered enterohepatic circulation of bile salts or transrepression by inflammatory signals (59). In summary, bile acids appear to protect the host against pathogens in an FXR-dependent manner by stabilizing the integrity of the intestinal barrier and modulating the immune function of resident macrophages. These findings provide a rationale for exploring FXR agonists with a view to developing a novel therapeutic strategy for inflammatory bowel disease.

6.3 Role of TGR5 in the modulation of immune function Bile acids such as taurolithocholic acid increase intracellular cAMP via a TGR5/adenylate cyclase–dependent pathway, thereby blocking the LPS-induced production of proinflammatory cytokines such as TNF-α, IL-1, and IL-6 from CD14+ human monocytes (15), rat alveolar macrophages (15), and rat Kupffer cells (34) as well as in human macrophages (authors’ unpublished observations) (fFigs. 6.1 and 6.2). Apart from the inhibition of inflammatory cytokine production, TGR5 ligands also impaired the phagocytic capability of monocytes (15). Induction of cholestatic conditions by bile duct ligation resulted in an upregulation of TGR5 expression in liver macrophages, which was postulated to mediate protective effects during obstructive jaundice by preventing inflammationinduced tissue damage through a bile acid–mediated feedback loop (34). TGR5⫺/⫺ mice consistently showed more severe liver necrosis and inflammation in an LPS-induced inflammation model compared with wild-type mice, suggesting that TGR5 indeed mediates protective effects (33). Apart from TGR5-mediated suppression of inflammatory cytokine production in macrophages, the protective effects of TGR5 activation may also be due to the induction of increased NO production in SECs, thereby protecting from oxidative stress and lipid peroxidation (35). In animal models of steatosis and obesity, these TGR5-mediated anti-inflammatory actions of bile acids mediate important hepatoprotective effects, since mice lacking the TGR5 gene, as compared with control animals, develop a pronounced nonalcoholic steatohepatitis in response to a high-fat diet (60). In addition, the development of atherosclerosis in Ldlr⫺/⫺Tgr5⫹/⫹ mice was substantially pronounced in Ldlr⫺/⫺Tgr5⫺/⫺ double-knockout mice, an observation that has also been ascribed to the suppressive effects of TGR5 on macrophages (61). However, despite the anti-inflammatory effects of TGR5, it has also been held responsible for the increased susceptibility to infection observed in patients with obstructive jaundice (10,33). The exact mechanism by which bile acids block TLR-induced proinflammatory cytokine production is not well characterized. Recently Wang et al. described an inhibition of LPS-induced NF-κB phosphorylation, blockade of p65 translocation, impairment of NF-κB DNA binding activity, and the transcription activity of NF-κB in response to a

6.4 Effects of bile acids on immunological function Suppression of phagocytic activity and of LPS-induced cytokine production (e.g. TNF, IL-6, IL-1) or iNOS expression

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Suppression of osteopontin-mediated activation of neutrophils by NKT-cells

Microglia

Neutrophil granulocytes Monocytes NKT-cells

T-cells Macrophages

Dendritic cells

Hepatocyte

Suppression of NK-cell activation

Suppression of T-cell activation by dendritic cells

Inhibition of STAT1/3-mediated IL-6 and type I IFN signalling and gene expression

Fig. 6.2: Schematic summary of the immunmodulatory effects of bile acids on different cell types. Bile acids are synthesized from cholesterol in the liver and are secreted into bile. In cholestatic syndromes bile acids accumulate and reach levels up to 300 μmol/l, thereby modulating immunological functions of different liver cells (hepatocytes, Kupffer cells) and of diverse immunocompetent cells such as monocytes, macrophages, T-cells, NKT cells, NK cells and microglia. On the other hand, bile acids inhibit IL-6- and IFNα- signaling in hepatocytes, which results in an anti-inflammatory and pro-viral state.

synthetic TGR5 agonist in murine Kupffer cells and bone marrow–derived macrophages. The molecular basis of this inhibition was suspected to be an enhanced interaction of ß-arrestin 2 with IκBα in response to a TGR5 agonist, thereby blocking NF-κB activity (33). Moreover, Pols et al. have demonstrated that activation of TGR5 in macrophages by a semisynthetic bile acid (INT-777) inhibits proinflammatory cytokine production by blocking NF-κB activity via a TGR5-induced cAMP pathway (61). This observation is in line with the fact that cAMP is thought to be an important modulator of monocytes/macrophage action, generating an anti-inflammatory state by diverse molecular mechanisms including transrepression of NF-κB or assembly of distinct multiprotein complexes (62).

6.4 Effects of bile acids on immunological function independently of bile acid receptors Apart from the receptor-mediated immunological actions of bile acids, several reports suggest that bile acids may also have effects that are possibly receptor-independent,

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although the question of whether such effects are truly independent from TGR5 and FXR has not been thorougly addressed. In vitro experiments have shown that hydrophobic bile acids inhibit IFN-α -induced expression, of IFN-stimulated genes (ISGs) encoding for antiviral effector molecules such as myxovirus resistance protein A (MxA), 2’-5’ oli goadenylate synthetase (OAS), and dsRNA-activated protein kinase (PKR). This inhibitory effect of bile acids on ISG gene expression has been suggested to be due to inhibition of the two receptor-associated Janus kinases Jak1 and Tyk2, which are important for the STAT-dependent signal transduction of type I interferons in hepatoma cells transfected with the sodium taurocholate cotransporter NTCP (63). A comparable effect of bile acids on IFN-induced ISG expression has been reported by another group analyzing the effect of bile acids on IFN signaling in three hepatoma cell lines, although in these cell lines inhibition of ISG expression was not due to impaired activation of STAT1 or protein kinase C (64). These inhibitory effects of bile acids on IFN-triggered ISG expression were not restricted to hepatocytes but have also been reported for NK cells and lymphocytes (65,66). Another interesting observation was that bile acids abolished the IL-6 signaling pathway in hepatocytes in a p38MAPK and caspase 3–dependent manner. Thus activation of caspase 3 leads to cleavage of the signal transducing subunit gp130 of the IL-6 receptor complex and subsequent abrogation of IL-6–induced STAT3–mediated signal transduction (48), an effect that could be also mimicked by stimulation with the ligand of the CD95 death receptor CD95L (49). Caspase-mediated receptor cleavage could be prevented by introducing a point mutation into a caspase cleavage site identified within position 800–806 (DHVDGGD) of the cytoplasmic tail of gp130 (49). In this context it is interesting to note that the gp130/IL-6 signaling pathway has been suggested to mediate hepatoprotective effects and to ameliorates liver injury in models of obstructive jaundice (67,68). Hence one may conclude that apart from the receptor-mediated immune-modulatory or anti-inflammatory effects of bile acids, hydrophobic bile acids in particular exert hepatotoxic effects by abrogating hepatoprotective signals.

6.5 Obstructive jaundice and its impact on immune function As outlined in section 6.1, patients with obstructive jaundice develop more severe perioperative complications after surgery, with increased morbidity and mortality, compared with noncholestatic patients (7–10), because the cholestatic patients tend to suffer from bacterial infections, sepsis, and impaired wound healing (10,11). These complications are due to complex alterations of innate and adaptive immunity observed in vivo during cholestatic conditions. Hence the ability of the reticuloendothelial system to clear phagocytes is substantially decreased in patients with obstructive jaundice, and these patients are also hypersensitive to endotoxins (10,69,70). Moreover, in bile duct–ligated (BDL) rats, by comparison with sham operated animals, levels of serum endotoxins, TNF-α, IL-6, and the expression of iNOS mRNA by Kupffer cells have been found to be increased. This increase in cytokine expression in BDL rats can be reversed by implementing internal biliary or external drainage (71). Besides, superoxide production was strongly suppressed in Kupffer cells from BDL rats compared with cells of sham operated animals (72). In contrast, the phagocytic ability of Kupffer cells in BDL rats was reported to be increased, although their capacity to kill bacteria is suggested to be impaired (16).

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Apart from altered macrophage function and inflammatory cytokine production, obstructive jaundice in the model of BDL rats also resulted in the impaired function of liver T cells, with reduced responsiveness to allogeneic and syngeneic antigen-loaded dendritic cells (73). In addition, under cholestatic conditions, altered proliferative responses among splenic lymphocytes and peripheral blood lymphocytes have been described (74,75). Moreover, it has recently been postulated that invariant NKTs suppressed the neutrophilic inflammatory response in a mouse model of cholestatic liver damage (76). Further on, experimental studies have shown different alterations of immune function, including suppression of NK cell activity (77), impairment of macrophage phagocytic ability and metabolic activity (78), and inhibition of neutrophil chemotaxis (79). However, the mediators of this immune dysregulation in obstructive jaundice are not clearly identified, although bile acids may be good candidates (73).

6.6 Role of bile acids and FXR in viral infections In the case of hepatitis C infection, it has been shown that bile acids have a prognostic impact on the success rate of IFN-α–dependent therapies (80,81). In replicon systems, bile acids trigger HCV genotype 1 RNA replication via FXR, and blockade of the FXR pathway by a pharmacological inhibitor blocks the enhancing effects of bile acids on viral replication (82,83). In the case of the hepatitis B virus (HBV) two FXR response elements located in enhancer II and the core promoter regions have been identified. FXR binding to both sides results in the increased synthesis of HBV pregenomic RNA and viral replication. This finding was confirmed by Kim et al., who showed that bile acids increase HBV expression and decrease susceptibility to IFN-α treatment (84). Bile acids have been further reported to promote norovirus replication, probably in an FXR-dependent manner (85,86). In contrast to this, replication of rotavirus has recently been described to be inhibited by bile acids such as chenodeoxycholic acid and synthetic FXR ligands, probably owing to a bile acid or FXR ligand–mediated downregulation of the lipid synthesis required for an undisturbed life cycle of rotavirus (87). Accordingly, own data suggest that hydrophobic bile acids block the replication of human and murine cytomegalovirus in different hepatocellular cells (unpublished data).

6.7 Concluding remarks Immunosuppression and infectious complications are well-known attendant phenomena of cholestasis and have been well documented by a variety of previous reports starting in the early 1980s. In these reports, the failure of antiviral therapy was consistently correlated with serum bile acid concentrations, suggesting that bile acids have immunomodulatory or immunosuppressive effects. Further analysis of this phenomenon revealed that bile acids suppress the inflammatory response of macrophages, affect interferon signaling in hepatocytes, and alter the effector functions of immune cells involved in innate and adaptive immunity. During the past decade it became increasingly evident that in this context FXR, a member of the nuclear receptor family, and the G protein–coupled receptor TGR5, which is expressed at the cellular surface, are both essentially involved in mediating the immunosuppressive and anti-inflammatory

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effects of bile acids. On a shorter time scale, these immunomodulatory activities of bile acids appear to be beneficial, since they temper the inflammatory response and therefore protect from inflammation-mediated tissue damage. On a larger time scale, however, these immunosuppressive actions may be detrimental, because they lead to an insufficiency of the anti-infectious effector mechanisms. A more detailed characterization of the molecular mechanisms involved in mediating the immunosuppressive or anti-inflammatory actions of FXR and TGR5 remain among the future challenges within this field, as such research will be required to identify appropriate targets for therapeutic intervention.

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64. Podevin P, Rosmorduc O, Conti F, et al. Bile acids modulate the interferon signalling pathway. Hepatology 1999;29:1840–7. 65. Podevin P, CalmusY, Bonnefi s MT, et al. Effect of cholestasis and bile acids on interferon-induced 2´-5´-adenylate synthetase and NK cell activities. Gastroenterology 1995;108:1192–8. 66. Podevin P, Blanc MC, Vaubourdolle M, et al. Bile acid inhibition of interferon activity in human lymphocytes: no evidence of oxidative stress. Eur. J. Clin. Invest. 1997;27:491–6. 67. Ezure T, Sakamoto T, Tsuji H, et al. The development and compensation of biliary cirrhosis in interleukin-6-deficient mice. Am. J. Pathol. 2000;156:1627–39. 68. Wuestefeld T, Klein C, Streetz KL, et al. Lack of gp130 expression results in more bacterial infection and higher mortality during chronic cholestasis in mice. Hepatology 2005;42:1082–90. 69. Pain JA, Collier DS, Ritson A. Reticuloendothelial system phagocytic function in obstructive jaundice and its modifi cation by a muramyl dipeptide analogue. Eur. Surg. Res. 1987;19:16–22. 70. Lago MTD, Jukemura J, Machado MCC, et al. Phagocytosis and production of H2O2 by human peripheral blood mononuclear cells from patients with obstructive jaundice. Pancreatology 2006;6:273–8. 71. Meng Y, Gong YC, Dou Y, et al. Changes of serum cytokines and expression of inducible nitric oxide synthase mRNA by Kupffer cells after relief from obstructive jaundice in rats. J. Gastroenterol. Hepatol. 2009;24:1064–9. 72. Sung JJ, Go My. Reversible Kupffer cell suppression in biliary obstruction is caused by hydrophobic bile acids. J. Hepatol. 1999;30:413–8. 73. Katz SC, Ryan K, Ahmed N, et al. Obstructive jaundice expands intrahepatic regulatory T cells, which impair liver T lymphocytes function but modulate liver cholestasis and fibrosis. J. Immunol. 2011;187:1150–6. 74. Pinto M, Kaplun A. Immune status in mice with experimental biliary obstruction. Clin. Immunol. Immunopathol. 1980;16:396–405. 75. Thompson RL, Hoper M, Diamond T, et al. Development and reversibility of T lymphocyte dysfunction in experimental obstructive jaundice. Br. J. Surg. 1990;77:1229–32. 76. Wintermeyer P, Cheng CW, Gehring S, et al. Invariant natural killer T cells suppress the neutrophilic inflammatory response in a mouse model of cholestatic liver damage. Gatroenterology 2009;136:1048–59. 77. Lane DR, Joshi P, Grogan JB, et al. Suppression of natural killer cells activity in biliary obstruction. Am. J. Surg. 1996;62: 259–62. 78. Ding JW, Nassberger L, Andersson R, et al. Macrophage phagocytic dysfunction and reduced metabolic response in experimental obstructive jaundice. Eur. J. Surg. 1994;160:437–42. 79. Andy OJ, Grogan JB, Griswold JA, et al. Peritoneal neutrophil chemotaxis is impaired in biliary obstruction. Am. Surg. 1991;58:28–31. 80. Serfaty L, Giral P, Loria A, et al. Factors predictive of the response to interferon in patients with chronic hepatitis C. J. Hepatol. 1994;21:12–17. 81. Jorquera F, Monte MJ, Guerra J, et al. Usefulness of combined measurement of serum bile acids and ferritin as additional prognostic markers to predict failure to reach sustained response to antiviral treatment in chronic hepatitis C. J. Gastroenterol. Hepatol. 2005;20:547–54. 82. Chang KO, George DW. Bile acids promote the expression of hepatitis C virus in repliconharboring cells. J. Virol. 2007;81:9633–40. 83. Scholtes C, Diaz O, Icard V, et al. Enhancement of genotype 1 hepatitis C virus replication by bile acids through FXR. J. Hepatol. 2008;48:192–9.

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84. Kim HY, Cho HK, Choi YH, et al. Bile acids increase hepatitis B virus gene expression and inhibit interferon-alpha activity. FEBS J. 2010;277:2791–802. 85. Chang KO, Sosnovtsev SV, Belliot G, et al. Bile acids are essential for porcine enteric calcivirus replication in association with down-regulation of signal transducer and activator of transcription 1. PNAS 2004;101:8733–8. 86. Chang KO. Role of cholesterol pathways in norovirus replication. J.Virol. 2009;83:8587–95. 87. Kim Y, Chang KO. Inhibitory effects of bile acids and synthetic farnesoid x receptor agonist on rotavirus replication. J. Virol. 2011;85:12570–7.

7 Bile acids as extrahepatic and interorgan signaling molecules Verena Keitel

7.1 Introduction Over the last decade bile acids have been increasingly recognized as signaling molecules with diverse endocrine and paracrine functions (1–3). Bile acids regulate bile acid, glucose, and lipid metabolism; induce energy expenditure; and modulate the inflammatory response. Furthermore, bile acids can induce programmed cell death (apoptosis) but can also promote cell proliferation, cell differentiation, and liver regeneration. Thus bile acid–dependent signaling pathways (see also Chapter 5) may play a role in the pathogenesis of metabolic disorders, such as type II diabetes, obesity, gallstone disease, atherosclerosis, and nonalcoholic steatohepatitis (NASH) (3). Furthermore, animal models have linked bile acid signaling pathways to the development of liver and gastrointestinal cancers (4–6). Bile acids can activate nuclear bile acid receptors, such as the farnesoid X receptor (FXR, NR1H4, Fig. 7.1, see also Chapter 4), which are ligand-activated transcription

Kidney Coactivator complex

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• Increased eNOS expression • Downregulation of endothelin-1

Fig. 7.1: Function of the farnesoid X receptor (FXR) in extrahepatic tissues. FXR is a ligandactivated transcription factor, which functions as heterodimer with the retinoid X receptor (RXR). Ligand binding to FXR initiates coactivator recruitment and transcription of FXR target genes (7,8).

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factors (7–10), as well as the plasma membrane-bound G protein–coupled bile acid receptor TGR5 (Gpbar-1) (11,12). Recently conjugated bile acids were identified as ligands for the G protein–coupled sphingosine-1-phosphate receptor 2 (S1P2); however, the role of S1P2 in bile acid signaling remains elusive (13). In addition, bile acids can bind and modulate integrin signaling in the liver and are modulators of muscarinic M2 receptors in fetal cardiomyocytes (14,15). Furthermore, bile acids have been demonstrated to activate different signaling cascades, such as kinase pathways and ion channels (see also Chapter 5) (14,16–18). The nuclear bile acid receptor FXR is highly expressed in hepatocytes and intestinal epithelial cells, but is also detectable in proximal tubules of the kidney (7,8,19,20); different peripheral blood mononuclear cells (21), endothelial cells (22), vascular smooth muscle cells (23) and adipocytes of white adipose tissue (24). Chenodeoxycholic acid (CDCA) and its conjugates are the most potent ligands for FXR, with an EC50 of approximately 5–10 μM (9,10,25). Expression of the membrane-bound bile acid receptor TGR5 has been detected in many tissues, including liver, gallbladder, intestine, kidney, spleen, and brain (11,12,26– 28). In liver TGR5 was localized in sinusoidal endothelial cells, Kupffer cells, and biliary epithelial cells (29–32). In the intestine TGR5 has been detected in enteroendocrine L-cells (33) as well as in neurons and astrocytes of the enteric nervous system (34). TGR5 is also present in CD14-positive monocytes (11), brown adipocytes, and skeletal muscle cells (35). Several unconjugated and conjugated bile acids are natural ligands for TGR5 (rank order of potency: lithocholic acid [LCA] > deoxycholic acid [DCA] > chenodeoxycholic acid [CDCA] > cholic acid [CA]), with taurolithocholic acid (TLCA) being the most potent agonist, having an EC50 of 0.29 μM (36). Activation of TGR5 by bile acids activates adenylate cyclase, thereby elevating intracellular cyclic AMP levels (11,12). Through activation of FXR and TGR5, bile acids modulate bile acid, glucose, lipid and energy homeostasis, as well as the immune response. Thus bile acid receptors have emerged as potential therapeutic targets for the treatment of liver and metabolic disorders. This chapter focuses on the endocrine functions of bile acids mediated by FXR and TGR5 in extrahepatic tissues. The role of FXR for the enterohepatic circulation of bile acids is discussed in Chapter 4 and bile acid signaling in the liver is reviewed in Chapter 5.

7.2 Bile acid–dependent modulation of glucose homeostasis A link between bile acid homeostasis and glucose metabolism was recognized in patients with type II diabetes who were treated for dyslipidemia with the bile acid sequestrant cholestyramine. Besides the expected reduction in cholesterol levels, cholestyramine induced a significant decrease in plasma glucose levels by 13% and a reduction in urinary glucose output (37). Furthermore, animal models of diabetes have been associated with altered bile acid composition and bile acid pool size (38,39). In primary hepatocytes FXR mRNA levels were increased by glucose treatment and repressed in the presence of insulin (40). Similar data was obtained with a genetic animal model for type II diabetes and obesity, the db/db mice, which showed increased FXR mRNA expression levels as compared to their wild-type litter mates (41). In this study treatment of wild-type and diabetic db/db animals with the synthetic FXR

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agonist GW4064 lowered plasma glucose levels significantly (41). However, the role of FXR in regulation of gluconeogenesis in liver is highly controversial (for a recent review, see reference 8). Although two studies showed that activation of FXR induces the expression of the rate-limiting enzyme of gluconeogenesis phosphoenolpyrovate carboxykinase (PEPCK) in primary hepatocytes and hepatoma cell lines (41,42), bile acids decreased PEPCK expression in an FXR-dependent and FXR-independent, SHPmediated mechanism in both hepatoma cells and mouse liver in several other studies (43–46). One explanation for this paradox is that bile acids mediate an indirect effect on gluconeogenesis and that FXR is only one of several signaling pathways involved. This hypothesis is supported by recent data on the role of fibroblast growth factor 19 for energy metabolism and glucose homeostasis (47,48). In the small intestine bile acids activate FXR, which in turn induces expression of fibroblast growth factor 19 (FGF19, the rodent orthologue is Fgf15) (49). In humans FGF19 serum levels peaked 90–120 minutes following the postprandial rise of serum bile acid levels (50). FGF19 is an important regulator of bile acid synthesis in the liver and thus bile acid homeostasis (see Chapter 4) (49). Furthermore, administration of FGF19 or overexpression of FGF19 in mice lowered serum glucose levels and improved insulin sensitivity in obese mice (47). FGF19 suppressed hepatic gluconeogenesis through inactivation of the transcription factor cAMP regulatory element–binding protein (CREB), which in turn downregulated the expression of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and genes involved in gluconeogenesis (PEPCK and glucose-6 phosphatase [G6Pase]) (48). Overexpression of PGC-1α restored the expression levels of PEPCK and G6Pase (48). Apart from gluconeogenesis FGF19 has been shown to modulate hepatic glycogen synthesis. The activity of glycogen synthase (GS) is negatively regulated through phosphorylation by glycogen synthase kinase 3 (GSK3), which is also inactivated by phosphorylation. FGF19 treatment promoted an increased phosphorylation of GSK3, thus inducing glycogen synthesis and liver weight in mice (51). Taken together, these data indicate that FXR modulates glucose homeostasis both directly through transcriptional activation of hepatic PEPCK as well as indirectly via upregulation of intestinal FGF19 expression. FGF19 does not only target its receptor in liver but also has effects on brain (47) and adipose tissue (52). Thus, the contribution of the liver versus extrahepatic tissues in FGF19 mediated metabolic effects has yet to be determined (48). TGR5 is expressed in an enteroendocrine cell line, where activation of the receptor promotes glucagon-like peptide-1 (GLP-1) secretion (53). Through several different mechanisms GLP-1 reduces glucose levels, protects pancreatic β cells from apoptosis, promotes pancreatic β cell proliferation, delays gastric emptying, and induces satiety, resulting in weight loss (54,55). GLP-1 receptor agonists are successfully used in the treatment of type II diabetes (54). TGR5 is expressed in intestinal L-cells, which are a major source of GLP-1 secretion (33). Activation of TGR5 with the TGR5 agonist 6α-ethyl 23(S)-methyl-cholic acid (INT-777, EMCA) induced intestinal GLP-1 release and increased serum insulin levels (56). Treatment of mice with a high fat diet and INT-777 improved insulin sensitivity in obese mice. Furthermore, stimulation of TGR5 in obese animals increased energy expenditure in brown adipose tissue resulting in a significant reduction of weight gain as well as hepatic steatosis as compared with vehicle-treated controls (56). The beneficial effects of the TGR5 agonist in obese mice

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were mediated through increased intestinal GLP-1 release as well as upregulation of type 2 deiodinase and activation of the mitochondrial respiratory chain in brown adipose tissue (56,57). An increase in the bile acid concentration in the large intestine seems to promote the activation of TGR5 in L-cells and the subsequent GLP-1 release. This hypothesis is underscored by the finding that inhibition of active bile acid reabsorption in the intestine with a selective inhibitor of the apical sodium-dependent bile acid transporter (ASBT, SLC10A2) enhanced intestinal GLP-1 secretion, thereby reducing serum glucose and HbA1c levels in diabetic rats (58). The activation of TGR5 in L-cells and the associated rise in GLP-1 release may also underlie the glucose lowering effect of bile acid sequestrants (59). However, it is unclear whether TGR5 is located in the apical membrane of L-cells and is activated by bile acids from the intestinal lumen or whether bile acids need to be absorbed (either actively via ASBT or passively) in order to activate TGR5 and facilitate GLP-1 secretion.

7.3 Impact of bile acids on energy expenditure Feeding of cholic acid to mice has been demonstrated to increase energy expenditure in brown adipose tissue, thereby preventing obesity and insulin resistance (35,56,60). TGR5 is expressed in tissues involved in thermogenesis and energy expenditure, such as brown adipose tissue of mice and human skeletal muscle. Activation of TGR5 in brown adipose tissue increased the expression of cAMP-regulated type 2 iodothyronine deiodinase, which converts thyroxine (T4) to active 3,5,3’-triiodothyronine and induced oxygen consumption (see Fig. 7.2). Treatment of isolated brown adipocytes or human skeletal muscle cells with bile acids increased deiodinase 2 (D2) activity and facilitated oxygen consumption. Targeted disruption of deiodinase 2 abolished this effect (35). Similar results were obtained with human skeletal muscle cells. In line with this, female TGR5 knockout mice gained significantly more weight on high fat diet as compared to wild-type litter mates (27). Bile acid pool size was also reduced in homozygous TGR5 knockout mice and seems to play a role in energy homeostasis (27). Activation of FXR apparently does not directly contribute to bile acid induced energy expenditure. Administration of the synthetic FXR agonist GW4064 to obese mice resulted in significant weight gain and increased insulin resistance of the treated animals (60). These effects could be attributed to the reduced bile acid pool size following GW4064 treatment and could be restored by additional feeding of cholic acid (60). Thus, reduction in bile acid pool size and systemic bile acid levels may compromise TGR5 activation in brown adipose tissue hence reducing energy expenditure (60).

7.4 Bile acid receptors and immune response Bile acids can compromise macrophage function by affecting phagocytic activity as well as cytokine production (61–65). FXR and TGR5 are both expressed on peripheral blood mononuclear cells (11,21). An anti-inflammatory function has been demonstrated for FXR in liver and intestine. FXR knockout mice have higher amounts of aerobic bacteria in their intestine as compared to wild-type litter mates (66). Bacterial overgrowth, mucosal injury and bacterial translocation were aggravated after bile duct

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ligation in both, FXR knockout and wild-type mice, however, the effects were more pronounced in FXR knockout mice. In wild-type mice administration of the synthetic FXR agonist (GW4064) prevented bacterial propagation, disruption of the epithelial barrier, and translocation of bacteria across the mucosa into lymph nodes (66). GW4064 itself had no bacteriostatic effect when incubated with ileal contents, indicating that the anti-inflammatory functions of FXR in intestine are mediated most likely through transcriptional upregulation of antimicrobial genes such as inducible nitric oxide synthase (iNOS), interleukin-18, and angiogenin 1 (66). The experimental setup did not discriminate whether the FXR-dependent upregulation of anti-inflammatory genes occurred in immune cells or in enterocytes (66). A recent study demonstrated that activation of FXR in enterocytes (HT29 and Caco2 cells) suppressed proinflammatory cytokine expression (interleukin-1β, interleukin-6, and macrophage attractant protein-1) and upregulated antimicrobial genes such as iNOS, angiogenin 1, and cathelicidin (67). Furthermore, treatment of mice with the FXR agonist obeticholic acid (INT-747) protected mice from dextran sodium sulfate (DSS)- or trinitrobenzenesulfonic acid (TNBS)–induced colitis (67). Treatment of isolated human mononuclear cells with the FXR agonist (INT-747) also resulted in decreased tumor necrosis factor-α (TNF-α) secretion and abolished differentiation of CD14-positive monocytes into dendritic cells (67). In the liver, FXR also exerts anti-inflammatory effects. FXR knockout mice have increased liver inflammation and spontaneously develop liver tumors (4,6). Activation of FXR in isolated hepatocytes and HepG2 hepatoma cells did not prevent nuclear factor-κB (NF-κB)-p65 translocation; however, it decreased NF-κB transcriptional activity (68). In turn, activation of NF-κB suppresses expression of FXR and its target genes (68,69), which may explain the downregulation of FXR target genes in liver during the acute-phase response. TGR5 is expressed in CD14-positive monocytes of the peripheral blood as well as in macrophages of several organs such as lung, liver (Kupffer cells) and intestine (11,29,70). Activation of TGR5 in isolated macrophages or Kupffer cells increased cAMP production and decreased expression of inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) (70,71). Stimulation of TGR5 resulted in decreased phosphorylation of IκBα, thus reducing nuclear translocation of p65 and NF-κB transcriptional regulation (71) (see Fig. 7.2). Whereas FXR mediates anti-inflammatory effects in both, parenchymal cells, such as hepatocytes and enterocytes, as well as in immune cells, TGR5 activates antiinflammatory pathways mainly in immune cells. Taken together, bile acids may exert anti-inflammatory effects through activation of both, FXR and TGR5, resulting in reduced NF-κB transcriptional activity. Chapter 6 includes a comprehensive overview of the immunomodulatory effects of bile acids.

7.5 Role of bile acid receptors in the cardiovascular system FXR is expressed in vascular endothelial cells. Activation of FXR resulted in a decreased expression of endothelin-1, one of the most potent vasoconstrictors, and an upregulation of endothelial NO synthase (eNOS), suggesting that FXR may regulate the expression of different vasoactive mediators (22,72). In the liver, TGR5 was detected in sinusoidal endothelial cells (SEC) but not in the endothelium of the portal vein, the hepatic veins, or the hepatic artery. Activation of TGR5 in SEC by bile acids leads to an upregulation of

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eNOS mRNA expression, an increased serine phosphorylation of eNOS, and a rise in nitric oxide production (30) (see Fig. 7.2). These data indicate that bile acids may modulate systemic vascular tone via FXR and hepatic microcirculation through activation of TGR5. Dyslipidemia and chronic inflammation are important mediators in the pathogenesis of atherosclerosis, which is now seen as a chronic inflammatory disease (73–75). The accumulation of apoplipoprotein B–containing lipoproteins in the vessel wall, the infiltration with monocytes, and the subsequent chronic inflammation represent important steps in atherogenesis (73–75). As described in section 7.4, both, FXR and TGR5 have anti-inflammatory functions and thus may be involved in the pathogenesis of atherosclerosis. Furthermore, FXR is an important regulator of cholesterol and triglyceride metabolism. Although FXR knockout mice have a proatherogenic serum lipid profile, they do not develop significant atherosclerosis (76–78). However, when FXR knockout mice were crossed with mice deficient for apolipoprotein E (ApoE), the double knockout mice presented with increased aortic plaque formation and reduced survival following treatment with a high-fat/highcholesterol diet (76). However, a study with female FXR/ApoE double-knockout mice showed reduced plaque formation as compared to ApoE single-knockout animals (79). Further studies with tissue-specific knockdown of FXR in endothelial cells, smooth muscle cells, and macrophages are required to clarify the role of FXR in atherogenesis (80). Activation of TGR5 in LDL receptor knockout mice resulted in decreased foam cell formation and reduced aortic plaque formation (57). These effects were abolished in TGR5-deficient LDL-receptor knockout mice (57). Treatment with the TGR5 agonist had no influence on cholesterol or triglyceride levels in any of the animals but reduced macrophage content and cytokine levels in aortic plaques (57) (see Fig. 7.2). FXR- and TGR5-independent bile acid effects have been described for endothelial cells, vascular smooth muscle cells as well as cardiomyocytes (for a recent review, see reference 80).

7.6 Role of bile acid receptors in the kidney FXR is expressed in renal proximal tubule cells (20), which are involved in bile acid excretion under cholestatic conditions. Bile duct ligation and bile acid feeding promote an upregulation of the organic anion transporter ABCC2 (MRP2) in the proximal tubules in rat kidneys (81–83). However, this adaptive response was independent of FXR since it was also observed in FXR knockout mice (83). The organic solute transporter (OSTα/ OSTβ) is located in the basolateral membrane of kidney proximal tubules and is upregulated under cholestatic conditions. This increase in expression of OSTα /OSTβ was dependent on FXR activation (84,85). Furthermore, similar to the situation in liver, FXR seems to be an important regulator of renal lipid metabolism. Activation of FXR with the agonist INT-747 in a mouse model of chronic nephropathy and diet-induced obesity (DBA/2J) suppressed the expression of the sterol-regulatory element binding protein (SREBP-1c), the master regulator of fatty acid metabolism, in kidney tissue (86). Inhibition of SREBP-1c and its target genes led to a decrease in fatty acid synthesis and an upregulation of fatty acid oxidation, resulting in reduced renal triglyceride levels (86). As expected, renal inflammation was also reduced by the FXR agonist (86). Taken together, FXR activa-

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Fig. 7.2: Expression and function of the membrane-bound bile acid receptor TGR5. Activation of TGR5 by bile acids leads to an activation of a stimulatory G protein and adenylate cyclase, resulting in an increase in intracellular cyclic AMP (11,12) and subsequent activation of protein kinase A (PKA). PKA serine/threonine phosphorylates the CD95 receptor (CD95-P) thus activating antiapoptotic signaling (30,90). PKA also serine phosphorylates endothelial NO synthase (eNOS-P), thereby promoting increased nitric oxide (NO) production (30). The activation of the cAMP response element binding protein (CREB) induces the upregulation of eNOS and deiodinase type 2 (D2) mRNA (35). Activation of TGR5 via cAMP also prevents translocation of NF-κB-p65 into the nucleus thus reducing NF-κB transcriptional activity (71). TGR5 is almost ubiquitiously expressed; however, the function of the receptor in various organs is just being elucidated.

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tion improved lipid metabolism, inflammation, fibrosis, and proteinuria in this animal model of nephropathy (86,87). TGR5 mRNA has also been detected in the kidney; however, it is unclear which renal cells express the receptor (12,27,28).

7.7 Bile acid receptors in the central and peripheral nervous system To date a role of FXR in nervous tissue has not been postulated. In contrast, TGR5 is expressed in neurons and astrocytes in the enteric nervous system as well as in the central nervous system (26,34). TGR5 was detected in neurons and astrocytes in the submucosal and myenteric plexus of the small and large intestine (34). Activation of TGR5 by bile acids delayed gastric emptying and transit time through the small intestine (34). This is line with recent data from a genetic analysis that showed an association of the C-allel of the common TGR5 single nucleotide polymorphism in exon 1 rs11554825 and faster small bowel transit time (88). This exon 1 SNP is located outside the coding sequence of TGR5; however, it has been associated with decreased TGR5 expression levels (89). In the central nervous system TGR5 is located in the plasma membrane of astrocytes and neurons. Besides bile acids, neuroactive steroids were identified as potent ligands for TGR5. Even nanomolar concentrations of 5ß-pregnan-3α-ol-20-one and micromolar concentrations of 5ß-pregnan-3α-17α-21-triol-20-one and 5α-pregnan-3α-ol-20-one (allopregnanolone) have led to a significant increase in intracellular cyclic AMP (cAMP) as measured by luciferase activity in TGR5-transfected cells. TGR5 stimulation in astrocytes and neurons promoted activation of adenylate cyclase, elevation of intracellular calcium levels, and the generation of reactive oxygen species (ROS) (26). Expression of TGR5 mRNA in isolated rat astrocytes was suppressed in the presence of neurosteroids or ammonia. Decreased TGR5 mRNA levels were measured in cerebral cortex from cirrhotic patients dying of hepatic encephalopathy (HE) when compared to brains from noncirrhotic patients, suggesting a role for TGR5 in the pathogenesis of HE (26). Treatment of astrocytes with ammonia for 72 hours resulted in a significant decrease in TGR5 mRNA and protein levels and impaired neurosteroid-induced calcium elevation (26). Whether TGR5 in the brain is activated by bile acids under physiological conditions is unclear.

7.8 Summary and future perspectives Bile acids are signaling molecules with hormone-like functions. Multiple bile acid effects are mediated through the nuclear receptor FXR (fFig. 7.1) and the G protein– coupled receptor TGR5 (fFigs. 7.2 and 7.3). Whereas FXR is highly expressed in tissues participating in the enterohepatic circulation of bile acids (e.g. liver, biliary tree, intestine), TGR5 is ubiquitously expressed. FXR has been identified as an important regulator of bile acid, triglyceride, and glucose metabolism and also exerts anti-inflammatory effects. In the liver, TGR5 has anti-inflammatory, antiapoptotic, and choleretic functions. TGR5-mediated extrahepatic bile acid effects comprise intestinal GLP-1 release, thereby modulating serum insulin and glucose levels as well as increased energy expenditure in brown adipose tissue and skeletal muscle. Additionally, TGR5 exerts anti-inflammatory actions.

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Fig. 7.3: Activation of TGR5 in the intestine stimulates glucagon-like peptide-1 (GLP-1) release. Increased GLP-1 secretion elevates insulin levels and protects pancreatic β cells from apoptosis. Furthermore, it can slow intestinal transit time and induce satiety. These effects together with the anti-inflammatory function of TGR5 can improve glycemic control, liver steatosis, and body weight (34,54–56,71).

The beneficial effects of both bile acid receptors on glucose homeostasis and their anti-inflammatory properties make them attractive therapeutic targets for metabolic diseases such as diabetes, obesity, atherosclerosis, and NASH.

7.9 References 1. Houten SM, Watanabe M, Auwerx J. Endocrine functions of bile acids. EMBO J. 2006;25:1419–25. 2. Keitel V, Kubitz R, Häussinger D. Endocrine and paracrine role of bile acids. World J. Gastroenterol. 2008;14:5620–9. 3. Thomas C, Pellicciari R, Pruzanski M, et al. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 2008;7:678–93. 4. Kim I, Morimura K, Shah Y, et al. Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice. Carcinogenesis 2007;28:940–6. 5. Modica S, Murzilli S, Salvatore L, et al. Nuclear bile acid receptor FXR protects against intestinal tumorigenesis. Cancer Res. 2008;68:9589–94. 6. Yang F, Huang X, Yi T, et al. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Cancer Res. 2007;67:863–7. 7. Kalaany NY, Mangelsdorf DJ. LXRS and FXR: the yin and yang of cholesterol and fat metabolism. Annu. Rev. Physiol. 2006;68:159–91. 8. Lefebvre P, Cariou B, Lien F, et al. Role of bile acids and bile acid receptors in metabolic regulation. Physiol. Rev. 2009;89:147–91. 9. Makishima M, Okamoto AY, Repa JJ, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362–5. 10. Parks DJ, Blanchard SG, Bledsoe RK, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365–8.

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11. Kawamata Y, Fujii R, Hosoya M, et al. A G protein-coupled receptor responsive to bile acids. J. Biol. Chem. 2003;278:9435–40. 12. Maruyama T, Miyamoto Y, Nakamura T, et al. Identification of membrane-type receptor for bile acids (M-BAR). Biochem. Biophys. Res. Commun. 2002;298:714–19. 13. Studer E, Zhou X, Zhao R, et al. Conjugated bile acids activate the sphingosine1-phosphate receptor 2 in primary rodent hepatocytes. Hepatology 2011;doi: 10.1002/ hep.24681. 14. Häussinger D, Kurz AK, Wettstein M, et al. Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis. Gastroenterology 2003;124:1476–87. 15. Sheikh Abdul Kadir SH, Miragoli M, bu-Hayyeh S, et al. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes. PLoSOne 2010;5:e9689. 16. Becker S, Reinehr R, Grether-Beck S, et al. Hydrophobic bile salts trigger ceramide formation through endosomal acidification. Biol. Chem. 2007;388:185–96. 17. Häussinger D, Kubitz R, Reinehr R, et al. Molecular aspects of medicine: from experimental to clinical hepatology. Mol. Aspects Med. 2004;25:221–360. 18. Kubitz R, Häussinger D. Osmoregulation of bile formation. Methods Enzymol. 2007;428:313–24. 19. Forman BM, Goode E, Chen J, et al. Identification of a nuclear receptor that is activated by farnesol metabolites. Cell 1995;81:687–93. 20. Suh JM, Yu CT, Tang K, et al. The expression profiles of nuclear receptors in the developing and adult kidney. Mol. Endocrinol. 2006;20:3412–20. 21. Schote AB, Turner JD, Schiltz J, et al. Nuclear receptors in human immune cells: expression and correlations. Mol. Immunol. 2007;44:1436–45. 22. He F, Li J, Mu Y, et al. Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells. Circ. Res. 2006;98:192–9. 23. Bishop-Bailey D, Walsh DT, Warner TD. Expression and activation of the farnesoid X receptor in the vasculature. Proc. Natl. Acad. Sci. USA 2004;101:3668–73. 24. Rizzo G, Disante M, Mencarelli A, et al. The farnesoid X receptor promotes adipocyte differentiation and regulates adipose cell function in vivo. Mol. Pharmacol. 2006;70:1164–73. 25. Wang H, Chen J, Hollister K, et al. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol. Cell. 1999;3:543–53. 26. Keitel V, Gorg B, Bidmon HJ, et al. The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain. Glia 2010. 27. Maruyama T, Tanaka K, Suzuki J, et al. Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice. J. Endocrinol. 2006;191:197–205. 28. Vassileva G, Golovko A, Markowitz L, et al. Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation. Biochem. J. 2006;398:423–30. 29. Keitel V, Donner M, Winandy S, et al. Expression and function of the bile acid receptor TGR5 in Kupffer cells. Biochem. Biophys. Res. Commun. 2008;372:78–84. 30. Keitel V, Reinehr R, Gatsios P, et al. The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology 2007;45:695–704. 31. Keitel V, Cupisti K, Ullmer C, et al. The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders. Hepatology 2009;50:861–70. 32. Keitel V, Ullmer C, Häussinger D. The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes. Biol. Chem. 2010;391:785–9. 33. Reimann F, Habib AM, Tolhurst G, et al. Glucose sensing in L cells: a primary cell study. Cell Metab. 2008;8:532–9.

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8 Disorders of bile duct development Aileen Raizner, Luca Fabris, and Mario Strazzabosco

8.1 Introduction The morphogenesis of the biliary tree is a complex process orchestrated by a series of transcription factors, growth factors, and cytokines variably interplayed through the different embryological steps. This process is under the control of specific genes whose defect results in a perturbed development leading to a spectrum of congenital liver disorders primitively targeting the biliary epithelium (congenital cholangiopathies). Secretory functions of the biliary epithelium can be variably altered and may contribute to the aberrant developmental processes. Congenital cholangiopathies account for a large proportion of liver transplants in the pediatric and young adult populations. Among them, polycystic/fibropolycystic liver disease and Alagille syndrome are the most clinically relevant because they can affect both pediatric and adult patients. Several genes regulating development are also involved in the regulation of biliary repair mechanisms induced by liver damage in adult life. Therefore unraveling the mechanisms of biliary development and the related molecular factors hindering this process ultimately paves the way toward the better care of patients with congenital and acquired cholangiopathies.

8.2 Morphogenesis of the intrahepatic bile duct epithelium: molecular players involved and their relationship with arterial morphogenesis Biliary ductal cells start to develop from the hepatocytes abutting the portal mesenchyme (1). Periportal hepatoblasts differentiate into biliary precursor cells, which essentially form a single-layered sheath of small, flat epithelial cells encircling the portal vein branches, called the ductal plate (“ductal plate stage”) (fFig. 8.1A). In the following weeks, discrete portions of the ductal plates are duplicated by a second layer of cells over variably long segments of their perimeter (double-layered ductal plate) (fFig. 8.1B), which then dilate to form tubular structures in the process of being incorporated into the mesenchyme of the nascent portal space (incorporating bile duct, “migratory stage”) (fFig. 8.1C). Once incorporated into the portal space, the immature tubules are remodeled into individualized bile ducts (incorporated bile duct, “bile duct stage”) (2) (fFig. 8.1D). During the development of the double-layered ductal plate, abnormal remodeling of the primitive ductal plate may occur. The most common type of this ductal plate malformation (DPM) is the von Meyenburg complex (VMC), or bile duct microhamartoma, an abnormally dilated biliary structure localized to the distal portions of the intrahepatic biliary tree (fFig. 8.1E). The progression of biliary tree morphogenesis and the factors involved in its various stages are depicted in fFig. 8.2. This process, entailing the progressive ramification of branching structures, is collectively called tubulogenesis. The tubulogenesis of intrahepatic bile ducts depends on a process of transient asymmetry. Developing bile ducts are, in fact, heterogeneous structures, since they are formed by ductal plate cells positive for

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B: Double layer ductal plate

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D: Incorporated bile duct

PV

PV

PV

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E: Biliary microhamartoma (von Meyenburg complex)

Fig. 8.1: The progressive stages of intrahepatic bile duct development. A. The development of the bile duct begins with the “ductal plate stage.” The periportal hepatoblasts organize into a single layer of flat epithelial cells of the ductal plate around the portal mesenchyme surrounding the portal vein (PV). B. Certain portions of the ductal plate then form a doublelayer ductal plate. C. The double layer ductal plate then dilates to form tubular structures while migrating directly into the portal mesenchyme in the “migratory stage.” D. Once incorporated into the portal space, the immature tubules are remodeled into symmetrical bile ducts. This is known as the “bile duct stage.” E. Malformations can occur during development of the double ductal plate, resulting in a von Meyenburg complex. A tissue section from a liver biopsy performed for diagnostic purposes in a patient with CHF stained by immunohistochemistry for cytokeratin 19 reveals irregularly shaped biliary structures or von Meyenburg complexes (microhamartomas) embedded in a fibrous stroma in the portal tracts. Ductal plate remnants are seen at the margins of the portal tract (⫻200).

the SRY-related HMG box transcription factor Sox 9 on the side facing the portal tract (cholangiocyte phenotype) and by ductal plate cells positive for the hepatocyte nuclear factor 4 (HNF-4) and the transforming growth factor receptor type II (TβRII) on the parenchymal side (hepatoblast phenotype) (3) (fFig. 8.2A). After formation of the lumen, the developing ducts become symmetrical, as cells with the hepatoblast phenotype are progressively replaced by those with the cholangiocyte phenotype (fFig. 8.2B). In the liver, tubulogenesis extends from the hilum to the periphery, ensuring a progressive elongation and ramification of the bile ducts (fFig. 8.2C). This process requires a mechanism able to orient cell mitoses along both a cross-sectional and a craniocaudal axis in a coordinated manner. Through this mechanism, called planar cell polarity (PCP), the epithelial cells orient the axis of cell division in such a way that growth of the epithelial duct is “polarized” within the plane of the cell sheet. By aligning the mitotic spindle along the tubular axis, a daughter cell may be inserted in such a manner as to

8.2 Morphogenesis of the intrahepatic bile duct epithelium Cholangiocyte precursor cells

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Fig. 8.2: (Continued ) The progressive development of tubulogenesis. A. The differentation of hepatoblasts to cholangiocyte precursor cells, which form the ductal plate (DP) surrounding the portal vein (PV) via DP asymmetry, with development along the portal and parenchymal sides. The factors involved in this initial stage of tubulogenesis are shown. B. The progressive development of a bile duct along the DP as it migrates into the portal tract mesenchyme, and the major factors involved in tubulogenesis. C. The stepwise progression of migration and associated arteriogenesis to elongation from the hilum to the periphery and the factors involved therein.

allow tubular elongation rather than an increase in the size of the lumen (4). This is a fundamental mechanism for the maintenance of the tubular architecture. Biliary morphogenesis is orchestrated by various signaling pathways elicited by functional interactions between the epithelial cells and the nonparenchymal cell compartment. A finely tuned and highly regulated process controls the transdifferentiation of liver progenitor cells or hepatoblasts into hepatocytes or cholangiocytes. Transforming growth factor β (TGF-β), particularly TGF-β2 and TGF-β3, has been implicated in playing a pivotal role in this process of differentiation via a gradient-dependent process driven by the portal mesenchyme. The phenotypic switch of hepatoblasts into the biliary lineage is essentially regulated by TGF-β (5). TGF-β signals through the TGF-β-receptor type II (TβRII), which is transiently expressed by the ductal plate, and then is switched off in the mature bile ducts. In addition to TGF-β, other molecular players crucially involved in the emergence of the biliary phenotype are the transcription factors forkhead box (Fox) m1 (6), and Hhex (7) (fFig. 8.2A). Once the fate of the biliary cells has been specified, other factors, particularly transcriptions factors HNF-6 and HNF-1β, which are selectively expressed by cholangiocytes, are called into play to modulate tubulogenesis. In fact, the inactivation of HNF-1β in mouse models leads to defective maturation of the primitive ductal structure (8). Further, the mutation in HNF-1β has been linked to the development of congenital cystic disease in mouse kidney by inhibiting the Pkhd1 gene (9). These transcription factors are both relevant for tubular morphogenesis and each takes on a distinct role in the process. While HNF-6 regulates the differentiation of hepatoblasts into the ductal plates, HNF-1β drives the following maturation of the primitive ductal plate (10). Functional defects in these factors lead to specific phenotypes, which have been considered to reclassify the malformations of the ductal plate (10). For example, a deletion of HNF-1β has been found to be associated with the development of cysts and abnormalities in PCP in fibropolycystic kidney disease (4). Mutation in Cys1, a common target for both HNF-6 and HNF-1β, also resulted in pathological duct expansion and disruption of polarity while maintaining normal differentiation and maturation, a phenotype similarly observed in disease models caused by defect in Pkhd1 (10). A crucial event in bile duct morphogenesis is the termination of tubulogenesis and development. Two transcription factors, forkhead box proteins (Fox) A1 and A2, have been identified as critical components of the machinery governing the termination of branching morphogenesis. They act by inhibiting IL-6, a strong inducer of cholangiocyte proliferation (11). An important regulator of the morphogenesis of intrahepatic biliary epithelium is Notch signaling (12). The Notch genes encode for four transmembrane receptors (Notch 1 to 4), which can interact with a number of ligands (Jagged-1; Jagged-2; and

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Delta-like 1, 3, and 4). Among them, Jagged1 and its receptor Notch2 play a pivotal role in both ductal plate remodelling (13,14) and differentiation of biliary epithelial cells (13). Jagged1 is expressed by mesenchymal cells in the nascent portal tract while Notch2 is expressed by peripheral hepatoblasts adjacent to the portal mesenchyme. To be functionally relevant, this interaction requires close contact of one cell type with the other (15). Following activation of Notch signaling, a number of transcription factors, including HNF-1β, are triggered to induce hepatoblast differentiation into a cholangiocyte (15). During this process, effector genes, including Hairy and Enhancer of Split homologs (Hes1 and Hey1), are induced by Notch via the recombination signal binding protein for immunoglobulin kappa J (RBPJκ). It has been suggested that Notch signaling via Hes1 may affect TGF-β content by establishing a parenchymal gradient along the radial axis (3). An additional effect of Notch signaling is the modulation of tubule formation and its appropriate branching, which also represents an important mechanism in biliary repair. This effect is dose-dependent, since the density of three-dimensional peripheral intrahepatic bile duct architecture depends on Notch gene dosage (16). On the other hand, the Jag1 gene dosage influences the proliferation of the primitive ductal structures (17). Deletion of Jag1 expression in portal mesenchymal cells leads to a maturational arrest of bile duct development at the ductal plate stage (18). The Wingless (Wnt)/β-catenin signaling is an additional signaling pathway critically involved in bile duct morphogenesis. Wnt/β-catenin activation halts the progression of hepatoblast development to hepatocytes and switches their differentiation to a biliary phenotype (19). In addition to interfering with hepatoblast maturation, β-catenin is involved in the following steps of biliary tubulogenesis, where survival, maturation, and expansion of the primitive ductal structures are required (20). Wnt may also act without signaling through β-catenin, a pathway known as noncanonical. Noncanonical Wnt was shown to control planar cell polarity (PCP) (21). Inversin, a gene encoding for a cilium-associated protein regulating the left-right symmetry of organs, has been identified as an important modulator of noncanonical Wnt signaling. Defective inversin function may cause cyst formation in both renal and bile duct epithelia (22) and is associated with a form of biliary atresia in rodents (23). The biliary tree develops in strict anatomical and functional relation with hepatic arteriogenesis (fFig. 8.2C). The developing intrahepatic bile ducts require a blood supply, which is provided by the peribiliary plexus (PBP), a network of capillaries that arises from the peripheral branches of the hepatic artery. PBP is crucial for maintaining the integrity and function of the biliary epithelium. Inactivation in mouse of Hnf-6 or Hnf-1ß resulted in anomalies of the hepatic arterial branches in addition to the expected bile duct abnormalities (24). A similar pattern has been observed in human liver diseases related to DPM (25). In these congenital cholangiopathies, the dysmorphic bile ducts are surrounded by an increased number of vascular structures with a pattern resembling a “pollard willow” (25). One of the signals linking ductal and arterial development in the liver is vascular endothelial growth factor (VEGF), which cooperates with Angiopoietin-1 (Ang-1). During their migration into the portal mesenchyme, developing ductal plates generate a VEGF gradient that signals endothelial cells to enable their recruitment. Ang-1 signaling from hepatoblasts acts in concert with VEGF from ductal plates to modulate the maturation of the hepatic artery terminations by recruiting mural pericytes to the nascent endothelial layer (26).

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8.3 Ductal plate malformation (DPM): definition, clinical heterogeneity, and classification based on animal models DPM is a pathogenetic mechanism characterized by a failure of ductal plate remodeling, which can occur at different time points of embryogenesis and results in a vast spectrum of congenital disorders. Phenotypic expression of these diseases varies greatly depending on the extent and degree of bile duct abnormalities. DPM is frequently associated with various syndromes, the majority of which are lethal at the embryonic stage. Those with severe DPM who survive beyond birth, die as neonates or in early childhood. On the other side of the clinical spectrum, there are patients who remain entirely asymptomatic throughout life, with abnormal findings becoming evident only upon liver biopsy. DPM has been implicated in fibrocystic liver diseases affecting different segments of the biliary tree in a time-dependent fashion. Mouse models have been utilized to study specific defects in biliary morphogenesis. Based on the phenotype of these models, a clearer understanding of DPM has emerged. DPMs have therefore been categorized into three main mechanisms (10,27). When HNF-6 is defective, the DPM develops at an early phase, deriving from altered differentiation of the biliary precursor cells. A deficiency of HNF-1β results in a later defect, induced by a defective maturation of the primitive ductal structures. Finally, if cystin-1 is defective, biliary differentiation proceeds properly but ductal expansion is compromised. From this point of view, cholangiopathies related to DPM can serve as disease models. Thus the pathogenetic mechanisms underlying DPM may shed light on the involvement of specific genes in the process of biliary repair. This would help to clarify the way in which genetic components may be involved in the reaction to damage occurring in adult life. Because many of the proteins encoded by genes defective in DPM are localized to the cilium, we will first briefly outline the pathophysiological role of this organelle, with particular attention to its involvement in fluid secretion. We will then illustrate the different conditions related to DPM.

8.4 Cilia in cholangiocytes: a multifunctional transducing system Nonmotile cilia are microscopic organelles found on epithelial cells, including those lining the renal tubules and bile ducts. Unlike hepatocyes, cholangiocytes possess a primary cilium consisting of a microtubular axoneme and a basal body microtubuleorganizing center from which the axoneme arises and extends from the apical plasma membrane into the lumen of the bile duct (28). The function of the primary cilia on cholangiocytes has gained increasing attention in the last few years. They possess pleiotropic biological functions, acting as mechano-, osmo-, and chemosensory organelles (29). In response to alterations in luminal flow, they have the ability to bend and induce an intracellular calcium ([Ca²+]i) influx (30). These sensory organelles may also detect changes in bile composition and osmolarity and activate signaling pathways (28). A new pathophysiological concept is that the primary cilia are intricately involved in the regulation of epithelial cell proliferation (31) (fFig. 8.3) and secretion (32). In addition to behaving as polyfunctional receptors, cilia are intimately involved in the morphogenesis of the biliary tree. Cilia contain several important proteins, which are variably involved in the regulation of critical cellular processes. These include cell proliferation, cytoskeletal arrangement, intraflagellar transport, and developmental signaling

8.4 Cilia in cholangiocytes: a multifunctional transducing system

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Cyclins

HIF1a VEGF

Fig. 8.3: The roles of PC-1 and PC-2 located on the primary cilium in the intracellular signaling pathway, resulting in proliferation in a cholangiocyte. With the inhibition of PC-2, there is a responsive downregulation of Ca²+ which subsequently upregulates AC6 and cAMP and the MEK/ERK1/2 pathway. The illustration also shows the regulatory pathway, via upregulation of pmTOR, and its stimulatory effect on HIF1α-dependent VEGF secretion to promote cellular proliferation.

pathways, particularly the canonical and noncanonical Wnt pathways. Among them, the best characterized are polycystin-1 (PC-1), polycystin-2 (PC-2), and fibrocystin/polyductin (FPC), whose genetic defect is associated with cystic diseases in different organs, including kidney, liver, and pancreas. Other cilia-related proteins whose defect results in cyst formation include cystin (responsible for kidney cysts and characterized in the cpk mouse model of PKD), nephrocystin (kidney cysts, liver fibrosis, and retinal dysplasia), inversin (kidney cysts, situs inversus, and biliary atresia–like liver lesions), and nephroretinin (kidney cysts and retinitis pigmentosa) (33). Other cilia-related proteins include those that are associated with Bardet-Biedl syndrome (characterized by kidney cysts, obesity, anosmia, retinal dystrophy, male infertility, situs inversus, and diabetes), oral-facial-digital syndrome I (characterized by kidney cysts and malformations of the oral cavity, face, and digits), and Meckel-Gruber syndrome (characterized by kidney and liver cysts, brain malformations, hydrocephalus, and polydactily) (33). Whether ciliary defects may actually be the underlying cause of abnormal differentiation of bile ducts is a debated issue. In fact, recent evidence indicates that ductal plate differentiation

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occurs at a point prior to ciliary assembly on biliary cells (34). It is noteworthy that the primary cilia on cholangiocytes are nonmotile, in contrast to those present on renal tubular and pulmonary epithelial cells. In addition to interfering with developmental processes, alterations of ciliary functions may affect a series of transport activities depending on the activation of regulatory pathways that converge on the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. Using microperfused rat intrahepatic bile duct units (IBDUs), Masyuk et al. (32) showed that alterations in luminal flow increased [Ca²+]i and suppressed forskolin-induced cAMP production. These changes were essentially abrogated by removing the cilia via chloral hydrate or downregulating PC-1, PC-2, and the adenylyl cyclase isoform 6 (AC6) by siRNAs (32). PC-2 is believed to be activated by PC-1 via its C-terminus and to function as a non-selective Ca²+ channel. AC6 is a Ca²+ inhibitable AC that interacts with PC-2. In mouse models of autosomal dominant polycystic kidney disease (ADPKD) with defective PC-2 function, ciliary dysfunction causes a decrease in the [Ca²+]i levels, which subsequently leads to an increase in AC6 activity and cAMP levels. In turn, cAMP stimulates cholangiocyte secretion via the Src/Ras/MEK/ERK1/2 pathway, a mechanism involved in the formation of liver cysts (35). On the other hand, PC-1 may also exert a direct transcriptional effect following the proteolytic cleavage and nuclear translocation of its carboxy-terminal tail to the nucleus (36). In addition, PC-1 has recently been characterized as a regulator of the signal transducer mTOR in renal tubular epithelial cells (37,38). As shown by Spirli et al. (39), mTOR in PC-2defective cholangiocytes can also be activated via Raf/ERK1/2 and in turn stimulate HIF-1α-dependent VEGF secretion. The constitutive activation of these pathways is crucial in promoting the progressive growth of renal cysts (37,39,40). Ciliary proteins are not strictly localized to the cilia. They are also expressed in the endoplasmic reticulum (ER), as seen with PC-2. The functional role of PC-2 in the regulation of the [Ca²+]i levels is also maintained at this level, where it interacts with ryanodine or InsP3 receptors. Defects in PC-2, via altered calcium homeostasis, lead to cyst development. In effect, defective PC-2 results in a decrease in both cytoplasmic and endoplasmic reticular Ca²+ levels as well as in an inhibition of store-operated calcium entry (SOCE), and a change in Ca²+ level triggers an AC6-dependent cAMP and PKA-dependent ERK1/2 phosphorylation upregulation, leading to the development of cysts (39). Notably, other proteins whose mutations are associated with polycystic liver diseases, including those encoded by PRKCSH and SEC63, are also expressed by the ER. Further, the inactivation of xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis associated with the ER, has also been associated with polycystic kidney and liver diseases (41). It is worth highlighting that ciliary proteins may interact with ER proteins to regulate certain mechanisms such as trafficking and quality control processes. In fact, PC-1 links PRKCSH and SEC63 functions, a mechanism behaving as a rate limiter in cyst formation (42).

8.5 DPM-related cholangiopathies As discussed in section 8.3, DPM is a fundamental pathophysiological mechanism derived from abnormal ductal plate remodeling, ultimately leading to aberrant intrahepatic biliary tree development. An array of congenital disorders are related to

Polycystin 1

Epithelial cell morphogenesis, differentiation, proliferation

Hepatic, pancreatic, arachnoid cysts, aortic aneurysms, cardiac valve defects , intracranial aneurysms

Primary cilium, plasma membrane

Renal cysts: Fluid-filled in all nephron segments, Poorly differentiated proliferative cells Hepatic cysts: Intrahepatic bile ducts throughout hepatic parenchyma Fluid-filled cysts with shortened, few, or absent cilia

Cell proliferation

Minimal

Rare

Preserved

Rare

Protein products

Protein function

Systemic manifestations

Subcellular localization

Morphology

Cystogenesis

Hepatic fibrosis

Cirrhosis

Hepatocellular function

Malignant progression (cholangiocarcinoma)

Increased

Preserved

Rare

Fibrosis

Rare

Preserved

Rare

Minimal

Disruption of cell adhesion

Multiple cysts throughout parenchyma, biliary cysts

Renal cysts: In collecting ducts, fluid-filled Liver: Bile duct microhamartomas, bile duct segmental dilations Cell proliferation

ER

Intracranial aneurysms

Translocation, folding membrane glycoproteins

Glucosidase IIβ subunit,* hepatocystin, SEC63•

N/A

PRKCSH, SEC63

PLD

Primary cilium, centrioles

Biliary dysgenesis, CHF, CD, portal hypertension

Proliferation, secretion, differentiation, tubulogenesis

Fibrocystin/Polyductin

Approximately 30% of children develop severe renal failure

PKHD1

ARPKD

冷

ADPKD, autosomal dominant polycytic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; CD, Caroli’s disease; CHF, congenital hepatic fibrosis; DPM, ductal plate malformation; ER, endoplasmic reticulum; PLD, polyccystic liver disease; *, protein encoded by PRKCSH; •, protein encoded by SEC63.

Primary cilium, ER

Polycystin 2

50% adequate function by 70 years

Failure by 70 years

Renal function

PKD2

PKD1

Gene mutations

ADPKD

Tab. 8.1: Comparison of ADPKD, ARPKD, and PLD

8.5 DPM-related cholangiopathies 139

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Tab. 8.2: The rare DPM-associated congenital disorders, their underlying genetic defects, and distinct hepatic and systemic manifestations Meckel’s syndrome

Jeune’s syndrome (asphyxiating thoracic dystrophy)

Joubert’s syndrome

Genetic defect

B9D1 (44) MKS1 (45) TMEM67 (46) CEP290 (47) RPGRIP1L (48) CC2D2A (49) TMEM216 (50)

WDR19 (51)

RPGIP1L (48,52) TMEM216 (50) CEP290 (53)

Pattern of inheritance

Autosomal recessive

Autosomal recessive

Autosomal recessive

Hepatic phenotype

Hepatic fibrosis, hepatic cysts, bile duct cysts

Cysts, bile duct hyperplasia, portal fibrosis, periportal fibrosis, cirrhosis

Biliary fibrosis

Organ involvement

Renal cysts, CNS anomalies (meningoencephalocele), postaxial polydactyly

Skeletal dysplasia (narrow thorax, short limbs), pancreatic cysts, pulmonary hypoplasia, renal cysts, renal insufficiency, retinal degeneration

Apnea, ataxia, CNS anomalies, oculomotor apraxia, microcystic renal disease, retinal defects, polydactyly

Embryonic lethality

Lethal

Lethal

Not lethal

DPM. fTab. 8.1 delineates the gene mutations, biliary morphology, and hepatic and systemic manifestations of the most clinically relevant DPM-related cholangiopathies featuring cyst formation. fTab. 8.2 shows the DPM-associated rare congenital disorders that have high pre- or perinatal mortality, namely Meckel’s, Jeune’s, and Joubert’s syndromes. Meckel syndrome is an autosomal recessive disorder characterized by the association of DPM with renal cysts, anomalies of the central nervous system, and postaxial polydactyly. Renal cystic dysplasia and hepatic fibrosis are generally associated in Meckel’s syndrome (43). Jeune’s syndrome, also known as asphyxiating thoracic dystrophy (ATD), is an autosomal recessive multisystemic disorder characterized by skeletal dysplasia, pulmonary hypoplasia, and renal, pancreatic, and hepatic disease. Although clinical manifestations of hepatic disease are rare, liver abnormalities associated with DPM are frequently observed at autopsy. In contrast with Meckel’s and Jeune’s syndromes, Joubert’s syndrome is not embryonically lethal. It is a multisystemic disorder characterized by abnormal development of the cerebellar vermis and is associated with oculomotor apraxia, polydactyly, renal disease, and biliary fibrosis.

8.5 DPM-related cholangiopathies

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141

8.5.1 Von Meyenburg complexes (VMCs) VMCs, also known as bile duct microhamartomas, are the most common DPMs. They are considered benign lesions and are characterized by dilated biliary structures with an irregular shape embedded in a dense, fibrous stroma. Typically VMCs are situated focally and can occasionally be found as unexpected histological findings at liver biopsies performed for other reasons. However, when present diffusely, they are associated with cystic lesions and are seen in congenital hepatic fibrosis (54). VMCs have also been noted to occur in patients with ADPKD and fibropolycystic liver diseases (55,56). Very rarely, DPMs are associated with cholangiocarcinoma, a liver malignancy arising from the biliary epithelium (57,58). fFig. 8.1E is a micrograph of bile duct hamartomas obtained by immunohistochemistry in the portal tracts in congenital hepatic fibrosis (CHF), with remnants of the DP apparent at the margins of the portal tract.

8.5.2 Autosomal recessive polycystic kidney disease (ARPKD), congenital hepatic fibrosis (CHF), and Caroli’s disease (CD) Three main histological components have been described in association with fibropolycystic diseases: ductal plate remnants, bile duct microhamartomas (VMCs), and biliary cysts (54). Given the close association of these bile duct abnormalities with peribiliary fibrosis, these disorders are known collectively as fibropolycystic liver diseases. Such diseases encompass multiple congenital disorders of the biliary epithelium, including ARPKD, CHF, and CD as well as Meckel’s, Jeune’s, and Joubert’s syndromes, which are rarer conditions. Biliary malformations, particularly in CHF and CD, are associated with the progressive deposition of peribiliary fibrosis, which is clinically responsible for portal hypertension even in the absence of frank evolution to biliary cirrhosis. In the context of CHF or CD, hepatocellular function is typically well preserved. In addition to portal hypertension, patients with CD are clinically characterized by recurrent cholangitis, chronic cholestasis, and intrahepatic cholelithiasis. Those with CD also have an increased risk of developing cholangiocarcinoma; the frequency is not well known, but it can approach approximately 10% (59). Mutations in PKHD1, a gene that encodes for the ciliary protein fibrocystin/polyductin (FPC), are responsible for causing CHF and CD. FPC is a large membrane receptor–like protein expressed by centromeres and the basal body of cilia, a subcellular organelle that originates from the mother centriole in the centrosome and is responsible for assembly of the cilium. Centrioles organize the mitotic spindle and serve as microtubule organizing centers, which is critical for the generation and maintenance of PCP. In fact, an anomalous cell division would result in tubular enlargement rather than tubule elongation. FPC is expressed in ductal epithelia as renal tubular and bile duct epithelial cells (60). Although the exact role of FPC is largely unknown, it is thought to be involved in a variety of functions from interactions with the extracellular matrix to tubulogenesis. Silencing Pkhd1 in cultured mouse renal tubular cells alters cytoskeletal architecture and loosens cell-cell and cell-matrix contacts (61). Recent data suggest that FPC plays a pivotal role in maintaining proper tubular architecture. In the kidney, Pkhd1 expression is directly regulated by Hnf-1β; its inhibition by mutations of HNF-1β generates renal cyst formation in mice (62). In the Pck rat, a model orthologue of ARPKD, FPC deficiency is strongly correlated with the loss of PCP at the renal level (4). However, the mechanistic

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relationships between biliary dysgenesis and portal fibrosis are still uncertain. In the same animal model, cholangiocytes may acquire mesenchyme-like features in response to TGF−β1 and contribute to progressive portal fibrosis by producing extracellular matrix components, including fibronectin and collagen, even in absence of full conversion to an activated fibroblast phenotype (63). Kidney cyst formation, resulting from mutations in Pkhd1, is sensitized by a reduced expression of PC-1, a different ciliary protein defective in ADPKD, indicating the presence of a genetic interaction between ARPKD and ADPKD (42).

8.5.3 Autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) ADPKD and PLD are genetic cholangiopathies inherited as an autosomal dominant trait characterized by the development of multiple cysts throughout the liver parenchyma (64). Both disorders lead to an important increase in liver mass without causing significant hepatic fibrosis, contrary to ARPKD and the related hepatic phenotypes. Despite the fact that there is extensive cyst substitution of liver parenchyma, liver function is generally well preserved and the development of portal hypertension is a rare event. Patients with these disorders are generally asymptomatic. However, complications, even severe, may occasionally occur, including cyst infections, bleeding, or a direct mass effect on the adjacent organs. Two causative genes of ADPKD have been identified, PKD1 and PKD2 (65,66). The PKD1 and PKD2 gene products are PC-1 and PC-2, respectively. PCs are membrane proteins located on the primary cilia of renal tubular and biliary epithelia. The precise function of these proteins has not yet been clearly defined. PC-1 and PC-2 are involved in the regulation of common signaling pathways mediating epithelial cell morphogenesis and proliferation (fFig. 8.3). In mice with a conditional liver-specific defect of PC-1 or PC-2, liver cysts develop progressively even when both proteins are deleted after birth. This suggests that PC-1 and PC-2 still play an important role in adult life (40,67). When PC function is defective, differentiating signals are lacking, thereby favoring the maintenance of an immature and proliferative phenotype by biliary epithelial cells, a functional hallmark of liver cystogenesis. A similar phenotypic and functional profile has been identified between cystic epithelial cells and ductal plate cells (68). This includes the ability to secrete a variety of cytokines and growth factors – such as IL-6 and IL-8 (69), IGF1 (70), VEGF, and Ang-1 (68) – in conjunction with the expression of a rich receptor machinery encompassing estrogen receptors, growth hormone receptors (70), CXCR2 (71), VEGFR-2, and Tie-2 (68). VEGF, in particular, has been implicated in ADPKD as a molecular driver of cystogenesis and cyst expansion through an autocrine effect on cholangiocyte growth as well as a paracrine effect on the pericystic vasculature. The MEK/ERK1/2/mTOR pathway is an important signaling cascade mediating most of these effects. It is overactive in cystic cholangiocytes from Pkd2-defective mice, where it causes an increased expression of hypoxia-inducible factor 1α–dependent VEGF and VEGFR-2 (67). Based on these observations, polycystic liver diseases are considered to be congenital diseases of cholangiocyte signaling (40). This concept has a significant translational meaning in that each pathway may serve as a potential target for therapeutic intervention to halt the progression of cystic disease. Using VEGFR-2 blocking agents or mTOR inhibitors, cystic growth may be inhibited by reducing epithelial proliferation and cyst vascularization in Pkd2KO mice (39,40,67).

8.5 DPM-related cholangiopathies

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143

In PLD, liver cysts are morphologically identical to those developing in ADPKD. Two causative genes have been described in PLD. These include PRKCSH, which encodes protein kinase C substrate heavy chain, also called hepatocystin or glucosidase IIβ (72), and SEC63, which encodes an enzyme involved in regulating the translocation, folding, and quality control of membrane glycoproteins (73). An additional molecular mechanism implicated in the development of renal and liver cysts depends upon a defect in xylosyl transferase 2, an enzyme responsible for initiating heparin sulfate and chondroitin sulfate biosynthesis, although a specific gene defect has not been linked to this mechanism (41). All of these proteins are expressed at the ER level. This supports the current view that in addition to ciliary proteins, ER-associated proteins are also involved in liver cystogenesis (74). However, there is a functional cross-talk between ER proteins and ciliary proteins in promoting the development of polycystic kidney and liver diseases. In mice, the gene products of Prkcsh and Sec63 interact with PC-1 and PC-2, a prerequisite to allow the adequate expression of this functional complex. PC-1 is a crucial player in this interaction, since there is a dose-response relationship between cyst expansion and levels of functional PC-1 following mutations in Prkcsh and Sec63 (42,75). Abnormal secretory functions are putatively involved in liver cystogenesis. fFig. 8.4 depicts the mechanisms and pathways involved in cyst progression. Different studies, starting from the early 1990s, have investigated the aberrant secretory functions of cyst epithelia as potential mechanism underpinning progressive cyst expansion, which is, in fact, the fundamental feature ultimately responsible for progression of the disease. In ADPKD patients, hepatic cysts have an enhanced ability to generate ion secretion under basal conditions and after stimulation with secretin (76). Increased secretion into the closed cyst induces increased intraluminal pressure, which stimulates cell proliferation by stretching the lining epithelial sheet. This mechanism may also contribute to cyst expansion. In cell culture models of epithelial cysts, increasing intraluminal pressure increased the rate of cell proliferation (77,78). In addition, stretch may stimulate apical secretion of purinergic agonists (79), which are critical in the regulation of cholangiocyte secretion and proliferation (80,81). Cultured epithelial cells of kidney cysts from both ARPKD and ADPKD release substantial amounts of ATP (82) and express P2X and P2Y purinergic receptors, along with strong Ca2+-stimulated Cl⫺ channel activation (83). These functions lead to the progressive accumulation of cystic fluid. Whether these findings apply to cholangiocyte biology is still uncertain. In fact, intracellular Ca2+ homeostasis is altered in cystic cholangiocytes, making it difficult to estimate the overall impact of purinergic activation in polycystic liver disease. On the other hand, there is important cross-talk between the cAMP- and the Ca2+-dependent pathways. An increase in cellular cAMP would promote an increase in CFTR-dependent secretion via a decrease in Ca2+-dependent inhibition of AC6. Notably, in two cases where ADPKD coexisted with cystic fibrosis, the prototypic disease with defective CFTR-dependent Cl⫺ secretion, the severity of the cyst phenotype was milder (84). In experimental polycystic kidney disease, small-molecule CFTR inhibitors were noted to be involved in slowing the progression of cyst growth (85,86). In spite of these observations supporting the presence of active fluid secretion in cystic kidney and liver diseases, there is no evidence showing that unregulated fluid secretion is a major mechanism responsible for cyst growth in the liver. Nevertheless, in light of increasing intraluminal pressure, there should be a subtle net difference between absorption and secretion to account for the slow growth of the cysts (87).

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VEGF VEGFR-2 VEGFR-1

IGF1

IL-6

GHR

CXCR2 VEGF IL-8

ATP P2Y2

Ang-1 ER VEGFR-2

Tie2

A

Apical Cl –

ATP

HCO3–

Cl –

cAMP앖 ERK1/2 MEK Ras앖

Ca2쎵앗

ATP

ATP Hѿ

Secretin B

Naѿ

Basolateral

(Continued )

8.6 Alagille’s syndrome (AGS)

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145

Fig. 8.4: (Continued ) Illustration of the various factors, including interleukins, receptors, and specific signaling pathways, involved in cystogenesis in a bile duct and the associated arteriogenesis. A. Illustrates the release of interleukins, IGF1, VEGF, and Ang-1 and the upregulation of multiple receptors (discussed in the text) which all function to promote cyst expansion. In addition, the autocrine effect of VEGF on cholangiocyte growth and its paracrine effect on the pericystic vasculature is depicted. B. A working model depicting the signaling pathways involved in cyst formation. An alteration in calcium homesostasis, particularly, its downregulation, secondary to a defect in PC-2 as described in the text, stimulates an increase in cAMP and subsequent upregulation of the ERK1/2/MEK/Ras pathway and an upregulation of various channels (as shown in the figure) contributing to the development of cyst formation. Intracellular ATP is involved in this signaling pathway as shown.

8.6 Alagille’s syndrome (AGS) Alagille’s syndrome is a complex multisystemic disorder inherited by autosomal dominant transmission. In contrast with fibropolycystic liver diseases, DPM is not the pathogenetic mechanism underlying the liver phenotype in AGS. Rather, liver disease in AGS is characterized by ductopenia. The main histological lesions featuring DPM – such as bile duct microhamartomas, ductal plate remnants, and biliary cysts – are typically absent. Ductopenia is associated with a wide range of extrahepatic manifestations, hence the term syndromic bile duct paucity (88). These manifestations have a major impact on the prognosis of the disease and may include cardiac valve defects (in 96% of patients), butterfly vertebrae (51%), posterior embyotoxon (78%), typical facies (96%), renal disease (40%), and stroke due to the rupture of cerebral aneurysms (14%) (89). The clinical phenotype of hepatic involvement is characterized by ductopenia associated with variable degrees of cholestasis, jaundice, and pruritus, which can be severe and difficult to treat. In spite of severe cholestasis, the development of portal fibrosis is not a prominent feature of AGS and progression to cirrhosis is uncommon. Nonetheless, there are rare cases that require liver transplantation mostly due to pruritus or failure to thrive (89). In 94% of AGS patients, the mutation is in the JAGGED1 gene (90), and less frequently in the NOTCH2 gene (91); however, the mechanism linked to the genetic defect is currently unknown. As previously described, Notch signaling is an evolutionary conserved pathway that is intricately involved in tubular ontogenesis. Notch signaling is necessary at varying stages of tubulogenesis. In zebrafish, Notch and Jagged mutations have been identified as pivotal factors disrupting biliary development (92). Further, using a mouse model of bile duct paucity, it has been shown that loss of Notch function results in defects specifically affecting the differentiation and morphogenesis of bile ducts (12,14,93). More specifically, in a recent comparison of Notch1 and Notch2 mutations in a mouse model, Notch2 specifically leads to defective tubulogenesis and elongation in a dosage-dependent manner (94). Specific genes, such as Fringe, can alter Notch signaling. As shown in Jag1/Fringe heterozygous mice, defects in Fringe genes which encode glycosyltransferase, result in significant bile duct proliferation, an essential step in tubulogenesis (95). In addition to tubulogenesis, Notch signaling is also involved in liver repair. Data from our group have recently addressed a specific role of Notch signaling in regulating the balance among the different cellular elements contributing to the hepatic reparative complex that is activated in response to liver damage. This

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balance is a determinant of the development of portal fibrosis. In fact, AGS is characterized by a marked reduction in the reactive ductal and hepatic progenitor cells. This is in sharp contrast with biliary atresia, a congenital cholangiopathy with similar levels of cholestasis but a much faster evolution to biliary cirrhosis. In biliary atresia, reactive ductal and hepatic progenitor cells are, by contrast, extensively represented (96). The starkly different phenotype of the hepatic reparative complex observed in AGS is likely related to a Notch-dependent block in cell fate determination upstream of HNF-1β. The Notch downstream effector RBP-Jk is central in this mechanism, given that progenitor cell activation and tubule formation are dramatically impaired following treatment with cholestatic agents in mice with a liver-specific defect in RBP-Jk (97). These data confirm that Notch signaling plays an essential role in liver repair by regulating the generation of biliary committed precursors as well as branching tubularization (97).

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37. Weimbs T. Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystic-1. Am. J. Physiol. Renal Physiol. 2007;293:F1423–F32. 38. Shillingford JM, Murcia NS, Larson CH, et al. The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease. PNAS 2006;103(14):5466–71. 39. Spirli C, Locatelli L, Fiorotto R, et al. Altered store operated calcium entry increases cAMP production and ERK1/2 phosphorylation in Polycystin-2 defective cholangiocytes. Hepatology 2011; doi: 10.1002/hep.24723[Epub ahead of print]. 40. Strazzabosco M, Somlo S. Polycystic liver diseases: congenital disorders of cholangiocyte signaling. Gastroenterology 2011;140:1855–9. 41. Condac E, Silasi-Mansat R, Kosanke S, et al. Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis. PNAS 2007;104(22):9416–21. 42. Fedeles SV, Tian X, Gallagher AR, et al. A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation. Nat. Genet. 2011;19;43(7):639–47. 43. Salonen R. The Meckel syndrome: clinicopathological findings in 67 patients. Am. J. Med. Genet. 1984;18(4):671–89. 44. Hopp K, Heyer CM, Hommerding CJ, et al. B9D1 is revealed as a novel Meckel syndrome (MKS) gene by targeted exon-enriched next generation sequencing and deletion analysis. Hum. Mol. Genet. 2011;20(13):2524–34. 45. Kyttälä M, Tallila J, Salonen R, et al. MKS1, encoding a component of the flagellar apparatus basal body proteome, is mutated in Meckel syndrome. Nat. Genet. 2006;38(2):155–7. 46. Smith UM, Consugar M, Tee LJ, et al. The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat. Nat. Genet. 2006;38(2):191–6. 47. Baala L, Audollent S, Martinovic J, et al. Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am. J. Hum. Genet. 2007;81(1):170–9. 48. Delous M, Baala L, Salonen R, et al. The ciliary gene RPGRIP1L is mutated in cerebellooculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. Nat. Genet. 2007;39(7):875–81. 49. Tallila J, Jakkula E, Peltonen L, et al. Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle. Am. J. Hum. Genet. 2008;82(6):1361–7. 50. Valente EM, Logan CV, Mougou-Zerelli S, et al. Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes. Nat. Genet. 2010;42(7):619–25. 51. Bredrup C, Saunier S, Oud MM, et al. Ciliopathies with skeletal anomalies and renal insuffi ciency due to mutations in the IFT-A gene WDR19. Am. J. Hum. Genet. 2011;89(5):634–43. 52. Wolf MT, Saunier S, O’Toole JF, et al. Mutational analysis of the RPGRIP1L gene in patients with Joubert syndrome and nephronopthisis. Kidney Int. 2007;72(12):1520–6. 53. Coppieters F, Lefever S, Leroy BP, et al. CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum. Mutat. 2010;31(10):1097–108. 54. Desmet VJ. Ludwig symposium on biliary disorders—part I: pathogensis of ductal plate abnormalities. Mayo Clinic Proceedings 1998;73(1):80–9. 55. Redston MS, Wanless IR. The hepatic von Meyenburg complex: prevalence and association with hepatic and renal cysts among 2843 autopsies. Mod. Pathol. 1996;9(3):233–7. 56. Tsui WMS. How many types of biliary hamartomas and adenomas are there? Advances in Anatomic Pathology 1998;5(1):16–20. 57. Jain D, Sarode VR, Abdul-Karim FW, et al. Evidence for the neoplastic transformation of Von-Meyenburg complexes. Am. J. Surg. Pathol. 2000;24(8):1131–9. 58. Song JS, Lee YJ, Kim KW, et al. Cholangiocarcinoma arising in von Meyenburg complexes: report of four cases. Pathol. Int. 2008;58:503–12.

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59. Abdalla EK, Forsmark CE, Lauwers GY, et al. Monolobar Caroli’s disease and cholangiocarcinoma. HPB Surgery 1999;11:271–7. 60. Ward CJ, Hogan MC, Rossetti S, et al. The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nat. Genet. 2002;30:259–69. 61. Mai W, Chen D, Ding T, et al. Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells. Mol. Biol. Cell 2005;16:4398–409. 62. Hiesberger T, Bai Y, Shao X, et al. Mutation of hepatocyte nuclear factor-1E inhibits Pkhd1 gene expression and produces renal cysts in mice. J. Clin. Invest. 2004;113(6):814–25. 63. Sato Y, Harada K, Ozaki S, et al. Cholangiocytes with mesenchymal features contribute to progressive hepatic fi brosis of the polycystic kidney rat. The Am. J. Pathol. 2007;171(6):1859–71. 64. Tahvanainen E, Tahvanainen P, Kääriäinen H, et al. Polycystic liver and kidney diseases. Ann. Med. 2005;37(8):546–55. 65. Igarashi P, Somlo S. Genetics and pathogenesis of polycystic kidney disease. J. Am. Soc. Nephrol. 2002;13:2384–98. 66. Wilson PD. Polycystic kidney disease: new understanding in the pathogenesis. IJBCB 2004;36:1868–73. 67. Spirli C, Okolicsanyi S, Fiorotto R, et al. Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cysts growth in polycysin-2 defective mice. Hepatology 2010;51(5):1778–88. 68. Fabris L, Cadamuro M, Fiorotto R, et al. Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases. Hepatology 2006;43(5):1001–12. 69. Nichols MT, Gidey E, Matzakos T, et al. Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid. Hepatology 2004;40(4):836–46. 70. Alvaro D, Onori P, Alpini G, et al. Morphological and functional features of hepatic cyst epithelium in autosomal dominant polycystic kidney disease. Am. J. Path. 2008;172(2):321–32. 71. Amura C, Brodsky K, Gitomer B, et al. CXCR2 agonists in ADPKD liver cyst fluid promote cell proliferation. Am. J. Physiol. Cell Physiol. 2008;294(3):C786–C96. 72. Drenth JPH, te Morsche RHM, Smink R, et al. Germline mutations in PRKCSH associated with autosomal dominant polycystic liver disease. Nat. Gen. 2003;33:345–7. 73. Davila S, Furu L, Gharavi AG, et al. Mutations in SEC63 cause autosomal dominant polycystic liver disease. Nat. Genet. 2004;36(6):575–7. 74. Janssen MJ, Waanders E, Woudenberg J, et al. Congenital disorders of glycosylation in hepatology: the example of polycystic liver disease. J. of Hepatology 2010;52:432–40. 75. Bergmann C, Weiskirchen R. It’s not all in the cilia, but on the road to it: genetic interaction network in polycystic kidney and liver diseases and how trafficking and quality control matter. J. Hepatol. 2011, doi:10.1016/j.jhep.2011.10.014 [Epub ahead of print]. 76. Everson GT, Emmett M, Brown WR, et al. Functional similarities of hepatic cystic and biliary epithelium: studies of fluid constituents and in vivo secretion in response to secretin. Hepatology 1990;11(4):557–65. 77. Vandenburgh HH. Mechanical forces and their second messengers in stimulating cell growth in vitro. Am. J. Physiol. 1992;262(3 Pt2):R350–R5. 78. Tanner GA, McQuillan PF, Maxwell MR, et al. An in vitro test of the cell stretch-proliferation hypothesis of renal cyst enlargement. J. Am. Soc. Nephrol. 1995;6(4):1230–41. 79. Roman RM, Fitz JG. Emerging roles of purinergic signaling in gastroeintestinal epithelial secretion and hepaotbiliary function. Gastroenterology 1999;116:964–79. 80. Zsembery A, Spirli C, Granato A, et al. Purinergic regulation of acid/base transport in human and rat biliary epithelial cell lines. Hepatology 1998;28(4):914–20.

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81. Schwiebert EM, Zsembery A. Extracellular ATP as a signaling molecule for epithelial cells. Biochem. Biophys. Acta 2003;1615(1–2):7–32. 82. Wilson PD, Hovater JS, Casey CC, et al. ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys. J. Am. Soc. Nephrol. 1999;10(2):218–29. 83. Persu A, Devuyst O. Transepithelial chloride secretion and cystogenesis in autosomal dominant polycystic kidney disease. Nephrol. Dial. Transplant. 2000;15:747–50. 84. Xu N, Glockner J, Rossetti S, et al. Autosomal dominant polycystic kidney disease coexisting with cystic fibrosis. J. Nephrol. 2006;19(4):529–34. 85. Yang B, Sonowane ND, Zhao D, et al. Small-molecule CFTR inhibitors slow cyst growth in polycystic kidney disease. J. Am. Soc. Nephrol. 2008;19(7):1300–10. 86. Li H, Findlay IA, Sheppard DN. The relationship between cell proliferation, Cl- secretion, and renal cyst growth: a study using CFTR inhibitors. Kidn. Int. 2004;66:1926–38. 87. Grantham JJ, Chapman AB, Torres VE. Volume progression in autosomal dominant polycystic kidney disease: the major factor determining clinical outcomes. Clin. J. Am. Soc. Nephrol. 2006;1:148–57. 88. Piccoli DA, Spinner NB. Alagille syndrome and the Jagged1 gene. Seminars in Liver Disease 2001;21(4):525–34. 89. Emerick KM, Rand EB, Goldmuntz E, et al. Features of Alagille syndrome in 92 patients: frequency and relation to prognosis. Hepatology 1999;29:822–9. 90. Warthen DM, Moore EC, Kamath BM, et al. Jagged 1 (JAG1) mutations in Alagille syndrome: increasing the mutation detection rate. Human Mutation 2006;27(5):436–43. 91. McDaniell R, Warthen DM, Sanchez-Lara PA, et al. NOTCH2 mutations cause Alagille syndrome, a heterogenous disorder of the Notch signaling pathway. The Am. J. Hum. Genet. 2006;79:169–73. 92. Lorent K, Yeo SY, Oda t, et al. Inhibition of Jagged-mediated Notch signaling disrupts zebrafi sh biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy. Development 2004;131(22):5753–66. 93. Tchorz JS, Kinter J, Müller M, et al. Notch2 signaling promotes biliary epithelail cell fate specification and tubulogenesis during bile duct development in mice. Hepatology 2009;50:871–9. 94. Geisler F, Nagl F, Mazur PK, et al. Liver-specific inactivation of notch2, but not notch1, comprises intrahepatic bile duct development in mice. Hepatology 2008;48(2):607–16. 95. Ryan MJ, Bales C, Nelson A, et al. Bile duct proliferation in Jag1/fringe heterozygous mice identifi es candidate modifi ers of the Alagille syndrome hepatic phenotype. Hepatology 2008;48(6):1989–97. 96. Fabris L, Cadamuro M, Guido M, et al. Analysis of liver repair mechanisms in Alagille syndrome and biliary atresia reveals a role for notch signaling. Am. J. Pathol. 2007;171(2):641–53. 97. Fiorotto R, Spirli C, Fabris L, et al. Defective progenitor cell activation and biliary tubule formation in liver conditional RPB-jk-knowckout mice exposed to cholestastic injuries reveals a key role for notch signaling in liver repair. Hepatology 2010;52:406A.

9 Mutations of the bile salt export pump (BSEP) and multidrug-resistance protein 3 (MDR3) Ralf Kubitz and Dieter Häussinger

9.1 Introduction Bile formation is an osmotic process involving uptake of bile constituents at the sinusoidal membrane, their intracellular biochemical modifications, and secretion into bile at the canalicular membrane. A major function of bile secretion is the removal of cholesterol, which is insoluble in water. Within bile, cholesterol is incorporated into mixed micelles, which contain large amounts of bile acids (BAs) and phospholipids (1). In order to eliminate 500 mg of cholesterol per day, about 12–20 g of BAs must be excreted into bile, necessitating effective BA and phospholipid transport mechanisms. Apart from cholesterol removal, BAs are involved in multiple biological processes such as liver regeneration (2), glucose homeostasis (3), atherosclerosis (4), and carcinogenesis (5). At high concentrations, BAs are toxic owing to their detergent properties (6), and hydrophobic BAs can activate proapoptotic pathways (7) (see also Chapter 5). BA homeostasis is maintained by the action of several transport systems (for details, see Chapter 1). In regard to the vectorial transport of BAs through the hepatocytes, canalicular secretion is regarded as the rate-limiting step (8–10) and involves several energy-dependent transporter proteins, including the bile salt export pump (BSEP/ ABCB11), multidrug-resistance protein 3 (MDR3/ABCB4), and the “familial intrahepatic cholestasis 1” protein (FIC1/ FIC1/ATP8B1). FIC1 belongs to the group of P-type ATPases, a large family of ion pumps of ubiquitous expression (11). Although the exact function of FIC1 is not yet unraveled, it has been suggested that FIC1 acts as a flippase, which transports phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of cell membranes (12). This inward transport of membrane lipids is essential for plasma membrane asymmetry, which may either maintain resistance of the canalicular membrane to the detergent effects of BAs (13) or may be a prerequisite for proper BSEP activity (13,14). This possible interplay between FIC1 and BSEP explains the development of cholestasis with elevated serum bile salt levels in patients with FIC1 mutations, similar to that in patients with BSEP mutations. Several diseases can be related to abnormalities of transporter proteins, which are localized in the canalicular membrane of hepatocytes (fFig. 9.1). Among these, BSEPand MDR3-related diseases have increasingly gained attention and are discussed in this chapter.

9.2 BSEP-related liver diseases 9.2.1 Expression and function of BSEP The bile salt export pump BSEP (gene symbol ABCB11) belongs to the MDR/TAP subfamily of ABC transporters together with P-glycoprotein (P-gp/ or MDR1, gene: ABCB1)

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PFIC-1 BRIC-1

FIC1

PFIC-2 BRIC-2 Dubin-Johnson

MRP2

ATP7B

M. Wilson

BSEP

MDR3

ABCB11

ABCB4

PFIC-3 LPAC ICP

ATP8B1

ABCG5/8 Sitosterolemia

ABCC2

Fig. 9.1: Canalicular transporter proteins associated with diseases. Model of hepatocytes with transporter proteins in the canalicular membrane. Mutations of ATP8B1 (=FIC1), a P-type ATPase, is associated with progressive familial intrahepatic cholestasis type 1 (PFIC-1) and benign recurrent intrahepatic cholestasis type 1 (BRIC-1). Mutations of the bile salt export pump BSEP (ABCB11) are related to PFIC-2 and BRIC-2. The Dubin-Johnson syndrome is caused by mutations of the bilirubin transporter multidrug resistance associated protein 2 (MRP2/ABCC2) and is characterized by conjugated hyperbilirubinemia. Wilson's disease is caused by mutations of the copper transporter ATP7B. The Multidrug resistance protein MDR3 (ABCB4), a floppase for phosphatidylcholine, is related to PFIC-3, the low phospholipid associated cholelithiasis (LPAC) syndrome and to intrahepatic cholestasis of pregnancy (ICP). Finally, mutations of the cholesterol transporter ABCG5/8 lead to sitosterolemia, characterized by increased absorption of plant sterols which leads to premature atherosclerosis.

and MDR3 (ABCB4). In humans, the ABCB11 gene is localized on the short arm of chromosome 2 (2q24) (15). It consists of one exon at the 5’-untranslated end of the mRNA and 27 coding exons, all together encoding a protein of 1.321 amino acids with a molecular mass of approximately 160 kDa. In humans, BSEP is the major canalicular bile salt transporter and therefore is responsible for the bile salt–dependent bile flow. It mainly transports monovalent BAs including taurine and glycine conjugates of primary BAs (cholic acid and chenodeoxycholic acid) as well as secondary BAs (deoxycholic acid) and ursodeoxycholic acid (16). Km-values of human BSEP, as determined in vesicles from transfected HEK293 cells, were 6.2 μM for TC, 6.6 μM for TCDC, 7.5 μM for GCDC, and 21.7 μM for GC. Calculated intrinsic clearance values (Vmax/Km) resulted in a rank order of TCDC > GCDC > TC > GC (17), suggesting a “better” elimination of chenodeoxycholate as compared with cholate derivatives. This seems reasonable because CDCA and its conjugates are potentially more toxic for hepatocytes in vivo (18) and in vitro (7,18) than other bile acids. Adequate regulation of transporter activity is crucial for BA levels in different compartments and includes transcriptional as well as posttranscriptional levels (8,10,19). At the level of transcription, BSEP expression is controlled in at least three different ways. BAs increase transcription of BSEP-mRNA via the farnesoid X receptor (FXR), which is the key regulator for BA homeostasis (20). In human hepatocytes, FXR forms heterodimers with RXRα upon BA binding and eventually transactivates the BSEP promoter (21). In line with this, Bsep expression in Fxr knockout mice is reduced

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to 30% under basal conditions and BA-dependent induction of Bsep is completely eliminated (22). The liver receptor homolog 1 (Lrh1) is another regulator of Bsep (23). In hepatocyte-specific Lrh1 knockout mice, reduced Bsep expression was observed, along with reduced cholic acid synthesis, which was not due to changes in Cyp7A1 expression, the key enzyme for bile salt synthesis (24). Furthermore, the nuclear factor erythroid 2–related factor 2 (Nrf2) regulates Bsep through musculoaponeurotic fibrosacroma (Maf) recognition elements (MARE) approximately 200 base pairs proximal to the transcription start site of the ABCB11 gene (25). Nrf2 senses oxidative stress (26,27) and mediates cellular detoxification mechanisms, including the upregulation of transporter proteins (28), which may be relevant for defense against oxidative stress induced by toxic BAs (29). BSEP is exclusively expressed in hepatocytes and is localized mainly at the canalicular membrane. Targeting of BSEP/Bsep to the canalicular membrane is partly stimulated by Erk, p38 MAP kinase, and protein kinase C (30,31) and is influenced by HCLS1associated protein X-1 (Hax1). Depletion of Hax1 or a dominant negative form of its interacting partner cortactin leads to an increase of BSEP at the apical membrane in MDCK cells (32). In line with this, Fyn-dependent phosphorylation of cortactin, which is induced by cell shrinkage, has been suggested to trigger endocytosis of Bsep and Mrp2 (33). Cortactin interacts with dynamin (34), and a tyrosine-based motif at the C-terminus has recently been shown to be essential for the dynamin- and clathrindependent endocytosis of BSEP (35), which may be another link between cortactin and transporter endocytosis.

9.2.2 Progressive familial intrahepatic cholestasis type 2 (PFIC-2) More than 40 years ago, severe cholestasis with low gamma-glutamyltranspeptidase (γ GT ) levels was observed in an Amish family descending from Jacob and Nancy Byler (36) and was termed Byler’s disease. The idiom “progressive familial intrahepatic cholestasis” (PFIC) was introduced (37) to describe cholestatic liver diseases leading to end-stage liver disease in childhood but that did not meet the criteria for other forms of neonatal or pediatric cholestasis such as biliary atresia, Alagille’s syndrome, or Aagenaes syndrome (38). Later, mutations of FIC1 were shown to be responsible for Byler´s disease (39–41), which was then named PFIC-1. It became apparent that several patients with low γ GT-cholestasis in childhood had a condition distinct from Byler's disease (39,42); therefore the term Byler-like syndrome came into use (43). Finally, mutations of ABCB11 (BSEP) were linked to Byler-like syndrome, now termed PFIC-2 (15,39). To date, more than 150 mutations have been associated with PFIC-2. Most PFIC-2 patients present within the first 6 months of life; typical clinical findings include jaundice, itching, and growth failure. PFIC patients have almost normal serum cholesterol levels (44), which are significantly lower than those in other forms of pediatric cholestatic liver disease. Pruritus is usually more severe in PFIC-2 (and PFIC-1) as compared with PFIC-3 (45). Patients with PFIC-2 have normal γ GT levels, attributed to the absence of toxic concentrations of free bile salts in the bile and thus preventing damage to cholangiocytes (46). A common histological feature of PFIC-2 is the presence of giant cell transformation. Furthermore, the development of hepatocellular carcinoma in young PFIC-2 patients has been observed (47,48). Both phenomena point to an interaction between elevated intracellular BA concentrations and cell division. BAs may

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act as carcinogens via the induction of reactive oxygen (49) and nitrogen species and possibly by selecting apoptosis-resistant cells (5). A common feature of PFIC-2 is reduced expression of BSEP at the canalicular membrane, which has been described by many groups (50–53). Significant downregulation of BSEP/Bsep has also been observed in models of cholestasis (54,55), in association with functional impairment of BSEP/Bsep. Whereas reduced protein expression is selfevident in cases of premature stop-codon and frame shift mutations, the reason for reduced or even absent transporter expression in the context of missense mutations may be more complex. Mutations may alter splicing efficiency, as shown for cystic fibrosis–relevant mutations (56). In line with this, it has been demonstrated by a minigene in vitro assay that missense such as c.1445A > G (p.D482G), c.1757C > T (p.T586I), c.3432C > A (p.S1144R), c.3458G > A (p.R1153H), c.3460T > C (p.S1154P), c.3691C > T (p.R1231W) and c.3692G > A (p.R1231Q) as well as synonymous variants of BSEP such as c.3084A > G (p.A1028A) induce differential splicing or exon skipping (57). Effective splicing in mammalian cells involves a complex splicing machinery guided to the splice sites by so-called exonic splicing enhancers (ESE). Nucleotide changes within ESE sequences, which consist of hexanucleotides, can interfere with the proper function of spliceosomes (58). Whether the missense mutations mentioned above and synonymous variants have the same consequences in vivo as shown in vitro awaits verification. Missense mutation may also induce the formation of misfolded proteins and their proteasomal degradation during the process of quality control in the endoplasmic reticulum (ER). It is believed that up to 30% of normal proteins are degraded by the ER-associated degradation (ERAD) pathway (59). In this context it is of interest that the very common polymorphism p.V444A of BSEP, which may be isoallelic with mutations, strongly increases ERAD, as shown in expression studies of cloned BSEP (60). A decrease of half-life may be associated with shortened residency of BSEP at the canalicular membrane. Expression of BSEP with the two common PFIC-2 mutations, p.E297G and p.D482G, in Madin-Darby canine kidney (MDCK) cells and treatment with 4-phenylbutyrate (4-PBA) increases BSEP-expression by prolonging its half-life at the canalicular membrane (61). Degradation of proteins may be initiated by short-chain ubiquitination. In BSEPE297G or BSEPD482G a higher proportion of short-chain ubiquitination was observed as compared with wild-type BSEP, which was suggested to reduce the half-life of mutated proteins (62). Our studies have identified several new mutations in the BSEP gene. These were detected in patients with PFIC-2 or BRIC-2 phenotypes and include p.M217R, p.W330R, p.W342G, p.G374S, p.A382G, p.G648V, p.R698C, p.T919del, p.G1032R, p.A1044P, p.S1120N, p.N1173D, and p.A1283V. These and many other mutations of BSEP have not yet been analyzed on a functional level; their pathophysiological relevance is assumed on the basis of the patients’ clinical phenotypes. It has been suggested that apart from reduced BSEP expression or activity, the selectivity of BSEP may also determine the severity of a mutation (63).

9.2.3 Benign recurrent intrahepatic cholestasis type 2 (BRIC-2) Some BSEP mutations are associated with milder disease progression and have been classified as “benign recurrent intrahepatic cholestasis type 2” (BRIC-2) (64) in order to distinguish them from BRIC-1, which is due to “mild” mutations of FIC1 (40,65).

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Whereas the clinical presentations of BRIC-1 and BRIC-2 are very similar, a higher incidence of gallstones was observed in BRIC-2 patients (64). BSEP mutations detected in BRIC-2 patients were p.E186G, p.E297G, p.R432T, p.A570T, p.T923P, p.A926P, p.G1004D, p.R1050C, and p.R1128H (64,66–68) (fFig. 9.2). BRIC-2 may be observed in patients with compound heterozygous or homozygous mutations, but some BRIC-2 patients have only one (heterozygous) PFIC-2 mutation. There might also be a transition between BRIC-2 and PFIC-2, as shown for a patient with compound heterozygosity for p.I498T and c.2098delA (69). We recently detected another homozygous mutation in siblings who presented with severe pruritus, elevated bile salt levels, and normal γ GT (70). This mutation (p.G374S) apparently resides within the sixth transmembrane domain close to the channel pore and is associated with a decreased transport rate (71). Interestingly, this mutation showed normal or even increased membrane expression when transfected into HepG2 cells (72). Otherwise, an inverse correlation between the plasma membrane expression of BSEP and the severity of phenotypes has been described: the two “BRIC mutations” p.A570T and p.R1050C had lower expression levels than the “ICP mutation” p.N591S but higher expression levels than the common “PFIC-2-mutations” p.D482G and p.E297G (73).

W342 G1004 G374 W330 M217 T923 G374

A926

A382

T919 G1032 K930

L199 R432

L1165 E186

P456 R1128

S1120

I610

A570 A1283 A

B

Fig. 9.2: Model of BSEP showing amino acid residues with known mutations. Model of BSEP with the putative transmembrane segments (α helices are shown in yellow). A. Positions of amino acid, which are mutated in BRIC-2 patients, are shown in red. B. Amino acid residues from new BSEP mutations (own studies) mutated in PFIC-2 and BRIC-2 patients. At red residues missense mutations were found (p.M217R, p.W330R, p.W342R, p.G374S, p.A382G, p.T919del, p.G1032R, p.S1120N, p.L1165del, p.A1283V) whereas at green residues, frame shift mutations were detected (p.L199Ifs14X, p.P456Pfs24X, p.I610Qfs45X, p.K930Efs92X).

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9.2.4 Intrahepatic cholestasis of pregnancy and BSEP Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic liver disease affecting between 0.1% (Canada), and 27.6% (Araucanians in Chile) of pregnancies (74). Pruritus is the leading symptom, whereas jaundice occurs in only about 10% of women with ICP (75). The onset of pruritus most frequently occurs during the third trimester of pregnancy (76). A hallmark of ICP is an increase in serum BA concentrations, and a cutoff of about 40 μmol/L of bile salts has been found to be predictive for ICP-related complications (74). The main risks associated with this condition are preterm birth and asphyxia with meconium staining of the amniotic fluid (74,75,77). Most cases of ICP resolve spontaneously within 2 weeks after delivery; however, some patients have elevated liver function tests independent of ICP; therefore follow up is mandatory until liver enzymes are completely normalized (78). Several “common” BSEP mutations – including p.E297G, p.D482G, and p.N591S – have been linked with ICP. In a large study of about 491 patients with ICP, heterozygosity of these common BSEP mutations was detected in about 1.4% of ICP patients (79). In addition, the C-allele of the very common BSEP polymorphism p.V444A (c.1331T > C, rs2287622, valine to alanine at position 444) was more frequent in patients with than without ICP (79–81). As in the case of ICP, contraceptive-induced cholestasis (CIC) is likely to be triggered by female hormones. In a rat model it was shown that estrogens are glucuronidated to become a substrate of the multidrug resistance–related protein 2 (Mrp2). Glucuronidated estrogens are secreted into bile, where they trans-inhibit BSEP (82). Interestingly, in CIC all patients were homozygous for the p.V444A polymorphism (81), suggesting a cumulative effect of this genetic variant. Which metabolites are responsible for ICP has not yet been fully elucidated. Apart from metabolites of estrogen, sulfated progesterone metabolites are also increased in ICP (83,84) and may add to BSEP inhibition (85).

9.2.5 Anti-BSEP antibodies and cholestasis Children with PFIC-2 often require liver transplantation. Some of these children develop recurrent cholestasis with a clinical appearance resembling PFIC. In such instances it could be demonstrated that the recurrence of cholestasis was due to the appearance of anti-BSEP antibodies, which occurred after liver transplantation (60,86,87). These antibodies bind to an extracellular loop of BSEP and thereby alter BA transport. So far we have demonstrated the development of anti-BSEP antibodies in five children of different ethnic backgrounds from Germany, Saudi Arabia, and Japan. In each case anti-BSEP antibodies reacted with the N-terminal half of BSEP (fFig. 9.3). Most likely the complete absence of BSEP expression in the native liver prevents the development of autotolerance to BSEP. After transplantation, BSEP is recognized as a foreign antigen and antibody development occurs. Affected PFIC-2 children with recurrent cholestasis after liver transplantation may be treated by a change of immune suppressants, plasmapheresis, or immune adsorption. Furthermore, B-cell depletion by rituximab (directed against the CD20 epitope) may also be effective.

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@hu IgG

MRP2

Merge

Fig. 9.3: Model of BSEP with epitopes of anti-BSEP antibodies. Children with PFIC-2 may develop recurrent cholestasis after liver transplantation. Some of these children develop anti-BSEP disease. These antibodies bind to BSEP within the canalicular membrane and can be detected with anti-human IgG antibodies (left panel, red) in co-localisation with canalicular proteins such as MRP2 (green). The epitopes of these antibodies detect BSEP at the first extracellular loop (right panel, epitopes of two patients shown in red and blue).

9.2.6 Other liver diseases related to BSEP Whereas BSEP mutations and genetic variants have been linked to PFIC, BRIC, and ICP, no association with primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) has been observed (88,89). However, the common BSEP polymorphism p.V444A may influence the course of several liver diseases, although it does not induce liver diseases by itself. p.V444A is not only associated with ICP but is also more frequent in patients with drug-induced liver injury (DILI). Allele frequency of c.1331T > C was 76% in DILI patients as compared with 59% in controls, with an odds ratio of 3 (90). The association of p.V444A with ICP and DILI may be explained by the decreased expression of BSEP in the presence of the polymorphism, as shown in a small cohort (57,91). Reduced BSEP function may not only be due to decreased expression but also to pharmacological interference involving drugs. The potency of pharmacological interaction of different drugs with human BSEP correlates with their hepatotoxicity in humans (92,93). p.V444A may aggravate the course of MDR3-dependent ICP (94) as well as LPAC or PFIC-3 (95). Furthermore, it may influence the development of fibrosis in patients with chronic hepatitis C infection and treatment responses to interferon/ribavirin. Patients infected with the hepatitis C virus, who are homozygous for valine of the p.V444A polymorphism, have less severe liver fibrosis (96), and a better treatment response was observed in a single cohort of 352 patients (78). This polymorphism, however, has a

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lower impact as a host factor than do IL-28B SNPs (97–100), and it is not yet clear whether the effect of p.V444A as a relevant host factor in HCV-infected patients is restricted to certain viral genotypes or if it is population-dependent (78,96).

9.3 MDR3-related liver diseases 9.3.1 Expression and function of MDR3 The multidrug-resistance protein 3 (MDR3; gene symbol ABCB4) belongs to the same subfamily of ABC transporters as BSEP (ABCB11). MDR3 is expressed at the canalicular membrane of hepatocytes (101). Whereas mRNA splice variants have been detected in other tissues, protein expression seems to be restricted to the liver (101). Here, three mRNA isoforms of MDR3 have been described (102), with the main isoform A consisting of 1279 amino acids. Isoform B contains 7 more amino acids (1286) located at the proximal end of the 25th coding exon of MDR3. In isoform C, the 22nd coding exon is missing, resulting in a truncated RNA with an open reading frame for 1222 amino acids. Isoform C can be detected in normal human liver; its function is unknown. MDR3 is a lipid translocator that acts as a floppase, transporting phospholipids from the inner to the outer leaflet of the canalicular membrane, where they are extracted from by BAs (103,104). The main substrate of MDR3 is phosphatidylcholine (PC) (103), which is needed for the formation of mixed micelles in bile. Apart from phosphatidylcholine, digoxin, paclitaxel, daunorubicin, vinblastine, and ivermectin have been shown to be transported by MDR3, although with a lower rate as compared with the multidrug-resistance protein MDR1/P-gp (105). Phosphatidylcholine protects bile ducts from the toxic/detergent action of BAs. Therefore, in situations of decreased MDR3 function, the detergent activity of BAs in bile increases, which is thought to be responsible for the observed rise in serum γ GT in the presence of MDR3 mutations (46,106). Hydrophobic BAs, especially, form mixed micelles upon addition of phospholipids (107); their detergent and proapoptotic properties may become relevant when micelle formation is disturbed. Mutations and genetic variations of MDR3 have been associated to a variety of inherited cholestatic liver diseases (46,108), including PFIC-3 with high γ GT levels (106), intrahepatic cholestasis of pregnancy (ICP) (109–113), low phospholipid–associated cholelithiasis (LPAC) syndrome (114–116), adult biliary cirrhosis (117), small-duct ductopenia (118), and drug-induced liver injury (DILI). Interestingly, the same mutation may be linked to liver diseases of varying severity even within the same family. Thus, apart from the individual genetic background, gender or environmental factor may influence the phenotypes of MDR3-related liver diseases.

9.3.2 Progressive familial intrahepatic cholestasis type 3 (PFIC-3) A subtype of PFIC patients have presented with high gamma-glutamyltranspeptidase (γ GT) cholestasis, and it was suspected that they were distinct from "classic" Byler disease patients. Based on similar histology in children with high γ GT cholestasis of a PFIC phenotype (119) and Mdr2 knockout mice (Mdr2 is the murine ortholog to human MDR3 [120]), mutations of MDR3 (ABCB4) were suspected to cause this type of PFIC.

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In the study of Deleuze et al. (119), absent MDR3-mRNA in one patient and decreased biliary phospholipid levels in another were indicative of the role of MDR3 in PFIC-3. The most prominent symptoms of PFIC-3 are jaundice, discolored stools, pruritus, and hepato- or splenomegaly (108). These symptoms may develop later as compared with disease progression in PFIC-1 and PFIC-2 patients. Intestinal bleeding due to portal hypertension and esophageal varices are late characteristics of PFIC-3, when cirrhosis has evolved (121). About 50% of PFIC-3 patients have progression and require liver transplantation, whereas the other half remarkably benefit from UDCA treatment (108), with a significant delay or even avoidance of the need for transplantation. Typically, patients with missense mutations have better responses to UDCA than those with premature stop codons or frame-shift mutations, which is probably explained by a complete loss of function in the latter group (108). In PFIC-3, ductal proliferation is a common feature, and in later stages portal fibrosis and eventually biliary cirrhosis develop. Whereas the absence of BSEP immunoreactivity or its significant reduction is a very common feature in patients with PFIC-2 (50,51,53), absence of MDR3 immunoreactivity is less regularly observed in PFIC-3 patients (46,51,108). Nevertheless, when MDR3 mutations are considered, MDR3 immunofluorescence of liver biopsies should be included in the workup (78). It has been proposed that a retrograde cholangiography is justified in children with suspected PFIC-3. The aim is to exclude sclerosing cholangitis and to collect bile samples for the determination of biliary bile salt and phospholipid concentrations (121). The ratios of BAs to phospholipids as well as cholesterol to phospholipids are increased about fivefold in PFIC-3, whereas they are decreased in PFIC-1 or -2 (121). Mdr2 knockout mice develop severe liver disease, as do PFIC-3 patients. However, Mdr2 knockout mice display changes similar to the histologic features of primary sclerosing cholangitis (PSC), which is not a feature of PFIC-3 (108). These mice frequently develop hepatocellular carcinoma (but not cholangiocarcinoma) (122), which has only rarely been observed in PFIC-3 (121). HCC development may become especially relevant when more specific therapies for PFIC-3 allow for longer transplant-free survival. In human fetal livers, significantly lower expression levels of MDR3 (and NTCP) in relation to BSEP were observed as compared with adult livers. At 16–20 weeks of gestation, MDR3-mRNA expression was only about 6%, whereas BSEP- and FIC1-mRNA expression at that time was already 34% and 37%, respectively, as compared with mRNA expression in adult livers (123). A low expression level at term may explain why the postpartum period is critical in children with MDR3 mutations. Some PFIC-3 patients seem to improve after initial cholestatic presentation and then to deteriorate again (78,108). It may be speculated that maturation of MDR3 expression is the reason for the observation that neonatal cholestasis spontaneously resolves in a high proportion of such patients (124).

9.3.3 Low phospholipid–associated cholelithiasis syndrome (LPAC) Stone formation in the context of MDR3 mutations is referred to as the LPAC syndrome. LPAC is characterized by early development of cholesterol gallstones and related complications, usually leading to cholecystectomy in patients below age 40. Another typical feature is recurrence of intraductal or intrahepatic cholesterol stones in spite of

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cholecystectomy (114–116). LPAC is associated with (mildly) elevated γ GT levels and alkaline phosphatase activity in serum, reflecting a cholestatic situation. If untreated, LPAC may progress to fibrosis and even necessitate liver transplantation (116), especially when biliary cirrhosis occurs due to cholangitis as a consequence of intraductal stone formation. Parents of patients with proven PFIC-3 often have gallstone disease or a history of ICP (108). However, there is only minor overlap between “LPAC mutations” and “PFIC-3 mutations,” and it has been assumed that LPAC patients are not at a particular risk to develop PFIC-3-like cholestasis in later life. Whether there is an increased risk of HCC development in LPAC patients is also not known. Because the identification of MDR3 mutations in patients with the LPAC phenotype has become possible only very recently, sufficient follow-up times are lacking. In Mdr2 knockout mice, not only phospholipids but also biliary cholesterol levels will be significantly reduced unless more hydrophobic BAs such as taurodeoxycholate are present (125). At the same time the cholesterol saturation index is elevated (126), which explains the increased risk of cholesterol gallstone formation in Mdr2 knockout mice and likewise in LPAC patients (116). Treatment of LPAC with ursodeoxycholic acid is effective in the majority of cases and should be initiated as early as possible (116). Typically, symptoms including “biliary pain” decrease within a few weeks, followed by the disappearance of gallstones. It has been suggested that this sequence of events indicates an inflammatory rather than a mechanical cause of biliary pain (121).

9.3.4 Intrahepatic cholestasis of pregnancy and MDR3 A genetic basis for ICP has long been suggested (127), and it has been observed that the mothers of PFIC-3 patients suffered from ICP during pregnancy (106,109). Genetic variants of the hepatic transporter genes can be detected in a minority of ICP cases (88), and it has been estimated that about 15% of ICP cases can be related to MDR3 (121). Some patients with MDR3-associated ICP may develop cholestatic liver disease and finally liver cirrhosis (117,118), which may correlate with the “severity” of the underlying mutation, as for example, described for p.G535D (117). Because several ICP patients have only a single heterozygous MDR3 mutation, ICP represents an autosomal dominant disease with low penetrance (78,113). In line with this, the mutation c.2362C > T (p.R788W) has caused mild liver disease (including ICP) in heterozygotes as well as severe liver disease with cirrhosis in homozygotes of the same family (118). The coincidental presence of the BSEP polymorphism p.V444A has been suggested to aggravate the course of MDR3-related ICP (94). In the latter case, homozygous p.S320F was detected. Apart from this mutation, other PFIC-3 and LPAC-relevant MDR3 mutations have been found in our studies (fFig. 9.4), such as p.L59R, p.S320F, p.E528D, p.R549H, p.P726L, p.A953D, p.A984T, p.S1076N, p.Q1181E, and p.H1238Y). The mutation H1238Y is of interest because it affects the histidine of the H-loop, a highly conserved amino acid responsible for the coordination of ATP binding to the nucleotide binding domain (128). In one third of ICP patients, increased γ GT levels are observed (129), and these patients are more likely to carry a mutation or genetic variant of MDR3 (94,109–113). However, even in severe MDR3-associated ICP, γ GT may be or become normal despite an elevated serum liver test (94,113,130).

9.3 MDR3-related liver diseases

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161

P726

TMDs

S320

A953 A984

L59

NBDs

Q1181

E528 S1076 H1238

R549

Fig. 9.4: Model of MDR3. Amino acid residues, which are mutated in patients with PFIC-3, LPAC or ICP patients and which were identified in own studies (p.L59R, p.S320F, p.E528D, p.R549H, p.P726L, p.A953D, p.A984T, p.S1076N, p.Q1181E and p.H1238Y) are shown in red. The α-helices form the transmembrane domains (TMDs, delineated by blue lines) and the nucleotide binding domains (NBDs, delineated by green lines) are shown.

9.3.5 Drug-induced liver injury and MDR3 In a small study of 36 patients with drug-induced liver injury (DILI), some MDR3 gene polymorphisms have been identified. These polymorphism were p.I764L (druginduced cholestasis) and p.L1082Q (drug-induced hepatocellular injury) (90). The association of MDR3 with DILI may be due to transport of certain drugs by MDR3 or inhibition of MDR3 by drugs (e.g., digoxin, paclitaxel, vinblastine, verapamil, and cyclosporine) (105).

9.3.6 Other liver diseases and MDR3 Patients with unexplained anicteric cholestasis (normal bilirubin but elevated AP or γ GT ⱖ 1.5 ULN) in whom common causes such as PBS, PSC, alcohol intake, nonalcoholic steatohepatitis (NASH), and granulomatous hepatitis have been excluded should be considered for MDR3 mutations. MDR3 mutations were detected in 11 patients (131). Although there was no direct proof of the pathophysiological relevance of these genetic variations, there is increasing evidence that MDR3 mutations are involved in otherwise unexplained cirrhosis. In contrast to unexplained cholestasis, the MDR3 mutations could not be detected at higher frequencies in patients with PBC or PSC (88,89). On the other hand, an association between defined haplo-/diplotypes (constructed from three single-nucleotide polymorphisms within the ABCB4 gene region) and disease progression was reported in a Japanese cohort of 148 PBC patients (132). The latter study did not focus on exonic (protein-modifying) variations, and the mechanistic relation between haplotype and disease progression remains hypothetical.

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9.4 Treatment of BSEP- and MDR3-associated liver diseases Surgical and medical treatment options are supportive, with the intention of preventing growth retardation and the complication of chronic liver diseases (133). This includes supplementation of fat-soluble vitamins (e.g., to prevent hemorrhage and rickets) and medium-chain triglycerides, which are absorbed independent of BAs (133,134). Surgical treatment options include the partial biliary external diversion (PEBD) (135–137), which improves clinical symptoms, growth, histology, and liver functions tests (137). PEBD can be applied in about 40% to 50% of PFIC patients and was reported to be successful in about 75% of patients (136). If successful, serum BA levels normally drop below 10 μmol/L, jaundice disappears, and transaminases normalize (136). PEBD can be performed less invasively as a laparoscopic procedure (138,139). UDCA treatment may be effective in some patients with PFIC-2 (78,140), PFIC-3 (78,109,141), or PFIC-1 (142), but failure of UDCA has also been reported for PFIC-1 (143). The responsiveness to UDCA therapy depends on the underlying genetic variant. For MDR3-related PFIC, the response rate was estimated to be 60% (121). Patients with missense mutations are better responders than those with premature stop codons, which is probably explained by a complete loss of function in the latter group (109). Apart from replacement of more toxic bile salts by UDCA (104), UDCA may induce choleresis by vesicular insertion of Bsep (30). In addition, UDCA is a potent inhibitor of hepatocyte apoptosis, which is induced by toxic BAs (144). Furthermore, Mdr2/MDR3 expression can be increased by UDCA; this has also been observed in response to statins or fibrates in rodent models (145) partly mediated by the nuclear receptor PPARα (146). However, whether PPARα is involved in fibrate-induced expression of human MDR3 is controversial (147,148), MDR3 activity may also be enhanced by posttranscriptional mechanisms involving increased transporter recruitment to the canalicular membrane (149), thereby explaining the beneficial effect of fibrates in cholestatic liver diseases such as PBC (150,151). The use of phenobarbital or cholestyramine seems not to be beneficial in BRIC patients (152), whereas rifampicin has been effective in PFIC patients (153). Treatment with chaperons may be feasible, and it has been shown that 4-phenylbutyrate increases the membrane expression of BSEP (61). Betain, which was shown to be antiapoptotic in BA-induced apoptosis (144), may also have chaperon properties; however, its clinical efficacy has not yet been tested.

9.5 Concluding remarks The bile salt export pump (BSEP/ABCB11) and the multidrug-resistance protein 3 (MDR3/ABCB4) are crucial for the canalicular secretion of BAs and phospholipids, respectively. Mutations of BSEP and MDR3 are associated with moderate to severe forms of cholestasis, including progressive familial intrahepatic cholestasis (PFIC) types 2 and 3, benign recurrent intrahepatic cholestasis (BRIC) type 2, intrahepatic cholestasis of pregnancy (ICP), and low phospholipid–associated cholelithiasis syndrome (LPAC). Detailed analyses of mutations and genetic variants in humans have enhanced our understanding of bile formation and will, in the future, open new therapeutic strategies for (cholestatic) liver diseases.

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63. Kubitz R, Brinkmeyer C, Sagir A, et al. Genetic variants of the bile salt export pump: inducers and modifiers of liver diseases. Dig. Dis. 2011;29:89–92. 64. van Mil SW, Van Der Woerd WL, Van Der BG, et al. Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology 2004;127:379–84. 65. Tygstrup N, Steig BA, Juijn JA, et al. Recurrent familial intrahepatic cholestasis in the Faeroe Islands. Phenotypic heterogeneity but genetic homogeneity. Hepatology 1999;29:506–8. 66. Kubitz R, Keitel V, Scheuring S, et al. Benign recurrent intrahepatic cholestasis associated with mutations of the bile salt export pump. J. Clin. Gastroenterol. 2006;40:171–5. 67. Chen HL, Liu YJ, Su YN, et al. Diagnosis of BSEP/ABCB11 mutations in Asian patients with cholestasis using denaturing high performance liquid chromatography. J. Pediatr. 2008;153:825–32. 68. Noe J, Kullak-Ublick GA, Jochum W, et al. Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis. J. Hepatol. 2005;43:536–43. 69. Lam CW, Cheung KM, Tsui MS, et al. A patient with novel ABCB11 gene mutations with phenotypic transition between BRIC2 and PFIC2. J. Hepatol. 2006;44:240–2. 70. Kubitz and Häussinger, unpublished results. 71. Kubitz, Stindt, Häussinger, unpublished results. 72. Kubitz, Weissenberger, Häussinger, unpublished results. 73. Lam P, Pearson CL, Soroka CJ, et al. Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases. Am. J. Physiol. Cell Physiol. 2007;293:C1709–C16. 74. Glantz A, Marschall HU, Mattsson LA. Intrahepatic cholestasis of pregnancy: relationships between bile acid levels and fetal complication rates. Hepatology 2004;40:467–74. 75. Rioseco AJ, Ivankovic MB, Manzur A, et al. Intrahepatic cholestasis of pregnancy: a retrospective case-control study of perinatal outcome. Am. J. Obstet. Gynecol. 1994;170:890–5. 76. Lammert F, Marschall HU, Glantz A, et al. Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and management. J. Hepatol. 2000;33:1012–21. 77. Bacq Y, Sapey T, Brechot MC, et al. Intrahepatic cholestasis of pregnancy: a French prospective study. Hepatology 1997;26:358–64. 78. Kubitz R, Bode J, Erhardt A, et al. Cholestatic liver diseases from child to adult: the diversity of MDR3 disease. Z. Gastroenterol. 2011;49:728–36. 79. Dixon PH, van MS, Chambers J, et al. Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy. Gut 2009;58:537–44. 80. Pauli-Magnus C, Lang T, Meier Y, et al. Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy. Pharmacogenetics 2004;14:91–102. 81. Meier Y, Zodan T, Lang C, et al. Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T>C polymorphism in the bile salt export pump. World J. Gastroenterol. 2008;14:38–45. 82. Stieger B, Fattinger K, Madon J, et al. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology 2000;118:422–30. 83. Meng LJ, Reyes H, Axelson M, et al. Progesterone metabolites and bile acids in serum of patients with intrahepatic cholestasis of pregnancy: effect of ursodeoxycholic acid therapy. Hepatology 1997;26:1573–9. 84. Meng LJ, Reyes H, Palma J, et al. Profi les of bile acids and progesterone metabolites in the urine and serum of women with intrahepatic cholestasis of pregnancy. J. Hepatol. 1997;27:346–57.

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85. Vallejo M, Briz O, Serrano MA, et al. Potential role of trans-inhibition of the bile salt export pump by progesterone metabolites in the etiopathogenesis of intrahepatic cholestasis of pregnancy. J. Hepatol. 2006;44:1150–7. 86. Jara P, Hierro L, Martinez-Fernandez P, et al. Recurrence of bile salt export pump deficiency after liver transplantation. N. Engl. J. Med. 2009;361:1359–67. 87. Maggiore G, Gonzales E, Sciveres M, et al. Relapsing features of bile salt export pump defi ciency after liver transplantation in two patients with progressive familial intrahepatic cholestasis type 2. J. Hepatol. 2010;53:981–6. 88. Pauli-Magnus C, Kerb R, Fattinger K, et al. BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 2004;39:779–91. 89. Poupon R, Ping C, Chretien Y, et al. Genetic factors of susceptibility and of severity in primary biliary cirrhosis. J. Hepatol. 2008;49:1038–45. 90. Lang C, Meier Y, Stieger B, et al. Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet. Genomics 2007;17:47–60. 91. Meier Y, Pauli-Magnus C, Zanger UM, et al. Interindividual variability of canalicular ATP-binding-cassette (ABC)-transporter expression in human liver. Hepatology 2006;44:62–74. 92. Morgan RE, Trauner M, van Staden CJ, et al. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 2010;118:485–500. 93. Ogimura E, Sekine S, Horie T. Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes. Biochem. Biophys. Res. Commun. 2011;416:313–7. 94. Keitel V, Vogt C, Häussinger D, et al. Combined mutations of canalicular transporter proteins cause severe intrahepatic cholestasis of pregnancy. Gastroenterology 2006;131:624–9. 95. Poupon R, Barbu V, Chamouard P, et al. Combined features of low phospholipidassociated cholelithiasis and progressive familial intrahepatic cholestasis 3. Liver Int. 2010;30:327–31. 96. Iwata R, Stieger B, Mertens JC, et al. The role of bile acid retention and a common polymorphism in the ABCB11 gene as host factors affecting antiviral treatment response in chronic hepatitis C. J. Viral Hepat. 2010;18:768–78. 97. Ge D, Fellay J, Thompson AJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009;461:399–401. 98. Suppiah V, Moldovan M, Ahlenstiel G, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat. Genet. 2009;41:1100–4. 99. Tanaka Y, Nishida N, Sugiyama M, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat. Genet. 2009;41:1105–9. 100. Rauch A, Kutalik Z, Descombes P, et al. Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. Gastroenterology 2010;138:1338–45. 101. Smit JJ, Schinkel AH, Mol CA, et al. Tissue distribution of the human MDR3 P-glycoprotein. Lab. Invest. 1994;71:638–49. 102. Van der Bliek AM, Baas F, Ten Houte de LT, et al. The human mdr3 gene encodes a novel P-glycoprotein homologue and gives rise to alternatively spliced mRNAs in liver. EMBO J. 1987;6:3325–31 138:1338-45.

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103. van Helvoort A, Smith AJ, Sprong H, et al. MDR1 P-glycoprotein is a lipid translocase of broad specifi city, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell 1996;87:507–17. 104. Oude Elferink RP, Paulusma CC. Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein). Pflugers Arch. 2007;453:601–10. 105. Smith AJ, van HA, van MG, et al. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping. J. Biol. Chem. 2000;275:23530–9. 106. De Vree JM, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc. Natl. Acad. Sci. USA 1998;95:282–7. 107. Donovan JM, Jackson AA, Carey MC. Molecular species composition of intermixed micellar/vesicular bile salt concentrations in model bile: dependence upon hydrophilic-hydrophobic balance. J. Lipid Res. 1993;34:1131–40. 108. Jacquemin E, De Vree JM, Cresteil D, et al. The wide spectrum of multidrug resistance 3 defi ciency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001;120:1448–58. 109. Jacquemin E, Cresteil D, Manouvrier S, et al. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999;353:210–11. 110. Dixon PH, Weerasekera N, Linton KJ, et al. Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking. Hum. Mol. Genet. 2000;9:1209–17. 111. Gendrot C, Bacq Y, Brechot MC, et al. A second heterozygous MDR3 nonsense mutation associated with intrahepatic cholestasis of pregnancy. J. Med. Genet. 2003;40:e32. 112. Wasmuth HE, Glantz A, Keppeler H, et al. Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene. Gut 2007;56:265–70. 113. Floreani A, Carderi I, Paternoster D, et al. Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations. Aliment. Pharmacol. Ther. 2006;23:1649–53. 114. Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001;120:1459–67. 115. Rosmorduc O, Hermelin B, Boelle PY, et al. ABCB4 gene mutation-associated cholelithiasis in adults. Gastroenterology 2003;125:452–9. 116. Rosmorduc O, Poupon R. Low phospholipid associated cholelithiasis: association with mutation in the MDR3/ABCB4 gene. Orphanet J. Rare Dis. 2007;2:29. 117. Lucena JF, Herrero JI, Quiroga J, et al. A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis. Gastroenterology 2003;124:1037–42. 118. Gotthardt D, Runz H, Keitel V, et al. A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults. Hepatology 2008;48:1157–66. 119. Deleuze JF, Jacquemin E, Dubuisson C, et al. Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996;23:904–8. 120. Smith AJ, De Vree JM, Ottenhoff R, et al. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (-/-) mice. Hepatology 1998;28:530–6. 121. Davit-Spraul A, Gonzales E, Baussan C, et al. The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects. Semin. Liver Dis. 2010;30:134–46.

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122. Katzenellenbogen M, Mizrahi L, Pappo O, et al. Molecular mechanisms of liver carcinogenesis in the mdr2-knockout mice. Mol. Cancer Res. 2007;5:1159–70. 123. Chen HL, Chen HL, Liu YJ, et al. Developmental expression of canalicular transporter genes in human liver. J. Hepatol. 2005;43:472–7. 124. Jacquemin E, Lykavieris P, Chaoui N, et al. Transient neonatal cholestasis: origin and outcome. J. Pediatr. 1998;133:563–7. 125. Oude Elferink RP, Ottenhoff R, van WM, et al. Uncoupling of biliary phospholipid and cholesterol secretion in mice with reduced expression of mdr2 P-glycoprotein. J. Lipid Res. 1996;37:1065–75. 126. Lammert F, Wang DQ, Hillebrandt S, et al. Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis. Hepatology 2004;39:117–28. 127. Holzbach RT, Sivak DA, Braun WE. Familial recurrent intrahepatic cholestasis of pregnancy: a genetic study providing evidence for transmission of a sex-limited, dominant trait. Gastroenterology 1983;85:175–9. 128. Kubitz, Engelmann, Schmitt, Häussinger, unpublished results. 129. Milkiewicz P, Gallagher R, Chambers J, et al. Obstetric cholestasis with elevated gamma glutamyl transpeptidase: incidence, presentation and treatment. J. Gastroenterol. Hepatol. 2003;18:1283–6. 130. Schneider G, Paus TC, Kullak-Ublick GA, et al. Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy. Hepatology 2007;45:150–8. 131. Ziol M, Barbu V, Rosmorduc O, et al. ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults. Gastroenterology 2008;135:131–41. 132. Ohishi Y, Nakamura M, Iio N, et al. Single-nucleotide polymorphism analysis of the multidrug resistance protein 3 gene for the detection of clinical progression in Japanese patients with primary biliary cirrhosis. Hepatology 2008;48:853–62. 133. De Bruyne R, Van Biervliet S, Vande Velde S, et al. Clinical practice : neonatal cholestasis. Eur. J. Pediatr. 2011;170:279–84. 134. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. J. Pediatr. Gastroenterol. Nutr. 2008;46:241–52. 135. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of intractable pruritus associated with intrahepatic cholestasis. Gastroenterology 1988;95:130–6. 136. Emond JC, Whitington PF. Selective surgical management of progressive familial intrahepatic cholestasis (Byler’s disease). J. Pediatr. Surg. 1995;30:1635–41. 137. Kurbegov AC, Setchell KD, Haas JE, et al. Biliary diversion for progressive familial intrahepatic cholestasis: improved liver morphology and bile acid profile. Gastroenterology 2003;125:1227–34. 138. Metzelder ML, Bottlander M, Melter M, et al. Laparoscopic partial external biliary diversion procedure in progressive familial intrahepatic cholestasis: a new approach. Surg. Endosc. 2005;19:1641–3. 139. Metzelder ML, Petersen C, Melter M, et al. Modified laparoscopic external biliary diversion for benign recurrent intrahepatic cholestasis in obese adolescents. Pediatr. Surg. Int. 2006;22:551–3. 140. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and progression between severe FIC1 and BSEP deficiencies. J. Hepatol. 2010;53:170–8. 141. Jacquemin E, Hermans D, Myara A, et al. Ursodeoxycholic acid therapy in pediatric patients with progressive familial intrahepatic cholestasis. Hepatology 1997;25:519–23. 142. Dinler G, Kocak N, Ozen H, et al. Ursodeoxycholic acid treatment in children with Byler disease. Pediatr. Int. 1999;41:662–5.

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143. Crosignani A, Podda M, Bertolini E, et al. Failure of ursodeoxycholic acid to prevent a cholestatic episode in a patient with benign recurrent intrahepatic cholestasis: a study of bile acid metabolism. Hepatology 1991;13:1076–83. 144. Graf D, Kurz AK, Reinehr R, et al. Prevention of bile acid-induced apoptosis by betaine in rat liver. Hepatology 2002;36:829–39. 145. Gupta S, Todd SR, Pandak WM, et al. Regulation of multidrug resistance 2 P-glycoprotein expression by bile salts in rats and in primary cultures of rat hepatocytes. Hepatology 2000;32:341–7. 146. Kok T, Bloks VW, Wolters H, et al. Peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated regulation of Mdr2 expression and function in mice. Biochem. J. 2003;369:539–47. 147. Roglans N, Vazquez-Carrera M, Alegret M, et al. Fibrates modify the expression of key factors involved in bile-acid synthesis and biliary-lipid secretion in gallstone patients. Eur. J. Clin. Pharmacol. 2004;59:855–61. 148. Shoda J, Okada K, Inada Y, et al. Bezafibrate induces multidrug-resistance P-Glycoprotein 3 expression in cultured human hepatocytes and humanized livers of chimeric mice. Hepatol. Res. 2007;37:548–56. 149. Shoda J, Inada Y, Tsuji A, et al. Bezafibrate stimulates canalicular localization of NBDlabeled PC in HepG2 cells by PPARalpha-mediated redistribution of ABCB4. J. Lipid Res. 2004;45:1813–25. 150. Kurihara T, Niimi A, Maeda A, et al. Bezafibrate in the treatment of primary biliary cirrhosis: comparison with ursodeoxycholic acid. Am. J. Gastroenterol. 2000;95:2990–2. 151. Kanda T, Yokosuka O, Imazeki F, et al. Bezafibrate treatment: a new medical approach for PBC patients? J. Gastroenterol. 2003;38:573–8. 152. Brenard R, Geubel AP, Benhamou JP. Benign recurrent intrahepatic cholestasis. A report of 26 cases. J. Clin. Gastroenterol. 1989;11:546–51. 153. Yerushalmi B, Sokol RJ, Narkewicz MR, et al. Use of rifampin for severe pruritus in children with chronic cholestasis. J. Pediatr. Gastroenterol. Nutr. 1999;29:442–7.

10 MRP2 (ABCC2) and disorders of bilirubin handling in liver Dietrich Keppler

10.1 Introduction Multidrug-resistance protein 2 (MRP2) is a member of the ATP-binding cassette (ABC) transporter family, and this efflux pump was the second member cloned from the MRP subfamily of ABC transporters (symbol ABCC2). Human MRP2 is well established as the ATP-dependent transporter of bilirubin glucuronides (1,2). Absence of a functionally active MRP2 protein in the hepatocyte canalicular membrane results in conjugated hyperbilirubinemia and represents the molecular basis of Dubin-Johnson syndrome (3). Only in recent years have the processes been elucidated that underlie the uptake of bilirubin and its glucuronic acid conjugates from blood into hepatocytes as well as the transport process mediating the efflux of these conjugates from hepatocytes into sinusoidal blood. Conjugated hyperbilirubinemia in cholestatic liver disease originates from the MRP3-mediated transport of bilirubin conjugates from hepatocytes into blood (4). Moreover, a better understanding has been reached of the interaction between canalicular efflux and sinusoidal efflux of bilirubin conjugates. Sinusoidal (basolateral) efflux allows not only for the renal elimination of the conjugates but also for their reuptake by neighboring and more downstream hepatocytes in the sinusoid. This reuptake of bilirubin glucuronides can be mediated with high affinity by the organic anion-transporting proteins OATP1B1 (5) and OATP1B3 (6).

10.2 The conjugate efflux pump MRP2 in the hepatocyte canalicular membrane MRP2 is exclusively localized in apical membrane domains of polarized cells and plays a decisive role in the terminal excretion of phase-2 conjugation products as well as in the transport of a broad spectrum of endogenous and xenobiotic anionic substances (for a recent review see reference 7). Prototypical substrates of recombinant human MRP2 include bisglucuronosyl bilirubin, monoglucuronosyl bilirubin (2), many glutathione conjugates such as leukotriene C4, and glucuronides such as 17ß-glucuronosyl estradiol (8). Synthetic substrates include bromosulfophthalein (9), cholecystokinin peptide (CCK-8) (10), cholyl-L-lysyl-fluorescein (11), fluo-3 (9,12), and methotrexate (13). The substrate specificity of rat Mrp2, which is similar to human MRP2 (7,8), had been indicated long before its cloning by the comparison of the hepatobiliary excretion of organic anions between normal and mutant rats (14–16). These mutants were subsequently shown to lack Mrp2 in their hepatocyte canalicular membrane (17–20). The molecular characterization of MRP2 (ABCC2) was initiated in 1995 by the cloning of a 347 bp cDNA fragment from rat liver using degenerate oligonucleotides complementary to ABCC1 and Leishmania ltpgpA (21). This fragment was distinct from rat and human Abcc1/ABCC1, was detectable only in the liver of normal but not of

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transport-deficient mutant rats (21), and was soon shown to be part of rat Abcc2 (18). The cDNAs encoding full-length rat Mrp2 (17–19) and human MRP2 were subsequently cloned (18,22,23), stably expressed in various cell lines, and functionally characterized (8,24). At present, MRP2 (ABCC2) orthologs have been cloned from more than 20 organisms, ranging from cellular slime molds, plants, bony fishes, and birds to many mammalian species (25). Thus, MRP2 is a phylogenetically very ancient efflux pump involved in detoxification. Genetic variants in mammals leading to an inactive MRP2 transporter are usually well compensated by alternative efflux pathways, including basolateral MRP3 (see section 10.6). Human MRP2 is composed of 1545 amino acids and contains 3 extracellular glycosylation sites (7) (fFig.10.1). The transport-active glycoprotein has been purified to homogeneity (26,27). The membrane topology analysis indicated an extracellular amino terminus and two ATP-binding domains (7,8,18). The total number of transmembrane-spanning helices is predicted to be 15, according to most analysis programs, and thus seems to differ from the 17 transmembrane-spanning helices of MRP1 topology (7). It should be noted in this context that MRP2 and MRP1 share an amino acid identity of only 50% (7). Specific antibodies have served to localize rat Mrp2 (18) and human MRP2 protein in the apical domain of various polarized cell types, including hepatocytes (23,28), kidney proximal tubule cells (29,30), human small intestine (31), colon (32), gallbladder (33), segments of bronchi (32), and placenta (34). MRP2 protein is absent or below

MSD0

MSD1

R100X

MSD2

R768W

1 OUT

I1173F R1150H

G676R

IN

1545 R393W

R1066X W709R R1310X Y1275X Q1382R R1392_M1393del2

Fig. 10.1: Predicted membrane topology of human MRP2 with amino acid changes in Dubin-Johnson syndrome (7). MSD0, MSD1, and MSD2 indicate the three membranespanning domains. Amino acids within nucleotide-binding domains are in black; predicted N-glycosylation sites are shown by tree-like structures. Reproduced with permission from Nies and Keppler 2007 (7).

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current detection limits in several other normal human cell types and tissues, including endothelial cells of the blood-brain barrier (35).

10.3 Formation of unconjugated bilirubin and its uptake into hepatocytes Bilirubin is the product of the catabolism of hemoglobin, other hemoproteins, and free heme (36,37). The major source of unconjugated bilirubin (UCB) are senescent erythrocytes in addition to tissue-derived hemoproteins. UCB is a potent antioxidant (38), but is potentially toxic at high concentrations (37,38). Under normal conditions, UCB is rendered nontoxic by tight binding to albumin in blood plasma and by subsequent uptake into hepatocytes, followed by conjugation with glucuronic acid and excretion into bile (37). Although uptake of UCB into hepatocytes is predominant (39,40), extrahepatic, particularly intestinal, uptake and glucuronidation seems to play important roles in preventing hyperbilirubinemia under some conditions (41). In normal human serum, only about 4% of bilirubin is conjugated (37). The mean concentration of total bilirubin in serum in the adult population is 0.62 mg/dL (10.6 μmol/L); it averages 0.52 mg/dL in women and 0.72 mg/dL in men (42). The mode of uptake of UCB into hepatocytes and other cell types has been unresolved for many years (43,44); suggestions have ranged from passive diffusion (44) to obligatory transporter-mediated uptake across the sinusoidal membrane (6). Four lines of recent evidence indicate that UCB is taken up into hepatocytes by members of the human organic anion-transporting polypeptide (OATP) family: (a) Initially, Cui et al. showed high-affinity uptake of labeled UCB by OATP1B1 into stably transfected HEK293 cells

Hepatocyte UDP GA UDP Bilirubin (B)

BGA

B

OATP1B1

Bile canaliculus

ATP BGA

UGT1A1

MRP2

OATP1B3 ATP MRP3

Blood BGA Reuptake by hepatocytes

Renal excretion

Fig. 10.2: Hepatocellular uptake of unconjugated bilirubin (B) by OATP1B1 and OATP1B3 is followed by glucuronic acid conjugation catalyzed by UGT1A1, yielding bilirubin bisglucuronide and monoglucuronide (BGA). Under normal conditions, bilirubin glucuronides are predominantly transported by MRP2 into the bile canaliculus. However, when MRP2 is deficient or rate-limiting, efflux of the conjugates by MRP3 into sinusoidal blood enables reuptake by more downstream hepatocytes in the sinusoid or by renal excretion.

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in the presence of albumin (6) and Briz et al. subsequently demonstrated uptake of UCB in Xenopus laevis oocytes expressing OATP1B1 or OATP1B3 (45). (b) A genetic polymorphism in the gene encoding human OATP1B1 and leading to impaired transport activity is associated with a significant increase in serum UCB (46). (c) Genomewide scans indicate highly significant associations of the human gene encoding OATP1B3 with unconjugated hyperbilirubinemia (47) and with total serum bilirubin (48). (d) Mice with disrupted genes encoding several hepatocellular Oatp transporters (of the 1a and 1b subfamilies of mouse Oatps) display a 2.5-fold increase in UCB and a more than 40-fold increase in total plasma bilirubin, 95% of which are conjugated (49,50). These different approaches and techniques support and confirm that UCB uptake into human hepatocytes is mediated by members of the OATP family, particularly by human OATP1B1 and OATP1B3 (fFig. 10.2). It is not known at present whether additional uptake transporters, such as human OATP2B1 (51), contribute further to this uptake. Moreover, a significant role for passive diffusion in the uptake of UCB in vivo is unlikely in view of the evidence summarized above in section 10.3, lines of evidence (a) - (d).

10.4 Formation of bilirubin glucuronides and their transport into bile by MRP2 The conjugation of bilirubin with glucuronic acids is catalyzed by the isoform 1A1 of uridine diphosphate glucuronosyltransferase (UGT1A1) in the endoplasmic reticulum and yields monoglucuronosyl and bisglucuronosyl bilirubin (37,52,53). UGT1A1 is abundantly expressed in human hepatocytes but also present in extrahepatic tissues such as the gastrointestinal tract (53). The glucuronidation of bilirubin is essential for its terminal excretion into bile, feces, and urine, which is mediated by efflux pumps of the MRP family (1,2,4). Bisglucuronosyl bilirubin accounts for about 80% of the conjugates eliminated into human bile under normal conditions, but the proportion of the monoglucuronide increases in states of a partial deficiency of hepatic glucuronidation, such as Crigler-Najjar syndrome type 2 and Gilbert syndrome (37). It may be noted that the monoglucuronide of bilirubin is by far the predominant conjugate in mouse bile and urine whereas bisglucuronosyl bilirubin only accounts for 10%–20% of the bilirubin conjugates (49). This indicates significant species differences in the substrate specificity of conjugation and transport. ATP-dependent transport of bilirubin glucuronides by recombinant human MRP2 assayed in inside-out membrane vesicles indicates Km values of 0.7 and 0.9 μM for monoglucuronosyl and bisglucuronosyl bilirubin, respectively (2). The Km values for recombinant rat Mrp2 are in a similar submicromolar concentration range (2). Thus bilirubin glucuronides represent the substrates with the highest affinity for MRP2 identified so far. This high affinity enables an efficient transport of bilirubin conjugates from hepatocytes not only into bile but also into urine and the intestinal lumen, where MRP2 has been localized in the apical membrane of epithelia.

10.5 Uptake of bilirubin glucuronides into hepatocytes The efficient uptake of bilirubin glucuronides from the blood circulation into hepatocytes mediated by OATP1B1 and OATP1B3 is a prerequisite of their subsequent

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elimination into bile via MRP2. In addition, it is important to appreciate this uptake process from (sinusoidal) blood into hepatocytes in view of the basolateral efflux of bilirubin glucuronides into sinusoidal blood followed by potential reuptake into neighboring, more downstream hepatocytes (see section 10.6) and because of the extrahepatic glucuronidation of bilirubin (53). Contrary to the difficult methodology of measurements of uptake transport of UCB, transport measurements of monoglucuronosyl bilirubin and bisglucuronosyl bilirubin are straightforward once the synthesis of the labeled conjugates has been achieved (1,2). Km values of 0.10 and 0.28 μM have been determined for the uptake of monoglucuronosyl bilirubin and bisglucuronosyl bilirubin by recombinant human OATP1B1 (6). High-affinity uptake of monoglucuronosyl bilirubin was also determined for OATP1B3 (6).

10.6 MRP3, a basolateral efflux pump, contributes to conjugated hyperbilirubinemia Conjugated hyperbilirubinemia results from bilirubin glucuronidation in hepatocytes or in extrahepatic tissues, such as the gastrointestinal tract (41,53). However, the glucuronides cannot exit from the cells across their basolateral membrane into blood unless there is sufficient expression of an efflux transporter. The molecular basis of the efflux of bilirubin glucuronides from hepatocytes into blood under normal and cholestatic conditions was not understood until recently. The cloning and localization of human MRP3 to the basolateral hepatocyte membrane (54) and the demonstration that it is an ATPdependent efflux transporter with a high affinity for monoglucuronosyl and bisglucuronosyl bilirubin (4) established that MRP3 is a key transporter for our understanding of conjugated hyperbilirubinemia (fFig.10.2). Human and rat MRP3/Mrp3 have also been localized to the basolateral membrane of epithelia of the intestinal tract, where it is abundantly expressed (55–57). By now, many nonbilirubin glucuronic acid conjugates of natural and synthetic compounds have been identified as MRP3 substrates (for review see reference 58). Bilirubin glucuronide efflux into sinusoidal blood via basolateral MRP3 of the hepatocyte has a compensatory function in cholestatic liver diseases and under conditions of MRP2 deficiency, leading to subsequent renal excretion; in addition, bilirubin glucuronides may also undergo reuptake by OATP1B1 and OATP1B3 into neighboring more downstream hepatocytes in the sinusoid. This concept was proposed earlier for bile acid cycling and percolation along the sinusoid (59), and a corresponding proposal has been made recently for bilirubin glucuronide reuptake on the basis of studies in mice with disrupted Oatp transporters of the 1a and 1b subfamilies and in patients with Rotor syndrome (50). This efflux and reuptake along the hepatic sinusoid may also serve to balance a high rate of uptake into some hepatocytes with the capacity of efflux into bile across the canalicular membrane by other more downstream hepatocytes (fFig. 10.2).

10.7 Genetic disorders and drug-induced inhibition of bilirubin uptake into hepatocytes A considerable number of genetic variants of the genes encoding the uptake transporters OATP1B1 and OATP1B3 have been identified in recent years (for recent reviews

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see references 60 and 61). Although mouse hepatic Oatps differ largely from human OATP1B1 and OATP1B3, it is important to note that genetic disruption of mouse Oatp transporters of the 1a and 1b subfamilies causes a massive increase in plasma bilirubin glucuronides and a significant increase in UCB (49,50). As mentioned in section 10.3, genomewide association studies have linked the human genes encoding OATP1B1 and OATP1B3 with unconjugated and conjugated hyperbilirubinemia (47,48). Human Rotor syndrome, a rare, benign hereditary disorder characterized by predominantly conjugated hyperbilirubinemia, was recently shown to be caused by a complete deficiency of OATP1B1 and OATP1B3 (50). Drug-induced hyperbilirubinemia can be caused by both patient susceptibility and drug properties. Up to 25% of patients receiving the HIV protease inhibitor indinavir develop hyperbilirubinemia (62–64). The Ki constant of OATP1B1 inhibition by indinavir was estimated to be 6.8 μM (64). Other drugs inhibiting OATP1B1-mediated transport with a Ki value of 0.2 μM include Rifamycin SV and cyclosporine (64). In the case of indinavir treatment, the risk of severe hyperbilirubinemia is associated, in addition, with genetic variants of the UDP-glucuronosyltransferase genes UGT1A3, UGT1A7, and UGT1*28 (63). Taken together, therapeutically relevant drug concentrations may be important contributing factors to reversible unconjugated hyperbilirubinemia (64). Moreover, several porphyrins were shown to interact with high affinity with OATP1B1, and inhibition of OATP1B1 may exacerbate clinical signs of porphyria by decreasing the hepatic clearance of porphyrins (65).

10.8 Genetic variants of the MRP2 (ABCC2) gene and MRP2 deficiency in Dubin-Johnson syndrome The Dubin-Johnson syndrome is a rare autosomal recessively inherited disorder characterized by a predominantly conjugated hyperbilirubinemia and deposition of a dark pigment in hepatocytes so that the liver appears dark blue or black (3,37,66,67). However, signs of severe liver disease are absent. The incidence of Dubin-Johnson syndrome ranges from 1:1,300 among Iranian Jews (68) to 1:300,000 in a Japanese population (69). The absence of a functionally active MRP2 protein from the human hepatocyte canalicular membrane has been recognized as the molecular basis of Dubin-Johnson syndrome (3,28), and functional tests have shown an impairment of the hepatobiliary elimination of organic anions (37), which is in line with the established transport function of MRP2 (7). Many sequence variants in the gene encoding human MRP2 have been identified in patients with Dubin-Johnson syndrome, including nonsense mutations leading to a premature stop codon (23), missense mutations (70,71), and a deletion mutation leading to the loss of two amino acids from the second nucleotidebinding domain (72) (fFig. 10.1; for review, see reference 7). Although all sequence variants associated with Dubin-Johnson syndrome result in the absence of a functionally active MRP2 protein from the canalicular membrane, their effects differ with regard to the synthesis and function of the respective MRP2 protein. Premature stop codons may cause rapid degradation of the mutated mRNA by nonsense-mediated decay, a mechanism that cotranslationally recognizes when a stop codon precedes the last splice site (73), thus leading to the apparent absence of the MRP2 protein in many variants of Dubin-Johnson syndrome (3,28,72). MRP2 sequence variants can lead to deficient

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protein maturation and impaired sorting as the key mechanism (74) or to an apically localized but functionally inactive MRP2 protein (71,75).

10.9 Impairment of MRP2 localization in the hepatocyte canalicular membrane Impaired MRP2 function and hyperbilirubinemia may have causes other than genetic variants of the gene encoding this drug and conjugate efflux pump. This may be exemplified by the essential role of radixin, a protein coupling Mrp2 to the actin cytoskeleton (76). Knockout of radixin in mice leads to hyperbilirubinemia and mild liver disease (76).This interaction of radixin with MRP2 may be similarly important for the targeting to the canalicular membrane in human hepatocytes (77). Modulation of the amount of the conjugate efflux pump in the apical membrane by recruitment of transporter molecules from intracellular pools or their retrieval into these pools has been extensively studied under various experimental conditions in rat liver and isolated hepatocytes. Insertion of Mrp2 into the canalicular membrane seems to depend on protein kinase C (78) or cyclic AMP (79). Retrieval of rat Mrp2 molecules from the canalicular membrane into the hepatocyte has been detected under various cholestatic conditions (80,81), after phalloidin treatment (82), after cytokine stimulation (83), and under hyperosmolar conditions (83–85). Under these experimental conditions, immunostaining of Mrp2 is no longer confined to the canalicular membrane but the transport protein seems to undergo endocytic retrieval into a subapical compartment. A comparable immunostaining, indicative of endocytic retrieval of transporter molecules, has been described in human liver diseases, including inflammation-induced cholestasis with hyperbilirubinemia (86), advanced stages of primary biliary cirrhosis (77), and obstructive cholestasis (87,88).

10.10 Reference 1. Jedlitschky G, Leier I, Buchholz U, et al. ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2. Biochem. J. 1997;327:305–10. 2. Kamisako T, Leier I, Cui Y, et al. Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinant human and rat multidrug resistance protein 2. Hepatology 1999;30:485–90. 3. Kartenbeck J, Leuschner U, Mayer R, et al. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 1996;23:1061–6. 4. Lee YM, Cui Y, König J, et al. Identifi cation and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3). Pharmacogenetics 2004;14:213–23. 5. König J, Cui Y, Nies AT, et al. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am. J. Physiol. Gastrointest. Liver Physiol. 2000;278:G156–64. 6. Cui Y, König J, Leier I, et al. Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J. Biol. Chem. 2001;276:9626–30. 7. Nies AT, Keppler D. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch.-Eur. J. Physiol. 2007;453:643–59.

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8. Cui Y, König J, Buchholz J, et al. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol. Pharmacol. 1999;55:929–37. 9. Cui Y, König J, Keppler D. Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2. Mol. Pharmacol. 2001;60:934–43. 10. Letschert K, Komatsu M, Hummel-Eisenbeiss J, et al. Vectorial transport of the peptide CCK-8 by double-transfected MDCKII cells stably expressing the organic anion transporter OATP1B3 (OATP8) and the export pump ABCC2. J. Pharmacol. Exp. Ther. 2005;313:549–56. 11. de Waart DR, Hausler S, Vlaming ML, et al. Hepatic transport mechanisms of cholylL-lysyl-fluorescein. J. Pharmacol. Exp. Ther. 2010;334:78–86. 12. Cantz T, Nies AT, Brom M, et al. MRP2, a human conjugate export pump, is present and transports Fluo-3 into apical vacuoles of HepG2 cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2000;278:G522–31. 13. Hooijberg JH, Broxterman HJ, Kool M, et al. Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. Cancer Res. 1999;59:2532–5. 14. Jansen PLM, Peters WHM, Lamers WH. Hereditary chronic conjugated hyperbilirubinemia in mutants rats caused by defective hepatic anion transport. Hepatology 1985;5:573–9. 15. Huber M, Guhlmann A, Jansen PL, et al. Hereditary defect of hepatobiliary cysteinyl leukotriene elimination in mutant rats with defective hepatic anion excretion. Hepatology 1987;7:224–8. 16. Oude Elferink RPJ, Meijer DKF, Kuipers F, et al. Hepatobiliary secretion of organic compounds: Molecular mechanisms of membrane transport. Biochim. Biophys. Acta 1995;1241:215–68. 17. Paulusma CC, Bosma PJ, Zaman GJR, et al. Congenital jaundice in rats with a mutation in a multidrug resistance associated-protein gene. Science 1996;271:1126–8. 18. Büchler M, König J, Brom M, et al. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMRP, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J. Biol. Chem. 1996;271:15091–8. 19. Ito K, Suzuki H, Hirohashi T, et al. Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am. J. Physiol. 1997;272:G16–22. 20. König J, Nies AT, Cui Y, et al. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim. Biophys. Acta 1999;1461:377–94. 21. Mayer R, Kartenbeck J, Büchler M, et al. Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J. Cell Biol. 1995;131:137–50. 22. Taniguchi K, Wada M, Kohno K, et al. A human canalicular multispecific organic anion transporter (cMOAT) overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res. 1996;56:4124–29. 23. Paulusma CC, Kool M, Bosma PJ, et al. A mutation in the human canalicular multispecifi c organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 1997;25:1539–42. 24. Evers R, Kool M, van Deemter L, et al. Drug export activity of the human canalicular multispecifi c organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA. J. Clin. Invest. 1998;101:1310–19. 25. National center for biotechnology information (accessed December 15, 2011, at http:// www.ncbi.nlm.nih.gov/gene?term=abcc2).

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26. Hagmann W, Nies AT, König J, et al. Purifi cation of the human apical conjugate export pump MRP2. Reconstitution and functional characterization as substrate-stimulated ATPase. Eur. J. Biochem. 1999;265:281–9. 27. Hagmann W, Schubert J, König J, et al. Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes. Biol. Chem. 2002;383:1001–9. 28. Keppler D, Kartenbeck J. The canalicular conjugate export pump encoded by the cmrp/cmoat gene. In: Boyer JL, Ockner RK, eds. Progress in Liver Diseases. Vol. 14. Philadelphia: Saunders, 1996:55–67. 29. Schaub TP, Kartenbeck J, König J, et al. Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules. J. Am. Soc. Nephrol. 1997;8:1213–21. 30. Schaub TP, Kartenbeck J, König J, et al. Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal-cell carcinoma. J. Am. Soc. Nephrol. 1999;10:1159–69. 31. Fromm MF, Kauffmann HM, Fritz P, et al. The effect of rifampin treatment on intestinal expression of human MRP transporters. Am. J. Pathol. 2000;157:1575–80. 32. Sandusky GE, Mintze KS, Pratt SE, et al. Expression of multidrug resistance-associated protein 2 (MRP2) in normal human tissues and carcinomas using tissue microarrays. Histopathology 2002;41:65–74. 33. Rost D, König J, Weiss G, et al. Expression and localization of the multidrug resistance proteins MRP2 and MRP3 in human gallbladder epithelia. Gastroenterology 2001;121:1203–8. 34. St-Pierre MV, Serrano MA, Macias RI, et al. Expression of members of the multidrug resistance protein family in human term placenta. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2000;279:R1495–503. 35. Nies AT. The role of membrane transporters in drug delivery to brain tumors. Cancer Letters 2007;254:11–29. 36. Berk PD, Howe RB, Bloomer JR, et al. Studies of bilirubin kinetics in normal adults. J. Clin. Invest. 1969;48:2176–90. 37. Chowdhury NR, Arias IM, Wolkoff AW, et al. Disorders of bilirubin metabolism. In: Arias IM et al., eds. The Liver, Biology and Pathobiology. 4th ed. Philadelphia: Lippincott, 2001:291–309. 38. Sedlak TW, Saleh M, Higginson DS, et al. Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proc. Natl. Acad. Sci. USA 2009;106:5171–6. 39. Arias IM, Johnson L, Wolfson S. Biliary excretion of injected conjugated and unconjugated bilirubin by normal and Gunn rats. Am. J. Physiol. 1961;200:1091–4. 40. Schmid R, Hammaker L. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 1963;42:1720–34. 41. Fujiwara R, Chen S, Karin M, et al. Reduced expression of UGT1A1 in intestines of humanized UGT1 mice via inactivation of NF- B leads to hyperbilirubinemia. Gastroenterology 2012;142:109–18. 42. Zucker SD, Horn PS, Sherman KE. Serum bilirubin levels in the U.S. population: gender effect and inverse correlation with colorectal cancer. Hepatology 2004;40:827–35. 43. McDonagh AF. Controversies in bilirubin biochemistry and their clinical relevance. Semin. Fetal Neonatal Med. 2010;15:141–7. 44. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–62. 45. Briz O, Serrano MA, Macias RI, et al. Role of organic anion-transporting polypeptides, OATP-A, OATP-C and OATP-8, in the human placenta-maternal liver tandem excretory pathway for foetal bilirubin. Biochem. J. 2003;371:897–905.

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46. Zhang W, He YJ, Gan Z, et al. OATP1B1 polymorphism is a major determinant of serum bilirubin level but not associated with rifampicin-mediated bilirubin elevation. Clin. Exp. Pharmacol. Physiol. 2007;34:1240–4. 47. Sanna S, Busonero F, Maschio A, et al. Common variants in the SLCO1B3 locus are associated with bilirubin levels and unconjugated hyperbilirubinemia. Hum. Mol. Genet. 2009;18:2711–18. 48. Johnson AD, Kavousi M, Smith AV, et al. Genome-wide association meta-analysis for total serum bilirubin levels. Hum. Mol. Genet. 2009;18:2700–10. 49. van de Steeg E, Wagenaar E, van der Kruijssen CM, et al. Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs. J. Clin. Invest. 2010;120:2942–52. 50. van de Steeg E, Stranecky V, Hartmannova H, et al. Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J. Clin. Invest. 2012;122:519–28. 51. Hagenbuch B, Gui C. Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family. Xenobiotica 2008;38:778–801. 52. Mackenzie PI, Bock KW, Burchell B, et al. Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet. Genomics 2005;15:677–85. 53. Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol. 2000;40:581–616. 54. König J, Rost D, Cui Y, et al. Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 1999;29:1156–63. 55. Rost D, Mahner S, Sugiyama Y, et al. Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 2002;282:G720–6. 56. Akita H, Suzuki H, Hirohashi T, et al. Transport activity of human MRP3 expressed in Sf9 cells: comparative studies with rat MRP3. Pharm. Res. 2002;19:34–41. 57. Scheffer GL, Kool M, de Haas M, et al. Tissue distribution and induction of human multidrug resistant protein 3. Lab. Invest. 2002;82:193–201. 58. Borst P, de Wolf C, van de Wetering K. Multidrug resistance-associated proteins 3, 4, and 5. Pflugers Arch. 2007;453:661–73. 59. Rius M, Hummel-Eisenbeiss J, Hofmann AF, et al. Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione. Am. J. Physiol. Gastrointest. Liver Physiol. 2006;290:G640–9. 60. König J. Uptake transporters of the human OATP family: molecular characteristics, substrates, their role in drug-drug interactions, and functional consequences of polymorphisms. In: Fromm MF, Kim RB, eds. Drug Transporters. Handb. Exp. Pharmacol., vol. 201, Heidelberg: Springer, 2011:1–28. 61. Degorter MK, Xia CQ, Yang JJ, et al. Drug transporters in drug efficacy and toxicity. Annu. Rev. Pharmacol. Toxicol. 2012;52:249–73. 62. Zucker SD, Qin X, Rouster SD, et al. Mechanism of indinavir-induced hyperbilirubinemia. Proc. Natl. Acad. Sci. USA. 2001;98:12671–6. 63. Lankisch TO, Behrens G, Ehmer U, et al. Gilbert's syndrome and hyperbilirubinemia in protease inhibitor therapy-an extended haplotype of genetic variants increases risk in indinavir treatment. J. Hepatol. 2009;50:1010–18. 64. Campbell SD, de Morais SM, Xu JJ. Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia. Chem. Biol. Interact. 2004;150:179–87. 65. Campbell SD, Lau WF, Xu JJ. Interaction of porphyrins with human organic anion transporting polypeptide 1B1. Chem. Biol. Interact. 2009;182:45–51.

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66. Dubin IN, Johnson FB. Chronic idiopathic jaundice with unidentified pigment in liver cells; a new clinicopathologic entity with report of 12 cases. Medicine (Baltimore) 1954;33:155–79. 67. Sprinz H, Nelson RS. Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells; report of four cases. Ann. Intern. Med. 1954;41:952–62. 68. Shani M, Seligsohn U, Gilon E, et al. Dubin-Johnson syndrome in Israel. I. Clinical, laboratory, and genetic aspects of 101 cases. Quarterly J. Med. 1970;39:549–67. 69. Kajihara S, Hisatomi A, Mizuta T, et al. A splice mutation in the human canalicular multispecifi c organic anion transporter gene causes Dubin-Johnson syndrome. Biochem. Biophys. Res. Commun. 1998;253:454–7. 70. Toh S, Wada M, Uchiumi T, et al. Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. Am. J. Hum. Genet. 1999;64:739–46. 71. Mor-Cohen R, Zivelin A, Rosenberg N, et al. Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome. J. Biol. Chem. 2001;276:36923–30. 72. Tsujii H, König J, Rost D, et al. Exon-intron organization of the human multidrugresistance protein 2 (MRP2) gene mutated in Dubin-Johnson syndrome. Gastroenterology 1999;117:653–60. 73. Thermann R, Neu-Yilik G, Deters A, et al. Binary specifications of nonsense codons by splicing and cytoplasmic translation. EMBO J. 1998;17:3484–94. 74. Keitel V, Kartenbeck J, Nies AT, et al. Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome. Hepatology 2000;32:1317–28. 75. Keitel V, Nies AT, Brom M, et al. A common Dubin-Johnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2). Am. J. Physiol. Gastrointest. Liver Physiol. 2003;284:G165–74. 76. Kikuchi S, Hata M, Fukumoto K, et al. Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nature Genet. 2002;31:320–5. 77. Kojima H, Nies AT, König J, et al. Changes in the expression and localization of hepatocellular transporters and radixin in primary biliary cirrhosis. J. Hepatol. 2003;39:693–702. 78. Beuers U, Bilzer M, Chittattu A, et al. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 2001;33:1206–16. 79. Roelofsen H, Soroka CJ, Keppler D, et al. Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets. J. Cell Sci. 1998;111:1137–45. 80. Trauner M, Arrese M, Soroka CJ, et al. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997;113:255–64. 81. Mottino AD, Cao J, Veggi LM, et al. Altered localization and activity of canalicular Mrp2 in estradiol-17beta-D-glucuronide-induced cholestasis. Hepatology 2002;35:1409–19. 82. Rost D, Kartenbeck J, Keppler D. Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis. Hepatology 1999;29:814–21. 83. Dombrowski F, Kubitz R, Chittattu A, et al. Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide. Biochem. J. 2000;348:183–8.

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84. Kubitz R, D'Urso D, Keppler D, et al. Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane. Gastroenterology 1997;113:1438–42. 85. Cantore M, Reinehr R, Sommerfeld A, et al. The Src family kinase Fyn mediates hyperosmolarity-induced Mrp2 and Bsep retrieval from canalicular membrane. J. Biol. Chem. 2011; 286:45014–29. 86. Zollner G, Fickert P, Zenz R, et al. Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology 2001;33:633–46. 87. Shoda J, Kano M, Oda K, et al. The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function. Am. J. Gastroenterol. 2001;96:3368–78. 88. Yamada T, Arai T, Nagino M, et al. Impaired expression of hepatic multidrug resistance protein 2 is associated with posthepatectomy hyperbilirubinemia in patients with biliary cancer. Langenbecks Arch. Surg. 2005;390:421–9.

11 Hepatobiliary transport during pregnancy: Crosstalk between transporters and hormones Roman Müllenbach and Frank Lammert

11.1 Introduction The liver is accessed by blood flow from both the portal and systemic circulation, and hormones regulate diverse hepatic functions. With the discovery of nuclear hormone receptors and other, membrane-bound receptors, the mechanisms underlying the hormonal regulation of liver function have been successfully investigated over the past decades. A substantial part of the progress toward hormonal regulation of liver function has been achieved by studying animal models incurring either chemical liver damage or bearing knockouts and/transgenes of various genes known to be involved in liver function (1–3). Besides being metabolically responsive to hormone signaling, the liver is the “central clearance station” for all blood-borne molecules, including hormones after chemical modification (glucuronidation and other conjugations). Hence, it is necessary for the liver to differentiate between hormones that need to be removed and others that mediate specific intrahepatic effects. During pregnancy, the most extreme physiological changes happen within a short time. The need to accommodate a non-self entity inside the body and at the same time provide a nutritionally sufficient environment poses several serious challenges. While the immune system must be suppressed to avoid an allergic reaction to the embryo itself as well as circulating embryonic cells carrying non-self, surface proteins, an efficient defense against viral infection, bacteria, and other exogenous threats must be maintained in order to ensure a successful reproductive cycle. At the same time, an ample supply of nutrients, ions, and all the other components needed to build a new organism must be maintained. The profound changes required to address this task are achieved by high doses of reproductive hormones, mainly estrogen and its endogenous relatives estradiol, estrone, and estriol. These molecules are produced by the ovaries and placenta and excreted primarily in urine after glucuronidation and sulfation (4,5). Around 50% of circulating estrogens undergo “enterohepatic circulation.” Following conjugation in liver, they are excreted in bile, and 80% of this fraction is subsequently reabsorbed after hydrolysis in the intestine.

11.2 Estrogens as cholestatic agents Abnormal retention of bromosulfophtalein (BSP) – an organic anion cleared from plasma mainly by the organic anion-transporting peptide (OATP1) and excreted into bile by the ATP-binding cassette transporter (ABCC2) (also known as multidrug resistance– associated protein [MRP2] or canalicular multispecific organic anion transporter [cMOAT]) – was shown in humans treated with natural estrogens in 1964 (6). The effect was immediate and reversible upon discontinuation of estrogen administration. Subsequent confirmation of estrogen as a cholestatic agent was helped by the observation

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that some women develop cholestasis outside pregnancy after administration of oral contraceptive steroids containing synthetic estrogens (7–9). These observations confirmed the suspicion for a role of estrogens or other pregnancy hormones as causative agents of intrahepatic cholestasis of pregnancy (ICP) (8). Such cholestasis usually starts in the last trimester of pregnancy, when the hormone levels are highest (10), and twin pregnancies, associated with more pronounced rises in hormone levels, show a higher incidence of ICP (11). The rapid resolution of ICP after delivery, in line with the reduction of hormone levels, was equally suggestive of a hormone-based etiology. Following observations in patients during pregnancy or under the influence of synthetic estrogens, the cholestatic effects of sex hormones were scrutinized in animal models, particularly the rat, over the course of the next 30 years. Estrogens, in particular glucuronides like estradiol-17β-glucuronide, were found to be cholestatic in animal models. Decreased uptake of bile salts at the basolateral membrane was suggested as a potential mechanism (12), caused by decreased fluidity of the sinusoidal membrane (13) in combination with increased permeability of tight junctions. It was assumed that a loss of Na+,K+-ATPase activity due to decreased membrane fluidity was reducing the sodium gradient required for sodium-dependent bile salt uptake into hepatocytes (13–16). However, these observations may be consequences rather than causes of cholestatic changes in the liver (17). To date, no transporter molecule from the sinusoidal membrane of hepatocytes has been associated with hormone-induced cholestatic liver disease in humans. The secretion and storage of bile involves the sequential action of several proteins required for the specific transport of the main constituents of bile – i.e., phosphatidylcholine, cholesterol, bile salts, and other organic anions. Critical transporters required to generate bile flow through the hepatocyte into the bile canaliculus are localized in the basolateral and canalicular membranes, physically and functionally distinct domains of the hepatocyte plasma membrane (fFig. 11.1). In experimental models of cholestasis in rats, inhibition of basolateral bile salt transport proteins (sodium-dependent taurocholate cotransporting polypeptide [NTCP] = solute carrier 10A1; organic anion cotransporting peptides [OATPs] = SLCOs, or solute carrier organic anion transporters) occurs at the transcriptional level (16). Biliary excretion of estradiol-17β-glucuronide is performed by the MRP2/ABCC2 transporter, a multispecific conjugate export pump, and causes a cholestatic phenotype by inhibition of the canalicular bile salt export pump (BSEP, ABCB11) only after its secretion into bile canaliculi, a phenomenon referred to as transinhibition (18). Estradiol-17β-glucuronide suppresses the expression of MRP2 at the posttranscriptional level (19). Hepatic modification and clearance of the main estrogen in pregnancy, estradiol, includes sulfation and glucuronidation (fFig. 11.1), typical phase II detoxification reactions, which help to minimize the cholestatic effects of estrogen (20). It is intriguing to notice that the estrogen conjugate that increases most during pregnancy, estriol-16αglucuronide, is itself a cholestatic agent in an animal model (21). A similar cholestatic mechanism for inhibiting the apical bile salt transport has been shown for another metabolite, estradiol-17-O-glucuronide (22). In contrast to ethinyl estradiol, which takes hours to show an effect, bile flow is reduced immediately following exposure to estrogen glucuronides in a dose-dependent manner (20). The use of animal models has enabled researchers to clarify in more detail the impact of estrogen and its dedicated receptors on the dysregulation of hepatobiliary transport.

11.3 Cholestatic activity of progesterone Portal vein

Hepatocyte Estriol-3-sulfate/ Estriol-3-sulfate, 16-D-glucuronide/ Estriol-16-Dglucuronide앖

Estriol앖

NTCP앗 Bile salts앖

Progesterone앖

Bile salts앖

Pregnanolone/ Pregnanediolsulfate/glucuronide N-acetylD-3-Oxosteroid-5b(a) glucosaminide앖 reductase 3a/b-Hydroxysteroid Pregnanolone/ dehydrogenase Pregnanediol앖

OATP앗

Sinusoidal uptake

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185

Bile duct

MRP2/ ABCC2앗 BSEP/ ABCB11앗 ???

MDR3/ ABCB4앗

Canalicular efflux

Fig. 11.1: The impact of pregnancy hormones on transporters involved in bile salt secretion.

A sophisticated mouse model of gonadectomized mice bearing subcutaneously implanted pellets that release synthetic estrogen derivatives (17β-estradiol) revealed a role for estrogen receptor (ER) alpha but not ER beta in biliary hypersecretion of cholesterol and subsequent gallstone formation (23,24). This effect was shown to be mediated by the sterol regulatory element binding protein 2 (SREBP2), which resulted in activation of SREBP2-responsive genes in the cholesterol synthesis pathway (25). Estrogen-induced cholestasis results in a downregulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, Oatp2, and Oatp4 may explain the unchanged sodium-independent transport of bile salts due to overlapping substrate specificity (26). Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF-1A, C/EBP, and PXR by estrogens. Milona et al. (27) have described how both the ablation and activation of FXR prevent the accumulation of hepatic bile salts in pregnancy.

11.3 Cholestatic activity of progesterone During pregnancy, 250 to 500 mg/day of progesterone is synthesized in the placenta. In the liver, progesterone is reduced to pregnanolone and pregnanediol (fFig. 11.1). Four different isomers (3α/3β, 5α/5β) are formed, which are metabolized by hydroxylation and conjugation with sulfate and glucuronic acid. Mono- or disulfated progesterone metabolites are the most prevalent steroids during pregnancy, with plasma levels of 10 to 15 μmol/L (28). Elevated levels of these sulfated metabolites, particularly the 3α/5α-isomers, have been reported in patients with ICP (28,29). The different ratio of 3α- and 3β-hydroxysteroids

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is most characteristic for ICP (30), and both biliary and fecal excretion of sulfated and glucuronidated progesterone metabolites is decreased in patients with ICP (31,32). A study from France reported that a majority of cases with ICP had taken oral, natural progesterone for risk of premature delivery (33). Administration of progesterone during pregnancy was shown to increase serum levels of bile salts during the third trimester (34,35). However, there is no mechanistic explanation for the impact of progesterone as yet, since the export of BSP was not impeded by progestins (8). The nuclear progesterone receptor (PR, NR3C3) is not expressed at detectable levels in the liver. Hence any transcriptional effect of progesterone is likely to be mediated either indirectly or via membrane bound progesterone receptor mPR, at least in rodents (36). Nongenomic effects of progesterone and its metabolites have also been observed in vitro and in mouse models (37,38).

11.4 Biochemical observations in symptom-free pregnant women Several studies have been able to show a slight cholestasis during pregnancy along with the associated hormonal and metabolic changes (39). Serum bile salt levels in most pregnant women stay within the normal range, with a slight increase during the third trimester (T, resulting in the amino acid exchange p.A444V, conveys a 2.8-fold risk of developing ICP (59). BRIC2 is a spectrum of intermittent cholestatic episodes precipitated by drug administration, viral infection, or pregnancy. The majority of BRIC2 patients described by van Mil et al. (60) were carriers of either homozygous or compound heterozygous functional ABCB11 variants. Screening the complete coding sequence of ABCB11 in ICP patients with normal GGT levels revealed functional variants of this gene in only a minority of cases (59,61). A similar paucity of functional variants in ICP patients was observed in the canalicular transporter underlying two subtypes of congenital familial cholestasis, PFIC1 and the less severe BRIC1: ATP8B1 is a P-type ATPase that flips phosphatidylserine from the outer leaflet of the hepatocyte membrane to the inner leaflet, (Fig. 11.2) thereby maintaining membrane asymmetry, which is required for normal transport activity of ABCB4 and ABCB11 (62,63). Although very rare cases of functional sequence variants in this gene have been reported for ICP patients (64), no evidence for a contribution of the locus to general population risk of ICP could be demonstrated to date. Based on the observations of familial clustering and ethnic variation, it is evident that ICP is caused by genetic factors, with the low penetrance in carriers of known variants pointing to a complex inheritance. This genetic heterogeneity was confirmed when Savander et al. (65) analyzed two extended Finnish pedigrees of ICP patients for haplotype transmission. Both segregation of haplotypes and multipoint linkage analysis excluded the ABCB4, ABCB11, and ATP8B1 loci in the studied pedigrees (65). Taken together, the three known transporters explain only a fraction of risk and heritability of ICP, and the search for the “missing heritability” of this complex disease is continuing (66,67). The next step toward the identification of as yet unknown contributors will be genomewide association studies or next-generation sequencing of severe cases to identify causative functional variants (68). The collection of affected patients to reach cohort size, which will allow identification of any locus outside the already known ABCB4 and ABCB11

11.7 Nuclear receptors and cholestasis of pregnancy

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gene loci, is ongoing. For the time being, studying the inheritance of risk in affected families might provide clues to modifier loci and the combined effect of alleles genetic risk factors (69–71). Furthermore, studies of knockout mice, either targeted or by random mutagenesis (72,73), might also reveal new candidate genes for cholestatic diseases, a recent example being the discovery of cholestatic properties in the Atp11c knockout mouse model (74).

11.7 Nuclear receptors and cholestasis of pregnancy The steady state of bile salts in the liver during cycles of feeding and fasting is maintained by an intricate network of nuclear receptors, a class of molecules that was originally described as “nuclear hormone receptors.” These molecules are characterized by their ability to regulate transcription in response to ligand binding at very low concentrations but also by their ability to interact and form complex regulatory networks (75). Hence it comes as no surprise that a profound change in the hormonal environment, as is encountered during pregnancy, causes major effects throughout the system. A study of the expression of various nuclear receptors in mouse livers during the last third of pregnancy (day 19 onward) reported decreased expression levels for all receptors involved in regulating fat and cholesterol metabolism (76). This is in line with the observation of increases in serum triglyceride and cholesterol levels during this period (fTab. 11.1). Differences between activation of the SHP gene promoter by SREBP1 between human and mouse cells were traced to species-specific differences in promoter sequences (77). Rather than weakening the significance of data from mouse models, these observations strengthen the potential role of sequence variants in regulatory regions in individual and species differences of metabolic regulation and their potential contribution to cholestatic phenotypes. Following the discovery of FXR (farnesoid X receptor [NR1H4]) as the central bile salt sensor of hepatocytes, regulating the synthesis and transport of bile salts, van Mil et al. (78) analyzed the entire coding length of the human NR1H4 gene in a cohort of 52 ICP patients to gauge the contribution of variations in this gene to the etiology of the disease. Functional variants affecting the trans-activation of the human ABCB11 promoter by FXR were found in a minority of cases, presenting a credible mechanism for the way in which rare variants in the central nuclear bile salt receptor can cause cholestasis in conditions of metabolic stress and high levels of reproductive hormones (79). The intricate crosstalk connecting the regulatory networks of nuclear receptors involves the atypical orphan nuclear receptor/repressor small heterodimer partner (SHP, NR0B2). Whereas this molecule functions as a corepressor in most downregulatory aspects of FXR (80–82), it is also directly afffected by ER alpha (83). Although these data are based on observations in the mouse model, they can serve as a good example of the way in which hormones of pregnancy interfere with complex metabolic pathways in the liver.

11.8 Gallstones and pregnancy Observations of increased gallstone frequencies in women as compared to men, and an increased gallstone risk following exposure to oral contraceptive steroids, have strengthened the hypothesis of a cholestatic or cholesterogenic effect of the molecules involved.

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Pregnancy is associated with an increased incidence of asymptomatic gallstones (84,85). Knockout of the mouse Abcb4 gene leads to formation of intrahepatic and gallbladder stones, resembling the human phenotype of patients with mutations of the orthologous ABCB4 gene (86). Although these observations offer a plausible role for common genetic factors in gallstone disease in women and increased serum bile salt levels during pregnancy, no such factors have been identified to date. Furthermore, common ICP risk alleles of the hepatocanalicular transporters ABCB4 (57,58) and ABCB11 (59) appear not to be overrepresented in patients with gallstones (87).

11.9 Summary Estrogen, progesterone, and their metabolites affect molecules involved in hepatic bile salt transport during pregnancy. On the sinusoidal side, the effects seems to be more secondary to changes in membrane fluidity, whereas the impact on the canalicular domain of the hepatocyte involves direct (transcriptional) and trans-inhibitory effects on specific export pumps. Nuclear receptor signaling in the liver is profoundly modified by reproductive hormones, which can contribute to cholestasis by unmasking existing imbalances caused by genetic variation. Transporters identified in congenital cholestasis serve as signposts for the identification of genetic risk factors in intrahepatic cholestasis of pregnancy.

11.10 References 1. Riant E, Waget A, Cogo H, et al. Estrogens protect against high-fat diet-induced insulin resistance and glucose intolerance in mice. Endocrinology 2009;150:2109–17. 2. Hewitt KN, Pratis K, Jones ME, et al. Estrogen replacement reverses the hepatic steatosis phenotype in the male aromatase knockout mouse. Endocrinology 2004;145:1842–8. 3. Toda K, Takeda K, Akira S, et al. Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator. J. Steroid Biochem. Mol. Biol. 2001;79:11–17. 4. Adlercreutz H. Hepatic metabolism of estrogens in health and disease. N. Engl. J. Med. 1974;290:1081–3. 5. Bolt HM. Metabolism of estrogens--natural and synthetic. Pharmacol. Ther. 1979;4:155–81. 6. Mueller MN, Kappas A. Estrogen pharmacology. I. The influence of estradiol and estriol on hepatic disposal of sulfobromophthalein (Bsp) in man. J. Clin. Invest. 1964;43:1905–14. 7. Kreek MJ, Sleisenger MH, Jeffries GH. Recurrent cholestatic jaundice of pregnancy with demonstrated estrogen sensitivity. Am. J. Med. 1967;43:795–803. 8. Kreek MJ. Female sex steroids and cholestasis. Semin. Liver Dis. 1987;7:8–23. 9. Dalen E, Westerholm B. Occurrence of hepatic impairment in women jaundiced by oral contraceptives and in their mothers and sisters. Acta Med. Scand. 1974;195:459–63. 10. Leslie KK, Reznikov L, Simon FR, et al. Estrogens in intrahepatic cholestasis of pregnancy. Obstet. Gynecol. 2000;95:372–6. 11. Gonzalez MC, Reyes H, Arrese M, et al. Intrahepatic cholestasis of pregnancy in twin pregnancies. J. Hepatol. 1989;9:84–90. 12. Forker EL. The effect of estrogen on bile formation in the rat. J. Clin. Invest. 1969;48:654–63.

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33. Bacq Y, Sapey T, Brechot MC, et al. Intrahepatic cholestasis of pregnancy: a French prospective study. Hepatology 1997;26:358–64. 34. Noblot G, Audra P, Dargent D, et al. The use of micronized progesterone in the treatment of menace of preterm delivery. Eur. J. Obstet. Gynecol. Reprod. Biol. 1991;40:203–9. 35. Erny R, Pigne A, Prouvost C, et al. The effects of oral administration of progesterone for premature labor. Am. J. Obstet. Gynecol. 1986;154:525–9. 36. Dressing GE, Goldberg JE, Charles NJ, et al. Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications. Steroids;76:11–17. 37. Vallejo M, Briz O, Serrano MA, et al. Potential role of trans-inhibition of the bile salt export pump by progesterone metabolites in the etiopathogenesis of intrahepatic cholestasis of pregnancy. J. Hepatol. 2006;44:1150–7. 38. Abu-Hayyeh S, Martinez-Becerra P, Sheikh Abdul Kadir SH, et al. Inhibition of Na+taurocholate Co-transporting polypeptide-mediated bile acid transport by cholestatic sulfated progesterone metabolites. J. Biol. Chem.;285:16504–12. 39. Fulton IC, Douglas JG, Hutchon DJ, et al. Is normal pregnancy cholestatic? Clin. Chim. Acta 1983;130:171–6. 40. Carter J. Serum bile acids in normal pregnancy. Br. J. Obstet. Gynaecol. 1991;98:540–3. 41. Lunzer M, Barnes P, Byth K, et al. Serum bile acid concentrations during pregnancy and their relationship to obstetric cholestasis. Gastroenterology 1986;91:825–9. 42. Heikkinen J, Maentausta O, Ylostalo P, et al. Changes in serum bile acid concentrations during normal pregnancy, in patients with intrahepatic cholestasis of pregnancy and in pregnant women with itching. Br. J. Obstet. Gynaecol. 1981;88:240–5. 43. Pascual MJ, Serrano MA, El-Mir MY, et al. Relationship between asymptomatic hypercholanaemia of pregnancy and progesterone metabolism. Clin. Sci. (Lond.) 2002;102:587–93. 44. Shneider BL, Fox VL, Schwarz KB, et al. Hepatic basolateral sodium-dependent-bile acid transporter expression in two unusual cases of hypercholanemia and in extrahepatic biliary atresia. Hepatology 1997;25:1176–83. 45. Jiang ZH, Qiu ZD, Liu WW, et al. Intrahepatic cholestasis of pregnancy and its complications. Analysis of 100 cases in Chongqing area. Chin. Med. J. (Engl.) 1986;99:957–60. 46. Roncaglia N, Arreghini A, Locatelli A, et al. Obstetric cholestasis: outcome with active management. Eur. J. Obstet. Gynecol. Reprod. Biol. 2002;100:167–70. 47. Abedin P, Weaver JB, Egginton E. Intrahepatic cholestasis of pregnancy: prevalence and ethnic distribution. Ethn. Health 1999;4:35–7. 48. Brites D, Rodrigues CM, van-Zeller H, et al. Relevance of serum bile acid profile in the diagnosis of intrahepatic cholestasis of pregnancy in an high incidence area: Portugal. Eur. J. Obstet. Gynecol. Reprod. Biol. 1998;80:31–8. 49. Rioseco AJ, Ivankovic MB, Manzur A, et al. Intrahepatic cholestasis of pregnancy: a retrospective case-control study of perinatal outcome. Am. J. Obstet. Gynecol. 1994;170:890–5. 50. Lee RH, Goodwin TM, Greenspoon J, et al. The prevalence of intrahepatic cholestasis of pregnancy in a primarily Latina Los Angeles population. J. Perinatol. 2006;26:527–32. 51. Jacquemin E, Cresteil D, Manouvrier S, et al. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999;353:210–11. 52. de Vree JM, Jacquemin E, Sturm E, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc. Natl. Acad. Sci. USA 1998;95:282–7. 53. Bull LN, van Eijk MJ, Pawlikowska L, et al. A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat. Genet. 1998;18:219–24. 54. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat. Genet. 1998;20:233–8.

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75. Bookout AL, Jeong Y, Downes M, et al. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 2006;126:789–99. 76. Sweeney TR, Moser AH, Shigenaga JK, et al. Decreased nuclear hormone receptor expression in the livers of mice in late pregnancy. Am. J. Physiol. Endocrinol. Metab. 2006;290:E1313–20. 77. Kim HJ, Kim JY, Park SK, et al. Differential regulation of human and mouse orphan nuclear receptor small heterodimer partner promoter by sterol regulatory element binding protein-1. J. Biol. Chem. 2004;279:28122–31. 78. Van Mil SW, Milona A, Dixon PH, et al. Functional variants of the central bile acid sensor FXR identifi ed in intrahepatic cholestasis of pregnancy. Gastroenterology 2007;133:507–16. 79. Mullenbach R, Lammert F. An update on genetic analysis of cholestatic liver diseases: digging deeper. Dig. Dis.;29:72–7. 80. Lu TT, Makishima M, Repa JJ, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell 2000;6:507–15. 81. Goodwin B, Jones SA, Price RR, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 2000;6:517–26. 82. Denson LA, Sturm E, Echevarria W, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–7. 83. Lai K, Harnish DC, Evans MJ. Estrogen receptor alpha regulates expression of the orphan receptor small heterodimer partner. J. Biol. Chem. 2003;278:36418–29. 84. Ko CW, Beresford SA, Schulte SJ, et al. Incidence, natural history, and risk factors for biliary sludge and stones during pregnancy. Hepatology 2005;41:359–65. 85. Tsimoyiannis EC, Antoniou NC, Tsaboulas C, et al. Cholelithiasis during pregnancy and lactation. Prospective study. Eur. J. Surg. 1994;160:627–31. 86. Lammert F, Wang DQ, Hillebrandt S, et al. Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis. Hepatology 2004;39:117–28. 87. Acalovschi M, Tirziu S, Chiorean E, et al. Common variants of ABCB4 and ABCB11 and plasma lipid levels: a study in sib pairs with gallstones, and controls. Lipids 2009;44:521–6.

12 Hepatobiliary transport and gallstone formation Henning Wittenburg

12.1 Introduction Detailed studies in human and model organisms have confirmed the pivotal role of hepatobiliary transport proteins in the pathophysiology of both pigment and cholesterol gallstone formation. Conditions that increase the biliary secretion of bilirubin via MRP2, such as hemolysis and induced enterohepatic circulation of bilirubin due to bile salt malabsorption (e.g., in Crohn’s disease and cystic fibrosis), predispose individuals to the formation of pigment gallstones. Cholesterol gallstones may form if the amount of cholesterol in gallbladder bile exceeds the maximal concentration that is soluble at the given concentration of bile salts and phospholipids. Conditions such as pregnancy, fasting, and insulin resistance increase cholesterol gallstone risk by promoting biliary cholesterol secretion via the heterodimeric ABCG5/ABCG8 transporter, whereas certain drugs (e.g., cyclosporine) also predispose individuals to cholesterol gallstone formation owing to inhibition of the bile salt export pump (BSEP). Furthermore, studies in the mouse model of cholelithiasis and in humans have identified genetic variations in the ABCG5/ABCG8 transporter genes predisposing to cholesterol gallstone formation. In addition, mutations in the transporter genes ABCB11 (encoding BSEP) and ABCB4 (encoding the phospholipid transporter MDR3) cause rare “oligogenic” forms of cholelithiasis, underscoring the importance of genetic variation in hepatobiliary transporter genes for perturbations in the physicochemical stability of bile.

12.2 Epidemiology and risk factors of cholelithiasis Gallstones are extraordinarily common in Europe and North and South America, with prevalence rates from cross-sectional ultrasound surveys surpassing 20% in selected populations (1,2). The majority of gallbladder stones remain asymptomatic. However, their inherent risk to cause pain and complications render gallstones the second most costly digestive disease, with direct costs being exceeded only by the costs associated with gastroesophageal reflux disease (3). The two major gallstone subtypes that can be distinguished are cholesterol gallstones and bilirubin or “pigment” gallstones (4). In Western populations cholesterol gallstones prevail over pigment stones (5). In contrast, in Asia and in Africa bilirubin gallstones are more common than cholesterol stones (6). Ultrasound-based studies have identified a wide range of prevalence rates of gallstones in ethnically distinct populations (4). Combined with the results from family studies of affected individuals, indicating an increased risk of cholelithiasis among their relatives, these observations support an inherited risk of gallstone formation (6). However, with the advent of a “Western” diet, high in calories, the dramatic increase in weight gain and of gallstone prevalence among American Indians point to the important role of environmental factors in individual gallstone risk (7). In addition to diet and obesity, other risk factors for cholelithiasis

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include the number of pregnancies and certain medications. Furthermore, gallstone risk is increased in patients with diabetes and insulin resistance suggesting cholelithiasis as an additional element of the metabolic syndrome. Finally, rapid weight loss and even moderate intentional weight loss associated with regain (“weight cycling”) is accompanied by a substantial risk of gallstone formation (4). Importantly, these environmental risk factors most likely apply to the common cholesterol gallstones only. In contrast, the risk of forming bilirubin gallstones is increased from hemolysis and hyperbilirubinemia, the induced enterohepatic circulation of bilirubin from Crohn’s disease, resection of the terminal ileum, and in patients with cystic fibrosis and liver cirrhosis (8). Since twins usually share similar environmental factors, the comparison of the concordance rates for cholelithiasis between dizygotic (genetically different) and monozygotic (genetically identical) twins makes possible the quantification of genetic and environmental factors for the manifestation of a disease. A twin study from Sweden including more than 43.000 twin pairs established the heritability of symptomatic cholelithiasis. From higher concordance rates among monozygotic as compared with dizygotic twins, the genetic factors were found to confer 25% (confidence interval 9%–40%) of the risk of symptomatic cholelithiasis, the remainder of the risk being accounted for by environmental factors (9). These findings confirm common cholelithiasis as a complex, multifactorial trait that stems from the influence of environmental factors in individuals carrying lithogenic alleles of genes predisposing to gallstone formation (“LITH genes”) (10). In addition to the common complex cholelithiasis, in rare instances mutations in single genes with a high penetrance confer an “oligogenic” cholelithiasis, with a substantially lower impact of environmental factors (10).

12.3 Hepatobiliary transporters and the pathophysiology of gallstone formation 12.3.1 Pigment gallstones Pigment gallstones are commonly classified as either “brown” or “black.” Brown pigment stones form secondary to infection in any part of the biliary tree and consist of unpolymerized calcium bilirubinate (the salt of unconjugated bilirubin) and calcium salts of long chain fatty acids (8). Brown pigment stones are not considered further in this chapter. Black pigment stones form in the gallbladder; they consist of polymerized and oxidized calcium bilirubinate and may contain calcium carbonate or calcium phosphate (8). The hallmark of black pigment stone formation is the secretion of excess conjugated bilirubin into bile (“hyperbilirubinbilia”). Bilirubin for canalicular secretion is principally derived from the degradation of heme protein, such as hemoglobin and myoglobin, and may increase from hemolysis and ineffective erythropoiesis, as in vitamin B12 and folate deficiency (8). Another cause of pigment stone formation is the induced enterohepatic circulation of bilirubin secondary to bile salt malabsorption due to Crohn’s disease of the terminal ileum or resection of the terminal ileum (11,12). Generally bile salts undergo a very efficient enterohepatic circulation following their uptake into the enterocytes of the terminal ileum by the sodium-dependent bile salt transporter ASBT, encoded by the SLC10A2 gene, and hepatocellular uptake by the basolateral sodium/

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taurocholate cotransporting polypeptide NTCP, encoded by the SLC10A1 gene (13). In contrast, in health, unconjugated bilirubin derived from the deconjugation of bilirubin conjugates by bacterial or mucosal β-glucoronidases does not undergo enterohepatic circulation but is reduced to urobilinoids and excreted via the feces. However, following ileal bile salt malabsorption, excess amounts of bile salts in the colon can bind calcium and prevent the formation of complexes of calcium and unconjugated bilirubin. This may ultimately lead to passive colonic absorption of unconjugated bilirubin solubilized by bile salts that are malabsorbed in the ileum and therefore have spilled into the colon (8,12). Following transport to the liver, unconjugated bilirubin is taken up in the hepatocytes by members of the organic anion transport protein family (OATP) (14). In hepatocytes, unconjugated bilirubin is conjugated to soluble mono- or diglucuronidated bilirubin by the enzyme UDP glucuronosyltransferase, encoded by the UGT1A1 gene (15). Glucuronidated bilirubins are secreted into bile via the multidrug resistance–associated protein 2 (MRP2), which is localized in the canalicular domain of the hepatocellular plasma membrane and encoded by the ATP-binding cassette (ABC) transporter gene ABCC2 (16). Accordingly, an increase in the amount of bilirubin transported to the liver – from chronic hemolysis, ineffective erythropoiesis, or induced enterohepatic cycling – results in more bilirubin conjugates secreted into bile and an increased risk of pigment gallstone formation. Excess bilirubin conjugates in bile are hydrolyzed to unconjugated bilirubin by endogenous β-glucoronidases, providing the physicochemical condition for the precipitation of calcium bilirubinate (8). Thus the reduced capacity of the liver to glucuronidate bilirubin in chronic liver diseases leads to the biliary secretion of more monoglucuronidated bilirubin via MRP2, which may be hydrolyzed more rapidly to unconjugated bilirubin. In addition, in cirrhosis the capacity to bind and solubilize calcium and unconjugated bilirubin is reduced owing to lower biliary bile salt and cholesterol levels and resulting in a predisposition for precipitation of calcium bilirubinate and hence bilirubin gallstone formation (4). In summary, hyperbilirubinbilia due to the increased secretion of bilirubin conjugates via the transport protein MRP2 and increased biliary secretion of monoglucuronidated bilirubins, which facilitate hydrolysis to unconjugated bilirubin, are pathophysiological key factors predisposing to pigment gallstone formation.

12.3.2 Cholesterol gallstones Following its secretion into bile by the heterodimeric transporter ABCG5/ABCG8, the almost insoluble cholesterol forms unilamellar vesicles with phospholipids (mostly phosphatidylcholines from de novo synthesis) that are secreted into bile by the multdrug-resistance protein 3 (MDR3) encoded by the ABCB4 gene. Subsequently cholesterol is solubilized in the gallbladder in mixed micelles containing phophatidylcholines and bile salts, which are secreted into bile by the BSEP, encoded by the ABCB11 gene (17). Prerequisite for the formation of cholesterol gallstones in the gallbladder is the disturbance of bile composition, with the amount of cholesterol exceeding the maximal concentration that is soluble at the existing concentration of bile salts and phospholipids (i.e., a cholesterol saturation index greater than 1). Additional pathophysiological key features of cholesterol gallstone formation are impaired motility of the gallbladder,

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leading to stasis of bile within the gallbladder, and a mucin gel in the gallbladder serving as a nucleation matrix for cholesterol monohydrate crystals, the principal component of cholesterol gallstones (18). Alterations in the function of hepatobiliary transport proteins change the composition of the bile and therefore may predispose to the formation of cholesterol gallstones. This explains the higher gallstone risk in several clinical conditions that lead to an increase in biliary secretion of cholesterol by ABCG5/ABCG8 and/or a decreased biliary secretion of bile salts or phospholipids by BSEP and MDR3, respectively (fFig. 12.1). One example is the increased gallstone risk in late pregnancy and postpartum (19). This finding, in part, is explained by supersaturation of the bile with cholesterol in the second and third trimesters of pregnancy owing to increased secretion rates of cholesterol relative to secretion rates of bile salts and phospholipids (20). These changes most likely occur because of high estrogen levels and insulin resistance (21,22). This concept is supported by an increased risk for cholecystectomy due to complications of gallstone disease in postmenopausal women taking hormone replacement therapy (23). The observation of an increased gallstone risk from insulin resistance may also be explained in part by alterations in biliary cholesterol secretion. Mice deficient for the hepatic insulin receptor share a number of features with the metabolic syndrome (24). In this mouse model, biliary cholesterol secretion was found to be increased owing to higher expression levels of ABCG5/ABCG8. Insulin resistance and higher expression levels of the biliary cholesterol transporter were linked to the transcription factor FoxO1, which is inhibited in health by insulin through phosphorylation. It has been confirmed that FoxO1 is a transcription factor for ABCG5/ABCG8, and these findings suggest that, in part, insulin resistance promotes cholesterol secretion and cholesterol gallstone formation by the reduced inactivation of FoxO1 (24).

Calcineurin inhibitors (Cyclosporine, Tacrolimus)

Decreased biliary bile salt secretion

Dieting, octreotide

Increased intestinal transit time

Cholelithiasis

Increased biliary cholesterol secretion Pregnancy

Insulin resistance Rapid weight loss

Fig. 12.1: Factors that influence cholesterol gallstone risk and their putative molecular mechanisms. For details and references, see text.

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Another condition associated with gallstone formation from increased cholesterol secretion is rapid weight loss (25). Weight loss results in the mobilization of cholesterol from peripheral tissues and is also associated with the increased hepatic synthesis of cholesterol. This leads to an increase in biliary cholesterol secretion relative to bile salt and phospholipid secretion and a significant increase in the cholesterol saturation index (26). These findings explain the significant risk of gallstone formation during rapid weight loss if no preventive measures are taken with the intake of ursodeoxycholic acid (27). In addition, dieting impairs the secretion of cholecystokinin from enteroendocrine cells in the small intestine, leading to gallbladder stasis and a decreased gut transit time, which results in higher cholesterol absorption (28). The same effects explain the increased gallstone risk from total parenteral nutrition and a therapy with the somatostatin analogue octreotide. Finally, certain medications influence biliary lipid composition by interfering with hepatobiliary transporters. Fibrates may increase hepatic cholesterol synthesis and biliary cholesterol secretion and, in addition, reduce the activity of cholesterol 7α-hydroxylase and therefore decrease bile salt synthesis. This, in turn, may stimulate biliary cholesterol secretion further by decreasing cholesterol degradation into bile salts, and both mechanisms may contribute to the supersaturation of bile with cholesterol (6). On the other hand, epidemiological studies suggest a decreased gallstone risk from statins that inhibit hepatic cholesterol synthesis (29). Furthermore, experimental studies suggest that inhibition of intestinal cholesterol uptake from the drug ezetimibe protects against gallstone formation (30). Last, the calcineurin inhibitors cyclosporine and tacrolimus inhibit BSEP and biliary bile salt secretion and are associated with a high incidence of gallstone formation (6). In summary, a prerequisite of cholesterol gallstone formation is the supersaturation of gallbladder bile with cholesterol relative to bile salts and phospholipids. Several conditions – such as rapid weight loss, pregnancy, insulin resistance, and certain medications – primarily increase biliary cholesterol secretion via the heterodimeric cholesterol transporter ABCG5/ABCG8 and predispose to cholesterol gallstone formation.

12.4 Genetic variation in hepatobiliary transporter genes and gallstone susceptibility The notion of a genetic predisposition to gallstone formation has led to numerous studies attempting to identify the underlying genes and polymorphisms. fFig. 12.2 summarizes lithogenic genes with relevance to hepatobiliary transport that are associated with pigment and cholesterol gallstone formation, respectively. Pigment stone formation is directly associated with genetic defects causing hemolytic anaemia (10). In addition, cystic fibrosis, an autosomal recessive disease due to mutations in the gene ABCC7 encoding the chloride ion transporter CFTR, is associated with gallstone formation. Among other cell types, CFTR is expressed in cholangiocytes and enterocytes. In a mouse model of cystic fibrosis it was recently confirmed that increased loss of fecal bile acid was associated with a more hydrophobic biliary bile salt pattern, increased bile flow, and biliary lipid secretion rates as well as higher biliary conjugated and unconjugated bilirubin levels. These findings suggest that because of the malabsorption of bile

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Hepatocyte

Bile Duct

Unconjugated Bilirubin

Hepatocyte

MDR3 (ABCB4)

UGT1A1

Phospholipids MRP2 (ABCC2) Conjugated Bilirubin

ABCG5 Cholesterol ABCG8 Bile salts BSEP (ABCB11)

CFTR (ABCC7) Cl앥

Cholangiocyte

Cholangiocyte

Fig. 12.2: Hepatobiliary transporters with relevance for the pathophysiology of pigment gallstone formation (left) and cholesterol gallstone formation (right). Transporters associated with oligogenic forms of cholelithiasis are indicated in checkered circles, full circles denote transporters associated with common complex cholelithiasis. Note: In addition to being a modifier gene for pigment gallstone formation, UGT1A1 appears to be associated with cholesterol gallstone susceptibility.

salts, the enterohepatic circulation of bilirubin leads to increased biliary secretion of bilirubin via MRP2. A lower pH in gallbladder bile in cystic fibrosis may facilitate deconjugation of bilirubin, and this may predispose to the formation of gallstones, which, in cystic fibrosis, are typically “mixed” and contain more cholesterol than usual pigment stones owing to an increased cholesterol saturation index (31,32). However, only up to 30% of patients with cystic fibrosis develop gallstones, indicating the presence of additional genetic variations that influence individual risk. One such “modifier gene” is the UGT1A1 gene, encoding the enzyme that catalyzes the formation of bilirubin glucuronides. A polymorphism in the promoter of UGT1A1 gene leads to intermittent jaundice but also higher proportional secretion of monoglucuronidated bilirubin, a common condition known as Gilbert’s syndrome (33). UGT1A1 polymorphism has been confirmed to increase the susceptibility to gallstone formation in patients with cystic fibrosis and hemolytic anemia, supporting the concept that modifier genes may play a role in oligogenic bilirubin gallstone susceptibility (34).

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One principal example of an oligogenic predisposition to gallstone formation is the “low phospholipid–associated cholelithiasis (LPAC) syndrome,” which is due to mutations in the ABCB4 gene encoding the phospholipid transporter MDR3. Patients harboring these mutations were of younger age, had a positive family history of gallstones and intrahepatic cholestasis of pregnancy, displayed recurrence of gallstones after cholecystectomy, and were found to have intrahepatic sludge or microlithiasis (35). In a limited number of patients with the LPAC syndrome, a low phospholipid concentration in bile was confirmed (36). In addition, the physicochemical consequences of mutations in the phospholipids transporter gene were substantiated in a mouse model of ABCB4-deficient mice that displayed spontaneous formation of needlelike crystals (as predicted in bile devoid of phospholipids) and gallstones (37). In addition to mutations in ABCB4, patients with a condition known as “benign recurrent intrahepatic cholestasis” (BRIC) seem to be predisposed to gallstone formation if BRIC was due to mutation in the ABCB11 gene encoding the bile salt export pump (BRIC2) but not in patients with BRIC1 due to mutations in the ATP8B1 gene encoding a canalicular aminophospholipid flippase (38). Even though mutations in ABCB4 and ABCB11 were shown to cause oligogenic cholelithiasis, these both genes have not to date been confirmed to be associated with common multifactorial cholelithiasis (35,39). Identification of the LITH genes underlying susceptibility to common cholelithiasis was facilitated by the results of genetic studies in inbred mice. That is, the mouse model of diet-induced cholelithiasis confirmed the genetic background of cholesterol gallstone susceptibility (40). This prompted a number of systematic mouse crosses in order to perform quantitative trait locus (QTL) mapping to locate genomic regions associated with gallstone susceptibility and identify the underlying Lith genes (41). The first QTL crosses identified Abcb11, encoding the BSEP, and Abcc2, encoding the bilirubin transporter MRP2 as candidate genes for the susceptibility loci termed Lith1 and Lith2 (42). However, additional studies in the mouse model did not unequivocally confirm Abcb11 as a Lith gene, and an association study in humans identified no association of variation in the human orthologous ABCB11 gene and cholelithiasis (39,43). Likewise, to date Abcc2 has not been confirmed as a Lith gene in the murine model of cholelithiasis. Furthermore, the orthologous ABCC2 gene does not appear to be a major LITH gene in humans (44). Nevertheless, polymorphisms of UGT1A1 were recently confirmed to not only increase the risk of pigment stone formation but also to be associated with cholelithiasis in a population of individuals with mostly cholesterol gallstones, indicating a link between cholesterol gallstone susceptibility and bilirubin metabolism (45). Another gene identified as a Lith gene from QTL mapping in the mouse model of cholelithiasis is Nr1h4, encoding the bile salt receptor FXR (46). FXR, among many other functions, controls bile acid synthesis and secretion by acting as a nuclear transcription factor that inhibits the expression of CYP7A1, the gene encoding the rate-limiting enzyme of bile acid synthesis, and that promotes bile acid secretion by increasing the expression of BSEP. Even though expression differences of Nr1h4 and its target genes in the mouse model support the gene as a Lith gene, the results of association studies in different human populations have been conflicting (47). Likewise, SLC10A2 encoding the apical sodium-dependent bile salt transporter ASBT on enterocytes is another gene that is supported as a LITH candidate gene by systematic functional data. However, the results

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Tab. 12.1: Lithogenic genes with relevance to hepatobiliary transporters and their putative molecular mechanisms. Gene (encoded protein)

Molecular mechanism

Bilirubin gallstones UGT1A1

Higher proportional secretion of monoglucuronidated vs. diglucuronidated bilirubin

ABCC7 (CFTR)

Induced enterohepatic circulation of bilirubin, “hyperbilirubinbilia,” lower pH in gallbladder bile

Cholesterol gallstones ABCG5/ABCG8

Increased biliary cholesterol secretion

UGT1A1

Formation of the bilirubin core of cholesterol gallstones

SLC10A2 (ASBT)

Impaired enterohepatic circulation of bile salts

NR1H4 (FXR)

Modification of bile salt synthesis and secretion

ABCB4 (MDR3)

Impaired biliary phospholipids secretion (“LPAC syndrome”)

ABCB11 (BSEP)

Impaired biliary bile salt secretion (“BRIC2”)

of genetic association studies for SLC10A2 confirm an association with cholelithiasis in only some but not other populations (48,49). The most convincing LITH genes for cholelithiasis to date are ABCG5/ABCG8. These genes were first identified as candidate genes for one of the susceptibility regions, named Lith9, from one of the QTL crosses for cholelithiasis in inbred mice (46). Subsequently Lith9 was confirmed in additional crosses, and the results of systematic molecular and physicochemical studies supported Abcg5/Abcg8 as the genes underlying the susceptibility locus (50). More recently, the first human genomewide association study for cholelithiasis identified ABCG8 as a LITH gene in humans (51), a finding that was confirmed in association studies in several human populations (52). Most likely an individual single nucleotide polymorphism resulting in the amino acid exchange p.D19H increases the gallstone risk. The ABCG5/ABCG8 heterodimer transports cholesterol as well as plant sterols and promotes biliary cholesterol and plant sterol secretion. Since the lithogenic 19H allele is associated with lower plasma plant sterol levels (53), the findings indicate an increased risk for common cholesterol gallstone formation from a genetically determined higher biliary cholesterol secretion, which may predispose to supersaturation of bile with cholesterol in concert with lithogenic environmental factors (4). Genes with lithogenic alleles that influence the risk of cholesterol and bilirubin gallstone formation and their putative molecular mechanisms are summarized in fTab. 12.1.

12.5 Concluding remarks Detailed studies in human and model organisms have confirmed the pivotal role of hepatobiliary transport proteins for the pathophysiology of both bilirubin and cholesterol gallstone formation (43). Specifically the risk of cholesterol gallstone formation

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is increased in conditions such as fasting and insulin resistance, which induce cholesterol hypersecretion via ABCG5/ABCG8, or by drugs such as cyclosporine, which impair bile salt secretion via BSEP. The risk of bilirubin gallstone formation is increased in patients with hemolysis and in conditions associated with induced enterohepatic bilirubin circulation (e.g., Crohn’s disease and cystic fibrosis) due to higher biliary secretion of conjugated bilirubin via MRP2. In addition, genetic studies in the mouse model of cholelithiasis and in humans have confirmed lithogenic alleles of hepatobiliary transport proteins, foremost ABCG5/ABCG8, indicating a primary role of genetic variation in hepatobiliary transporters for the predisposition to gallstone formation (52). Accordingly, hepatobiliary transporter and their regulating transcription factors, such as FXR and FoxO1, are promising pharmaceutical targets for the treatment and prevention of cholelithiasis. However, such strategies await systematic studies in both model organisms and humans.

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16. Nies AT, Keppler D. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 2007;453:643–59. 17. Wittenburg H, Carey MC. Biliary cholesterol secretion by the twinned sterol halftransporters ABCG5 and ABCG8. J. Clin. Invest. 2002;110:605–9. 18. Portincasa P, Moschetta A, Palasciano G. Cholesterol gallstone disease. Lancet 2006;368:230–9. 19. Ko CW, Beresford SA, Schulte SJ, et al. Incidence, natural history, and risk factors for biliary sludge and stones during pregnancy. Hepatology 2005;41:359–65. 20. Kern F, Jr., Everson GT, DeMark B, et al. Biliary lipids, bile acids, and gallbladder function in the human female. Effects of pregnancy and the ovulatory cycle. J. Clin. Invest. 1981;68:1229–42. 21. Wang HH, Afdhal NH, Wang DQ. Estrogen receptor alpha, but not beta, plays a major role in 17beta-estradiol-induced murine cholesterol gallstones. Gastroenterology 2004a;127:239–49. 22. Ko CW, Beresford SA, Schulte SJ, et al. Insulin resistance and incident gallbladder disease in pregnancy. Clin. Gastroenterol. Hepatol. 2008;6:76–81. 23. Cirillo DJ, Wallace RB, Rodabough RJ, et al. Effect of estrogen therapy on gallbladder disease. JAMA 2005;293:330–9. 24. Biddinger SB, Haas JT, Yu BB, et al. Hepatic insulin resistance directly promotes formation of cholesterol gallstones. Nat. Med. 2008;14:778–82. 25. Stokes CS, Krawczyk M, Lammert F. Gallstones: environment, lifestyle and genes. Dig. Dis. 2011a;29:191–201. 26. Broomfi eld PH, Chopra R, Sheinbaum RC, et al. Effects of ursodeoxycholic acid and aspirin on the formation of lithogenic bile and gallstones during loss of weight. N. Engl. J. Med. 1988;319:1567–72. 27. Shiffman ML, Kaplan GD, Brinkman-Kaplan V, et al. Prophylaxis against gallstone formation with ursodeoxycholic acid in patients participating in a very-low-calorie diet program. Ann. Intern. Med. 1995;122:899–905. 28. Wang HH, Afdhal NH, Gendler SJ, et al. Targeted disruption of the murine mucin gene 1 decreases susceptibility to cholesterol gallstone formation. J. Lipid Res. 2004b;45:438–47. 29. Bodmer M, Brauchli YB, Krahenbuhl S, et al. Statin use and risk of gallstone disease followed by cholecystectomy. JAMA 2009;302:2001–7. 30. Wang HH, Portincasa P, Mendez-Sanchez N, et al. Effect of ezetimibe on the prevention and dissolution of cholesterol gallstones. Gastroenterology 2008;134:2101–10. 31. Freudenberg F, Broderick AL, Yu BB, et al. Pathophysiological basis of liver disease in cystic fi brosis employing a DeltaF508 mouse model. Am. J. Physiol. Gastrointest. Liver Physiol. 2008;294:G1411–20. 32. Freudenberg F, Leonard MR, Liu SA, et al. Pathophysiological preconditions promoting mixed "black" pigment plus cholesterol gallstones in a DeltaF508 mouse model of cystic fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 2010;299:G205–14. 33. Strassburg CP. Hyperbilirubinemia syndromes (Gilbert-Meulengracht, Crigler-Najjar, Dubin-Johnson, and Rotor syndrome). Best Pract. Res. Clin. Gastroenterol. 2010;24:555–71. 34. Wasmuth HE, Keppeler H, Herrmann U, et al. Coinheritance of Gilbert syndromeassociated UGT1A1 mutation increases gallstone risk in cystic fibrosis. Hepatology 2006;43:738–41. 35. Rosmorduc O, Hermelin B, Boelle P-Y, et al. ABCB4 gene mutation-associated cholelithiasis in adults. Gastroenterology 2003;125:452–9. 36. Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defects in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001;120:1449–67.

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37. Lammert F, Wang DQ, Hillebrandt S, et al. Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis. Hepatology 2004;39:117–28. 38. van Mil SW, van der Woerd WL, van der Brugge G, et al. Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology 2004;127:379–84. 39. Schafmayer C, Tepel J, Franke A, et al. Investigation of the Lith1 candidate genes ABCB11 and LXRA in human gallstone disease. Hepatology 2006b;44:650–7. 40. Khanuja B, Cheah YC, Hunt M, et al. Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice. Proc. Natl. Acad. Sci. USA 1995;92:7729–33. 41. Lyons MA, Wittenburg H. Cholesterol gallstone susceptibility loci: a mouse map, candidate gene evaluation, and guide to human LITH genes. Gastroenterology 2006;131:1943–70. 42. Paigen B, Schork NJ, Svenson KL, et al. Quantitative trait loci mapping for cholesterol gallstones in AKR/J and C57L/J strains of mice. Physiol. Genomics 2000;4:59–65. 43. Stokes CS, Lammert F. Transporters in cholelithiasis. Biol. Chem. (in press) 2011b. 44. Wittenburg, Stumvoll, Tönjes, unpublished results. 45. Buch S, Schafmayer C, Volzke H, et al. Loci from a genome-wide analysis of bilirubin levels are associated with gallstone risk and composition. Gastroenterology 2010;139:1942–51. 46. Wittenburg H, Lyons MA, Li R, et al. FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice. Gastroenterology 2003;125:868–81. 47. Kovacs P, Kress R, Rocha J, et al. Variation of the gene encoding the nuclear bile salt receptor FXR and gallstone susceptibility in mice and humans. J. Hepatol. 2008;48:116–24. 48. Renner O, Harsch S, Schaeffeler E, et al. A variant of the SLC10A2 gene encoding the apical sodium-dependent bile acid transporter is a risk factor for gallstone disease. PLoS One 2009;4:e7321. 49. Tönjes A, Wittenburg H, Halbritter J, et al. Effects of SLC10A2 variant rs9514089 on gallstone risk and serum cholesterol levels - meta-analysis of three independent cohorts. BMC Med. Genet. 2011;12:149. 50. Wittenburg H, Lyons MA, Li R, et al. Association of a lithogenic Abcg5/Abcg8 allele on Chromosome 17 (Lith9) with cholesterol gallstone formation in PERA/EiJ mice. Mamm. Genome 2005;16:495–504. 51. Buch S, Schafmayer C, Volzke H, et al. A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease. Nat. Genet. 2007;39:995–9. 52. Krawczyk M, Wang DQ, Portincasa P, et al. Dissecting the genetic heterogeneity of gallbladder stone formation. Semin. Liver Dis. 2011;31:157–72. 53. Gylling H, Hallikainen M, Pihlajamaki J, et al. Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity. J. Lipid Res. 2004;45:1660–5.

13 Molecular basis of primary biliary cirrhosis Simon Hohenester and Ulrich Beuers

13.1 Introduction Primary biliary cirrhosis (PBC) (1) is an immune-mediated chronic progressive inflammatory liver disease that leads to the destruction of small interlobular bile ducts and progressive cholestasis. Without medical treatment, PBC commonly progresses to fibrosis and cirrhosis of the liver, necessitating liver transplantation. PBC predominantly affects middle-aged women; it is characterized by biochemical markers of cholestasis, serum antimitochondrial autoantibodies (AMA), and lymphocytic infiltration of the portal tracts of the liver (2). Histologically, the hallmark of the disease is damage to biliary epithelial cells and loss of small intrahepatic bile ducts accompanied by significant portal tract infiltration with CD4+ and CD8+ T-cells, B-cells, macrophages, eosinophils and natural killer cells (3,4). The combination of focal duct obliteration with granuloma formation (“florid duct lesion”) is considered typical of PBC but is not frequently found. (Stage 1: portal lymphocellular inflammation with or without granulomatous destruction of bile ductules; portal fibrosis. Stage 2: portal and periportal inflammation (interface hepatitis) and bile duct proliferation; periportal fibrosis. Stage 3: septal fibrosis. Stage 4: cirrhosis.) Although there has been tremendous progress in unraveling potential pathophysiological factors in PBC over recent years (5), debate about the actual impact of each of the identified genetic and environmental associations is still ongoing. It is the aim of this chapter to review the current knowledge of the major pathological features of PBC.

13.1.1 Epidemiology of PBC PBC occurs in individuals of all ethnic origins and accounts for up to 2.0% of deaths from cirrhosis (6). Incidence and prevalence vary strikingly in different geographic regions (as does the quality of epidemiological studies related to PBC), ranging from 0.7 to 49 and 6.7 to 402 per million, respectively (7–14). The highest incidence and prevalence rates are reported from the United Kingdom (7,12), Scandinavia (8), Canada (9), and the United States (10,13), all in the northern hemisphere, whereas the lowest are found in Australia (11). There is no clear evidence to support or exclude the concept of “a polar-equatorial gradient,” as has been reported for other autoimmune conditions (15). Median survival in untreated individuals has been reported to be 7.5 to 16 years (1,16), but this has largely improved since the introduction of ursodeoxycholic acid (UDCA) therapy and liver transplantation. Most patients treated with UDCA monotherapy at an early stage of the disease generally respond well and have a normal life expectancy (17–20). However, one third of patients do not respond to UDCA monotherapy to an adequate degree.

13.1.2 Diagnosis in clinical practice Increased awareness of the condition and the increasing availability of diagnostic tools, in particular serological testing, have led to the more frequent and earlier diagnosis of

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Female preponderance 9:1 Age at manifestation 40–60 Survival (without treatment) 7.5–16 years Cholestatic pattern Autoantibodies

AP, GGT앖 AMA (anti-PDC-E2)

Signs & symptoms

Fatigue Pruritus Jaundice “Dry eye, dry mouth”

Fig. 13.1: Characteristics of primary biliary cirrhosis (PBC). PBC affects the small interlobular bile ducts, leading to progressive cholestasis, liver fibrosis, and cirrhosis.

PBC (21). More than half of the patients diagnosed with PBC today are asymptomatic at presentation (22,23). Asymptomatic patients generally attract attention by findings of elevated serum alkaline phosphatase (AP) and/or total serum cholesterol during a routine checkup. According to current consensus (24), a diagnosis of PBC is made when biochemical markers of cholestasis, particularly alkaline phosphatase, are elevated persistently for more than 6 months in the presence of high titers of serum AMA and the absence of an alternative explanation (25,26) (fFig. 13.1). Compatible histological findings confirm the diagnosis and allow staging before therapeutic intervention, but in many cases histological workup is not necessary to diagnose PBC. For a more in-depth discussion of the diagnostic approach in PBC (fFig. 13.1) and a discussion of the accompanying biochemical and serological alterations, the reader is referred to recent guidelines and reviews (24,27).

13.1.3 Clinical features of PBC At diagnosis, the majority of patients are asymptomatic and generally present for workup after detection of elevated serum levels of AP or cholesterol (28,29). In symptomatic patients, fatigue and pruritus are the most common complaints; these have been reported in 21% and 19% of patients, respectively, at initial presentation (23,30). During the course of the disease, the prevalence of these symptoms rises to up to 80% and 70%, respectively, sometimes heavily impairing quality of life and interfering with daily activities (31–33) (fFig. 13.1).

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13.1.4 Pharmacological therapy 13.1.4.1 Treatment with ursodeoxycholic acid Administration of ursodeoxycholic acid (UDCA) is the therapeutic mainstay in PBC (24). UDCA has been shown to improve serum biochemical markers such as bilirubin, GGT, cholesterol, and IgM levels (34–39) as well as AP, the most important prognostic marker (40). UDCA may slow the histological progression to liver cirrhosis (38,41,42), improve quality of life, and extend survival free of transplant as well as overall survival (17–20,43). Therefore the survival of patients with early-stage disease who respond to UDCA therapy is similar to that of control populations (44). The mechanisms of action of UDCA in chronic cholestasis are manifold and are discussed in detail elsewhere (27,45) (fFig. 13.2). About one third of patients are not sufficiently controlled with UDCA monotherapy (18,19), which is driving the search for additional therapeutic approaches.

13.1.4.2 Immunosuppressants and novel pharmacological approaches Corticosteroids and other immunosuppressive agents have been evaluated for therapeutic use in PBC and were found to be partially beneficial. Serious side effects of long-term glucocorticoid treatment can outweigh the potential benefit. In this respect, the introduction of budesonide, a corticosteroid with an extensive first-pass metabolism, has been a promising innovation. In two short-term trials in early-stage PBC, the beneficial effects of budesonide in addition to UDCA clearly outweighed the moderate side effects (46,47). Other immunosuppressive agents – including azathioprine, cyclosporine, mycophenolate mofetil, methotrexate, and drugs with antifibrotic properties including penicillamine, colchicine, and silymarin – have not been shown to markedly improve the natural history of the disease or were associated with significant toxicity during long-term treatment (1,48–56). Novel concepts for medical therapy of PBC, in combination with UDCA or alone, have recently been proposed, particularly for use in patients with incomplete responses to UDCA (fFig. 13.2).

Primary biliary cirrhosis: Immunologic bile duct injury

Therapy Budesonide?

Aggravation of bile duct injury by hydrophobic bile acids Cholestasis with retention of hydrophobic bile acids in liver

Ursodeoxycholic acid

Liver cell damage, apoptosis, necrosis, fibrosis, cirrhosis

Nuclear receptor agonist? • FXR agonists? • PPAR agonists?

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Fig. 13.2: Pathogenesis and therapy of PBC.

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The farnesoid X receptor (FXR) agonist 6-ethyl CDCA or obeticholic acid (OCA) has been tested in monotherapy and in addition to UDCA, and has beneficially affected the important prognostic marker AP (57,58). Results of pending phase III trials are thus eagerly awaited. An antiretroviral strategy has been tested in PBC: lamivudine in combination with zidovudine normalized AP and reduced bile duct injury in a 1-year pilot trial including 11 patients (59). This finding, however, awaits confirmation by a high-quality randomized placebo-controlled study. The peroxisome proliferator-activated receptor D (PPARD) agonist bezafibrate was reported to improve serum liver tests in PBC (60) and should undergo more extended evaluation in patients with PBC who have responded inadequately to UDCA.

13.2 Pathogenesis of PBC PBC is often referred to as a model autoimmune disease, being one of the first conditions in which the presence of autoantibodies in serum was identified and in which the antigen specificity was characterized (3). The predominant autoreactive antibodies in PBC are AMAs, which show an outstandingly high sensitivity and specificity, rendering them virtually pathognomonic in PBC. Targets of AMA are all members of the family of 2-oxo-acid dehydrogenase complexes (2-OADC), including the best-characterized epitope in PBC, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) (61), localized within the inner mitochondrial matrix. The autoreactive CD4+ and CD8+ T cells infiltrating the liver in PBC and the dominant autoreactive B-cell epitope all recognize the same PDC-E2 domain (62–64). Accumulating data suggest that the breakdown of self-tolerance is initiated by environmental trigger(s) acting on a genetically susceptible individual (65), as outlined in this chapter.

13.2.1 Genetic factors/susceptibility Genetic factors have an impact on PBC pathogenesis that is stronger than that in nearly any other autoimmune disease (66,67), with a concordance rate of about 60% in monozygotic twins (68,69). Furthermore, the relative risk of a first-degree relative of a PBC patient is 50- to 100-fold higher than that for the general population (70), yielding a prevalence rate up to 5%–6% in this group (10,71,72).

13.2.1.1 HLA family PBC is associated with the DRB1*08 family of alleles, although marked variation is observed among different ethnic groups. Association with the DRB1*0801-containing haplotype is seen in populations of European origin, whereas an association is seen with the DRB1*0803 allele in populations of Asian origin. A protective association has been described with DRB1*11 and DRB1*13; but once again, significant population differences are observed (73–82). Both associations of PBC with the DRB1*08 allele as well as the protective association of DRB1*11 and DRB1*13 were recently confirmed in the largest series ever reported, including 664 unrelated patients from Italy (83). The odds ratio (OR) for developing PBC was 3.3 for DRB1*08-positive subjects, whereas it was reduced to 0.3 for subjects positive for DRB1*11, to 0.7 for DRB1*13, and

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to 0.1 for carriers of both DRB1*11 and DRB1*13. This study highlights the relevance of geographic variation, with marked differences in allele association between northern and southern Italy.

13.2.1.2 Other genes associated with immunity Allelic variations of tumor necrosis factor D (TNF-D) and of cytotoxic T-lymphocyte antigen 4 (CTLA-4), a key regulator of the adaptive immune system, are associated with PBC (84–90). Poupon et al. confirmed a potential role of TNF-D and CTLA-4 variants in the pathogenesis of PBC (91) and showed a strong association of the allelic variant TNF-D rs1799724 (C/T) with disease progression. Various associations with other loci have been described in individual populations, mostly of limited size. However, the vast majority have not been confirmed in independent cohorts and to date none of the genetic associations described in PBC have been proved sufficiently (65,68,88).

13.2.1.3 X-chromosomal influence It remains speculative whether the female preponderance (gender ratio up to 10:1) reflects an X chromosome–linked locus of susceptibility or a gender-specific exposure to environmental triggers such as cosmetics (92) or nail polishes (93), as discussed further on. A pathomechanistic role for X-chromosomal genes was supported, however, by the increased frequency of X-chromosome monosomy in PBC (94) as well as reports of PBC in patients with Turner’s syndrome (95). Estrogen signaling might offer an alternative explanation for female preponderance. Studies on polymorphisms in estrogen receptor genes have revealed associations with the disease, at least in some populations (96). At the tissue level, cholangiocytes from PBC patients in the earliest disease stages (but not cholangiocytes from normal controls) express estrogen receptors (97). Agents (such as tamoxifen) able to modulate estrogen receptor–mediated responses have therefore been proposed as novel therapies targeting cholangiocellular homeostasis, and case reports support this approach (98,99).

13.2.2 Exogenous factors triggering disease Epidemiological identification of disease ‘‘hot spots’’ in former industrial and/or coal mining areas (100) and toxic sites (101) gave rise to the hypothesis of chemical environmental factors involved in the pathogenesis of PBC (65). Furthermore, hormone replacement therapy, frequent use of nail polish (93), and smoking (93,102) are associated with an increased risk of PBC. Such xenobiotics may contribute to the pathogenesis of PBC by triggering autoimmune reactions (67,103). The Gershwin group identified a potential pathogenetic pathway of xenobiotics when they modified the main antigen identified so far in PBC, PDC-E2. By replacing lipoic acid, a residue on PDC-E2, by different xenobiotics, reactivity of PBC sera against the epitope was greatly enhanced. One of the most potent xenobiotics in this study was 2-nonynoic acid. Interestingly, the methyl ester of 2-nonynoic acid has a violet-melon-cucumber-peachlike scent (92) and is used as an ingredient in perfumes. It is ranked 2324th out of 12,945 chemical compounds in terms of occupational exposure, with an 80% female preponderance due to its use in cosmetics (104,105). These findings have resulted in the development of an inducible animal model of PBC in C57BL/6 mice: 2-octynoic acid coupled to BSA after a short follow up

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of 8–12 weeks induced manifest autoimmune cholangitis, typical AMA, increased liver lymphoid cell numbers, an increase in CD8+ liver-infiltrating cells as well as elevated levels of serum tumor necrosis factor D and interferon J. Remarkably, unlike many other animal models of PBC, the reported immunogenic response was liver-specific and no inflammation was found in organs other than liver (106). However, the model is still incomplete, most importantly lacking the development of liver fibrosis. Infectious agents have long been suspected of triggering PBC (107). In support of this hypothesis, several bacteria were identified as containing proteins displaying a high degree of sequence homology with human PDC-E2, potentially inducing crossreactive antibodies. In favor of a bacterial etiology, recent data suggest that Toll-like receptor ligands induce an augmented inflammatory response in PBC. The presence of cross-reactive antigens in a proinflammatory environment could thus be able to break tolerance (108,109). Epidemiological and experimental data have further supported this hypothesis. The incidence of urinary tract infections, often induced by Escherichia coli, is high in PBC patients (110,111), and a history of urinary tract infections increases the risk of PBC (93). Another candidate microorganism for the induction of PBC is Novosphingobium aromaticivorans (112). Antibodies against lipoylated bacterial proteins of this ubiquitous organism are present in high titers in patients with PBC but not in healthy subjects. Lactobacilli and Chlamydia, which express proteins with some structural homology with PDC-E2, have also been implicated as putative pathogens, as have Helicobacter pylori and Mycobacterium gordonae (113–117). However, these intriguing associations need further confirmation. Additional objective data are warranted, obtained either from prospectively followed cohorts or through case-control epidemiological approaches, to confirm a role for bacteria in triggering PBC (65). Accumulating but controversially discussed data support the role of a human retrovirus, mouse mammary tumor virus (MMTV), as another infectious trigger of PBC (118–121). A viral trigger could provide explanations for some key phenomena in PBC. PBC can recur rapidly after transplantation (122), with high AMA titers, aberrant expression of the AMA reactive protein on cholangiocytes (123), and histological evidence of disease (124). Furthermore, potent immunosuppressive therapy following transplant is associated with earlier and more aggressive recurrence (125). Because MMTV replication is regulated by a progesterone-responsive element in the promoter region, its association with PBC would also offer an alternative explanation for the female preponderance (126). In a small nonrandomized pilot study, antiretroviral therapy improved inflammatory scores, normalized AP, and reduced bile duct injury in patients with PBC (59). However, these findings await confirmation in a randomized controlled trial, and other groups have raised methodological and mechanistic doubts regarding the role of MMTV in PBC (127–129).

13.2.3 Endogenous factors perpetuating autoimmunity 13.2.3.1 Biliary cell turnover and exposure of autoantigens Increasing evidence suggests that cholangiocellular senescence contributes to the development of PBC. Senescent biliary epithelia seem to support a proinflammatory environment (130), and cholangiocellular apoptosis leads to exposure of otherwise unexposed antigens, triggering autoimmunity. Biliary epithelia in patients with PBC seem to be under significantly increased apoptotic stress compared to healthy controls as

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well as to patients with other causes of inflammatory reactions in the liver, such as chronic viral hepatitis or PSC (131,132). Staining of cholangiocytes from PBC livers with monoclonal antibodies against the mitochondrial PDC-E2 autoantigen showed a specific reaction at the apical surface that was not found in controls (133,134). It was subsequently demonstrated that the apical staining was due to a complex between (auto-)antimitochondrial IgA and PDC-E2, giving rise to speculations that IgA might be a player in the immune-mediated destruction of biliary epithelia (135,136). In other cell types autoantibody recognition of PDC-E2 is abrogated during apoptosis, probably by glutathiolation of the lysine-lipoyl moiety of PDC-E2, but the antigenicity of PDCE2 persists in the apoptotic cholangiocytes, in which glutathiolation does not occur (137–140). Supporting the significance of this observation, Lleo et al. demonstrated that apotopes of biliary epithelial cells induce inflammatory cytokine secretion from macrophages of PBC patients in the presence of AMAs from PBC sera (141). If cholangiocellular apoptosis is a crucial feature in the pathogenesis of PBC, which factors influence cholangiocyte homeostasis? Cholangiocytes are continuously exposed to millimolar concentrations of proapoptotic, hydrophobic bile salts and must have evolved protective mechanisms to evade their devastating effects. We recently put forward the “biliary HCO3⫺ umbrella hypothesis” (fFig. 13.3). We assume that cholangiocytes (and hepatocytes) might prevent the uncontrolled penetration of apolar bile acids from bile into the intracellular space – a prerequisite for their toxic effect – via apical release of high quantities of HCO3⫺ in order to keep the pH above the apical membrane high and bile salts deprotonated and polarized (142) (fFig. 13.3). The first

HO

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H2O + CO2

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Fig. 13.3: Pathogenesis of PBC: a defect of the biliary bicarbonate umbrella? In PBC, expression of the anion exchanger 2 (AE2) and biliary bicarbonate secretion are impaired (144,145). This may lead to a defect in the protective biliary bicarbonate umbrella, thus facilitating passive absorption of proapoptotic apolar bile acids and subsequent cholangiocyte damage (142,143). Figure modified from Beuers (142).

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data from our lab confirm this hypothesis and furthermore highlight the crucial role of anion exchanger 2 (AE2) in the apical HCO3⫺ secretion of human cholangiocytes (143). These new findings offer a mechanistic pathway for the previously suggested role of AE2 in PBC: AE2 expression and HCO3⫺ secretion were found to be low in PBC livers (144,145) and were improved by UDCA therapy (144). Furthermore, it was shown that corticosteroids in addition to UDCA enhanced the expression of AE2 isoforms in human cholangiocytes (146) and improved prognostic markers of PBC in a 3-year placebocontrolled trial including 36 patients with PBC (147). Most interestingly, the AE2 variant rs2303932 (Adenine instead of Thymine) showed a strong association with disease progression in a large cohort study. Multivariant Cox regression revealed that this AE2 variant was an independent prognostic factor for disease progression in PBC under UDCA treatment (91). The role of AE2 variants has since been confirmed in independent cohorts (148).

13.2.3.2 Imbalance in T-cell regulation and control of inflammation In PBC, reduced systemic and tissue levels of Tregs are found, potentially resulting in an insufficient suppression of immune reactions against self (149). This is mimicked in two mouse models with T-regulatory deficiency, which spontaneously develop a PBC-like lymphoid cholangitis together with positivity for anti-PDC-E2 (150,151). One of the models involved transgenic disruption of the IL-2 receptor alpha, which is highly expressed on Tregs. A role for IL-2 signaling defects in the development of PBC was further supported by a report of PBC-like liver disease in a child with an inborn deficiency of IL-2 receptor alpha (152). Despite their great diagnostic importance, a pathogenetic relevance of AMAs has been controversially discussed in the past. Moritoki et al. demonstrated that B-cell depletion in another T regulatory–deficient model of dnTgfb-R2 mice effectively reduced AMA levels and ameliorated liver disease (153). The same group, however, also demonstrated that B-cell depletion preceding the injection of 2-octynoic acid coupled to BSA exacerbated the previously described liver phenotype (154), indicating that either the timing of B-cell depletion or the underlying cause of disease critically define the role of B cells in disease progression. Meanwhile, in a pilot study, rituximab was able to transiently lower AMA levels and improve alkaline phosphatase serum concentrations in PBC patients insufficiently responding to UDCA (155). Additional evidence suggests a role for the innate immune system in the development of PBC. In affected subjects, increased expression of the hepatic Toll-like receptor 3 (TLR-3) was found compared with liver tissues from controls (156). When stimulated by the TLR-3 ligand poly I:C, monocytoid cells from PBC patients secrete proinflammatory cytokines (108). Long-term administration of poly I:C to mice was found to induce AMAs associated with lymphocyte infiltrations of bile ducts (157). Coadministration of poly I:C in the established AMA-positive model of 2-octyonic acid injection exacerbated the systemic inflammatory response and is the first ever PBC mouse model showing a fibrotic response (158). Further evidence suggests a role for TLR-4 and natural killer cells (159).

13.3 Concluding remarks The available data make it attractive to speculate that a defective biliary HCO3⫺ umbrella due to impaired AE2 expression/function may play a central role in the pathogenesis

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of PBC, leading to cholangiocyte sensitization to bile acid–induced apoptosis, autoantigen presentation by apoptotic cholangiocytes, immune stimulation in susceptible individuals, and chronic fibrosing cholangitis, slowly leading to cirrhosis and portal hypertension. The beneficial effect of UDCA, a biliary HCO3⫺ secretagogue, supports this concept. The underlying molecular mechanisms have yet to be further unraveled.

13.4 References 1. Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N. Engl. J. Med. 2005;353:1261–73. 2. Nishio A, Keeffe EB, Gershwin ME. Immunopathogenesis of primary biliary cirrhosis. Semin. Liver Dis. 2002;22:291–302. 3. Yeaman SJ, Kirby JA, Jones DE. Autoreactive responses to pyruvate dehydrogenase complex in the pathogenesis of primary biliary cirrhosis. Immunol. Rev. 2000;174:238–49. 4. Terasaki S, Nakanuma Y, Yamazaki M, et al. Eosinophilic infiltration of the liver in primary biliary cirrhosis: a morphological study. Hepatology 1993;17:206–12. 5. Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis: Convenient and inconvenient truths. Hepatology 2008; 47:737–45. 6. Kaplan MM. Primary biliary cirrhosis. N. Engl. J. Med. 1987;316:521–8. 7. Hamlyn AN, Sherlock S. The epidemiology of primary biliary cirrhosis: a survey of mortality in England and Wales. Gut 1974;15:473–9. 8. Danielsson A, Boqvist L, Uddenfeldt P. Epidemiology of primary biliary cirrhosis in a defined rural population in the northern part of Sweden. Hepatology 1990;11:458–64. 9. Witt-Sullivan H, Heathcote J, Cauch K, et al. The demography of primary biliary cirrhosis in Ontario, Canada. Hepatology 1990;12:98–105. 10. Kim WR, Lindor KD, Locke GR, 3rd, et al. Epidemiology and natural history of primary biliary cirrhosis in a US community. Gastroenterology 2000;119:1631–6. 11. Watson RG, Angus PW, Dewar M, et al. Low prevalence of primary biliary cirrhosis in Victoria, Australia. Melbourne Liver Group. Gut 1995;36:927–30. 12. James OF, Bhopal R, Howel D, et al. Primary biliary cirrhosis once rare, now common in the United Kingdom? Hepatology 1999;30:390–4. 13. Hurlburt KJ, McMahon BJ, Deubner H, et al. Prevalence of autoimmune liver disease in Alaska Natives. Am. J. Gastroenterol. 2002;97:2402–7. 14. Lazaridis KN, Talwalkar JA. Clinical epidemiology of primary biliary cirrhosis: incidence, prevalence, and impact of therapy. J. Clin. Gastroenterol. 2007;41:494–500. 15. Karvonen M, Tuomilehto J, Libman I, et al. A review of the recent epidemiological data on the worldwide incidence of type 1 (insulin-dependent) diabetes mellitus. World Health Organization DIAMOND Project Group. Diabetologia 1993;36:883–92. 16. Lee YM, Kaplan MM. The natural history of PBC: has it changed? Semin. Liver Dis. 2005;25:321–6. 17. Corpechot C, Carrat F, Bahr A, et al. The effect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis. Gastroenterology 2005;128:297–303. 18. Pares A, Caballeria L, Rodes J. Excellent long-term survival in patients with primary biliary cirrhosis and biochemical response to ursodeoxycholic Acid. Gastroenterology 2006;130:715–20. 19. Corpechot C, Abenavoli L, Rabahi N, et al. Biochemical response to ursodeoxycholic acid and long-term prognosis in primary biliary cirrhosis. Hepatology 2008;48:871–7. 20. Kuiper EM, Hansen BE, de Vries RA, et al. Improved prognosis of patients with primary biliary cirrhosis that have a biochemical response to ursodeoxycholic acid. Gastroenterology 2009;136:1281–7.

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14 The molecular basis of primary sclerosing cholangitis Johannes Roksund Hov, Erik Schrumpf, and Tom Hemming Karlsen

14.1 Introduction Primary sclerosing cholangitis (PSC) is a chronic inflammatory bile duct disease characterized by macroscopic biliary strictures and in most cases ultimately cholestatic liver cirrhosis. The molecular basis of the bile duct injury in PSC is not well defined, but several hypotheses have been proposed (fTab. 14.1). These relate to the concept of aberrant T-cell homing, autoimmunity, the effect of toxic bile acids, and the potential “leakage” of proinflammatory molecules from the gut. To what extent each of these mechanisms may contribute in the pathogenesis of PSC is unknown. The goal of this chapter is to integrate knowledge from genetics, experimental studies, and animal models and point to possible molecular mechanisms that can explain some of the clinical and pathologic characteristics of PSC. This discussion proceeds along four axes believed to constitute important elements of PSC (fFig. 14.1).

14.1.1 Clinical characteristics of PSC PSC is a rare disease that predominantly affects young adult males and has an uneven geographic distribution. Population-based studies in northern Europe, North America, and New Zealand report the highest prevalence and annual incidence, in the range of 3.85–16.2 and 0.41–1.3, respectively, per 100,000 individuals, whereas the prevalence in southern Europe and Asia is probably much lower. PSC has a variable course but in general leads to end-stage liver disease after a median of 10–15 years (1). No medical therapy has been shown to slow its progression to end-stage liver disease. Therefore PSC is a common cause of liver transplantation. PSC recurs after liver transplantation in approximately 15%–20% of patients (2). PSC is strongly associated with inflammatory bowel disease (IBD). IBD in PSC is most often classified as ulcerative colitis, but certain clinical characteristics suggest that PSC-related IBD may represent a separate IBD phenotype (3). In northern Europe, up to 80% of PSC patients have concomitant IBD, while lower frequencies are reported in Italy, Spain, and Japan (4). PSC patients have a high risk of cholangiocarcinoma, which affects 4%–13% of these patients (5). In addition, there is a high risk of cancer of the gallbladder (6) and of the large intestine in patients with colitis (5). A minority of patients have normal cholangiography and are classified as small-duct PSC, which has a less severe course (7). Also, approximately 10% of patients have histologic features of autoimmune hepatitis (8), and 20%–25% have other concomitant autoimmune diseases (9,10). Patients with elevated IgG4 may represent a separate disease entity (11), but the diagnostic criteria for this have not yet been precisely defined.

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Tab. 14.1: Leading Hypotheses on the Molecular Basis of Primary Sclerosing Cholangitis Pathogenetic Mechanism

Possible Molecular Mechanisms Involved

The “aberrant homing” hypothesis

– The MAdCAM-1 adressin on sinusoidal endothelium in PSC enables homing of α4β7 T cells to PSC livers, attracted by increased production of the CCL25 chemokine (52,54).

The “autoimmunity” hypothesis

– Shared susceptibility with other autoimmune diseases (9, 10). – Oligoclonal T cells react toward unknown antigen (25,48). – Autoantibodies target possible antigens in cholangiocytes or in neutrophils (mimicking antigens of gut microbiota) (49–51). – Strong associations with certain MHC variants favor immune responses against particular antigen(s) (37).

The “toxic bile” hypothesis

– Lack of phospholipids in the bile due to ABCB4 defects causes insufficient mixed micelle formation and toxic effects of nonmicellar bile acids (78). May affect the degree of progression (84). – Genetic variants in SXR/PXR could affect the ability to detoxify bile acids and consequently disease progression (88). – CFTR and TGR5 defects may weaken the bicarbonate layer of the biliary epithelium protecting against toxic bile acids (68,80,82).

The “leaky gut” hypothesis

– Cholangiocytes express PRRs for PAMPs translocated from the gut (66,67): Peptidoglycan causes PSC-like changes in rats with small bowel bacterial overgrowth (57); Lipopolysaccharide induces sclerosing cholangitis in Cftr ⫺/⫺ mice with colitis (74). – Antibiotics improve cholangitis in the animal models and hepatic biochemistries in PSC patients (99). – Increased numbers of innate immune cells in PSC livers (22,55,56).

MHC: major histocompatibility complex; PAMP: pathogen-associated molecular pattern; PPR: pattern recognition receptor; PSC: primary sclerosing cholangitis.

14.1.2 Pathologic characteristics of PSC PSC is diagnosed by cholangiography (except for small-duct disease), which typically shows multifocal strictures and segmental dilatation. No histologic features are diagnostic of PSC, but the tissue may exhibit characteristic features. In a classic paper from 1981, Ludwig et al. suggest an order of histologic alterations: bile duct proliferation, periductal inflammation, periductal fibrosis, ductal obliteration, and loss of bile ducts, which may all be present concomitantly (12). The ductal changes may be associated with portal edema, mild portal and periportal hepatitis, cholestasis, periportal and septal fibrosis, and eventually cirrhosis. The typical PSC lesion reflects “onion skin” periductal fibrosis, but this is not always present. Cholangiectasias – that is, excessively dilated tubular or saccular segments – are also seen in PSC livers (13), suggesting that the disease process leads to complex alterations in the extracellular matrix involving stricturing and dilatation. Dysplasia of the biliary epithelium is present in more than a third of explanted PSC

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Fibrosis / cirrhosis Cancer development

Toxic bile duct injury

Immune mediated bile duct injury

Fig. 14.1: Four important components of the disease process in PSC: (a) Portal inflammation and association with genes of the immune system highlight the role of immune-mediated bile duct injury. (b) Increased toxicity of bile or loss of protective mechanisms may cause toxic bile duct injury. (c) The development of liver cirrhosis ultimately causes end-stage liver disease and the need for liver transplantation. (d) The development of cancer in PSC is related to inflammation and bile acid toxicity but may also be influenced by host factors contributing to PSC per se. © Kari C. Toverud CMI (certified medical illustrator). Printed with permission.

livers and in livers with cholangiocarcinoma, lower-grade neoplasia elsewhere in the liver is also often seen (14).

14.1.3 Overview of genetics in PSC Genetic risk factors are important in PSC, as shown by a 9–39 times increased risk of the disease in siblings (15). This degree of heritability is compatible with PSC being a complex trait wherein several genes interplay with the environment to cause disease. Several genetic risk factors have been identified in PSC through genetic association studies (16–19). These provide clues to the pathogenesis of PSC, but several limitations must be kept in mind in discussing the observed genetic associations. The most common type of genetic variant affects only one nucleotide, so-called single-nucleotide polymorphisms (SNPs). In genetic association studies, genetic variants are related to a phenotype, typically a categorical trait, comparing the frequency of the variants between the categories. A genetic variant associated with disease is regarded as a genetic risk factor. In genomewide association studies (GWAS), which are hypothesis-free, genotyping of SNPs covering variation in most of the genome is performed. However, since the genome DNA is inherited in larger “blocks,” genetic variants are correlated (i.e., are in so-called linkage disequilibrium) and it may be difficult to decide which variant or gene is related to the phenotype. Given the large number of variants investigated in genetic studies, the strength of the statistical association is critical. In a rare disease like PSC, so far only a limited number of variants satisfy strict criteria for “genomewide significance” (P < 10⫺8). Both the statistical strength and uncertainty regarding which gene is primarily associated at a locus should be taken into account in discussing the genetic associations detected so far (fTab. 14.2).

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Tab. 14.2: Genetic Findings in PSC Related to the Disease Processes Shown in fFig. 14.1a Locus

Likely Gene

Possible Roleb

6p21 MHC

Many

Immune, cancer

3p21

MST1

Immune

2q13

BCL2L11

Immune

1p36

TNFRSF14

Immune

4q27

IL2

Immune

9q35

CARD9

Immune

2p15

REL

Immune

16p13

CLEC16A

10p15

Other Genes

Association

Other Associated Conditions

Reference

Robustc

Most immune– diseases

(28)

Robustc

UC, CD

(16,17)

Robustc

Non-Hodgkin lymphoma

(17)

MMEL1

Robustc

UC, MS, RA, CeD, PBC

(19)

IL21

Robust

CeD, alopecia, vitiligo, RA, UC, CD, T1D

(18,19)

Robust

CD, UC, AS

(18,19)

Robust

CeD, RA, CD, UC, psoriasis

(18,19)

Immune

Robust

CeD, MS, T1D, RA, SLE, PBC

(19)

IL2RA

Immune

Suggestive

Alopecia, RA, MS, vitiligo, IBD, T1D

(17)

13q31

GPC6

Toxic

Suggestive

MS, lung cancer, height

(16)

19q13

FUT2

Immune

Suggestive

CD

(19)

2q35

TGR5

Immune, toxic

Suggestive

UC

(16,61)

7q31

CFTR

Immune, toxic

Inconsistent

3q12

PXR/SXR

Fibrosis

Progressiond

PBC, NASH

(88)

7q21

ABCB4

Toxic, fibrosis

Progressiond

PBC

(84)

12p13

NKG2D

Cancer

CCAe

GPX1

PUS10

GPC5

IL8RA, ARPC2

(69–72)

(97)

a The associations are listed according to (tentatively) decreasing statistical strength. bRefers to components of the pathogenesis shown in fFig. 14.1. cGenomewide significant association (P < 5 ⫻ 10⫺8). dAssociated with progression of PSC and not susceptibility. eAssociated with cholangiocarcinoma in PSC and not susceptibility. CCA, cholangiocarcinoma; CD, Crohn’s disease, CeD, celiac disease; MS, multiple sclerosis; NASH, nonalcoholic steatohepatitis; PBC, primary biliary cirrhosis; RA, rheumatoid arthritis; T1D, type 1 diabetes; UC, ulcerative colitis.

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14.1.4 Environmental risk factors in PSC There is a paucity of data on environmental risk factors in PSC, but several studies suggest that smoking is protective (20). This is in contrast to the negative effects in other inflammatory disorders but is in line with the effects in ulcerative colitis (UC). The molecular mechanisms are not known, and nicotine treatment is not beneficial. There is limited evidence suggesting a direct role of microbial pathogens in PSC.

14.2 Immune-mediated bile duct injury Mild to moderate inflammation of the portal areas is a typical finding in PSC. The inflammatory infiltrates are dominated by CD4-positive T cells but also consist of CD8-positive T cells, γδ T cells, neutrophils, macrophages, and natural killer (NK) cells (21–25). The T cell dominance suggests that adaptive immune responses are important. This is supported by the identification of genetic associations with T cell–related genes in PSC (26) and the fact that many of these associations overlap with other autoimmune diseases. However, as is evident from the following, a dysregulation of innate immune responses in both immune cells and cholangiocytes may be of pathogenetic importance, and the close relationship to IBD further suggests the involvement of both innate and adaptive immune responses.

14.2.1 Adaptive immune responses to foreign or self in PSC PSC has often been classified as an autoimmune liver disease. Circumstantial evidence for autoimmunity in PSC include (a) association with other autoimmune diseases in the patients and relatives (9,10); (b) Infiltration of T cells in the portal tracts (21–25); (c) The presence of autoantibodies (27), and (d) The association with genetic variants in the major histocompatibility complex (MHC) (28). The associations with MHC variants and other immune-related genes provide the strongest evidence for a role of adaptive (autoimmune) responses in the development of PSC.

14.2.1.1 The major histocompatibility complex (MHC) and antigen presentation By far the strongest genetic risk factors in PSC are located in the MHC (also called the human leukocyte antigen complex, HLA) on chromosome 6p21, which was discovered in 1982 (28). About 28% of the 252 expressed genes in this region have immune-related functions, including antigen presentation, cytokines, and complement factors. The genetic architecture characterized by many strongly correlated potential candidate genes makes exact identification of the disease genes extremely difficult. In northern European populations, PSC is strongly associated with the conserved socalled ancestral AH8.1 haplotype, which include the HLA-B*08:01 and DRB1*03:01 variants (28–33). AH8.1 is associated with numerous autoimmune diseases and harbors several variants with proinflammatory functional consequences (34). Which AH8.1 variants contribute to the autoimmune phenotype has not been defined. In diseases with well-defined MHC associations, the classic MHC class I and II genes are often implicated. The association with the MHC in PSC is strongest in the MHC class I subregion, near HLA-B (17). The classical HLA class I molecules (e.g., HLA-B and HLA-C) serve as peptide-presenting molecules to CD8-positive T cells. It could be

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speculated that these are involved in host control of viral infections. Another possible explanation for the HLA class I association in PSC relates to the additional role of the class I molecules as ligands for NK cell receptors (see section 14.2.2.2). Importantly, in controlling for the class I associations in PSC, residual associations are present at class II, suggesting the presence of additional effects in this region. The MHC class II genes have been the focus of many studies. In addition to associations with the HLA-DRB1*03:01, the haplotype carrying DRB1*13:01 has been associated with PSC risk (31,35). PSC risk may also be associated with haplotypes carrying DRB1*15 (serologically DR2, which includes both DRB1*15 and DRB1*16) (29,31,36). Protective effects have been found for haplotypes carrying DRB1*04, DRB1*07 and DRB1*11 (31,33,35). All the above HLA-DRB1 alleles are highly correlated with different HLADQ genes, which are genetically difficult to separate. Thus the class II associations are complex. In a recent study, amino acid positions 37 and 86 encoded by HLA-DRB1 were found to be key determinants of the electrostatic properties of pocket P9 in the HLA-DR molecule, suggesting that molecular structures allowing particular peptides to be presented to T cells could explain the association in PSC (37).

14.2.1.2 Non-MHC genes related to the adaptive immunity Several genetic associations outside the MHC substantiate a role of T cell regulation and break of tolerance in PSC. At the IL2RA and IL2/IL21 loci, associations have been observed in multiple autoimmune diseases besides PSC (17–19,26). Taken together, the findings suggest a role of IL-2 signaling and regulatory T cells (Tregs). Tregs are CD4positive cells that express the transcription factor FOXP3 (transcription factor forkhead box P3) and CD25 (encoded by IL2RA). Tregs depend on IL-2 for development and activity and are key regulators of tolerance by suppressing autoreactive lymphocytes (38). IL-2 is produced in effector T cells and binds an IL-2 receptor complex consisting of three subunits, of which CD25 is critical for high-affinity binding. Loss of FOXP3 or IL2RA function leads to severe lymphoproliferative and autoimmune syndromes, illustrating the immunoregulatory function of Tregs (39–42). Interestingly, both Foxp3⫺/⫺ and Il2ra⫺/⫺ mice exhibit T cell–mediated cholangitis as well as antimitochondrial antibodies and are considered models of primary biliary cirrhosis (PBC). Still, the genetic associations in PSC and the presence of colitis in IL2RA-deficient mice suggest that this mouse model could be relevant also in PSC. Genetic associations with PSC near the BCL2L11 (BCL2-like 11) were recently observed in PSC (17). BCL2L11 encodes the proapoptotic molecule BIM (Bcl-2 interacting mediator of cell death), which is expressed by lymphoid, myeloid, and epithelial cells. Bcl2l11⫺/⫺ mice exhibit abnormally high numbers of most leukocytes and acquire a syndrome of systemic autoimmunity, probably related to the role of BIM in apoptosis of autoreactive leukocytes and in the termination of T cell responses (43). Interestingly, Bcl2l11⫺/⫺ mice exhibit a mononuclear cell infiltration around bile ducts (17). Several autoimmune diseases are associated with genetic variation at 2p15 and 1p36, where REL (reticuloendotheliosis viral oncogene homolog) and TNFRSF14 (tumor necrosis factor receptor superfamily member 14) are the most obvious candidate genes, respectively. The REL gene encodes the c-REL protein, which belongs to the NF-κB family. c-REL is expressed in B and T lymphocytes, macrophages and dendritic cells, and is required for normal function of these cells. The role of REL in autoimmune disease is not

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clear, but it is of interest that c-REL may be involved in the development of Tregs (44). TNFRSF14 is expressed on hematopoietic cells, in particular T cells, and is a receptor for the proinflammatory TNF-related cytokine LIGHT (45). TNFRSF14 may also have inhibitory effects on B and T cells when stimulated by BTLA (B- and T-cell attenuator) (46). Interestingly, Btla⫺/⫺ mice develop an autoimmune hepatitis-like disease including pronounced portal inflammation with a mononuclear infiltrate (47).

14.2.1.3 Specific immune responses/antigens When a disease is classified as autoimmune, it is implied that specific immune responses against host targets do occur. In a single study performed in PSC, the T cells in the portal tracts exhibited a restricted (oligoclonal) T cell–receptor repertoire, suggesting that T cells with receptors for specific antigens predominate (25). Also, a study of intraepithelial T cells of the common bile duct did observe oligoclonality of the T cell receptors, which were stable over time (48). Several autoantigens have been suggested in PSC (27). Antibodies reacting against biliary epithelium have been detected, but the antigens are poorly defined (49,50). The most commonly detected autoantibodies in PSC are atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA). In PSC, atypical pANCA could represent antibodies against the nuclear membrane protein tubulin beta 5, which is similar to the Escherichia coli protein FtsZ (51). Thus autoimmunity could be speculated to be induced by immune responses against gut bacteria and cross-reactivity with self-antigens.

14.2.1.4 Aberrant T-cell homing An open question is where the initiation of adaptive immune responses in PSC takes place. A proportion of the portal tract T cells in PSC carry the α4β7 homing integrin, suggesting that the lymphocytes were primarily activated (imprinted) in the intestine (52). One theory of PSC pathogenesis suggests that these are recruited to the liver because of aberrantly expressed addressins and chemokines (53). Expression of the addressin for the α4β7 integrin, MAdCAM-1 (mucosal addressin cellular adhesion molecule-1), is normally confined to the intestine. In PSC and other inflammatory liver diseases, MAdCAM-1 is also expressed on sinusoidal and portal vein endothelium (52). The α4β7 T cells express CCR9 (chemokine receptor 9) and may be attracted to the liver by the increased production of CCL25 (chemokine ligand 25) in PSC (54). The recruitment of memory T cells imprinted in the gut may explain the inconsistent temporal relationship between IBD and PSC, but it could be argued that the MAdCAM-1 and CCL25 expression may represent physiologic and not necessarily aberrant phenomena.

14.2.2 Innate immune responses in PSC Inflammation is initiated by the innate immune system, which comprises the nonadaptive immune responses. The close relationship between PSC and IBD led to the early theory that bacterial products from the (inflamed) gut induce inflammation in the biliary tract. The hypothesis suggests that pathogen-associated molecular patterns (PAMPs, bacterial products) bind pattern recognition receptors (PRRs) on innate immune cells like dendritic cells, macrophages, and even cholangiocytes. Although the liver is normally tolerant toward food antigens, for example, from the gut, in PSC, these

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PAMPs cause immune activation and the release of proinflammatory cytokines, leading to cholangitis. Because NK cells and Kupffer cells are present in increased number in PSC livers (55,56), these cell types may also be involved in the pathogenesis. A rat model of small intestinal bacterial overgrowth proves the principle that bacterial products from the gut may cause sclerosing cholangitis (57). This model was associated with increased TNF levels, as also observed in human PSC, and was treatable by antibiotics. However, bacterial overgrowth or increased intestinal permeability is not common in PSC patients (58), yet genetic associations in PSC as well as mouse models suggest a role for proinflammatory alterations in innate immune cells in the pathogenesis.

14.2.2.1 Macrophages Thus far the strongest genetic risk factor in PSC outside the MHC complex is located at chromosome 3p21 (17). The most strongly associated variant affects MST1, which encodes the macrophage stimulating protein (MSP). MSP is a preprotein. After activation, MSP inhibits lipopolysaccharide- and cytokine-induced macrophage activation via the RON/Src tyrosine kinase (59). The PSC-associated variant has a 10-fold lower affinity to its receptor (60), suggesting that the variant has a proinflammatory effect. A suggestive PSC association has been observed at 2q35, where the membranebound bile acid receptor TGR5 (GPBAR1, G protein–coupled bile acid receptor 1) is one of the candidate genes (16,61). Whether or how the associated common TGR5 variant affects TGR5 function is not known. However, several rare mutations negatively affecting the function of TGR5 have also been detected in PSC and could be speculated to contribute to disease development. TGR5 is strongly expressed in Kupffer cells and other macrophages, and bile acid stimulation of TGR5 inhibits the release of cytokines after activation (62), most likely mediated via NF-κB (63). Thus, the TGR5 association could also have proinflammatory effects on macrophages. At chromosome 9q34, CARD9 is of particular interest (18,19). CARD9 protein is expressed in myeloid cells and is an important downstream mediator of signaling from PRRs like NOD2 and toll-like receptors. The association is also present in IBD and is linked with altered gene expression. Taken together, the association provides the first genetic link to dysregulated immune responses against the microbiota in PSC.

14.2.2.2 Natural killer cells NK cells are cytotoxic immune-modulating lymphocytes. A balance of activating and inhibitory signals regulates their activation state. Whereas the focus of MHC studies in PSC has often been antigen presentation, another possible explanation for the strong MHC class I association in PSC relates to the role of the class I molecules as ligands for NK cell receptors. The HLA-C and HLA-B genes encode molecules that not only present peptides to T cells but also act as ligands for killer immunoglobulin–like receptors (KIRs). These ligands may be classified as weak or strong inhibitory ligands for KIRs. The weak inhibitors are associated with PSC risk whereas the strong inhibitors are protective (36,64), suggesting that increased activity of NK cells, or subsets of T cells carrying KIRs, are involved in PSC. Genetic variation in the nearby MICA gene, which encodes a ligand for the activating NK cell receptor NKG2D, has also been associated with PSC (30,32), but this observation may relate to strong linkage disequilibrium (LD) (36,64).

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Given the complexity of the MHC associations, functional studies of KIR-expressing lymphocytes in PSC are necessary to further elucidate the role of these cells.

14.2.2.3 Biliary innate immunity The biliary epithelium actively participates in inflammation (65). Cholangiocytes express PRRs and may be activated by PAMPs in the bile (66,67). “Reactive” cholangiocytes release inflammatory cytokines affecting leukocytes and parenchymal cells as well as mediators inducing local reparative processes and fibrosis (65). Although these are general features of the biliary epithelium in cholangiopathies, PSC could involve more specific alterations in cholangiocytes, like the observed upregulation of PRRs when cholangiocytes are exposed to serum antibodies from PSC patients (67). Cystic fibrosis may be accompanied by hepatobiliary pathology similar to PSC (68), but a role of CFTR (cystic fibrosis transmembrane conductance regulator) defects in PSC is controversial (69–72). Mutations in CFTR are usually thought to cause toxic bile duct damage (see section 14.3), but it has been shown that the induction of colitis in Cftr⫺/⫺ mice causes cholangitis mediated via the PRR TLR4 and NF-κB (73,74). Thus loss of CFTR function changes the innate immunity of the biliary epithelium and increases the susceptibility to bile duct pathology induced by lipopolysaccharide from the gut. This gut-liver link could be relevant for PSC, given the reduced CFTR function reported in PSC patients, even in the absence of CFTR mutations (69,70). The association with FUT2, which encodes an enzyme (galactoside 2-alpha-L-fucosyltransferase 2) involved in protein glycosylation, could also be related to the innate immunity of the bile ducts and the gut. Nonfunctional FUT2-enzyme result in an inability to synthesize blood antigens on the mucosa, referred to as nonsecretor status. Nonsecretor status may affect susceptibility to infectious agents (75), possibly by altering the binding of pathogen to receptors on mucosal surfaces. Secretor status has been shown to influence the composition of the colonic microbial community (76), suggesting that the effect of gene-microbiota interactions should be investigated in PSC.

14.3 Toxic bile duct injury Bile acids accumulate in the liver during cholestasis and their toxic effects may contribute to the development of cirrhosis. However, toxic effects on bile ducts could also be speculated to be a primary event in PSC, either because of altered composition of the bile or because of increased vulnerability of the bile duct epithelium.

14.3.1 Bile composition One example of altered bile composition causing disease is the destructive cholangitis with periductal fibrosis seen in lithocholic acid fed mice (77). Another example is caused by ABCB4 mutations. ABCB4 is a canalicular phospholipid floppase important for biliary phospholipid excretion, and loss of phospholipids leads to insufficient formation of mixed micelles of phospholipids and bile acid. Homozygous mutations lead to toxic effects of nonmicellar bile acids and a PSC-like disease in mice and progressive familial intrahepatic cholestasis type 3 in humans (78). Heterozygosity or less severe ABCB4 mutations may contribute to milder cholestatic phenotypes with portal fibrosis

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(79), which in a minority of cases may resemble PSC. No studies support ABCB4 as a major determinant of PSC susceptibility, but ABCB4 variants have been suggested to modify disease progression in PSC (see section 14.4).

14.3.2 The biliary epithelium One hypothesis explaining increased vulnerability of the biliary epithelium discusses possible defects of a biliary bicarbonate “umbrella” (80). Cholangiocytes secrete bicarbonate, contributing to bile flow, proper viscosity and alkalinization. Keeping the bile alkaline just above the apical membrane could be one way to protect against bile acid toxicity. Elements involved in bicarbonate formation and excretion could cause cholangiopathies. In this regard, the role of the bile acid receptor TGR5 is of particular interest. TGR5 is expressed in the primary cilium of cholangiocytes and may possibly act as a sensor for bile acids (81). TGR5 colocalizes with the chloride transporter CFTR, and specific stimulation of TGR5 activates CFTR, indicating a role of TGR5 in the regulation of cholangiocyte secretion of fluids and electrolytes (82). Defects in the CFTR/TGR5 axis could therefore be speculated to be related to PSC. Suggestive associations in the GPC5/GPC6 genes at chromosome 13 could contribute to a proinflammatory and vulnerable biliary epithelium (16). The causative variant(s) in these large neighboring genes are not known, but Gpc6 silencing in a murine cholangiocyte cell line upregulates the expression of the nuclear bile acid receptor farnesoid X receptor (NR1H4) as well as several proinflammatory mediators.

14.4 Fibrosis and cirrhosis Whether the fibrotic process in PSC is a primary event or secondary to (localized) cholestasis is not known. The distribution of disease in PSC livers is nonuniform (patchy). There is therefore no good correlation between disease duration and histologic stage as determined by transcutaneous biopsy (12). Still, the degree of stricturing, exemplified by, for example, the presence of a dominant stenosis, influences the disease course. In chronic liver diseases, hepatic stellate cells (HSC) are thought to be the main source of collagen, while portal myofibroblasts around portal tracts seem to be of particular importance in fibrosis in cholestatic liver disease (83). In Abcb4⫺/⫺ mice, increased toxicity of bile leads to disruption of epithelial tight junctions, leakage of bile acids, activation of portal myofibroblasts, periductal fibrosis, and obliterative cholangitis (78). Effects of ABCB4 variants on progression or disease stage in PSC and PBC suggest that ABCB4 function may modify the disease process (84,85). Similar observations have been made for of the PXR/SXR (pregnane X receptor/steroid and xenobiotic receptor) gene in PSC, PBC, and noncholestatic liver disease (86–88). Activation of PXR is important for upregulation of bile acid detoxification mechanisms, may inhibit HSC proliferation and has anti-inflammatory effects via the NF-κB pathway (89,90). Activated and proliferating cholangiocytes may undergo neuroendocrine differentiation (69) and may also directly stimulate the fibrotic process by releasing fibrogenic mediators (83). Interestingly, lipopolysaccharide has been shown to disrupt cholangiocyte tight junctions in vitro (91), and it could be speculated that proinflammatory stimuli from the gut directly influence fibrosis due to bile acid leakage.

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14.5 Cancer development Cholangiocarcinoma is caused by malignant transformation of cholangiocytes, a sequential process via low- and high-grade dysplasia to cancer (14). This malignant transformation is caused by accumulating somatic mutations and epigenetic alterations, but host susceptibility may play a role.

14.5.1 Malignant transformation of cholangiocytes Somatic mutations in cholangiocarcinomas have been identified in genes implicating neoplastic features like autonomous proliferation, evasion of apoptosis, escape from senescence, and tissue invasiveness and metastasis (5). Bile acids have been implicated in multiple gastrointestinal cancers and may contribute to the carcinogenesis in PSC by stimulating the production of reactive oxygen species in cholangiocytes, leading to DNA damage (92). Inflammatory cytokines may contribute similarly by stimulating inducible nitric oxide synthase activity, which generate reactive nitric oxide species, known to cause DNA damage as well as inactivation of DNA repair enzymes (93). Interleukin-6 (IL-6) is produced at high levels by cholangiocarcinoma cells, and autocrine effects of IL-6 may play a key role in tumor development (94). Via the actions of STAT-3, IL-6 upregulates molecules associated with inhibition of apoptosis and stimulation of proliferation. STAT-3 activation is normally kept in check by negative feedback mediated via SOCS-3, but in cholangiocarcinomas epigenetic alterations leads to SOCS-3 silencing and unrestricted STAT-3 activity. Overall, this renders the cholangiocarcinoma cells resistant to apoptosis, a situation worsened by further increases in IL-6 production stimulated by other inflammatory cytokines.

14.5.2 Genetic predisposition to cholangiocarcinoma in PSC The risk of cholangiocarcinoma in PSC is not related to disease stage or duration. In fact, 30%–50% of cholangiocarcinomas in PSC are diagnosed within 1–2 years after PSC diagnosis and less than 10% after 10 years, suggesting a role of host factors (1,95). Cancer predisposition could, for example, be related to defective DNA repair systems. Hypomorphic mutations have been detected in such enzymes in PSC but at low frequencies (96). Still, these could be of importance in the individuals in question. The body continuously fights neoplasia through immunosurveillance by, for instance, T cells and NK cells. A statistical association has been observed between genetic variants of the activating NK cell receptor NKG2D and cholangiocarcinoma in PSC (97). Interestingly, homozygosity for the nonrisk variants was associated with an almost complete protection. In the same study, a suggestive protective association with MICA, which encodes an NKG2D ligand expressed during cellular stress, was also observed. Similar MICA associations have been observed for hepatocellular carcinoma in chronic hepatitis C infection (98).

14.6 Concluding remarks Recent advances in the study of the molecular basis of PSC have highlighted genetic defects affecting T cell activity and tolerance. The strong MHC associations in PSC also

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points to an important role of adaptive immunity, and further studies of the MHC in PSC seem crucial to understanding the initiating events of the disease. Bile duct pathology is seen in several animal models with defects in PSC-associated T cell–related genes. This could imply that the biliary tract is a “weak spot” among the mucosal surfaces, critically dependent on T cell–mediated immune tolerance. Several mechanisms have been proposed to explain the relationship between PSC and IBD, including liver homing of T cells imprinted in the gut. Animal models show that increased PAMP load from the gut as well as altered innate immunity of the bile ducts may lead to cholangitis. Of interest is the existence of shared susceptibility genes between IBD and PSC as well as more disease-specific variants (fFig. 14.2), which in part could explain the link between the diseases. Of these, proinflammatory genetic variants affecting the innate immune responses of NK cells and macrophages could also contribute to a proinflammatory hepatic environment. The vulnerability of the bile duct epithelium may involve not only innate immunity but also defects related to bile acid homeostasis and specific protective measures against toxic bile (e.g., the biliary bicarbonate “umbrella”). Whether bile acid toxicity could be of primary importance in PSC or mainly modifies resistance to the injury caused by immunologic injuries is an open question. The sum of bile acid toxicity and chronic inflammation is important to the neoplastic process in PSC, but genetic variation affecting cancer immunosurveillance has been implicated, and it is likely that other host factors are also important. In conclusion, several disease mechanisms likely contribute to the bile duct injury in PSC. Although autoimmunity is highlighted in this text, other pathogenetic mechanisms

IBD UC HNF4A, CDH1, GNA12, LAMB1, IL7R, PRDM1 ++

IL23R, STAT3, IL10, NKX2-3, ORMDL3, TYK2, ICOSLG ++

CD NOD2, IRGM, ATG16L1, CCR6, ERAP2, MUC1 ++

6p21 (HLA) 3p21, CARD9 IL2RA, REL IL2/IL21

2q35, TNFRSF14

FUT2

PSC BCL2L11, GPC6, CLEC16A

Fig. 14.2: Genetic overlap between inflammatory bowel disease (IBD) and primary sclerosing cholangitis (PSC). The figure shows the genetic associations in PSC listed in fTab. 14.2 and how they relate to some of the at least 47 genetic associations in ulcerative colitis (UC) and 71 associations in Crohn´s disease (CD). Genetic susceptibility to these overlapping phenotypes seems to be divided in shared and disease-specific genetic risk factors.

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are also likely to be important and may in some patient subgroups represent the initiating events.

14.7 References 1. Broome U, Olsson R, Loof L, et al. Natural history and prognostic factors in 305 Swedish patients with primary sclerosing cholangitis. Gut 1996;38:610–5. 2. Bjoro K, Brandsaeter B, Foss A, Schrumpf E. Liver transplantation in primary sclerosing cholangitis. Semin Liver Dis 2006;26:69–79. 3. Loftus EV, Jr., Harewood GC, Loftus CG, et al. PSC-IBD: a unique form of inflammatory bowel disease associated with primary sclerosing cholangitis. Gut 2005;54:91–6. 4. Broome U, Bergquist A. Primary sclerosing cholangitis, inflammatory bowel disease, and colon cancer. Semin Liver Dis 2006;26:31–41. 5. Lazaridis KN, Gores GJ. Primary sclerosing cholangitis and cholangiocarcinoma. Semin Liver Dis 2006;26:42–51. 6. Said K, Glaumann H, Bergquist A. Gallbladder disease in patients with primary sclerosing cholangitis. J Hepatol 2008;48:598–605. 7. Bjornsson E, Olsson R, Bergquist A, et al. The natural history of small-duct primary sclerosing cholangitis. Gastroenterology 2008;134:975–80. 8. Alvarez F, Berg PA, Bianchi FB, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999;31:929–38. 9. Saarinen S, Olerup O, Broome U. Increased frequency of autoimmune diseases in patients with primary sclerosing cholangitis. Am J Gastroenterol 2000;95:3195–9. 10. Lamberts LE, Janse M, Haagsma EB, van den Berg AP, Weersma RK. Immune-mediated diseases in primary sclerosing cholangitis. Dig Liver Dis 2011;43:802–6. 11. Mendes FD, Jorgensen R, Keach J, et al. Elevated serum IgG4 concentration in patients with primary sclerosing cholangitis. Am J Gastroenterol 2006;101:2070–5. 12. Ludwig J, Barham SS, LaRusso NF, Elveback LR, Wiesner RH, McCall JT. Morphologic features of chronic hepatitis associated with primary sclerosing cholangitis and chronic ulcerative colitis. Hepatology 1981;1:632–40. 13. Ludwig J, MacCarty RL, LaRusso NF, Krom RA, Wiesner RH. Intrahepatic cholangiectases and large-duct obliteration in primary sclerosing cholangitis. Hepatology 1986;6: 560–8. 14. Lewis JT, Talwalkar JA, Rosen CB, Smyrk TC, Abraham SC. Precancerous bile duct pathology in end-stage primary sclerosing cholangitis, with and without cholangiocarcinoma. Am J Surg Pathol 2009;34:27–34. 15. Bergquist A, Montgomery SM, Bahmanyar S, et al. Increased risk of primary sclerosing cholangitis and ulcerative colitis in first-degree relatives of patients with primary sclerosing cholangitis. Clin Gastroenterol Hepatol 2008;6:939–43. 16. Karlsen TH, Franke A, Melum E, et al. Genome-wide association analysis in primary sclerosing cholangitis. Gastroenterology 2010;138:1102–11. 17. Melum E, Franke A, Schramm C, et al. Genome-wide association analysis in primary sclerosing cholangitis identifies two non-HLA susceptibility loci. Nat Genet 2011;43:17–9. 18. Janse M, Lamberts LE, Franke L, et al. Three ulcerative colitis susceptibility loci are associated with primary sclerosing cholangitis and indicate a role for IL2, REL, and CARD9. Hepatology 2011;53:1977–85. 19. Folseraas T, Melum E, Juran BD, et al. Novel Susceptibility Loci for Primary Sclerosing Cholangitis Identified by Genome-Wide Association and Replication Analysis. J Hepatol 2011;54:S46.

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20. Mitchell SA, Thyssen M, Orchard TR, Jewell DP, Fleming KA, Chapman RW. Cigarette smoking, appendectomy, and tonsillectomy as risk factors for the development of primary sclerosing cholangitis: a case control study. Gut 2002;51:567–73. 21. Whiteside TL, Lasky S, Si L, Van Thiel DH. Immunologic analysis of mononuclear cells in liver tissues and blood of patients with primary sclerosing cholangitis. Hepatology 1985;5:468–74. 22. Hashimoto E, Lindor KD, Homburger HA, et al. Immunohistochemical characterization of hepatic lymphocytes in primary biliary cirrhosis in comparison with primary sclerosing cholangitis and autoimmune chronic active hepatitis. Mayo Clin Proc 1993;68:1049–55. 23. Ponsioen CY, Kuiper H, Ten Kate FJ, van Milligen de Wit M, van Deventer SJ, Tytgat GN. Immunohistochemical analysis of infl ammation in primary sclerosing cholangitis. Eur J Gastroenterol Hepatol 1999;11:769–74. 24. Senaldi G, Portmann B, Mowat AP, Mieli-Vergani G, Vergani D. Immunohistochemical features of the portal tract mononuclear cell infiltrate in chronic aggressive hepatitis. Arch Dis Child 1992;67:1447–53. 25. Broome U, Grunewald J, Scheynius A, Olerup O, Hultcrantz R. Preferential V beta3 usage by hepatic T lymphocytes in patients with primary sclerosing cholangitis. J Hepatol 1997;26:527–34. 26. Karlsen TH, Kaser A. Deciphering the genetic predisposition to primary sclerosing cholangitis. Semin Liver Dis 2011;31:188–207. 27. Hov JR, Boberg KM, Karlsen TH. Autoantibodies in primary sclerosing cholangitis. World J Gastroenterol 2008;14:3781–91. 28. Schrumpf E, Fausa O, Forre O, Dobloug JH, Ritland S, Thorsby E. HLA antigens and immunoregulatory T cells in ulcerative colitis associated with hepatobiliary disease. Scand J Gastroenterol 1982;17:187–91. 29. Donaldson PT, Farrant JM, Wilkinson ML, Hayllar K, Portmann BC, Williams R. Dual association of HLA DR2 and DR3 with primary sclerosing cholangitis. Hepatology 1991;13:129–33. 30. Wiencke K, Spurkland A, Schrumpf E, Boberg KM. Primary sclerosing cholangitis is associated to an extended B8-DR3 haplotype including particular MICA and MICB alleles. Hepatology 2001;34:625–30. 31. Spurkland A, Saarinen S, Boberg KM, et al. HLA class II haplotypes in primary sclerosing cholangitis patients from five European populations. Tissue Antigens 1999;53:459–69. 32. Norris S, Kondeatis E, Collins R, et al. Mapping MHC-encoded susceptibility and resistance in primary sclerosing cholangitis: the role of MICA polymorphism. Gastroenterology 2001;120:1475–82. 33. Wiencke K, Karlsen TH, Boberg KM, et al. Primary sclerosing cholangitis is associated with extended HLA-DR3 and HLA-DR6 haplotypes. Tissue Antigens 2007;69:161–9. 34. Candore G, Lio D, Colonna Romano G, Caruso C. Pathogenesis of autoimmune diseases associated with 8.1 ancestral haplotype: effect of multiple gene interactions. Autoimmun Rev 2002;1:29–35. 35. Donaldson PT, Norris S. Evaluation of the role of MHC class II alleles, haplotypes and selected amino acid sequences in primary sclerosing cholangitis. Autoimmunity 2002;35:555–64. 36. Hov JR, Lleo A, Selmi C, et al. Genetic associations in Italian primary sclerosing cholangitis: heterogeneity across Europe defines a critical role for HLA-C. J Hepatol 2010;52:712–7. 37. Hov JR, Kosmoliaptsis V, Traherne JA, et al. Electrostatic modifications of the human leukocyte antigen-DR P9 peptide-binding pocket and susceptibility to primary sclerosing cholangitis. Hepatology 2011;53:1967–76. 38. Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 2010;33:153–65.

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injury in genetically susceptible rats with experimental intestinal bacterial overgrowth. J Clin Invest 1992;90:1313–22. Bjornsson E, Cederborg A, Akvist A, Simren M, Stotzer PO, Bjarnason I. Intestinal permeability and bacterial growth of the small bowel in patients with primary sclerosing cholangitis. Scand J Gastroenterol 2005;40:1090–4. Correll PH, Morrison AC, Lutz MA. Receptor tyrosine kinases and the regulation of macrophage activation. J Leukoc Biol 2004;75:731–7. Gorlatova N, Chao K, Pal LR, et al. Protein Characterization of a Candidate Mechanism SNP for Crohn’s Disease: The Macrophage Stimulating Protein R689C Substitution. PLoS One 2011;6:e27269. Hov JR, Keitel V, Laerdahl JK, et al. Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis. PLoS One 2010;5:e12403. Keitel V, Donner M, Winandy S, Kubitz R, Haussinger D. Expression and function of the bile acid receptor TGR5 in Kupffer cells. Biochem Biophys Res Commun 2008;372:78–84. Wang YD, Chen WD, Yu D, Forman BM, Huang W. The G-Protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-kappaB) in mice. Hepatology 2011;54:1421–32. Karlsen TH, Boberg KM, Olsson M, et al. Particular genetic variants of ligands for natural killer cell receptors may contribute to the HLA associated risk of primary sclerosing cholangitis. J Hepatol 2007;46:899–906. Lazaridis KN, Strazzabosco M, Larusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 2004;127:1565–77. Harada K, Ohira S, Isse K, et al. Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells. Lab Invest 2003;83:1657–67. Karrar A, Broome U, Sodergren T, et al. Biliary epithelial cell antibodies link adaptive and innate immune responses in primary sclerosing cholangitis. Gastroenterology 2007;132:1504–14. Colombo C. Liver disease in cystic fibrosis. Curr Opin Pulm Med 2007;13:529–36. Sheth S, Shea JC, Bishop MD, et al. Increased prevalence of CFTR mutations and variants and decreased chloride secretion in primary sclerosing cholangitis. Hum Genet 2003;113:286–92. Pall H, Zielenski J, Jonas MM, et al. Primary sclerosing cholangitis in childhood is associated with abnormalities in cystic fibrosis-mediated chloride channel function. J Pediatr 2007;151:255–9. Henckaerts L, Jaspers M, Van Steenbergen W, et al. Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis. J Hepatol 2009;50:150–7. Girodon E, Sternberg D, Chazouilleres O, et al. Cystic fibrosis transmembrane conductance regulator (CFTR) gene defects in patients with primary sclerosing cholangitis. J Hepatol 2002;37:192–7. Blanco PG, Zaman MM, Junaidi O, et al. Induction of colitis in cftr-/- mice results in bile duct injury. Am J Physiol Gastrointest Liver Physiol 2004;287:G491–6. Fiorotto R, Scirpo R, Trauner M, et al. Loss of CFTR affects biliary epithelium innate immunity and causes TLR4-NF-kappaB-mediated inflammatory response in mice. Gastroenterology 2011;141:1498–508, 508 e1–5. Thorven M, Grahn A, Hedlund KO, et al. A homozygous nonsense mutation (428G-->A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections. J Virol 2005;79:15351–5.

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95. Boberg KM, Bergquist A, Mitchell S, et al. Cholangiocarcinoma in primary sclerosing cholangitis: risk factors and clinical presentation. Scand J Gastroenterol 2002;37:1205–11. 96. Forsbring M, Vik ES, Dalhus B, et al. Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma. Carcinogenesis 2009;30:1147–54. 97. Melum E, Karlsen TH, Schrumpf E, et al. Cholangiocarcinoma in primary sclerosing cholangitis is associated with NKG2D polymorphisms. Hepatology 2008;47:90–6. 98. Kumar V, Kato N, Urabe Y, et al. Genome-wide association study identifies a susceptibility locus for HCV-induced hepatocellular carcinoma. Nat Genet 2011;43:455–8. 99. Farkkila M, Karvonen AL, Nurmi H, et al. Metronidazole and ursodeoxycholic acid for primary sclerosing cholangitis: a randomized placebo-controlled trial. Hepatology 2004;40:1379–86.

15 Drug-induced cholestatic liver injury Christiane Pauli-Magnus and Bruno Stieger

15.1 Introduction Drug-induced live injury (DILI) is a form of acquired liver disease and constitutes an important clinical entity. On the one hand, the liver serves as an important barrier beyond the gut wall, preventing the entry of potentially harmful xenobiotics into the body; on the other hand, it plays a central role in the detoxification and elimination of metabolic end products and xenobiotics. This is exemplified by the observation that drugs with significant hepatic metabolism are at a higher risk for inducing DILI (1). Despite the expression of a sophisticated arsenal of enzymes for the detoxification of substances, DILI is often observed in patients with additional risk factors such as lifestyle (e.g., excessive alcohol consumption), female gender, and/or predisposing genetic factors. The exact overall incidence of DILI is not known, and it should be kept in mind that diagnosis of DILI tends to be missed (2). The incidence of serious DILI is high in hospitalized patients and accounts for up to 0.3% of deaths in inpatients (3), whereas adverse drug reactions could account for over 50% of patients presenting with acute liver failure (4). In patients with severe DILI, transplant-free survival is low. An overall survival of 66% is seen in such patients after liver transplantation (5). DILI presents as drug-induced cholestasis, drug-induced hepatitis, or mixed liver injury (6). In addition, DILI may lead to injuries of bile ducts, alone or in combination with cholestasis (7,8). The proportion of DILI cases presenting with cholestasis or mixed liver injury varies and has been reported as ranging from 17% to close to 50% (8). The reason for this rather large range may relate to the fact that most drugs can cause mild, asymptomatic cholestasis leading to minor elevations of serum liver parameters; hence DILI may not be recognized as such. Importantly, the occurrence of DILI is also significant during drug development, leading often to attrition of drugs being developed or even to withdrawal of drugs from the market (9,10).

15.2 Diagnostic criteria of drug-induced cholestasis Diagnosis of DILI, including drug-induced cholestasis, necessitates a detailed clinical assessment of the patient. It should be kept in mind that individual drugs may lead to drug-specific clinical phenotypes, which may make it challenging to arrive at a correct diagnosis (8,11–13). In addition to chronologic criteria, diagnosis of DILI and druginduced cholestasis includes careful exclusion of other causes for liver disease, such as infectious or autoimmune conditions, bile duct obstruction, and malignant disease. Also extrahepatic signs of adverse drug reactions, such as rash, may favor the diagnosis of DILI (3,8,11–15). For the specific identification of patients with drug-induced cholestasis, the classification system established by the Council for International Organization of Medical Sciences (6) should be considered in addition. Elevation of alkaline phosphatase and of bile salts in plasma is associated with bland cholestasis. These two laboratory values rapidly revert to normal after discontinuation of the causative drug.

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Mild characteristics of DILI may even be asymptomatic and disappear as a consequence of adaptation (16).

15.3 Hepatocellular drug concentration 15.3.1 Drug uptake systems As outlined elsewhere in this book, hepatocytes contain a complex array of transporters aimed at maintaining ongoing bile salt secretion by the liver. The bile salt uptake system sodium-taurocholate cotransporting polypeptide (NTCP) is a minor drug transporter (17), while the three hepatocellular members of the organic anion transporting polypeptide (OATPs) – OATP1B1, OATP1B3, and OATP2B1 – are predominantly drug transporters (18–21). In addition, hepatocytes also express members of the SLC22A family encoding organic anion transporters (OATs) (22) and organic cation transporters (OCTs) (23), which are also major drug transporters (fFig. 15.1). NTCP is a sodiumdependent electrogenic transporter capable of mediating uphill (concentrative) transport of its substrates. OATs act as organic anion exchangers (24); for example, in the liver, OAT7, exchanges butyrate for estrone-3-sulfate (25). In contrast, OCTs utilize the inside negative membrane potential to transfer their cationic substrates (26), which could lead to an intracellular accumulation of these substrates. The driving force of OATPs is not yet understood in full detail (21). Taken together, OATPs are very probably bidirectional transporters acting as anion exchangers.

MRP2 쎵

NTCP

2 Na cBS앥

cBS앥

cBS앥 cBS앥

OATPs drugs BSEP

OATs MRP3

MRP4

OCTs

Fig. 15.1: Drug transport and drug induced cholestasis in hepatocytes. Drugs are taken up from the portal blood plasma by members of the OATP, OAT or OCT families. They may inhibit bile salt secretion into the canaliculus by direct inhibition of the bile salt export pump (BSEP). Alternatively, they may inhibit BSEP indirectly by a mechanism requiring canalicular secretion, e.g. mediated by MRP2. In either case, this will lead to an intracellular accumulation of bile salt. Excess bile salts may either be exported by the salvage systems MRP4 and MRP3 or may impair cellular processes such as for example mitochondria and consequently become cytotoxic (see text for details). uBA = unconjugated bile acid; cBS = conjugated bile salt.

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Evidence has been presented that OATPs mediate substrate uptake in exchange for glutathione (27), glutathione-conjugates (28), and bicarbonate (29,30). Glutathione faces an in-to-out gradient in hepatocytes. As a counter ion, it could provide the driving force accumulating OATP substrates in hepatocytes against a concentration gradient. In humans, coadministration of the OATP-inhibitor rifampicin (31) with the antidiabetic glibenclamide leads to an increase of the serum glibenclamide concentration (32). This indicates that glibenclamide could be an OATP substrate. In a rat study, a 50-fold liver–serum difference of glibenclamide concentration has been reported (33). Similarly, the OATP1B3 and NTCP substrate indocyanine green (34) is 10-fold more concentrated in hepatocytes than in the plasma of rats (35). The transport capacity of OATPs can also be modulated from the extracellular site by endo- and xenobiotics. Prostaglandins (36), the steroid metabolites estrone-3-sulfate and estradiol-17βglucuronide (37,38), the drug clotrimazole (39) and herbal components (40,41) have been reported to activate the transport activity of OATPs. This activation is substratedependent, which can be explained by the presence of more than one substrate binding site on OATPs (40,42). Low extracellular pH stimulates many OATPs (30), which could add an additional mechanism of functional modulation of OATPs. All these different and sometimes subtle OATP transport properties may have different impacts on OATP-dependent drug uptake into hepatocytes, depending on the physiologic state of the liver as well as on polypharmacy. Clinical experience clearly shows that DILI including drug-induced liver injury correlates with drug dosing, which in turn affects intracellular drug concentrations (1).

15.3.2 Drug and drug metabolite export systems Next to uptake mechanisms, intracellular drug metabolism as well as the export of drugs and/or their metabolites from hepatocytes influence intracellular drug concentrations. Located in the canalicular membrane, multidrug-resistance protein 1 (MDR1, ABCB1) contributes significantly to the export of drugs from hepatocytes. MDR1 has a very broad substrate specificity, preferentially transporting neutral or positively charged, rather bulky substrates and mediating the transport of numerous drugs such as anticancer drugs, immunosuppressants, antifungal drugs, antihistamines, and protease inhibitors (43,44). The multidrug resistance–associated protein 2 (MRP2, ABCC2) and ABCG2, formerly called breast cancer resistance protein or BCRP (ABCG2), are also expressed in the canalicular membrane and together transport a long list of drugs and drug metabolites into bile (44–48). In addition to biliary export, hepatocytes can transport drugs and/or their metabolites back into the portal blood plasma across the basolateral plasma membrane. This exit function is maintained by MRP3 (ABCC3) and MRP4 (ABCC4), which mediate the transport of drugs as well as of drug metabolites (49,50) (fFig. 15.1). In summary, the interplay of hepatocellular uptake and export of drugs and their metabolites is very complex and affects intracellular drug concentrations. In addition, metabolites produced by phase I and II reactions are potential inhibitors of export systems and, after their excretion, also of uptake transporters. This generates a very complex interplay of different factors influencing hepatic drug disposition, which is tightly interlinked and also regulated at the transcriptional level (51).

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15.4 Hepatic bile salt accumulation Inhibition of the bile salt export pump (BSEP) leads to retention of bile salts in hepatocytes. Consequently bile flow diminishes, which presents a form of acquired cholestasis. Ongoing BSEP inhibition will lead to clinical cholestasis, usually bland cholestasis.

15.4.1 Direct BSEP inhibition Cyclosporine is known to cause cholestasis in susceptible patients (52) and to inhibit ATP-dependent bile salt transport into isolated rat canalicular plasma membrane vesicles (52,53). Cyclosporine is a competitive inhibitor of the cloned rat Bsep (54). Comparison of the inhibition of ATP-dependent taurocholate transport into isolated canalicular plasma membrane vesicles with Bsep expressed in the Sf9 cell system has revealed comparable Ki values (54). This confirmed the validity of the Sf9 cell expression system. Drugs inhibiting human, rat, mouse, or dog BSEP include bosentan, cyclosporine, glibenclamide, rifampicin, rifamycin, and troglitazone (54–60) (fFig. 15.1). The list of drugs reported to inhibit BSEP transport activity is by now rather long (61,62). Drugs having a low IC50 value for BSEP inhibition show an association with the clinical phenotype of DILI (61). In another study, comparison of Cmax values of drugs with IC50 values of BSEP inhibition showed no correlation (62). This indicates that other factors, such as the intrahepatic accumulation of drugs mediated by hepatocellular drug uptake, are important contributors to the development of drug-induced cholestasis. Comparison of a larger number of BSEP inhibitors between rat and human isoforms showed a very good correlation of IC50 values (62). This supports comparable kinetic properties of the different BSEP isoforms (59).

15.4.2 Indirect BSEP inhibition Bosentan is a dual endothelin receptor antagonist, which is taken up into hepatocytes by OATP1B1 and OATP1B3 (63). In clinical trials, bosentan revealed a potential for asymptomatic elevation of transaminases and of serum bile salts with normal serum bilirubin levels (56), suggesting a specific interaction of bosentan with BSEP. The proposed BSEP inhibitory potential of bosentan was confirmed in the Sf9 cell system where both, bosentan and its metabolites are good BSEP inhibitors (56,59). This pathophysiological concept is further supported by the observation that the bosentan-induced elevation of bile salts is dose-dependent and that patients receiving the BSEP inhibitor glibenclamide in addition to bosentan had a higher incidence of DILI (56). Additional investigations of the mechanism of bosentan-induced cholestasis in rats confirmed serum increase of serum bile salts, which was more pronounced after coadministration of glibenclamide (56). Intriguingly, instead of the expected reduction of bile flow expected in a cholestatic condition, bosentan leads to the stimulation rather than inhibition of bile flow in rats (64). This choleretic effect of bosentan depends on the functional activity of Mrp2 (64) (fFig. 15.1), which has been confirmed in the Sf9 cell expression systems. Here, bosentan stimulates the transport activity of rat and human MRP2 (65), thereby increasing bile flow without altering bile salt output but reducing biliary phospholipid output (64). This reduced phospholipid output might lead to an alteration of the properties of the canalicular membrane – for example, phospholipid asymmetry which may have a

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negative impact on hepatocytes (66). PKI166 as a new HER1/HR2 inhibitor has also been reported to inhibit BSEP indirectly (67). Oral contraceptives have been reported to cause acquired cholestasis (68,69). In a rat model of estrogen-induced cholestasis, administration of estradiol-17β-glucuronide (E17βG) leads to acute cholestasis (70). Interestingly, E17βG does not inhibit rat Bsep expressed in the Sf9 cell system. However, if in addition Mrp2 is expressed in the same Sf9 cell vesicles, E17βG is a time- and dose-dependent inhibitor of Bsep mediated bile salt transport (54) (fFig. 15.1). This complements the observation that in rats, canalicular Mrp2 expression is needed for E17βG to develop its cholestatic potential (71). Other groups have reported the same observation and extended it to progesterone sulfate (72,73). These findings suggest a trans-inhibition of BSEP by these steroid metabolites, which must be secreted into the canaliculus prior to inhibition of BSEP.

15.4.3 Intracellular action of bile salts Inhibition of BSEP will lead to an accumulation of bile salts in hepatocytes. Hepatocytes have detergent properties; consequently increased bile salt concentrations can damage mitochondria (74) (fFig. 15.1). Persistent intracellular accumulation of bile salts will induce cytotoxicity and ultimately liver disease (2,75). The antidiabetic drug troglitazone was withdrawn from the market in the wake of severe hepatotoxicity. The exact mechanism of troglitazone-induced DILI still remains unclear, but mitochondrial toxicity has emerged as an accepted concept (76,77). Troglitazone in rats is mainly metabolized to troglitazone sulfate, which is excreted into bile (57). Also, administration of troglitazone to rats leads to a reduction in bile flow (78), and troglizone and its sulfate are potent inhibitors of rat and human BSEP (57,76). It is therefore conceivable that troglitazone and its metabolite lead to an intracellular accumulation of bile salts, which aggravate the toxic effects of troglitazone to mitochondria. In summary, in susceptible patients BSEP may be subject to simple competitive inhibition of the Michaelis-Menten type or be affected by sometimes rather complex indirect mechanisms of BSEP functional alteration (79). Hence identification of the mechanism of BSEP inhibition may need study with specifically expressed BSEP together with animal studies.

15.5 Susceptibility to drug-induced cholestasis Drug-induced cholestasis due to BSEP inhibition is recognized as an important clinical entity. However, the risk for drug-induced cholestasis is low (80). Nevertheless, identification of potential risk factors might additionally lower the risk for drug-induced cholestasis. New risk factors will have to be identified in human studies, which for ethical reasons will be mainly of a retrospective nature. The use of animal models for the identification of new risk factors may not provide the necessary evidence, since such findings cannot always be translated into human terms. This is illustrated by progressive familial intrahepatic cholestasis type 2, which is a severe form of human liver disease and is due to mutations in the ABCB11 gene rendering human BSEP nonfunctional (81), whereas mice with a disrupted Abcb11 gene display a mild phenotype despite the absence of canalicular taurocholate secretion (82). One possible way of identifying susceptibility

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factors would be genomewide association studies, which, for example, have successfully identified a genetic variant of OATP1B1 as a risk factor for statin-induced myopathy (83). Such studies have not been reported for hepatocellular drug uptake systems and drug-induced cholestasis.

15.5.1 Role of drug uptake systems Publications on the identification of variants of drug uptake transporters into hepatocytes are numerous and have recently been summarized (84). In summary, both genetic variants with reduced transport activity as well as studies of protein expression have been published. In general, variants published to date have shown a reduced transporter function (84), which in principle would more likely be protective than a risk factor. However, both OATPs and OCTs (85–88) have been shown to display rather large interindividual variability of protein expression in human liver samples. It is conceivable that individuals with higher uptake transporter expression could import drugs into hepatocytes at a faster rate and thereby would be more prone to DILI.

15.5.2 Role of BSEP It seems very likely that BSEP itself should constitute a major susceptibility factor for drug-induced cholestasis, as it is the sole canalicular bile salt exporter, as outlined elsewhere in this book. In particular, careful investigation of human BSEP expression in a liver bank revealed that protein expression levels of BSEP in healthy liver tissue are subject to considerable interindividual variability (89). It can therefore be speculated that individuals with low BSEP protein levels could be at a higher risk for drug induced cholestasis (79). Furthermore, the analysis of the BSEP protein expression pattern in the above mentioned liver bank has revealed that individuals carrying the homozygous c.1331C variant of ABCB11, which is the most common ABCB11 polymorphism identified so far, tended to have very low BSEP protein levels (89). Consequently the c.1331C allele could be a potential susceptibility factor for drug-induced cholestasis. An association of the c.1331C allele was indeed observed in a cohort of 23 patients with drug-induced cholestasis but not in 13 patients with drug-induced hepatocellular injury and was significantly overrepresented in the cholestasis group (90). Similarly, in patients with intrahepatic cholestasis of pregnancy, the c.1331C allele is significantly overrepresented (91,92). This association was, however, not found in a Swedish cohort of 52 patients with acquired cholestasis of pregnancy, which may also have methodologic reasons (93). Recently the same allelic variant was found to be associated with progression to cirrhosis in patients with hepatitis C (94) and less often with a sustained viral response to therapy (95) in patients with viral genotype 2 and 3. The underlying mechanism may be related to higher intracellular bile salt levels in patients having the c.1331C allele of ABCB11 (96).

15.5.3 Role of efflux transporters for drugs and drug metabolites Thus far no studies specifically investigating the role of MRP2 as a susceptibility factor for drug-induced cholestasis have been reported. However, a potential role of MRP2 in the development of DILI is supported by the association of certain ABCC2 haplotypes with DILI caused by drugs, herbal remedies, or diclofenac (97,98). Further supporting

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a role of MRP2 is its association with altered drug disposition by ABCC2 variants (84). Pharmacogenetic investigations of a potential link of genetic variants of ABCG2, MRP3, and MRP4 to alterations in drug disposition or drug efficacy have yielded conflicting results (84), and the role of these transporters in drug-induced cholestasis remains to be investigated. The central role of MDR1 in drug disposition has triggered numerous pharmacogenetic studies. The results reported so far are highly inconsistent or even contradictory (84). From this perspective, an association of ABCB1 polymorphisms with drug-induced cholestasis is not likely, particularly since quite a few studies have involved cyclosporine. Clearly more work is needed here, which is hampered by the (luckily) very low incidence of drug-induced cholestasis.

15.6 Concluding remarks Drug-induced cholestasis is a rare but important clinical entity. A great deal of mechanistic understanding of this disease has already been achieved, clearly delineating a pathophysiologic key role of the functional interplay of different hepatocellular uptake and excretion systems for bile salts and xenobiotics. However, strongly prognostic risk factors have not yet been identified. One of the strongest (if not the strongest) genetic risk factors for DILI is the HLA-B*5701 genotype for flucloxacillin-induced DILI (99). However, this genotype accounts for only a small fraction of flucloxacillin-induced DILI. The low incidence of drug-induced cholestasis makes studies of this disease and in particular the identification of risk factors difficult. Clearly, it will be necessary to combine data from many centers involved in the care of patients with drug-induced cholestasis if we are to advance our understanding of risk factors.

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93. Wasmuth HE, Glantz A, Keppeler H, et al. Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene. Gut 2007;56:265–70. 94. Iwata R, Baur K, Stieger B, et al. A common polymorphism in the ABCB11 gene is associated with advanced fibrosis in hepatitis C but not in non-alcoholic fatty liver disease. Clin. Sci(Lond). 2011;120:287–96. 95. Iwata R, Stieger B, Mertens JC, et al. The role of bile acid retention and a common polymorphism in the ABCB11 gene as host factors affecting antiviral treatment response in chronic hepatitis C. J. Viral Hepat. 2011;18:768–78. 96. Stieger B, Geier A. Genetic variations of bile salt transporters as predisposing factors for drug-induced cholestasis, intrahepatic cholestasis of pregnancy and therapeutic response of viral hepatitis. Expert Opin. Drug Metab. Toxicol. 2011;7:411–25. 97. Choi JH, Ahn BM, Yi J, et al. MRP2 haplotypes confer differential susceptibility to toxic liver injury. Pharmacogenet. Genomics. 2007;17:403–15. 98. Daly AK, Aithal GP, Leathart JB, et al. Genetic susceptibility to diclofenac-induced hepatotoxicity: contribution of UGT2B7, CYP2C8, and ABCC2 genotypes. Gastroenterology 2007;132:272–81. 99. Daly AK, Donaldson PT, Bhatnagar P, et al. HLA-B*5701 genotype is a major determinant of drug-induced liver injury due to flucloxacillin. Nat. Genet. 2009;41:816–9.

16 Bile acids and receptors: Therapeutic relevance Anna Baghdasaryan, Thierry Claudel, and Michael Trauner

16.1 Introduction Bile acids (BAs) have been recently identified as highly potent signaling molecules with promising therapeutic properties for a range of hepatic and extrahepatic (e.g., metabolic) disorders (1). BAs such as ursodeoxycholic acid (UDCA) have been used therapeutically for decades and novel BA derivatives are under intensive investigation. Multiple actions of BAs in different tissues are now known to be mediated through activation of dedicated BA receptors, such as the nuclear BA receptor farnesoid X receptor (FXR: NR1H4) and the membrane-bound BA receptor TGR5 (also called GPBAR1 or M-BAR/BG37), which is the focus of this overview. Besides FXR and TGR5, BAs are able to activate other nuclear receptors, such as pregnane X receptor and vitamin D receptor; however, that is beyond the scope of this article (it is reviewed in reference 2).

16.2 Nuclear and membrane BA receptors: general concepts FXR (NR1H4 also known as BAR) was first cloned in 1995 (3,4), and soon thereafter BAs were identified as endogenous and physiologically relevant FXR ligands (5–7). Two FXR isotypes have been identified: FXRα and FXRβ. Whereas both FXRα and FXRβ are functionally active in rodents, only FXRα is functional in humans, while FXRβ is present as a pseudogene (also called ψFXRβ, NR1H5P) (8). Importantly, endogenous BAs differ in their ability to activate FXR. In humans the most potent FXRα activator is the primary BA chenodeoxycholic acid (CDCA) and its conjugates with an EC50 of 5–10 μM (5–7), whereas cholic acid (CA), lithocholic acid (LCA), and deoxycholic acid (DCA) are less potent, and hydroxymetabolites of CDCA, such as muricholic acid (MCA), are not FXR activators. The relevance of FXR is evident from FXR⫺/⫺ mice, which showed high hepatic BA and serum ALT levels (9) and are susceptible to BA-induced liver damage, inflammation, gallstone formation, steatosis, dyslipidemia, altered glucose homeostasis with insulin resistance, disturbed liver regeneration, and defective defense against bacterial overgrowth (9–16). Hence it becomes clear that pharmaceutical FXR activation may be an attractive strategy to treat disorders in which impaired bile, lipid, and glucose homeostasis as well as inflammation and carcinogenesis play critical role. In addition to FXR, a membrane-bound BA-specific receptor TGR5 (also called GPBAR1 or M-BAR/BG37) was discovered in 2002 (17). The most potent endogenous TGR5 activator is LCA, with an EC50 of 0.58 μM, followed by DCA, with an EC50 of 1.25 μM (18), whereas other BAs are less potent for TGR5 activation. Interestingly, gallbladder epithelium and intestine showed high TGR5 expression (19,20). In the liver, sinusoidal endothelial cells, Kupffer cells, and intrahepatic bile ducts show positive TGR5 staining, whereas hepatocytes and the quiescent stellate cells are negative (21–23). TGR5 expression in sinusoidal, inflammatory, and bile duct cells makes it attractive target for treating liver diseases associated with inflammation and bile duct damage.

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16.3 Therapeutic potential of BAs Ursodeoxycholic acid (UDCA) is used for the treatment of cholestatic liver diseases such as primary biliary cirrhosis (PBC) (see Chapter 13) and intrahepatic cholestasis of pregnancy (see Chapter 11). The molecular effects mediated by UDCA are discussed in Chapter 5. Notably, UDCA has poor affinity for BA-dedicated receptors such as FXR and TGR5 (5,6,18,24), is only a weak ligand for glucocorticoid receptor (GR) (24), and may indirectly (after enzymatic modification to LCA by intestinal flora) activate PXR (25,26). Thus interaction of UDCA with multiple receptors may explain part of its therapeutic effects in a wide range of cholestatic and metabolic diseases (e.g., fatty liver). NorUDCA is a side chain shortened modification of UDCA, with relative resistance to amidation, and represents a promising treatment for cholangiopathies, as revealed in the Mdr2 (human MDR3) knockout mouse model of primary sclerosing cholangitis (PSC) (27). norUDCA has anticholestatic, antifibrotic, and anti-inflammatory effects and undergoes cholehepatic shunting, which, in addition, induces bicarbonate-rich and potentially less toxic bile flow (28,29). Moreover, norUDCA also induces phase I and II detoxification enzymes and alternative basolateral efflux systems, resulting in alternative renal BA excretion (27,28) and restoring deranged hepatic lipid metabolism in these mice (30). Notably, so far no NR target of norUDCA has been identified; most likely the therapeutic effects of norUDCA are not mediated by BA receptors such as FXR and TGR5. Preliminary data suggest that norUDCA also reduces hepatic TG content in ApoE⫺/⫺ mice on a western diet; in addition, norUDCA reduced aortic plaques surface area and aortic staining for macrophage markers. The potential mechanisms of norUDCA action may include effects on lipoprotein composition, foam cell formation, and hepatic lipid metabolism (31). These properties make norUDCA a very attractive therapeutic candidate for cholestatic and metabolic liver diseases. Phase I trials have been successfully completed and the results of phase II trials are eagerly awaited.

16.4 Role of BA receptors in BA homeostasis and bile production: therapeutic implications in cholestasis Under cholestatic conditions or in situation of altered protective mechanisms, high concentrations of intrahepatic and systemic BAs (32,33) can solubilize lipid layers, leading to damage of cell membranes and subcellular organelles (34–38). Therefore inhibition of BA synthesis as well as stimulation of BA detoxification and export systems represent attractive strategies to reduce potentially toxic BA concentrations and cell damage in cholestasis. An important mechanism by which FXR can decrease intracellular BA load is the repression of BA synthesis via a feedback mechanism involving FXR-mediated SHP induction in the liver and fibroblast growth factor Fgf15 (human homologue FGF19) upregulation in the intestine (39–41). Interestingly, juvenile-onset cholestasis in FXR/SHP double knockout mice was, in addition to impaired sterol metabolism, associated with BA accumulation in serum, liver, and bile owing to complete lack of a BA synthesis negative feedback loop (42). Besides its role in BA synthesis, FXR activation promotes BA hydroxylation by inducing CYP3A4, sulfoconjugation by inducing SULT2A1, and glucuronidation by inducing UGT2B4 gene expression (2,43). Subsequently, FXR promotes excretion of biliary constituents (44) by inducing BSEP, MDR3 (Mdr2 in rodents)

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and MRP2 gene expression, thus contributing to the regulation of BA-dependent and BA-independent bile flow. Furthermore, FXR activation downregulates the sinusoidal BA uptake transporter NTCP (45) and induces alternative export transporters OSTα and OSTβ, which are highly expressed in the liver and intestine (46–48). Thus FXR activation can decrease the intracellular BA load by turning on several adaptive mechanisms. Multiple FXR agonists have been synthesized as potent pharmaceutical targets to treat cholestatic liver diseases. Among these, obeticholic acid or 6-ethylchenodeoxycholic acid (6-ECDCA), also called INT-747, a semisynthetic derivative of CDCA, has been shown to be approximately 100-fold more potent than CDCA (49). This compound has shown beneficial effects in experimental models of cholestasis. Furthermore, combination therapy of INT-747 together with standard UDCA treatment has been used in phase II clinical trial in PBC patients not responding to UDCA treatment. The combination of INT-747 and UDCA showed a significant reduction of biochemical parameters of liver damage and cholestasis, such as ALT and ALP, after short- and long-term administration (50,51). However, high-dose INT-747 administration was associated with itching. This recent double-blind, placebo-controlled study used INT-747 as a monotherapy in PBC patients. The first results from this trial reported a significant reduction of serum ALT as well as ALP and GGT levels after 12 weeks of treatment. In line with the preceding study, itching was the most common side effect in nonresponders to UDCA, especially at a higher dose (52). Finally, a novel role of FXR in bile secretion was provided by using a novel FXR/TGR5 dual agonist INT-767 in the Mdr2⫺/⫺ (Abcb4⫺/⫺) animal model of sclerosing cholangitis (53). INT-767 is a semisynthetic 23-sulfate-derivative of INT-747 (6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt) (54), which, in contrast to INT-747, is hydrophilic and a more potent FXR activator. When treated with INT-767, Mdr2⫺/⫺ mice showed significantly reduced serum ALT levels, decreased portal inflammation, and biliary fibrosis (53). In addition, INT-767 decreased biliary BA output by inhibiting endogenous BA synthesis. Interestingly, these effects were associated with an FXR-dependent induction of HCO3⫺-rich bile flow. Vectorial transport of HCO3⫺ from cells first requires accumulation of intracellular HCO3⫺ by input transporters and HCO3⫺ generation via the carbonic anhydrase (CA) pathway. Besides their catalytic activity, membrane-bound isoforms of CA may be involved in promoting HCO3⫺ output because of their localization and interaction with the HCO3⫺ transporter (55). Promotion of HCO3⫺-rich bile flow by INT-767 was associated with an FXR-dependent induction of hepatocellular membrane-bound CA 14, suggesting a novel role of FXR in regulating hepatocellular HCO3⫺ output and BA-independent bile flow. However, the potential impact of cholangiocytes as additional contributors to HCO3⫺-rich bile secretion cannot be ruled out; future studies must address their role in INT-767–induced choleresis. A potential role of FXR in promoting BA-independent bile flow is further supported by induction of VPAC-1 gene expression in human gallbladder by CDCA and the selective FXR agonist GW4064 (56). VPAC-1 is a receptor of vasoactive intestinal peptide (VIP), the most potent secretagogue in biliary epithelium (57). Given the fact that both FXR and VPAC-1 were shown to be expressed in the human intrahepatic biliary tree (56), it is attractive to speculate that FXR activators will also have choleretic effects at the intrahepatic bile duct level. In contrast to FXR, TGR5 was not detected in hepatocytes and its role in regulating BA homeostasis is not fully understood. Controversial studies have shown a decreased total

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BA pool size (58) and increased CYP7A1 gene expression (20) in TGR5-deficient mice. This discrepancy may reflect different experimental conditions used in each specific study. Importantly, TGR5 polymorphisms were detected in PSC patients, indicating a potential role of this receptor in cholangiocyte pathophysiology (59,60). Secretory and transport systems of cholangiocytes mediate modifications of primary canalicular bile, resulting in bicarbonate-rich ductal bile secretion. Ductal function is regulated by several factors – including hormones, innervation, and bile content – to assure the proper final bile composition. Unconjugated BAs can enter cholangiocytes from the bile duct lumen by passive diffusion, whereas conjugated BAs are transported via an apical Na+dependent BA transporter ASBT (61,62). Basolateral release of BAs from cholangiocytes is then mediated by a truncated isoform of the same transporter (63). Despite the fact that ASBT is regulated by FXR and that low FXR levels were determined in intrahepatic and extrahepatic biliary tree, the precise molecular regulation of cholangiocellular BA transport is poorly studied. Transport of biliary HCO3⫺ is another important function of cholangiocytes. HCO3- output is mediated by Cl-/HCO3⫺ anion exchanger 2 (AE2) and can be stimulated by increased levels of intracelluar cyclic AMP (cAMP). This transporter was detected in cholangiocytes as well as hepatocytes, and its deficiency resulted in a PBC-like phenotype in mice (64). AE2 gene expression was found to be reduced in liver biopsies from PBC patients, whereas beneficial effects of UDCA treatment were associated with restored AE2 expression (65,66). In addition, the combination of UDCA and glucocorticoids induced an upregulation of the AE2 alternate promoter in human liver cells in vitro and in vivo in mice (67). Importantly, bile ducts (in the human and rat, to lesser degree in the mouse) express TGR5 at the apical membrane, specifically on cholangiocellular cilia (23), suggesting an important role of this receptor in sensing the luminal BA concentrations and composition to adapt ductular secretion. If so, TGR5 activation by luminal BAs would induce cholangiocellular secretion via cAMP-induced activation of CFTR, Cl- export, and its subsequent exchange for HCO3⫺ through AE2. Induction of Cl- secretion in human gallbladder epithelia via cAMP and CFTR-mediated mechanisms would further support this hypothesis (19). Surprisingly, the selective TGR5 agonist INT-777 failed to induce HCO3⫺ output and bile flow in healthy mice as well as in the Mdr2⫺/⫺ model (53). Lack of choleretic effects by INT-777 were most likely due to the limited bioavailability of this compound or relatively low TGR5 expression and its low activation by INT-777 in mouse cholangiocytes. Since INT-777 already increased serum liver tests at the administered dose, other TGR5 activators with high bioavailability and low toxicity should be used to evaluate the role of TGR5 in biliary secretion. An interesting concept beyond the receptor-mediated alterations of bile flow and composition was developed based on hypercholeretic effects of some BAs such as UDCA and nor-UDCA (29,68,69). Because choleretic effects of these BAs were higher from what could be expected based on their osmotic activity, a “cholehepatic shunt” hypothesis has been suggested (29). According to this concept, BA protonation in bile duct lumen contributes to the HCO3⫺ anion formation. Cholangiocellular uptake of protonated BAs (by passive diffusion or through ASBT-mediated uptake), transport into periductal vessels and hepatocellular reuptake would subsequently cause BA resecretion and lead to increased HCO3⫺ production and bile flow. BAs that have no FXR or TGR5 agonistic activity may be beneficial in the treatment of cholestasis by promoting bile flow through cholehepatic shunting. However, induction of bile flow may be deleterious in obstructive cholestasis independent of the molecular mechanisms involved.

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16.5 Role of BA receptors for targeting hepatic inflammation and fibrosis Cholestatic liver injury is associated with hepatic inflammation and fibrosis. Inflammation may occur prior to or evolve as a consequence of cholestasis and, if persistent, may further promote liver damage and tumor development. Besides their detergent properties and proinflammatory effects, BAs can also act as anti-inflammatory agents through FXR and TGR5 activation. The role of FXR in inflammation became clear with the demonstration of increased baseline hepatic inflammation and spontaneous development of liver tumors in FXR-deficient mice (9,70). Moreover, compared with wild-type mice, FXR-deficient mice showed increased proinflammatory cytokine production in an experimental model of endotoxin-induced liver damage (71). Molecular mechanisms behind the anti-inflammatory effects of FXR were linked to FXR interference with the NF-κB inflammatory signaling pathway and subsequent inhibition of proinflammatory mediator production in hepatocytes (71). In addition, FXR agonists were reported to have direct antifibrotic effects in hepatic stellate cells (HSC) through the FXR-SHP regulatory cascade (72,73). These findings are highly controversial, since both FXR and SHP expression turned out to be very low or undetectable in profibrogenic cells such as HSC and periductal myofibroblasts (74). Beneficial effects of FXR on hepatic inflammation and fibrosis were further confirmed by using FXR and TGR5 agonists in an Mdr2⫺/⫺ animal model of chronic cholangiopathy. Interestingly, anti-inflammatory and antifibrotic effects of the dual FXR/TGR5 agonist INT-767 in this study were found to be secondary to an FXR-mediated reduction of bile toxicity, as reflected by an inhibition of BA synthesis and reduction of biliary BA output as well as the simultaneous induction of HCO3⫺-rich bile flow (53). In addition to FXR, BAs may have anti-inflammatory effects via TGR5 activation. Specifically, TGR5 activation by BAs inhibited proinflammatory cytokine release and reduced phagocytic activity in rabbit alveolar macrophages, human monocytic leukemia cells, and isolated rat Kupffer cells (21,75). Besides this, TGR5 activation in sinusoidal endothelial cells (SECs) suggested an important role for this receptor in nitric oxide (NO) production via cAMP-mediated induction of endothelial nitric oxide synthetase (eNOS) (22), indicating a beneficial role for the liver microcirculation. Interestingly, recent studies demonstrated that besides FXR, TGR5 activation also interferes with the NF-κB signaling pathway to protect against endotoxin-induced liver injury and atherosclerosis in mice (76,77). TGR5⫺/⫺ mice showed significantly increased serum ALT and AST as well as hepatic MCP-1, iNOS, and IFN-γ gene expression after LPS injection. Moreover, TGR5 activation by 23(S)-mCDCA synthesized in the same laboratory, inhibited LPS-induced proinflammatory cytokine expression in vivo and in vitro through TGR5-dependent suppression of NF-κB inhibitor IκBα phosphorylation, and enhanced interaction of β-arrestin2 with IkBα (77). However, the selective TGR5 agonist INT-777 failed to inhibit proinflammatory cytokine as well as procollagen gene expression in an Mdr2⫺/⫺ mouse model of chronic liver disease (53). This discrepancy is likely to be due to different experimental setup (e.g., administered dose, duration, sex) and additionally reflects different pathogenetic mechanisms of liver injury in each model. Finally, INT777 was found to counteract inflammation by repressing macrophage accumulation and inflammation in atherosclerotic lesions in LDLR⫺/⫺ murine model of atherosclerosis

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via TGR5-cAMP-mediated NF-κB inhibition. Importantly, beneficial effects of INT-777 were abrogated in mice deficient for both LDLR and TGR5 and in LDLR⫺/⫺ mice transplanted the bone marrow from TGR5⫺/⫺ mice (76). In addition, INT-777 repressed proinflammatory cytokine production in macrophages via TGR5-cAMP-mediated NF-κB inhibition in vitro. Together, these findings established beneficial role of TGR5 in counteracting inflammation.

16.6 Role of BA receptors in the pathogenesis and treatment of gallstone disease Gallstone disease is one of the most common digestive diseases, with a prevalence ranging from 5%–20% (78–80). Increased biliary cholesterol secretion, high BA hydrophobicity, gallbladder inflammation, as well as impaired gallbladder motility and some biliary proteins have been recognized as pathophysiologically relevant factors for gallstone development (78,81–83). In physiologic conditions, BAs, cholesterol, and phospholipids (PL) are solubilized because of tightly balanced concentrations in bile. Alterations of biliary BA concentration lead to reduced cholesterol solubilization, whereas disturbed PL excretion alters the process of cholesterol inclusion into mixed micelles. As a result, bile becomes supersaturated with cholesterol, which easily precipitates and predisposes to gallstone formation. Although FXR plays an important role in regulating BA and PL export, its role in human lithogenesis remains controversial owing to genetic variations in different ethnic populations and the importance of other risk factors (84). However, low FXR levels were found in patients with gallstone disease (85) and were associated with repressed expression of PGC-1α (a PPARγ coactivator), which has been shown to activate FXR (86). MDR3 deficiency and polymorphisms in BSEP and CYP7A1 are rare; when present, however, they lead to a high prevalence of gallstone formation (87–90). In mice, in contrast to humans, the role of FXR in gallstone disease was clearly demonstrated after feeding on a lithogenic diet. FXR-deficient mice developed gallbladder inflammation, increased biliary cholesterol, and a BA hydrophobicity index after 1 week of experimental feeding, whereas FXR⫹/⫹ mice were not affected. These alterations resulted from a significantly lower expression of FXR target genes BSEP and Mdr2 and increased expression of BA synthesis genes CYP7A1 and CYP8B1 in a situation of unchanged cholesterol export by ABCG5/ABCG8 (14). Furthermore, treatment with the FXR agonist GW4064 resulted in an improved cholesterol saturation index and reduced gallbladder wall inflammation in the gallstone-susceptible wild-type C57L model (14). Based on these findings, it is attractive to speculate that FXR activators will increase cholesterol solubilization in bile through increased BA and PL secretion and may be beneficial in human gallstone disease. BAs such as CDCA and UDCA were used to dissolve gallstones in humans long before their molecular mechanisms were elucidated. However, FXR-mediated CYP7A1 inhibition and reduced BA synthesis could also have the opposite effect due to i) decreased cholesterol utilization for BA synthesis and increased biliary excretion and ii) imbalanced BA and cholesterol excretion leading to cholesterol precipitation. In contrast to FXR, TGR5 activation is less likely to be favorable in treating gallstone disease. Importantly, TGR5-deficient mice were protected against lithogenic diet–induced gallstone formation (20). Moreover, gallbladder smooth muscle function was shown to

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be inhibited by hydrophobic BAs through TGR5 activation (91). Gallbladder contractility is an important function that promotes bile release from gallbladder after food consumption. Its dysfunctions lead to biliary stasis and may promote cholesterol precipitation and further growth of cholesterol microcrystals (81,82). However, no correlation of TGR5 protein expression with gallstone development was observed in humans when gallbladders from patients with chronic gallstone disease were compared with control gallbladder tissue (19). Together, our current knowledge indicates that TGR5 activation may not be beneficial for treatment of disorders affecting the gallbladder and biliary tree. However, the potential of TGR5 activators to increase cAMP-mediated biliary HCO3⫺ secretion and bile flow still remains an attractive hypothesis to increase bile flow and thereby flush the biliary system, which may decrease gallstone formation at least in the bile ducts. Finally, besides hepatic and gallbladder factors, intestinal BA and lipid homeostasis has been suggested in several studies to play a role in gallstone disease. Specifically, repressed FXR as well as OSTα and OSTβ expression were described in a subgroup of nonobese gallstone patients. In addition, intestinal FXR target Fgf15 was found to be involved in gallbladder smooth muscle relaxation and gallbladder filling via binding to FGF receptors in gallbladder, thus opposing cholecystokinin effects (92). Together these findings suggest that intestinal dysregulation of FXR and its target genes may contribute to gallstone formation by changing the BA pool and gallbladder contractility. Further studies are required to clarify direct mechanisms of intestinal effects on gallstone formation.

16.7 BA receptors in intestine: therapeutic implications for the gut-liver axis and inflammatory bowel disease High expression levels of FXR and TGR5 in intestine emphasize the role of BAs in intestinal pathophysiology. Obstructive cholestasis has been shown to increase intestinal permeability and bacterial overgrowth in humans and mice, effects that were counteracted by oral BA administration (93,94). A recent study has demonstrated the role of FXR in animal models of dextran sodium sulfate (DSS)–induced colitis and trinitrobenzenesulfonic acid (TNBS)-induced colitis. In both models, epithelial degeneration, goblet cell loss, and intestinal inflammation were restored by a treatment with the FXR agonist INT-747 in wild-type mice, whereas no effect was observed in FXR-deficient mice (95). Moreover, intestinal permeability as measured by plasma fluorescein isothiocyanate (FITC)–conjugated dextran release was decreased by INT-747, indicating improved intestinal barrier function. In line with these findings, proinflammatory gene expression in colons as well as in human polymorphonuclear cells (PMNCs), dendritic cells, and mononuclear cells from the intestinal lamina propria of patients with inflammatory bowel disease was repressed by INT-747. Importantly, INT-747 increased the expression of antibacterial genes such as cathelicidin and iNOS in the colon and ANG1 in the ileum. The same group showed later inhibitory effects of proinflammatory cytokines TNF-α/IL-1β on expression of FXR target genes such as IBABP, SHP, and FGF15 in vitro and by DSS in vivo (96) despite unchanged FXR expression. Furthermore, the authors showed a direct inhibitory effect of NF-κB inflammatory mechanism on FXR activity without alteration on gene expression. Hence this study confirmed a direct interaction between FXR and NF-κB, as in the case of anti-inflammatory effects in the liver. Moreover, the anti-inflammatory effects of intestinal FXR are supported by inhibition of

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bacterial overgrowth and regulation of genes involved in antimicrobial defense by FXR ligands without bacteriostatic effects per se (12). A recent study showed that intestinal FXR activation plays a protective role in liver injury (97). Specifically, selective intestinal FXR overexpression decreased liver injury in experimental model of total biliary obstruction and in Mdr2⫺/⫺ model of sclerosing cholangitis. As expected, FXR activation in gut repressed hepatic CYP7A1, reduced the BA pool size, and modified the BA pool’s content. In addition, cholestasis-associated mucosal injury was decreased in intestinal FXR overexpressing mice after common bile duct ligation (CBDL). In line with these findings an additional model of chemically induced liver injury by ANIT administration confirmed the hepatoprotective role of intestinal FXR. These findings may have major implications for treating complications of cholestasis and liver cirrhosis. BA-mediated TGR5 activation in intestinal enteroendocrine cells has been shown to stimulate cAMP-mediated induction of glucagon-like peptide-1 (GLP-1), an important regulator of insulin secretion and glucose homeostasis (see below) (98). Additionally, TGR5⫺/⫺ mice developed altered mucosal histology, increased intestinal permeability, and signs of colitis associated with altered cellular distribution of junctional proteins zonulin 1, occludin and E-cadherin in experimental models of DSS- and TNBS-induced colitis (99). In these models, ciprofloxacin-mediated repression of INF-γ, Il-1β, TNF-α, IL-6, and IL-1β was abrogated in TGR5⫺/⫺ mice, indicating that together with FXR, TGR5 may be involved in protecting against intestinal inflammation. In line with these findings, colonic TGR5 expression was increased in patients with Crohn’s disease (99), and TGR5 polymorphisms were described in PSC patients (59), which often is accompanied by ulcerative colitis. However, TRG5 was shown to be expressed in intestinal inhibitory motoneurons (rather than enterocytes), indicating that it may be involved in reduced intestinal motility, prolonging passage time, and increasing the risk for bacterial overgrowth/translocation (100).

16.8 Role of BAs in lipid metabolism: therapeutic implications for atherosclerosis and nonalcoholic fatty liver disease (NAFLD) The liver is one of the main organs involved in lipid homeostasis. As such it is an important site of lipoprotein synthesis and is involved in cholesterol, PL, and fatty acid (FA) homeostasis. In a process known as reverse cholesterol transport, a subclass of high-density lipoprotein (HDL) transports cholesterol from peripheral tissues back to the liver. Cholesterol transfer from HDL into hepatocytes involves two pathways: a) direct interaction of cholesteryl ester (CE) containing HDL with the hepatic SR-B1 or b) lowdensity lipoprotein (LDL) receptor (LDLR)–mediated uptake of CE from LDL. CE transfer protein (CETP) is present in humans but not in mice and mediates CE transport from HDL to very low density lipoprotein (VLDL) or LDL prior to its hepatocellular uptake. Hepatic cholesterol is then used either for BA synthesis or is excreted as free cholesterol into the bile, leading to an efficient reduction of serum cholesterol levels. Interestingly, BA loss in patients with ileal resection was accompanied by increased levels of serum HDL and its structural protein ApoAI (101–103). Accordingly, HDL cholesterol was decreased in cholestatic patients, whereas partial biliary diversion restored serum lipid levels (104). Furthermore, BA sequestrants, which irreversibly bind BAs in

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the intestine and block their absorption, induced CYP7A1 and hepatic cholesterol catabolism, which caused compensatory induction of LDLR for cholesterol uptake and efficiently lowered serum LDL cholesterol. In contrast, BA sequestrants increased HDL cholesterol (105–109), whereas BA supplementation was shown to decrease serum HDL (110) and triglyceride (TG) levels (111–114). In line with this, diet-induced hypercholesterolemia was prevented in CYP7A1 transgenic mice by increased cholesterol catabolism via BA synthesis and induced biliary cholesterol secretion through a BA-FXR-ABCG5/G8 mechanism (115). These reports clearly demonstrate a repression of serum HDL levels by BAs in men. By using BA feeding in human-ApoAI transgenic mice, ApoAI repression was found to be mediated through a negative FXR response element, indicating that FXR activation may have proatherogenic effects in men (116). In contrast, FXR-mediated induction of SR-B1 and reverse cholesterol transport were shown to mediate antiatherogenic effects of FXR in mice (117). Interestingly, FXR⫺/⫺ mice showed proatherogenic serum lipid profile after western diet feeding without development of atherosclerotic lesions. However, LDLR/FXR double-knockout male mice were protected from atherosclerosis by mechanisms not studied in detail (118). Finally, CDCA treatment increased CETP activity in patients with mitochondrial CYP27A1 mutations (119), which manifest with high serum cholesterol levels and CE deposition in peripheral tissues (cerebrotendinous xanthomatosis). However, whether BA-mediated activation of CETP was FXR-mediated remains unknown. These findings may have major implications for understanding atherosclerosis as “liver disease of the heart” (120), in particular in association with NAFLD as an independent risk factor for atherosclerosis (121,122). Besides its effects on HDL homeostasis, FXR plays an important role in TG metabolism, which may have major implications for understanding and treating NAFLD. TGs are secreted from liver in form of VLDL-TG, which in peripheral tissues such as adipose tissue and muscle are hydrolyzed by lipoprotein-lipase (LPL). ApoCII is a well-known co-activator of LPL, whereas ApoCIII acts as an LPL inhibitor. FXR was found to increase ApoCII and repress ApoCIII, thus leading to increased LPL activity and promoting VLDL hydrolysis. In addition to increased VLDL/TG hydrolysis, through SHP-mediated mechanisms, FXR was proposed to interfere with the LXR/LRH1-mediated trans-activation of SREBP-1c, a master regulator of FA and TG biosynthesis (123), which controls AcetylCoA carboxylase 1 (ACC1), ACC2, and fatty acid synthase (FAS). Furthermore, LDLR was recognized to be induced by FXR, promoting hepatic ApoB/LDL particle uptake and cleavage. Thus BAs act on both TG synthesis and TG clearance. Hyperlipidemia, especially hypertriglyceridemia, obesity, and diabetes are important during the development NAFLD. NAFLD is a general term used for hepatic disorders ranging from simple fatty liver to nonalcoholic steatohepatitis (NASH), liver cirrhosis and hepatocellular carcinoma (124). Mutations of the FXR target SHP were found in Japanese patients with hypertriglyceridemia and mild obesity (125), and SHP polymorphisms were reported to influence birth weight and body mass index (BMI) (126,127). However, murine SHP deficiency showed no hypertriglyceridemia or obesity despite lower hepatic TG content after dietary CA supplementation (128,129). In mice, FXRdeficiency results in increased serum lipid levels (15) and hepatic steatosis, whereas INT-747 decreased liver steatosis and excess fat deposition in muscle while it improved insulin tolerance in a leptin-deficient mouse NAFLD model (99). In line with this, another study found increased expression of proinflammatory TNF-α as well as profibrogenic TGF-β and type 1 collagen in LDLR⫺/⫺FXR⫺/⫺ double-knockout mice after a

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high-fat feeding, whereas LDLR⫺/⫺ mice demonstrated only hepatic fat accumulation and cell ballooning without inflammation (130). Moreover, the synthetic FXR agonist WAY-362450 decreased hepatic inflammation and fibrosis in a methionine choline– deficient diet-induced NAFLD model (131). Collectively these studies indicate that FXR activation may have beneficial effects in NASH. A phase II double-blind placebo controlled multicenter clinical trial has been started to evaluate the therapeutic effects of the FXR agonist obeticholic acid (INT-747) in adult NASH patients with type 2 diabetes (FLINT trial). The first results were analyzed after 6 weeks of treatment and showed an improved steady-state glucose disposal rate, weight loss, increased serum FGF19, and improved fibrosis markers (assessed by Enhanced Liver Fibrosis (ELF) test) (132). Since hepatic steatosis is a predictive factor for the development of insulin resistance and since hypertriglyceridemia and ApoCIII are independent risk factors for cardiovascular disease, multistep effects of BAs on serum TG levels and hepatic lipid metabolism make FXR agonists attractive as potential drugs to treat the metabolic syndrome. However, the development of BA receptor modulators with selective target properties due to specific cofactor recruitment might be a solution to overcome undesirable effects such as a decrease in the levels of HDL.

16.9 Role of BAs in hepatic glucose metabolism and beyond Despite early findings that BA sequestrants improved the glycemic control in dyslipidemia and type 2 diabetes (133), the role of FXR in regulating glucose homeostasis remains controversial. FXR appears to have an important role in controlling glucose homeostasis through pancreatic as well as peripheral effects. FXR-deficient mice demonstrated insulin resistance and impaired glucose homeostasis owing to increased gluconeogenesis in the liver and repressed glucose uptake in muscle, resulting in hyperglycemia (13). In line with these findings, FXR activation by GW4064 as well as constitutive hepatic FXR overexpression improved serum glucose levels as well as insulin sensitivity in diabetic db/db mice (16). Incubation with GW4064 promoted adipocyte differentiation and insulin-dependent glucose uptake in 3T3-L1 cells in vitro. In addition, FXR was found to be positively regulated by glucose in cultured hepatocytes (134) and FXR expression was restored by insulin in a model of experimental diabetes (134), indicating an important link between glucose and BA homeostasis. However, different studies showed opposing effects of BAs on gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-controlling enzyme in gluconeogenesis. BA-mediated PEPCK inhibition observed in mice was demonstrated to be HNF4α− but not FXR-mediated in vitro (135). In contrast, repression of PEPCK and glucose-6-phosphatase by BAs in vivo was linked to the FXR-SHP-HNF4α pathway (136), whereas another study demonstrated FXR-mediated PEPCK activation via mechanisms involving SHP and TRB3 (137), an important activator of gluconeogenesis under fasting conditions. These findings were supported by increased hepatic glycogen levels upon FXR activation (16,134). Interestingly, the FXR target FGF19 was shown to play an important role in normalising serum glucose levels and postprandial glycogen content in the liver via insulin-independent mechanisms (138). Importantly, reduced postprandial serum glucose levels by FGF19 were found to be mediated through increased hepatic glycogen synthesis, resulting in decreased hepatic glucose output. Unlike insulin, FGF19 did not increase hepatic TG or SREBP-1c

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expression. In addition, FGF19 repressed hepatic gluconeogenesis through cAMP response element-binding (CREB) inactivation (139). FGF19/15 expression increases in parallel to BA concentrations in the gut and may ensure postprandial control of glucose metabolism subsequent to insulin (139). Together, these studies show that the BA-FXRFGF19 cascade may act in parallel with and independent of insulin to regulate postprandial glucose metabolism. Interestingly, a role of FXR in regulating glucose homeostasis was suggested by demonstrating FXR expression in pancreatic β cells and stimulation of glucose-dependent insulin secretion by the selective FXR ligand INT-747 in human and murine β cells. In addition, the FXR agonist INT-747 reduced blood glucose levels and glycosuria in a mouse model of nonobese diabetes (NOS) (140). However, no significant increase of serum insulin levels was determined even if the glucose-to-insulin ratio was dramatically increased. Importantly, recent data suggest that highly potent FXR activators may be deleterious for metabolic syndrome, since they suppress BA synthesis and reduce BA pool size, with a subsequent impact on the BA-TGR5-D2 pathway and energy expenditure (see below), leading to weight gain and insulin resistance (141). Metabolic disorders such as type II diabetes mellitus, obesity, hyperlipidemia, and atherosclerosis share dysregulation of cellular metabolism as a critical pathogenetic mechanism. Mitochondria are central metabolic organelles in the cells and their dysfunction has been reported to increase the risk of metabolic disorders (142). Thyroid hormone is essential in controlling mitochondrial activity. Specifically it induces oxygen consumption and activates carbohydrate, fat, and protein metabolism as well as energy production. In this context, improvement of mitochondrial function and oxidative phosphorylation will improve metabolic disorders. Interestingly, BA feeding prevented and reversed diet-induced obesity in mice (143). These effects were mediated through TGR5-dependent activation of deiodinase 2 (D2), an enzyme that catalyzes transformation of inactive thyroxin (T4) into metabolically active triiodthyronine (T3). Active T3 stimulates local mitochondrial activity and oxidative phosphorylation upon binding to the T3 receptor. TGR5 is expressed in murine brown adipose tissue and human skeletal muscle, where the BA-TGR5-cAMP-D2 cascade induced FA oxidation and uncoupled proteins that promote increased energy expenditure and weight loss. These effects were associated with improved insulin sensitization in experimental models of dietary and genetic diabetes (143). Thus BAs may play a critical role in regulating metabolic control in humans through TGR5-cAMP-D2 signaling. Importantly, D2 gene polymorphism is associated with 20% less glucose disposal in obese women and higher insulin resistance in type II diabetes (144,145). Finally, TGR5 activation in enterohepatic SRC-1 cells increased GLP-1 production, which may add to improved glucose homeostasis and insulin sensitivity (98). In line with these findings, BA sequestrants improved insulin sensitivity and lowered fasting glucose (146), likely due to shifting BA signalling to the colon and increasing GLP-1 release in vivo (147–149). Thus the BA-TGR5-GLP-1 pathway may in part explain the antidiabetic properties of BA-binding resins. Interestingly, TUDCA was shown to improve glucose homeostasis in obese and diabetic mice by reducing stress on the endoplasmic reticulum (ER) (150). However, the role of TGR5 in TUDCA-mediated glycemic control is unknown. Notably, TGR5⫺/⫺ mice failed to develop hyperglycemia or fatty liver under either a control diet or a high-fat diet even if an insulin tolerance test showed gender-specific differences under the high-fat diet (151). Since the most potent physiologic TGR5 ligand, taurolitocholic aicd (TLCA), is rapidly metabolized in the intestine and liver because of its toxicity and other BAs are less

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potent, it is likely that TGR5 is not efficiently activated by circulating BAs in physiologic conditions; pharmaceutical TGR5 activators may be more efficient in activating TGR5 in vivo. Currently several synthetic TGR5 agonists are available. Whether they can be useful in the treatment of metabolic disorders has yet to be proven in clinical trials. In summary, the significant impact of FXR and TGR5 on diverse metabolic pathways emphasizes the important role of these receptors in metabolic disorders such as obesity, insulin resistance, NAFLD, and atherosclerosis. Owing to the high prevalence of cardiovascular diseases, NAFLD has been recognized as an independent risk factor for ischemic stroke, cardiomyopathy, and myocardial infarction (121,122,152,153). Based on available data, it is plausible to assume that TGR5 is a promising target for the treatment of diabetes, obesity, and metabolic diseases. In addition, FXR modulators may be beneficial by targeting several aspects of metabolic diseases.

16.10 BA receptors in hepatobiliary and colorectal cancer High concentrations of BA have been associated with several tumors, especially in the gastrointestinal tract (154). In addition, changes in bile composition as well as FXR expression have been detected in colorectal adenocarcinoma (155,156), indicating that BAs play a role in tumorigenesis. Chronic inflammation often predisposes to cancer development in various tissues. “Toxic bile” is believed to initiate and maintain portal inflammation, fibrosis, and spontaneous tumor development in animal models of chronic cholestasis (157). Since both FXR and TGR5 have shown anti-inflammatory effects in various cells, activation of these receptors may become an important strategy to stop inflammation-associated tumor development. Indeed, FXR-deficient mice displayed high serum and hepatic BA levels, increased hepatic proinflammatory cytokine gene expression, as well as enhanced hepatocyte proliferation and spontaneous tumor development (9,70). Moreover, FXR-deficient mice had significantly altered liver regeneration after partial hepatectomy (11), indicating that FXR is required for the hepatocyte proliferation. Indeed, dual FXR/TGR5 agonist induced hepatocellular proliferation in healthy and cholestatic mice in an FXR-dependent manner. For the first time, another study has shown induced cell proliferation, inflammation, and tumor development in the intestine of FXR-deficient mice (158). To study the role of FXR in intestinal tumorigenesis, the authors used two common murine models of tumorigenesis in FXR wildtype and FXR⫺/⫺ mice: APCmin mice and azoxymethan (AOM) treatment. Importantly, FXR deficiency increased adenoma as well as adenocarcinoma multiplicity and size in both the models. Furthermore, the cell-cycle regulator Fox1b was found to be induced by FXR activation, leading to restored liver regeneration in aging liver (159). In line with these findings, FXR expression was reduced in cell lines derived from human colorectal cancer (160) as well as human colon cancer (156). Reduced FXR expression was recently shown in cholangiocellular carcinoma (CCC) (161), where TGR5 was dramatically increased (162). To what extend TGR5 is involved in pathogenesis of CCC is currently unknown, but preliminary data suggest that it may mediate the resistance of cancer cells to apoptosis (162). It is clear that the roles of FXR and TGR5 in tumor development must be studied in greater detail. Although induced hepatocellular proliferation may be beneficial after liver resection, FXR-mediated hepatocellular proliferation may promote the development of

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hepatocellular carcinoma. Since the majority of metabolic disorders that would benefit from FXR activation need long-term therapy, induced cell proliferation may become a limiting factor for the use of these compounds in humans.

16.11 BA receptors beyond the liver and gastrointestinal tract The role of BAs as extrahepatic and interorgan signaling molecules is discussed in more detail in Chapter 7 of this book. FXR has been found in lung endothelial cells, and its role was tested in LPS-induced mouse model of acute lung injury. Interestingly, FXR⫺/⫺ mice were more prone to LPS-induced lung endothelial permeability and pneumonia with impaired lung repair (163). Importantly, FXR was found to be expressed in breast cancer (164), and its activation induced cancer cell apoptosis. Moreover, FXR activation inhibited tamoxifen-resistant breast cancer cell growth through the downregulation of HER2 expression (165). Notably, activation of TGR5 may also have undesirable effects in various organs. As such, the development of acute reflux-induced pancreatitis was directly associated with BA-mediated TGR5 activation in pancreatic acini in mice (166). TGR5 activation promoted the production of reactive oxygen species in astrocytes (167) and activated AKT signaling in cardiomyocytes (168). Finally, TGR5-activation by BAs was linked to increased hepatocellular apoptosis (169) through activation of c-Jun N-terminal kinase ( JNK) signaling pathways, indicating that TGR5 might have a critical role in cell death and cancer development. Future studies will have to determine whether TGR5-mediated alterations in cell function and inflammation may be involved in hepatic encephalopathy, cirrhotic cardiomyopathy, and renal failure in patients with end-stage liver disease. Importantly, such extrahepatic “off-target effects” must be considered in developing BA receptor ligands as therapeutics in patients with liver disease.

16.12 Concluding remarks From what is currently known, it is clear that the targeting of BA receptors is likely to have therapeutic relevance for a wide range of disorders (fFig. 16.1). In this respect, the targeting of FXR is likely to be beneficial via several mechanisms: Protection against intracellular accumulation of potentially toxic BAs Induction of biliary HCO3⫺ output and bile flow (a mechanism also shared by other therapeutic approaches such as norUDCA independent of FXR) Anti-inflammatory and antifibrotic effects through direct or indirect mechanisms Biliary cholesterol solubilization by increased biliary BA and PL excretion Protection against intestinal inflammation and bacterial translocation in IBD Effects on hepatic lipid and glucose metabolism In addition, TGR5 is a promising target for metabolic diseases such as diabetes, obesity and atherosclerosis as well as other chronic diseases accompanied with inflammation. TGR5-mediated beneficial actions may involve: Increased metabolic activity and energy expenditure Improved glycemic control Repressed inflammation

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BA metabolism

HDL metabolism TG metabolism

Synthesis uptake

Apo A-I

PLTP

Glycogen synthesis

Interaction with NF-kB

ApoCIII

Gluconeogenesis

Intestinal permeability

SREBP1c

Alternative transport

Inflammation

Apo CII

HL

Canalicular export

Glucose metabolism

Detoxification Low HDL

Low intracellular BA

Low serum TGs

Insulin resistance

Anti-inflammatory

FXR BAs TGR5

Energy expenditure

NF-kB activation

GLP-1

Pro-inflammatory cytokines

Insulin sensitivity

Anti-inflammatory

Fig. 16.1: Potential therapeutic implications for FXR and TGR5. FXR activation may counteract cholestasis by inhibiting BA uptake and synthesis and inducing export and detoxification systems. Reduction of TGs through FXR-mediated inhibition of hepatic FA synthesis and VLDL production together with inhibition of LPL inhibitor ApoCIII and ApoCII induction will contribute to VLDL hydrolysis and may have additional anti-atherosclerotic effects. By inducing hepatic glycogen synthesis and inhibiting gluconeogenesis, FXR activation may improve glycemic control in diabetic patients. Anti-inflammatory effects of FXR may be beneficial in several disorders accompanied by chronic inflammation. However, FXR activation inhibits ApoAI and reduces serum HLD levels, which should be considered when targeting FXR. TGR5 activation is promising in obesity and diabetes. By inducing mitochondrial activity in adipose tissue and skeletal muscle, TGR5 activation induces energy expenditure and weight loss, which may have major implication to improve metabolic control. Moreover, TGR5 activation induces GLP-1 release from enteroendocrine cells, which may have major implication to treat insulin resistance and diabetes. In addition, inhibition of NF-κB signaling and repression of pro-inflammatory cytokine production may be beneficial in chronic inflammation.

However, both FXR and TGR5 may cause undesirable effects due to their wide expression in the body and the complexity of their functions. Therefore a critical evaluation of benefits and risks is required in targeting FXR or TGR5 in human. FXR activation by non-BA ligands may be beneficial to overcome adverse effects of BAs and their derivatives.

16.13 References 1. Thomas C, Pellicciari R, Pruzanski M, et al. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov. 2008;7(8):678–93.

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100. Poole DP, Godfrey C, Cattaruzza F, et al. Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system. Neurogastroenterol. Motil. 2010;22(7):814–25, e227–8. 101. Buchwald H, Varco RL, Matts JP, et al. Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N. Engl. J. Med. 1990;323(14):946–55. 102. Grundy SM, Ahrens EH, Jr., Salen G. Interruption of the enterohepatic circulation of bile acids in man: comparative effects of cholestyramine and ileal exclusion on cholesterol metabolism. J. Lab. Clin. Med. 1971;78(1):94–121. 103. Heaton KW. Disturbances of bile acid metabolism in intestinal disease. Clin. Gastroenterol. 1977;6(1):69–89. 104. Melter M, Rodeck B, Kardorff R, et al. Progressive familial intrahepatic cholestasis: partial biliary diversion normalizes serum lipids and improves growth in noncirrhotic patients. Am. J. Gastroenterol. 2000;95(12):3522–8. 105. Angelin B. 1994 Mack-Forster Award Lecture. Review. Studies on the regulation of hepatic cholesterol metabolism in humans. Eur. J. Clin. Invest. 1995;25(4):215–24. 106. Angelin B, Einarsson K, Hellstrom K, et al. Effects of cholestyramine and chenodeoxycholic acid on the metabolism of endogenous triglyceride in hyperlipoproteinemia. J. Lipid Res. 1978;19(8):1017–24. 107. Buchwald H, Stoller DK, Campos CT, et al. Partial ileal bypass for hypercholesterolemia. 20- to 26-year follow-up of the first 57 consecutive cases. Ann. Surg. 1990;212(3): 318–29; discussion 29–31. 108. Duane WC, Hartich LA, Bartman AE, et al. Diminished gene expression of ileal apical sodium bile acid transporter explains impaired absorption of bile acid in patients with hypertriglyceridemia. J. Lipid Res. 2000;41(9):1384–9. 109. Molgaard J, von Schenck H, Olsson AG. Comparative effects of simvastatin and cholestyramine in treatment of patients with hypercholesterolaemia. Eur. J. Clin. Pharmacol. 1989;36(5):455–60. 110. Brensike JF, Levy RI, Kelsey SF, et al. Effects of therapy with cholestyramine on progression of coronary arteriosclerosis: results of the NHLBI Type II Coronary Intervention Study. Circulation 1984;69(2):313–24. 111. Bateson MC, Maclean D, Evans JR, et al. Chenodeoxycholic acid therapy for hypertriglyceridaemia in men. Br. J. Clin. Pharmacol. 1978;5(3):249–54. 112. Begemann F. Influence of chenodeoxycholic acid on the kinetics of endogenous triglyceride transport in man. Eur. J. Clin. Invest. 1978;8(5):283–8. 113. Camarri E, Marcolongo R, Fici F. Hypotriglyceridemic effect of chenodeoxycholic acid after a short time of administration. Int. J. Clin. Pharmacol. Biopharm. 1978;16(11): 527–8. 114. Camarri E, Marcolongo R, Zaccherotti L, et al. The hypotriglyceridemic effect of chenodeoxycholic acid in type IV hyperlipemia. Biomedicine 1978;29(6):193–8. 115. Li T, Matozel M, Boehme S, et al. Overexpression of cholesterol 7alpha-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis. Hepatology 2011;53(3):996–1006. 116. Claudel T, Sturm E, Duez H, et al. Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element. J. Clin. Invest. 2002;109(7):961–71. 117. Zhang Y, Yin L, Anderson J, et al. Identification of novel pathways that control farnesoid X receptor-mediated hypocholesterolemia. J. Biol. Chem. 2010;285(5):3035–43. 118. Zhang Y, Wang X, Vales C, et al. FXR deficiency causes reduced atherosclerosis in Ldlr-/- mice. Arterioscler. Thromb. Vasc. Biol. 2006;26(10):2316–21.

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119. Kinoshita M, Kawamura M, Fujita M, et al. Enhanced susceptibility of LDL to oxidative modification in a CTX patient:- role of chenodeoxycholic acid in xanthoma formation. J. Atheroscler. Thromb. 2004;11(3):167–72. 120. Davis RA, Hui TY. 2000 George Lyman Duff Memorial Lecture: atherosclerosis is a liver disease of the heart. Arterioscler. Thromb. Vasc. Biol. 2001;21(6):887–98. 121. Targher G. Non-alcoholic fatty liver disease, the metabolic syndrome and the risk of cardiovascular disease: the plot thickens. Diabet. Med. 2007;24(1):1–6. 122. Targher G. Non-alcoholic fatty liver disease as a determinant of cardiovascular disease. Atherosclerosis 2007;190(1):18–9; author reply 20–1. 123. Watanabe M, Houten SM, Wang L, et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c. J. Clin. Invest. 2004;113(10):1408–18. 124. Angulo P. Nonalcoholic fatty liver disease. N. Engl. J. Med. 2002;346(16):1221–31. 125. Nishigori H, Tomura H, Tonooka N, et al. Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects. Proc. Natl. Acad. Sci. USA 2001;98(2):575–80. 126. Hung CC, Farooqi IS, Ong K, et al. Contribution of variants in the small heterodimer partner gene to birthweight, adiposity, and insulin levels: mutational analysis and association studies in multiple populations. Diabetes 2003;52(5):1288–91. 127. Mitchell SM, Weedon MN, Owen KR, et al. Genetic variation in the small heterodimer partner gene and young-onset type 2 diabetes, obesity, and birth weight in U.K. subjects. Diabetes 2003;52(5):1276–9. 128. Kerr TA, Saeki S, Schneider M, et al. Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis. Dev. Cell 2002;2(6):713–20. 129. Wang L, Lee YK, Bundman D, et al. Redundant pathways for negative feedback regulation of bile acid production. Dev. Cell 2002;2(6):721–31. 130. Kong B, Luyendyk JP, Tawfik O, et al. Farnesoid X receptor deficiency induces nonalcoholic steatohepatitis in low-density lipoprotein receptor-knockout mice fed a high-fat diet. J. Pharmacol. Exp. Ther. 2009;328(1):116–22. 131. Mencarelli A, Renga B, Distrutti E, et al. Antiatherosclerotic effect of farnesoid X receptor. Am. J. Physiol. Heart. Circ. Physiol. 2009;296(2):H272–81. 132. Sanyal AJ, S. M, Henry RR, et al. A new therapy for nonalcoholic fatty liver disease and diabetes? INT-747-the first FXR hepatic therapeutic study. Hepatology Supplement, The 60th Annual Meeting of the American Association for the Study of Liver Diseases:The Liver Meeting 2009;Volume 50(Supplement 4):389A-90A. 133. Garg A, Grundy SM. Cholestyramine therapy for dyslipidemia in non-insulindependent diabetes mellitus. A short-term, double-blind, crossover trial. Ann. Intern. Med. 1994;121(6):416–22. 134. Duran-Sandoval D, Mautino G, Martin G, et al. Glucose regulates the expression of the farnesoid X receptor in liver. Diabetes 2004;53(4):890–8. 135. De Fabiani E, Mitro N, Gilardi F, et al. Coordinated control of cholesterol catabolism to bile acids and of gluconeogenesis via a novel mechanism of transcription regulation linked to the fasted-to-fed cycle. J. Biol. Chem. 2003;278(40):39124–32. 136. Yamagata K, Daitoku H, Shimamoto Y, et al. Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1. J. Biol. Chem. 2004;279(22):23158–65. 137. Stayrook KR, Bramlett KS, Savkur RS, et al. Regulation of carbohydrate metabolism by the farnesoid X receptor. Endocrinology 2005;146(3):984–91. 138. Kir S, Beddow SA, Samuel VT, et al. FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis. Science 2011;331(6024):1621–4. 139. Potthoff MJ, Boney-Montoya J, Choi M, et al. FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway. Cell Metab. 2011;13(6):729–38.

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140. Renga B, Mencarelli A, Vavassori P, et al. The bile acid sensor FXR regulates insulin transcription and secretion. Biochim. Biophys. Acta. 2010;1802(3):363–72. 141. Watanabe M, Horai Y, Houten SM, et al. Lowering bile acid pool size with a synthetic farnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energy expenditure. J. Biol. Chem. 2011;286(30):26913–20. 142. Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science 2005;307(5708):384–7. 143. Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 2006;439(7075):484–9. 144. Canani LH, Capp C, Dora JM, et al. The type 2 deiodinase A/G (Thr92Ala) polymorphism is associated with decreased enzyme velocity and increased insulin resistance in patients with type 2 diabetes mellitus. J. Clin. Endocrinol. Metab. 2005;90(6):3472–8. 145. Mentuccia D, Proietti-Pannunzi L, Tanner K, et al. Association between a novel variant of the human type 2 deiodinase gene Thr92Ala and insulin resistance: evidence of interaction with the Trp64Arg variant of the beta-3-adrenergic receptor. Diabetes 2002;51(3):880–3. 146. Kobayashi M, Ikegami H, Fujisawa T, et al. Prevention and treatment of obesity, insulin resistance, and diabetes by bile acid-binding resin. Diabetes 2007;56(1):239–47. 147. Chen L, McNulty J, Anderson D, et al. Cholestyramine reverses hyperglycemia and enhances glucose-stimulated glucagon-like peptide 1 release in Zucker diabetic fatty rats. J. Pharmacol. Exp. Ther. 2011;334(1):164–70. 148. Chen L, Yao X, Young A, et al. Inhibition of Apical Sodium-Dependent Bile Acid Transporter (Asbt) as a Novel Treatment for Diabetes. Am. J. Physiol. Endocrinol. Metab. 2011. 149. Shang Q, Saumoy M, Holst JJ, et al. Colesevelam improves insulin resistance in a dietinduced obesity (F-DIO) rat model by increasing the release of GLP-1. Am. J. Physiol. Gastrointest. Liver Physiol. 2011;298(3):G419–24. 150. Ozcan U, Yilmaz E, Ozcan L, et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006;313(5790):1137–40. 151. Vassileva G, Hu W, Hoos L, et al. Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice. J. Endocrinol. 2010;205(3):225–32. 152. Adams LA, Lymp JF, St Sauver J, et al. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 2005;129(1):113–21. 153. Ekstedt M, Franzen LE, Mathiesen UL, et al. Long-term follow-up of patients with NAFLD and elevated liver enzymes. Hepatology 2006;44(4):865–73. 154. Bernstein H, Bernstein C, Payne CM, et al. Bile acids as endogenous etiologic agents in gastrointestinal cancer. World J. Gastroenterol. 2009;15(27):3329–40. 155. Debruyne PR, Bruyneel EA, Karaguni IM, et al. Bile acids stimulate invasion and haptotaxis in human colorectal cancer cells through activation of multiple oncogenic signaling pathways. Oncogene 2002;21(44):6740–50. 156. De Gottardi A, Touri F, Maurer CA, et al. The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer. Dig. Dis. Sci. 2004;49(6):982–9. 157. Trauner M, Fickert P, Halilbasic E, et al. Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases. Wien. Med. Wochenschr. 2008;158(19–20):542–8. 158. Maran RR, Thomas A, Roth M, et al. Farnesoid X receptor deficiency in mice leads to increased intestinal epithelial cell proliferation and tumor development. J. Pharmacol. Exp. Ther. 2009;328(2):469–77.

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159. Chen WD, Wang YD, Zhang L, et al. Farnesoid X receptor alleviates age-related proliferation defects in regenerating mouse livers by activating forkhead box m1b transcription. Hepatology 2010;51(3):953–62. 160. Lax S, Schauer G, Prein K, et al. Expression of the nuclear bile acid receptor/farnesoid X receptor is reduced in human colon carcinoma compared to nonneoplastic mucosa independent from site and may be associated with adverse prognosis. Int. J. Cancer 2011. 161. Dai J, Wang H, Shi Y, et al. Impact of bile acids on the growth of human cholangiocarcinoma via FXR. J. Hematol. Oncol. 2011;4:41. 162. Keitel V, Reinehr R, Reich M, et al. The Membrane-bound bile acid receptor TGR5 (Gpbar-1) is highly expressed in intrahepatic cholangiocarcinoma. Hepatology Supplement, The 62nd Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2011;54(Supplement 4). 163. Zhang L, Li T, Yu D, et al. FXR Protects Lung from Lipopolysaccharide-Induced Acute Injury. Mol. Endocrinol. 2011. 164. Swales KE, Korbonits M, Carpenter R, et al. The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression. Cancer Res. 2006; 66(20):10120–6. 165. Giordano C, Catalano S, Panza S, et al. Farnesoid X receptor inhibits tamoxifenresistant MCF-7 breast cancer cell growth through downregulation of HER2 expression. Oncogene 2011;30(39):4129–40. 166. Perides G, Laukkarinen JM, Vassileva G, et al. Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1. Gastroenterology 2010;138(2):715–25. 167. Keitel V, Gorg B, Bidmon HJ, et al. The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain. Glia 2010;58(15):1794–805. 168. Desai MS, Shabier Z, Taylor M, et al. Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis. Hepatology 2010; 51(6):2097–107. 169. Yang JI, Yoon JH, Myung SJ, et al. Bile acid-induced TGR5-dependent c-Jun-N terminal kinase activation leads to enhanced caspase 8 activation in hepatocytes. Biochem. Biophys. Res. Commun. 2007;361(1):156–61.

17 Analysis of bile acids by tandem mass spectrometry Diran Herebian and Ertan Mayatepek

17.1 Introduction Mass spectrometry (MS) is used as a detection technique to analyze individual or classes of metabolites quantitatively (concentration) and qualitatively (structure). A tandem mass spectrometer (MS/MS) is one of several different types of mass spectrometers. It consists of two mass spectrometers in series connected by a chamber known as collision cell. The target analytes (precursor ions) are selected in the first mass spectrometer (specific mass-to-charge ratio) and fragmented in the collision cell; then a specific fragment ion (product ion) of the desired analyte is isolated in the second mass spectrometer. In combination with UHPLC and electrospray ionization technique, the UHPLC/ ESI-MS/MS has become a very powerful instrument capable of analyzing both small and large molecules of various polarities in a complex biological sample. There are four different data acquisition modes that are commonly used in a tandem mass spectrometer: selected reaction monitoring (SRM), parent ion scan, daughter ion scan, and neutral loss. By using these different acquisition modes of a tandem mass spectrometer one can generate qualitative and quantitative metabolite profiling of certain classes of compounds (1,2). Bile acid (BA) analysis in biological fluids is generally a three-step process comprising extraction, separation, and detection of target analytes. For the extraction step, two techniques can be used: liquid-liquid extraction (LLE) or solid-phase extraction (SPE). Application of SPE columns in steroid analyses is now the most widely used extraction technique to avoid the negative effects of coeluting matrix components on the ionization efficiency of analytes. For the second step, BAs and their conjugates are separated by high-performance liquid chromatography (HPLC) or ultraperformance liquid chromatography (UPLC). HPLC/UPLC hyphenated to a tandem mass spectrometer (UHPLC-MS/MS) using electrospray ionization (ESI) in negative or positive mode has become the method of choice because of this platform’s high sensitivity, selectivity, and accuracy. In addition, some specific analytical problems, such as nonlinearity of calibration curves or poor repeatability, can be resolved by LC-MS/MS. Furthermore, UHPLC-MS/MS experiments allow for the enhancement of sensitivity using the selected reaction monitoring mode for the identification and determination of selected BAs and their metabolites in biological matrices (3–5). BAs are formed in the liver from cholesterol and excreted in human bile as glycine and taurine conjugates. Cholic acid (CA) and chenodeoxycholic acid (CDCA), called primary BAs, undergo further metabolism by intestinal bacterial enzymes during enterohepatic circulation. This dehydroxylation process generates secondary BAs, namely deoxycholic acid (DCA) and lithocholic acid (LCA). fFig. 17.1 displays the molecular structures of BAs and their glycine and taurine conjugates (6). Hepatobiliary diseases disturb the enterohepatic circulation of bile salts, resulting in quantitative and qualitative concentration changes in serum, plasma, urine, and bile. Classic methods of measuring BAs in clinical laboratories have focused on total serum

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17 Analysis of bile acids by tandem mass spectrometry R4

R1

COR5

R3 H R2

Bile acids

R1

R2

R3

R4

R5

OH OH OH OH OH

H H H H H

a-OH a-OH

OH H OH H H

OH OH OH OH OH

Unconjugates CA CDCA DCA UDCA LCA

H b-OH H

Glycine conjugates Taurine conjugates

NHCH2COOH NH(CH2)2SO3H

Fig. 17.1: Chemical structures of bile acids and their conjugates.

concentrations. By profiling BA metabolites, we can gain more clinically relevant information related to hepatobiliary diseases (ratio of glycine/taurine; mono-, di-, and trihydroxylated BA classes; response to ursodeoxycholic acid [UDCA]). The metabolism of BAs in the liver is complex and includes many biochemical reactions, such as hydrolysis, oxidation and reduction, isomerization steps, hydroxylation, and dehydroxylation reactions. Additional conjugation reactions can be performed with glucuronic acid, sulfuric acid, glucose, or N-acetylglucosamine. The multiple isobaric isomers of BAs, their presence in very low concentrations (μmol/L-nmol/L) in biological fluids (except in gallbladder), and differences in physicochemical properties make their separation by liquid chromatography indispensable (7).

17.2 Experimental procedures In the following, the mass spectrometry method for the analysis of BAs developed and performed in our laboratory is described. Following are the abbreviations used for bile acids and their conjugates: cholic acid (CA), muricholic acid (MCA), hyocholic acid (HCA), taurocholic acid (TCA), glycocholic acid (GCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), lithocholic acid (LCA), taurolithocholic acid (TLCA), lithocholic acid-3-sulfate (LCA-3S), hyodeoxycholic acid (HDCA), dehydrocholic acid (DHCA), and d4-cholic acid (d4-CA).

17.2.1 Sample extraction Solid-phase extraction cartridges HR-X (Macherey-Nagel, Düren, Germany) were used for sample extraction of BAs and their conjugates. Bile samples were diluted 100-fold

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with LC-MS water; then 100 μL of diluted bile samples were spiked with 1 μg/mL internal standard (d4-CA), vortexed, and loaded onto SPE cartridges preconditioned with 1 mL of MeOH followed by 1 mL of H2O. Finally, the loaded cartridges were washed with 3 mL of H2O and eluted with 1 mL of MeOH. The eluate was evaporated under a nitrogen stream and reconstituted in 1 mL of 80% MeOH. Serum/plasma samples (1 mL) were then spiked with 0.1 μg/mL internal standard (d4-CA) and prepared in the same way as bile samples. After evaporation by the nitrogen stream, the samples were reconstituted in 100 μL of 80% MeOH. Urine samples were treated as serum samples and used only for qualitative analysis.

17.2.2 Instruments The system consisted of an HPLC Waters Alliance 2795 separation module (Waters, Milford, UK) coupled to a Quattro Micro triple quadrupole mass spectrometer (Micro Mass, Manchester, UK). Electrospray ionization was performed in the negative ionization mode. The following optimal conditions were found: capillary voltage 3 kV, ion source temperature 120°C, desolvation temperature 375°C, desolvation gas flow 660 L/h, cone gas flow 60 L/h. Chromatographic separation was performed on an analytical HPLC Phenomenex Luna C18 column (2.1 × 150 mm, 3 μm) coupled to a guard column Phenomenex Gemini C18 (4.0 × 3 mm, 5 μm). The mobile phase consisted of water containing 0.01% formic acid and 5 mM ammonium acetate (eluent A) and methanol (eluent B). Sample elution was run isocratically in a ratio of 18:82 (A:B, v/v) over 15 minutes at a flow rate of 0.2 mL/min. Injection volume was 10 μL and the column was maintained at room temperature. Cone voltage (CV) and collision energy (CE) were tuned to optimize the transition of the molecular ion to the most abundant daughter ion. Unconjugated BAs were detected unfragmented (CE 12 eV, CV 50 V). Taurine- and glycine-conjugated BAs were analyzed using their specific fragment ions at m/z 80 (CE 65 eV, CV 75 V) and m/z 74 (CE 50 eV, CV 40 V). Analytes were detected in the selected reaction mode (SRM); see fTab. 17.1. Quantification analysis was performed using standard calibration curves in up to seven different concentration ranges (0.25, 0.5, 1, 5, 10, 15, and 20 μg/mL for bile samples and 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 μg/mL for serum/plasma samples) including the internal standard (8). The UPLC-MS/MS system was composed of a Waters Acuity UPLC-H Class Sample Manager coupled to a Waters TQ Detector (Waters, Eschborn, Germany). Electrospray ionization was performed in the negative ionization mode. The following optimal conditions were established: capillary voltage 3.2 kV, ion source temperature 120°C, desolvation temperature 275°C, cone gas flow 50 L/h, and desolvation gas flow 600 L/h. Analytes were separated on an Acuity UPLC BEH C18-column (1.7 μm, 100 mm ⫻ 2.1 mm; Waters, Eschborn, Germany) and thermostatted at T = 40°C. A gradient at a flow rate of 0.350 mL/min was achieved with mobile phase A, composed of water and acidified with 0.1% formic acid, and mobile phase B, composed of acetonitrile acidified with 0.1% formic acid. Following a 5-μL injection, analytes were eluted and separated on an isocratic 40% mobile phase B for 3 minutes, followed by a linear gradient to 90% of mobile phase B from 3 to 7 minutes and from 7 to 8 minutes at 90% isocratically. Subsequently, a linear gradient was applied from 90% to 40% of mobile phase B over 2 minutes in order to reequilibrate the system. The overall run time of this assay was 10 minutes. Parameters of both of the gradient systems mentioned above can be altered

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17 Analysis of bile acids by tandem mass spectrometry

Tab. 17.1: Mass spectrometric parameters of the studied bile acids and their conjugates in human and rodent samples Bile acid

-1

MW

[M-H] (m/z)

SRM

CA

408

407

407 / 407

α–MCA

408

407

407 / 407

β–MCA

408

407

407 / 407

ω–MCA

408

407

407 / 407

CDCA

392

391

391 / 391

DCA

392

391

391 / 391

HDCA

392

391

391 / 391

UDCA

392

391

391 / 391

DHCA

402

401

401 / 401

LCA

376

375

375 / 375

d4–CA

412

411

411 / 411

CA

465

464

464 / 74

α–MCA

465

464

464 / 74

β–MCA

465

464

464 / 74

ω–MCA

465

464

464 / 74

CDCA

449

448

448 / 74

DCA

449

448

448 / 74

HDCA

449

448

448 / 74

UDCA

449

448

448 / 74

LCA

433

432

432 / 74

CA

515

514

514 / 80

α–MCA

515

514

514 / 80

β–MCA

515

514

514 / 80

ω–MCA

515

514

514 / 80

CDCA

499

498

498 / 80

DCA

499

498

498 / 80

HDCA

499

498

498 / 80

UDCA

499

498

498 / 80

LCA

483

482

482 / 80

Unconjugates

Glycine conjugates

Taurine conjugates

-1

MW: molecular weight; m/z: mass-to-charge; [M-H] : deprotonated molecular ion; SRM: selected reaction monitoring, d4–CA: deuterated CA as internal standard

17.3 Applications

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281

according to the lifetime of the used column and the number of injections. Concentrations were calculated using the QuanLynx or TargetLynx (MassLynx 4.1, Waters) software.

17.2.3 Method validation The limit of detection (LOD) and the limit of quantification (LOQ) of the different BAs were calculated using signal-to-noise ratios of 3 and 10, respectively. Linearity of response was tested by analyzing standard samples. Calibration curves were determined by least square regression and were linear for the lower (0.1–4 μmol/L) and higher (0.5– 40 μmol/L) calibration range, respectively. The precision and accuracy of the method were studied under two conditions: intra-assay (within days) and interassay (between days). Intra-assay precision was determined by analyzing five replicates of each analyte; and for the inter-assay this was done once on 5 different days. Coefficients of variation were between 2.1% and 5.3% for intra-assay analyses, whereas the values for interassay analyses were between 1.8% and 7.4%. Accuracy values for intra-assays were between 96% and 98%; for inter-assays they were between 94 and 98%. Recoveries of different BAs after solid-phase extraction were greater than 92%. No significant matrix effects (e.g., in terms of ion suppression) were found in sample extracts of SPE by using the T-valve infusion.

17.3 Applications 17.3.1 Analysis of serum samples The biliary BA composition in preprandial serum samples was analyzed by HPLC-MS/ MS in 40 healthy adults. Results of the mean BA concentrations are presented in fTab. 17.2. This method detects a total number of 18 BAs including the deuterated internal standard d4-CA. The four unconjugated trihydroxylated and dihydroxylated BAs (CA, CDCA, DCA, and UDCA) were detected in nearly all serum samples. Monohydroxylated BA LCA was not detectable in any sample. The major BAs were glycine conjugates, followed by unconjugates and taurine conjugates. GCDCA and TCDCA were the major BAs of their conjugation class. TUDCA was not detectable, whereas GUDCA and UDCA were found in some samples at very low concentrations. The conjugates of LCA, namely GLCA and TLCA, were not present in any sample. The mean ratio for trihydroxylated BA/dihydroxylated BA was 0.61 and for glycine conjugates/ taurine conjugates (G/T) 8.15. The total average concentration of all measured BAs of the 40 healthy adults was 1.42 μM; it was 0.202 μM for the minimal detected total concentration and 6.187 μM for the maximal concentration. fFig. 17.2 shows a serum profile of BAs of a patient suffering from a cholestatic disease. Elevated BA conjugates are predominantly found in comparison with the unconjugated BA. The total concentration of serum BA was 51 μM, compared with 1.42 μM in control subjects. Serum TCDCA and GCDCA were the major BAs, with a concentration of 18.95 and 18.53 μM, followed by TCA and GCA, with a concentration of 5.61 and 6.09 μM, respectively. The ratio of trihydroxylated BA/dihydroxylated BA was 0.31 and represents nearly 50% of the ratio in control subjects, whereas the ratio of G/T was 1.00. DCA and its conjugates were not detected, indicating an interruption of the enterohepatic circulation. Serum UDCA concentration and its conjugates did not

282

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17 Analysis of bile acids by tandem mass spectrometry

Tab. 17.2: Bile acid concentrations in human serum of healthy adults (n = 40) Compound

MEAN ± SD [μM]

Range (min/max) [μM]

CA

0.323 ± 0.652

0/3.344

CDCA

0.080 ± 0.124

0.002/0.516

DCA

0.122 ± 0.112

0.003/0.594

UDCA

0.036 ± 0.067

0/0.368

LCA

0

0/0

TCA

0.017 ± 0.035

0/0.180

TCDCA

0.054 ± 0.065

0/0.273

TDCA

0.023 ± 0.035

0/0.141

TLCA

0

0/0

TUDCA

0

0/0

GCA

0.199 ± 0.174

0.03/0.815

GCDCA

0.401 ± 0.361

0.057/2.018

GDCA

0.124 ± 0.102

0/0.590

GLCA

0

0/0

GUDCA

0.043 ± 0.060

0/0.284

LCA-3S

0

0/0

DHCA

0.002 ± 0.013

0/0.085

Unconjugated

0.562 ± 1.30

0.026/3.946

Taurine-BA

0.094 ± 0.112

0/0.503

Glycine-BA

0.766 ± 0.557

0.137/2.72

Total

SD = standard deviations

differ from those of the controls. In case of unconjugated BA, CDCA concentration was elevated (1.56 μM); however, that of CA was not (0.62). Serum TCA, TLCA, GLCA, and LCA-3S were not detected at all, which likewise may indicate a reduced enterohepatic circulation and dehydroxylation of CDCA.

17.3.2 Analysis of bile samples BAs and their conjugates from bile samples are usually present at very high concentrations (mmol/L) and hence must be diluted (1:100 to 1:1000) before separation and detection by LC-MS/MS. As depicted in fFig. 17.3, a bile sample of a healthy human subject revealed all the conjugates of primary and secondary BAs. Bile GCA, GCDCA, TCA, and TCDCA are found as major BAs in this profile, whereas GDCA, GUDCA, TDCA, and TUDCA are detected at lower concentrations. The total concentration of these detected six conjugates was 13 mmol/L. The unconjugated trihydroxy compound

冷

283

20 18 16 14 12 10 8 6 4 2 0

CA CD CA DC A U DC A TC TC A DC A TD CA TU DC A G C G A CD CA G DC G A U DC A DH CA

mM

17.3 Applications

BA

CA

G DC A

G CA G U DC A G CD CA

TD CA

TC DC A

TU DC A

16000 14000 12000 10000 8000 6000 4000 2000 0

TC A

mM

Fig. 17.2: Comparison of bile acid concentrations in serum from healthy subjects (black columns) and a cholestatic patient (gray balks).

BA

Fig. 17.3: Comparison of bile acid concentrations in bile from a healthy subject (black balks) and a patient with primary sclerosing cholangitis (gray balks).

CA showed a concentration of about 117 μM. The G/T ratio including UDCA derivatives was 1.28; without UDCA it was 1.27. fFig. 17.3 also includes a BA profile from a bile sample of a patient with primary sclerosing cholangitis (PSC) under UDCA treatment. The obtained profile was dominated by GUDCA as major product of UDCA therapy. Conjugates of primary BA were found at a total concentration of 20 mmol/L. The total concentration of GUDCA and TUDCA was 18 mmol/L. Bile DCA and its glycine and taurine metabolites were not detected, indicating a disruption of the enterohepatic circulation. The G/T ratio including UDCA was 3.12; without UDCA it was 2.13.

17.3.3 Treatment with UDCA Bile salts undergo enterohepatic circulation, which is mediated by efficient transport through the hepatocytes and enterocytes. The resulting bile pool cycles between liver and intestine. The primary BAs, CAs, and CDCAs are converted to secondary BAs, DCAs, and LCAs by bacterial enzymes by dehydroxylation processes. The toxic derivative of CDCA, namely LCA, undergoes a double conjugation during hepatocyte transport. The

284

冷

17 Analysis of bile acids by tandem mass spectrometry 25.00

mmol/L

20.00 15.00 10.00

LCA

DCA

CDCA

UDCA

CA

DHCA

GLCA

GDCA

GCDCA

GUDCA

GCA

LCA-S

TLCA

TDCA

TCDCA

TCA

0.00

TUDCA

5.00

BA

Fig. 17.4: Serum bile acid profile comprising LCA and its metabolites of a cholestatic patient treated with UDCA.

448.8

Scan ES1.36e8

528.7

%

100

498.7 464.8

651.8 530.6

0

701.80

m/z 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

Fig. 17.5: Urinary mass spectrum of a cholestatic patient treated with UDCA.

first conjugation is an amidation reaction at C-24 with taurine/glycine; the second reaction comprises esterification with a sulfate group at C-3, preventing intestinal absorption and favoring rapid elimination through the bowl. Formation and changes in LCA levels (LCA, TLCA, GLCA, and LCA-3-S) can provide more insight into the pathophysiology of the disease. All four metabolites were detected at low concentrations in the serum sample of a patient who was treated with UDCA (see fFig. 17.4). Oral administration of UDCA to patients with cholestatic diseases results in the amelioration of pruritus and serum liver values. UDCA and its conjugates TUDCA and GUDCA are always present at higher concentration in serum samples during treatment. In urine, UDCA can be detected as a double-conjugated compound, namely GUDCA-N-acetylglucosamine (m/z 651) or TUDCA-N-acetylglucosamine (m/z 701). The peak at m/z 528 in fFig. 17.5 corresponds to the double-conjugated dihydroxy BA with glycine and sulfate (GUDCA-sulfate). Further molecular ions at m/z 448, 464, 471, 480, 498, 514, and 530 correspond to gly-dihydroxylated, gly-trihydroxylated, sulfate-dihydroxylated,

冷

17.3 Applications

285

gly-tetrahydroxylated, tauro-dihydroxylated, tauro-trihydroxylated, and tauro-tetrahydroxylated BAs. This indicates that the effects of UDCA, as hepatoprotective drug, on the BA pool can be monitored by mass spectrometry during treatment.

17.3.4 Analysis of bile acids in infants Infants with cholestatic hepatobiliary diseases show raised concentrations of total serum BAs. Qualitative analysis of urinary samples, as a noninvasive tool, by mass spectrometry can distinguish between cholestatic and noncholestatic conditions in infants. The full scan, as shown in fFig. 17.6, revealed the presence of the following prominent molecular ions: 448.8, 464.8, 480.8, 498.7, 514.8, and 530.6 (m/z). They correspond to glycine- or taurine-conjugated dihydroxy BAs (m/z 448.8 and 498.7, respectively), trihydroxy BAs (m/z 464.8 and 514.8, respectively) and tetrahydroxy BAs (m/z 480.8 and 530.6, respectively). In addition, in this scan the presence of glycodihydroxy monosulfate (m/z 528.8) could be confirmed. Full scans of healthy subjects did not show any of the prominent ions mentioned above. By using the full scan and eventually the precursor ion mode, it is therefore possible to distinguish cholestatic patients from those without cholestasis. Qualitative analysis of urinary BAs und their intermediates are of great importance for the diagnosis of inborn errors of BA metabolism. For quantitative determination, it is recommended to analyze serum or plasma samples of affected patients by the selected reaction monitoring mode (9,10).

514.8

100

Scan ES5.24e7

448.8

%

464.8

530.6 498.7

528.8

480.8 0 360

380

400

420

440

460

480

500

520

540

560

580

600

620

640

660

680

m/z 700

Fig. 17.6: Typical urinary mass spectrum of a cholestatic infant.

17.3.5 Analysis of bile acids in rodents Experimental studies of BA metabolism in hepatobiliary diseases are in general performed by using rodents as model systems. Major primary BAs (e.g., in mice or rats) are CA (3α,7α,12α-trihydroxy), α-MCA (3α,6β,7α-trihydroxy) and β-MCA (3α,6β,7β-trihydroxy). Intestinal bacteria convert CA into DCA, CDCA into LCA and UDCA, α-MCA into HCA (hyocholic acid), and β-MCA into HDCA and ω-MCA (3α,6α,7β-trihydroxy). BAs in mice or rats are mainly conjugated with taurine before excretion into bile. Simultaneous analysis of these BAs and their taurine and glycine conjugates by LC-MS/MS is necessary

286

冷

17 Analysis of bile acids by tandem mass spectrometry

12.07

100

CDCA %

UDCA

MRM of 12 Channels ES391 쏜 391.3 3.36e6 12.31

DCA

HDCA

6.82

8.23

Time

0 1.00

2.00

3.00

4.00 3.87

100

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

%

8.19

GHDCA

9.12

GCDCA

14.00

MRM of 12 Channels ES448.1 쏜 73.6 3.91e5

3.43

GUDCA

13.00

GDCA

0

Time 1.00

2.00

100

3.00 3.15

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

%

TUDCA

THDCA

8.44

TCDCA

14.00

MRM of 12 Channels ES498 쏜 79.5 1.55e5

3.51 7.55

13.00

TDCA

0

Time 1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

Fig. 17.7: Elution profile of the isobaric isomers of bile acids and their conjugates.

to provide more scientific understanding on the metabolic profile of BAs in liver, bile, and intestine. The choice of the separation column and composition of the mobile phase are highly essential to separate isobaric compounds such as α-, β-, ω-MCA, HCA, and CA (11). These trihydroxylated BAs have the same molecular weight; however, they differ in their molecular structures (OH-groups in different positions). This is also true for the dihydroxylated BAs (UDCA, HDCA, CDCA, and DCA). fFig. 17.7 presents a typical elution profile of unconjugated and conjugated tri- and dihydroxylated BAs in order of decreasing hydrophilicity of the mobile phase.

17.4 Summary Tandem mass spectrometric instruments in combination with HPLC/UPLC offer excellent detection and separation options for profiling BAs and their conjugates in biological samples (e.g., serum, plasma, urine, or bile). This hyphenated technique provides accurate quantitative and qualitative analyses. BAs are often present in unconjugated, single-conjugated, or double-conjugated forms, which makes the use of appropriate separation columns inevitable. BA profiles of affected patients with cholestatic liver diseases combined with biochemical parameters, clinical symptoms, and further diagnostic tools can give us a deeper insight into the pathophysiology of these conditions. LC-MS/MS methods involving BAs can be also extended to metabolic or

17.5 References

冷

287

pharmacokinetics studies in animal models, providing a broad spectrum of useful scientific and clinical information.

17.5 References 1. Ho CS, Lam CWK, Chan MHM et al. Electrospray ionisation mass spectrometry: principles and clinical applications. Clin. Biochem. Rev. 2003;24: 3–12. 2. Watson JT, Sparkman OD. Introduction to mass spectrometry: instrumentations, applications and strategies for data interpretation. 4th edition, John Wiley & Sons Ltd, West Sussex, England, 2009. 3. Want EJ, Coen M, Masson P et al. Ultra performance liquid chromatography-mass spectrometry profiling of bile acid metabolites in biofluids: application to experimental toxicology studies. Anal. Chem. 2010;82: 5282–9. 4. Ye L, Liu S, Wang M, et al. High-performance liquid chromatography-tandem mass spectrometry for the analysis of bile acid profiles in serum of women with intrahepatic cholestasis of pregnancy. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 860:10–7. 5. Lu X, Zhao X, Bai C, et al. LC-MS-based metabonomics analysis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008;866:64–76. 6. Mukhopadhyay S, Maitra U. Chemistry and biology of bile acids. Current Science 2004; 12: 1666–83. 7. Sjövall J, Griffi ths WJ, Setchell KDR, et al. Analysis of Bile Acids. In: Steroid analysis. Makin HLJ, Gower DB, 2nd ed. Springer Netherlands, 2010, 837–966. 8. Qiao X, Ye M, Liu CF, et al. A tandem mass spectrometric study of bile acids: Interpretation of fragmentation pathways and differentiation of steroid isomers. Steroids, doi:10.1016/j. steroids.2011.11.008. 9. Yousef IM, Perwaiz S, Lamireau T, et al. Urinary bile acid profile in children with inborn errors of bile acid metabolism and chronic cholestasis; screening technique using electrospray tandem mass-spectrometry (ES/MS/MS). Med. Sci. Monit. 2003;9(3):MT21–31. 10. Herebian D, Mayatepek E. Inborn errors of bile acid metabolism and their diagnostic confirmation by means of mass spectrometry. J. Ped. Sci. 2011;3:e68. 11. Alnouti Y, Csanaky IL, Klaassen CD. Quantitative-profiling of bile acids and their conjugates in mouse liver, bile, plasma, and urine using LC-MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008;873:209–17.

Index

ABCG2 24 ABCG5/ABCG8 24, 197, 202, 258, 261 Alagille syndrome 131, 145 alkaline phosphatase 208 anion exchanger 2 (AE2) 214, 256 anti-BSEP antibodies 156 anti-mitochondrial antibodies 207, 210, 212, 213, 214 apical sodium-dependent bile acid transporter (ASBT, SLC10A2) 3, 74, 78, 94, 256 apolipoprotein E 122 apoptosis 87, 91, 117 atherogenesis 108, 122, 264 atorvastatin 7 ATP binding cassette transporter 23, 171 B-cell 103 benign recurrent intrahepatic cholestasis (BRIC) 154, 155, 187 bile acid — anti-inflammatory effects 11, 103, 107, 110, 122 — apoptosis 87, 117 — carcinogenesis 117, 233, 257, 264 — cell proliferation 93, 117 — enterohepatic circulation 73, 74, 77 — fluorescent 5, 9 — glucose homeostasis 262 — interferon signaling 109 — lipid homeostasis 260 — pool 76, 259, 263 — secretion 77 — sequestrant 118, 263 — signaling 85, 118 — synthesis 73, 76, 254, 261 bile duct ligation 51

— bile salt export pump 57 — Kupffer cell 110 bile salt export pump (BSEP, ABCB11) 8, 9, 11, 24, 54, 77, 85, 151, 152 184, 188, 254, 258 biliary atresia 6 bilirubin 171 bosentan 10, 244 breast cancer resistance protein (BCRP, ABCG2) 37–39 bromosulphophthalein 5 bumetanide 6 Byler’s disease 153 caspase 3 110 CD95 87 — internalization 88 — oligomerization 88, 91 — serine/threonine phosphorylation 88, 90 — tyrosine phosphorylation 88, 91 ceramide 56 cholangiocarcinoma 95, 141, 225, 233, 264 cholangiocyte 93, 131, 134, 138, 212, 229 — apoptosis 93, 212 — proliferation 145 — secretion 94, 138, 213, 256 cholecystokinin 199 choleresis 85, 87, 255, 256 cholestasis 2, 8, 80, 91, 92, 122, 145, 146, 156, 177, 184, 187, 207, 208, 231, 232, 264 cholesterol 7α hydroxylase 37, 79, 199, 256, 258, 260, 261 cilia 135, 136, 143, 232, 256 c-Jun-N-terminal kinase 88, 92, 265 clotrimazole 243 colitis 108, 223, 259 Crigler-Najjar syndrome 174

290

冷

Index

c-Src-kinase 86, 92 cyclosporine A 34, 199, 244 CYP7A1. See cholesterol 7α hydroxylase cyst formation 142, 143 cystic fibrosis 199 cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7) 94, 143, 231, 232, 256 cytokine 91, 120, 177, 214, 227, 231, 233, 257, 260 daunomycin 39 deiodinase 2 (D2) 120, 263 diabetes type II 118, 262 diclofenac 246 DNA-binding domain (DBD) 71, 72 doxorubicin 37 drug-induced liver injury 161 — bile salt export pump 244, 246 — incidence 241 — MRPs 243 — uptake transporter 242, 246 Dubin-Johnson syndrome 176 ductal plate 131, 135, 136, 140 ductopenia 145, 207 endothelial NO synthase (eNOS) 90, 121, 257 energy expenditure 120, 263 enterocyte 118, 121 epidermal growth factor receptor (EGFR) 86, 88, 91 estrogens 5, 10, 11, 54, 183, 243, 245 estrogen receptor (ER) 185, 211 exonic splicing enhancer 154 ezetimibe 199 farnesoid X receptor (FXR) 6, 71, 73, 90, 117, 189, 232, 253 agonist 79, 80, 119, 120, 122, 210, 255, 259, 261, 262, 263 knockout 57, 106 target gene 76, 259 — HCV replication 111 — Hepatitis B 111

— pro-inflammatory cytokine 106, 107 — responsive element (FXRE) 73 FGF. See fibroblast growth factor fibrates 199 fibroblast growth factor 2, 77, 119, 254, 262 fibrocystin/polyductin (FPC) 137, 141 focal adhesion kinase (FAK) 86 Forkhead box proteins 134 FXR. See farnesoid X receptor gallbladder 172, 259 gallstone 195 — benign intrahepatic recurrent cholestasis 201 — cholesterol 195, 197 — cystic fibrosis 199 — diet 195 — environment 196 — epidemiology 195 — formation 79, 95, 189, 258 — hemolysis 197 — hyperbilirubinemia 196 — lith genes 196 — pigment 195 — twins 196 — weight cycling 196 gamma-glutamyltranspeptidase 187, 255 Gilbert’s syndrome 174, 200 glibenclamide 244 glucagon-like peptide-1 119, 120, 260, 263 glucose homeostasis 119 hepatic encephalopathy 124, 265 hepatic stellate cells 91, 92, 232, 257 hepatoblast 135 hepatocyte nuclear factor 77, 132, 134, 136, 141, 185, 262 hepatocyte swelling 87 human leukocyte antigen complex (HLA) 210, 227 hyperbilirubinemia 173, 174, 175 hypercholanemia 6 hyperosmolarity 54

Index

indocyanine green 6 insect cell 39 insulin resistance 198 integrin 55, 85 interleukin-2 214, 228 interleukin-6 110, 121, 233, 260 intrahepatic cholestasis of pregnancy (ICP) 12, 156, 160, 184, 186 ivermectine 34 kidney 122, 172 Kupffer cells 90, 103, 105, 108, 118, 230, 253, 257 ligand-binding domain (LBD) 72 lipopolysaccharide 58, 106 Lith genes 201 liver cirrhosis 78, 209, 223, 231, 260 liver receptor homolog 1 (Lrh1, NR5A2) 57 liver regeneration 53 liver X receptor 71, 76 low phospholipid associated cholelithiasis syndrome (LPAC) 159, 160, 201 lysophosphatidylcholine 39 major histocompatibility complex (MHC) 227, 230, 233 Meckel syndrome 140 metabolite profiling 277 microlithiasis 201 mitogen activated protein kinase 55, 86, 88, 138 mitoxantrone 37, 40 mixed micelles 1, 32, 158 multidrug resistance-associated protein 4 (MRP4/ABCC4) 12 multidrug resistance-associated protein 2 (MRP2, ABCC2) 12, 24, 54, 77, 122, 171, 183, 197, 255 multidrug resistance protein 3 (MDR3, ABCB4) 24, 32, 74, 158, 187, 190, 231, 232, 254, 258, 260 — ATPase activity 36

冷

291

— drug induced liver injury 161 — expression 158, 159 — function 158 — intrahepatic cholestasis of pregnancy 160 — isoforms 158 multidrug resistance-associated protein 3 (MRP3, ABCC3) 12, 171 NADPH oxidase (Nox) 56, 87 Na+/ K+-ATPase 49 NBD. See nucleotide binding domain nervous system 124 Notch 134, 145 nuclear factor-κB (NF-κB) 91, 106, 109, 121, 228, 230, 231, 232, 257, 258, 259 nuclear liver receptor homolog-1 (LRH-1) 76 nuclear receptor 71, 72, 105, 106, 183, 186, 189 nucleotide binding domain (NBD) 23 obstructive jaundice 103, 110 octreotide 199 organic anion-transporting protein (OATP) 2, 7, 8, 171, 173, 183, 185 organic solute transporter (OSTα /OSTβ) 12, 74, 78, 122, 255, 259 osmolarity 54 osteopontin 107 oxidative stress 54, 57 paclitaxel 34 PBC. See primary biliary cirrhosis Pglycoprotein 34 phalloidin 58 phorbolester 54 phosphatidylinositol 3-kinase (PI3K) 86, 88, 89, 94 phosphoenolpyruvate carboxykinase (PEPCK) 119, 262 phospholipid 34, 78, 258 polycystic kidney disease 141, 142

292

冷

Index

polycystin (PC) 137, 138, 142 pregnane X receptor (PXR) 232, 253 primary biliary cirrhosis (PBC) 8, 157, 207, 209, 228, 254, 255 primary sclerosing cholangitis (PSC) 8, 157, 223, 229, 232, 256, 260 progesterones 185, 186, 245 progressive familial intrahepatic cholestasis (PFIC) 6, 8, 24, 77, 78, 153, 154, 158, 159, 162, 187, 231 protein kinase A 88, 94, 138 protein kinase C 54, 87 reactive oxygen species 87, 124 retinoid X receptor (RXR) 72, 73 rosuvastatin 5, 7 Rotor syndrome 176 signal transducer and activator of transcription (STAT) 88, 233 sinusoidal endothelial cells (SEC) 89, 118, 253 small heterodimer partner (SHP) 77, 189, 254, 261 sodium-taurocholate cotransporting protein (NTCP, SLC10A1) 3–7, 49–53, 74, 78, 85, 184, 186, 255 sphingomyelinase 56 Src kinase 56 sterol-regulatory element binding protein (SREBP) 122, 185, 189, 261 tacrolimus 199 tandem mass spectrometer 277 taurolithocholate-3 sulfate 9

tauroursodesoxycholate. See ursodeoxycholic acid T cells 207, 210, 227, 233 — homing 229 — regulatory T cells (Tregs) 214, 228 TGR5 (Gpbar-1) 90, 108, 118, 124, 230, 232, 253, 257, 265 TGR5 agonist 119, 256, 258, 264 thymeleatoxin 54 transforming growth factor-β 134, 135, 142, 261 transporter retrieval 50, 54, 58 troglitazone 245 tumor necrosis factor-α 121, 211, 229, 259 UDP glucuronosyltransferase 174, 197 ursodeoxycholic acid (UDCA) 54, 85, 90, 94, 207, 209, 214, 253, 254, 255 vascular endothelial growth factor (VEGF) 135, 138, 142 vinblastine 34 viral hepatitis 111 von Meyenburg complex (VMC) 131, 141 Wingless (Wnt)-signaling 135 Xenopus laevis 3, 38 zonal regulation 53 zonula occludens protein 1 58