Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications [1 ed.] 9781620813409, 9781620813065

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

CELL BIOLOGY RESEARCH PROGRESS

OSTEOCLASTS

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MORPHOLOGY, FUNCTIONS AND CLINICAL IMPLICATIONS

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Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

CELL BIOLOGY RESEARCH PROGRESS

OSTEOCLASTS MORPHOLOGY, FUNCTIONS AND CLINICAL IMPLICATIONS

ALEXANDER J. BROWN Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

AND

JOHANNA S. WALKER EDITORS

Nova Science Publishers, Inc. New York

Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

vii Modulation of Osteoclast Differentiation and Function in Rheumatoid Arthritis Roya Lari Osteoclast Formation and Function and Its role in Skeletal Bone Disease Monique Bethel, Angela Bruzzaniti, and Melissa A. Kacena The High Osteoclastogenic Potential of Human Osteosarcoma Cells: Reciprocal Interaction between MG63 Osteoblast-Like Cells and Osteoclast Precursors J. Costa-Rodrigues and M. H. Fernandes

1

31

53

Chapter 4

Osteoclast Biology in Paget’s Disease of Bone Stephen McManus, Lyne Bissonnette, and Sophie Roux

77

Chapter 5

Osteoclasts: The Major Actors in Bone Resorption Lucia D’Amico and Ilaria Roato

95

Chapter 6

Role of the Immuno-Skeletal Interface in Physiological and Pathological Osteoclast Regulation M. Neale Weitzmann

113

Multiple Functions of Osteoclasts and Potential Usefulness of Phosphatidylserine-Containing Liposomes on Bone Diseases Zhou Wu and Hiroshi Nakanishi

131

Chapter 7

Chapter 8

Chapter 9

Bone Formation and Osteoclastic Resorption after Implantation of B -Tricalcium Phosphate (B -TCP) Takaaki Tanaka, Masaaki Chazono, Seiichiro Kitasato, Atsuhito Kakuta, and Keishi Marumo Osteoclasts of Patients with Neurofibromatosis 1 (NF1) Eetu Heerva and Juha Peltonen

Index

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153 159

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PREFACE Osteoclasts are highly specialized cells, derived from the hematopoietic lineage, that resorb mineralized bone matrix. Osteoclast precursors can be recruited to the bone remodeling sites by specific cytokines in the microcellular environment, including some secreted by osteoblasts, fibroblasts, and osteocytes. In this book, the authors present current research in the study of the morphology, functions and clinical implications of osteoclasts. Topics include osteoclast formation and function and its risk in skeletal bone disease; the high osteoclastogenic potential of human osteosarcoma cells; osteoclast biology regarding Paget's disease and p62 mutations; the role of immuno-skeletal interface in the regulation of osteoclast formation; and osteoclasts in patients with neurofibromatosis 1. Bone-resorbing osteoclasts belong to the monocyte / macrophage lineages. Osteoclast progenitors migrate to the bone surface where they differentiate and fuse to form multinucleated osteoclasts. The bone microenvironment, including the presence of stromal cells (pre-osteoblast precursors) and mature osteoblasts, appears to be essential for osteoclast differentiation. Osteoblasts/stromal cells contribute to osteoclast differentiation by supplying receptor activator of nuclear factor- B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, in inflammatory pathological situations, such as RA other cell types such as synovial fibroblasts and activated T cells are also involved in the production of osteoclastic regulatory factors, contributing to the balance between the osteoclast-osteoblast functions. This review provides an overall picture of the effects of inflammatory factors on osteoclast differentiation and functions Chapter 1 - Rheumatoid arthritis (RA) is an autoimmune disease that characterized by synovial inflammation and bone and cartilage erosion. Several distinct patterns of bone loss have been shown in individuals with RA. These include juxta-articular osteopenia and focal erosions of subchondral bone and at joints in areas of direct inflammation. Also, the inflammatory processes in RA may cause slow induction of generalized osteopenia and osteoporosis of the axial and appendicular skeleton. The consequences of this bone loss are painful joint deformities, progressive functional disability, increased risk of bone fractures, and increased mortality rates. Studies have indicated that osteoclasts are the major cell type in bone resorption in RA. Chapter 2 - Osteoclasts are highly specialized cells, derived from the hematopoietic lineage, that resorb mineralized bone matrix. Osteoclast precursors can be recruited to the bone remodeling sites by specific cytokines in the microcellular environment, including some secreted by osteoblasts, fibroblasts, and osteocytes. Once the mononuclear osteoclast

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Alexander J. Brown and Johanna S. Walker

precursors converge on the bone surface, they fuse into large, multinucleated cells, a process that results in a highly efficient bone-resorbing cell. Cytoskeletal rearrangement then occurs, polarizing the cell such that the area of the cell directly in contact with the bone surface forms a sealing zone surrounding the ruffled border membrane which provides a large surface area for resorption. In the normal physiologic state, bone resorption and formation are tightly linked. However, several diseases have been identified where dysregulated bone resorption leads to bone of abnormal quality. Overly active osteoclasts have been implicated in the bone loss that occurs in a diverse set of diseases including osteoporosis, cancer, and inflammatory arthritis. Conversely, in osteopetrosis, underactive osteoclasts can lead to abnormally dense bone. In either situation, high or low bone mass, the poor bone quality leads to increased risk of fracture. Chapter 2 reviews the current knowledge of the cellular events that recruit osteoclasts to sites of bone remodeling, initiate differentiation and fusion, and induce the cytoskeletal rearrangements that prepare the cell for bone resorption. Also, the chapter examines how disruptions in any one or several of these processes create abnormalities in osteoclast morphology and function and contribute to skeletal disease in humans. Chapter 3 - Bone is in a continuous remodeling process which involves a complex and coordinated activity of osteoblastic and osteoclasticcells. Unbalances in this equilibrium leads to bonemetabolic pathological conditions, occurring in a variety of clinical situations, including bone primary tumors. Osteosarcoma, the most frequent bone oncologic disease, although with a variable behavior, affects bone metabolic activities, leading to a disturbed bone structure. Osteosarcoma is usually associated to the formation of woven bone, but in the last years it become evident that patients with this pathology frequently display a high bone turnover, with a progressive weakening of its structure. Osteosarcoma cells are osteoblast-like cells that display many osteoblastic features. Due to that, osteosarcoma cell lines are widely used as osteoblastic models for in vitro studies. Among them, MG63 cell line is by far the most used cell line in that context. Although the role of osteoblasts in osteoclastogenesis is well documented, the influence of MG63 osteosarcoma cells on osteoclast development remains poorly elucidated. In the last few years, the authors have been interested in this issue, aiming to characterize the osteoclastogenic potential of MG63 cells regarding both paracrine and direct cell-to-cell mechanisms. To address this, human osteoclast precursors from peripheral blood mononuclear cells were cultured in the presence of conditioned media from MG63 cell cultures or co-cultured with MG63 cells, in several experimental conditions. The osteoclastogenic response was compared with that achieved by human osteoblastic bone marrow cells (hBMC), in similar conditions. The influence of some osteoclastogenic signaling pathways was also addressed. MG63 cell line was able to elicit a high degree of osteoclast differentiation. Regarding paracrine mediated mechanisms, conditioned media from MG63 cell culturesinduced a significant osteoclastogenic response. Also, in co-cultures of osteoclast precursors and MG63 cells, osteoclastogenesis proceeded to a high extent in the presence of a low number of MG63 cells. In both situations, the osteoclastogenic response was higher than that elicited by hBMC, in similar experimental conditions. The intracellular mechanisms involved in the osteoclastogenesismediated by MG63 cells and hBMCrevealed some important differences, namely regarding the relative relevance of MEK and NFkB signaling pathways, and PGE2 production. Also, it was observed that the presence of osteoclastic cells modulate the osteoblastic behavior of MG63 cells.

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Preface

ix

In conclusion, MG63 cell line has a high osteoclastogenic potential and, compared to hBMC, differences were found regarding the pattern and intensity of the osteoclastic response, as well as in the involved intracellular pathways. Results suggest that understanding the molecular details of the osteoclastichyperactivation induced by osteosarcoma cells can open new perspectives for the design of therapeutic approaches for thistype of bone metabolic disorders. Chapter 4 - Paget’s disease of bone (PDB) is characterized by focal and disorganized increases in bone turnover. Because the initial phase of PDB involves excessive bone resorption, osteoclasts have been identified as the cells primarily affected in PDB. Pagetic osteoclasts are both larger and more numerous than normal osteoclasts. They are overactive and hypersensitive to osteoclastogenic factors, and resistant to apoptosis. Although a viral etiology has been suggested for Paget’s disease, several studies have revealed a marked genetic component. The discovery of mutations of the SQSTM1 (Sequestosome1, p62) gene in numerous patients has identified the protein p62 as an important modulator of bone turnover. p62 mediates several diverse cell functions, including the control of NFsignaling, protein trafficking and autophagy. Since SQSTM1 mutations do not fully explain the osteoclast phenotype of PDB, the contribution of other osteoclast-related genes, viruses or environmental factors may be involved. Chapter 4 reviews the most recent advances in osteoclast biology regarding PDB with a particular attention to the impact of the p62 mutations. Chapter 5 - The Osteoclast (OC), the exclusive bone resorptive cell, is a member of the monocyte/macrophage family and polykarion that can be generated by cytokine-driven proliferation and differentiation of monocyte precursors, which can circulate within the hematopoietic cell pool or be resident in a number of tissues. The maintenance of adequate bone mass is dependent upon the controlled and timely removal of old, damaged bone. This complex process is performed by the OCs, thus an increase of OC activity is observed in many pathologies characterized by bone loss, such as osteoporosis, hyperparathytoidism, rheumatoid arthritis, bone metastasis, periprosthetic osteolysis in aseptic loosening of arthroplasty and also in pediatric diseases like phenilketonuria and 21-hydroxylase deficiency. A primary mediator of osteoclastogenesis is the RANK-RANKL-OPG system, but also other factors may promote OC activation, according to the different pathologies. Anyhow the last result is the bone loss, primarily due to an expansion of the osteoclastic pool only partially or not compensated by a stimulation of bone formation. This review summarizes the main mechanisms promoting osteoclastogenesis in diseases characterized by bone loss, focusing on factors and cytokines involved in this process and on the interaction between OCs and T cells. Chapter 6 - The immuno-skeletal interface is a centralization of shared cells and cytokine effectors that serve critical functions within both the immune and skeletal systems. The precursors of the osteoclasts, the cells that resorb bone, have long been recognized to derive from monocytes/macrophages, cells central to both innate and adaptive immunity. Furthermore, osteoclast differentiation and function is potently responsive to a host of cytokine mediators secreted by multiple immune-related cell types during physiological immune renewal and pathological immune activation. Among these factors are receptor activator of NF(RANKL), the key osteoclastogenic cytokine and osteoprotegerin (OPG) its physiological decoy receptor, as well as a host of inflammatory cytokines that directly and/or indirectly amplify osteoclast formation and activity. As a consequence of this

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close association between the immune and skeletal systems, responses by the immune system to a wide range of pathological and inflammatory stimuli potently upset physiological bone turnover initiating a wave of inappropriate osteoclastogenesis and an upswing in bone resorption. Such disturbances to the immuno-skeletal interface may underlie the bone loss associated with numerous diverse pathological conditions ranging from postmenopausal osteoporosis to autoimmune and/or inflammatory states such as rheumatoid arthritis, inflammatory bowel disease, periodontal infection and in ”inflammaging” the generalized inflammatory state associated with aging. Paradoxically, not only do inflammatory states drive bone loss, but disruptions to basal immune function characteristic of immunodeficiency also alter physiological osteoclastogenesis and may be central to the high rates of skeletal deterioration associated with HIV-infection. This chapter examines the current understanding of the role of the immuno-skeletal interface in the regulation of osteoclast formation and function in the context of normal and pathological immune responses. Chapter 7 - Osteoclasts (OCs) are well known as the bone resorption cells in normal bone remodeling and pathological bone loss by increasing their number and resorptive activity. They are derived from myeloid osteoclast precursors (OPs) under the influence of the surrounding cells. There is growing evidence that OCs have multiple functions besides bone resorption. OCs secrete mediators to work as functional antigen-presenting cells in immune responses, affect angiogenesis by regulating the function of capillaries’ endothelial cells, and regulate hematopoietic stem cell (HSCs) functions. Furthermore, OCs can phagocytoze apoptotic bone cells. Phagocytosis of apoptotic cells causes phagocytes to secrete antiinflammatory mediators to control their functions in autocrine and paracrine manners. Posphatidylserine-containing liposomes (PSLs) are known to mimic the effects of apoptotic cells on phagocytes. The authors have found that phagocytosis of PSLs by OPs can regulate their secretion of anti-inflammatory mediators, including prostaglandin E2 and transforming growth factor-1, to inhibit OCs formation and inflammatory bone loss. In addition, PSLs promote the maturation of osteoblasts. Therefore, PSLs may provide potential pharmacological interventions against bone diseases through the regulation of these multiple functions of OCs. Chapter 8 - The mechanism of bone substitute resorption involves two processes: solution-mediated disintegration and cell-mediated disintegration. An example of the first process is calcium sulfate resorption. In previous studies, the main -tricalcium phosphate (TCP) resorption process was considered to be cell-mediated disintegration by tartrateresistant acid phosphatase (TRAP)-positive cells. Thus, osteoclast-mediated resorption of TCP may be important for enabling bone formation. In order to address a role of osteoclast in -TCP resorption deeply, two different experimental models were used. 1. Cylindrical -TCP blocks with 75% porosity were implanted in rabbit cancellous bone defects with or without bisphosphonate treatment. 2. -TCP blocks with or without bisphosphonate treatment were soaked with bone marrow cells obtained from femora of a 6-week-old Fisher rat, and were implanted into 12-week-old Fisher rats subcutaneously. The results showed that local application of bisphosphonate reduced the number of osteoclasts on the surface of -TCP. Inhibition of osteoclast formation resulted in reducing -TCP resorption and bone formation. Thus, these results suggest that osteoclast-mediated resorption plays an important role in TCP resorption and bone formation.

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Chapter 9 - Neurofibromatosis 1 (NF1) is an autosomal dominant neuro-skeletal cutaneous syndrome with an incidence of around 1/3000. Low bone mineral density (BMD) and osteoporosis/osteopenia are often associated with NF1. Bone dynamics include continuous bone formation and bone resorption, and imbalance in bone turnover may lead to low BMD. Bone is resorbed by osteoclasts which have been characterized in NF1 using peripheral blood-derived osteoclast differentiation assays. Peripheral blood mononuclear cells were isolated from patients with NF1, age, and gender-matched controls, and these cells were cultured into mature osteoclasts. The results showed that NF1 osteoclasts are more numerous, resorb larger amounts of bone, and display aberrant morphology compared to controls. NF1 osteoclasts also tolerate apoptotic signals, caused by serum deprivation or bisphosphonates, drugs used to treat osteoporosis. Taken together with the fact that osteoblast-mediated bone formation is impaired in NF1, this chapter provides insight on how mutation in the NF1 gene affects bone health, and these results may partially explain the low BMD in NF1.

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In: Osteoclasts Editors: A. J. Brown and J. S. Walker

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Chapter 1

MODULATION OF OSTEOCLAST DIFFERENTIATION AND FUNCTION IN RHEUMATOID ARTHRITIS Roya Lari Department of Biology, Ferdowsi University of Mashhd, Mashhad, Iran

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INTRODUCTION Rheumatoid arthritis (RA) is an autoimmune disease that characterized by synovial inflammation and bone and cartilage erosion. Several distinct patterns of bone loss have been shown in individuals with RA. These include juxta-articular osteopenia and focal erosions of subchondral bone and at joints in areas of direct inflammation. Also, the inflammatory processes in RA may cause slow induction of generalized osteopenia and osteoporosis of the axial and appendicular skeleton. The consequences of this bone loss are painful joint deformities, progressive functional disability, increased risk of bone fractures, and increased mortality rates. Studies have indicated that osteoclasts are the major cell type in bone resorption in RA. Bone-resorbing osteoclasts belong to the monocyte / macrophage lineages. Osteoclast progenitors migrate to the bone surface where they differentiate and fuse to form multinucleated osteoclasts. The bone microenvironment, including the presence of stromal cells (pre-osteoblast precursors) and mature osteoblasts, appears to be essential for osteoclast differentiation. Osteoblasts/stromal cells contribute to osteoclast differentiation by supplying receptor activator of nuclear factor- B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, in inflammatory pathological situations, such as RA other cell types such as synovial fibroblasts and activated T cells are also involved in the production of osteoclastic regulatory factors, contributing to the balance between the osteoclast-osteoblast functions. This review provides an overall picture of the effects of inflammatory factors on osteoclast differentiation and functions.

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OSTEOCLASTS IN RHEUMATOID ARTHRITIS In normal bone turnover, key communication between osteoclasts and the bone-forming osteoblasts occurs, and key cytokines controlling osteoclast development are macrophagecolony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) [1-2]. Bone-resorbing osteoclasts belong to the myelomonocytic lineage and can be generated from monocytes or even macrophage populations [3-4]. Osteoclast progenitors migrate to the bone surface where they differentiate and fuse to form multinucleated osteoclasts, but how commitment is established is only partly understood. The bone microenvironment, including the presence of stromal cells (pre-osteoblast precursors) and mature osteoblasts, appears to be essential for osteoclast differentiation [60]. Osteoblasts/stromal cells contribute to osteoclast differentiation by supplying RANKL and M-CSF (or CSF-1) [5-7]. Osteoclastogenesis has been shown to be directly and indirectly regulated by the effects of growth factors, cytokines and hormones acting on osteoclast precursors and osteoblasts, and a number of recent studies have shown an effect of these factors on osteoclastogenesis in pathological states. Rheumatoid arthritis (RA) is an autoimmune disease which characterized by synovial inflammation and bone and cartilage erosion [8] Several distinct patterns of bone loss have been shown in individuals with RA. These include juxta-articular osteopenia and focal erosions of subchondral bone and at joints in areas of direct inflammation. Also, the inflammatory processes in RA may cause slow induction of generalized osteopenia and osteoporosis of the axial and appendicular skeleton [9-10]. The consequences of this bone loss are painful joint deformities, progressive functional disability, increased risk of bone fractures, and increased mortality rates. The existence of multinuclear osteoclast-like cells at the site of arthritis in humans has been reported in earlier studies [11]. Several lines of evidence have shown that bone loss in RA is uniquely mediated by osteoclasts [12-13]. The transcription factor c-fos is known to be a key regulator of osteoclastogenesis since c-fos knockout mice (c-fos -/-) develop osteopetrosis due to a lack of osteoclasts [14]. When transgenic mice that express human TNF (hTNFtg), which suffer with severe destructive arthritis, were crossed with c-fos deficient mice, all clinical features of arthritis, such as joint swelling and inflamed tissue, progressed equally in both hTNFtg and c-fos -/- hTNFtg crosses. The cartilage destruction was similar in both groups but the c-fos -/- , hTNFtg crosses were fully protected against bone resorption [15]. Similar results were observed in RANKL knockout mice which are protected from bone erosion in a serum transferred model of arthritis [16]. Also, treatment of hTNFtg mice with OPG greatly reduced bone resorption [1718]. Therefore, it has been proposed that in RA the osteoclast is the major cell responsible for bone erosion and RANKL plays a key role in osteoclastogenesis. To extend these findings to humans, it has been shown that RA patients have high serum levels of OPG and sRANKL. Anti-TNF- treatment reduces the levels of OPG and sRANKL in serum but does not affect the ratio of these factors [19]. Studies indicate that osteoclast precursors at the site of inflammation have high osteoclastogenic potential [20-22]. Although the number of TRAP osteoclasts was relatively similar in monocytes cultured from RA patients and normal controls, monocyte derived-osteoclasts from RA patients showed greater bone resorption [23].

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Modulation of Osteoclast Differentiation and Function in Rheumatoid Arthritis

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In RA synovial fibroblasts as well as activated T cells contribute to the production of RANKL and OPG at the site of inflammation. These cells along with synovial macrophages also regulate osteoclast differentiation by generating a variety of osteoclast regulatory factors such as GM-CSF, TGF-, TNF-and IL-1. These factors in turn can have an effect on the production of RANKL and OPG at the site of inflammation. Also they can directly regulate proliferation of osteoclast precursors, as well as the differentiation and function of osteoclast cells.

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Figure 1. Osteoclastogenesis in rheumatoid arthritis.

Recent studies have shown that synovial macrophages are able to differentiate into osteoclasts [21-22, 24-26], suggesting the important role of this mechanism in the pathogenesis of bone destruction [20, 22]. Therefore it is possible that osteoclast precursors in inflammatory conditions may have different functional properties in comparison to osteoclast precursors under physiological conditions [27].In RA many cell types directly or indirectly participate in the pathogenic osteoclast differentiation and pathological bone erosion. Among these cells, activated T cells, synovial fibroblasts and stromal cells are the main source of RANKL in RA joints [28]. These cells produce a variety of cytokines and growth factors that can regulate osteoclast formation, activity, and survival (Fig1). This chapter provides an overall picture of the effects of inflammatory factors on osteoclast differentiation and functions.

GROWTH FACTORS Macrophage Colony Stimulating Factor macrophage colony-stimulating factor (M-CSF), also known as CSF-1, is produced by different type of cells such as monocytes, fibroblasts, endothelial and osteoblast cells [30-31]. It is widely known that M-CSF is essential for osteoclast differentiation. The op/op mouse, which has a mutation in the M-CSF gene, has an osteopetrotic condition with loss of bone

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marrow cavity and induction of bone density. In op/op mouse deficiency of synovial macrophages and bone osteoclasts has been shown, which is due to the lack of functional activity of M-CSF [32]. Moreover, these animals demonstrated a 10-fold reduction in the number of marrow cells [33]. In op/op mice osteoclast numbers are decreased, impairing bone resorption. Either injection of recombinant human M-CSF [34-36] or transduction of human M-CSF cDNA [37] into op/op mice reversed all the osteopetrotic abnormalities in these mice, including osteoclast differentiation and function, the restoration of the bone marrow cavity and the restoration of normal gross morphology [36-37]. Interestingly, osteopetrosis in these mice appears to recover with age, suggesting an alternative mechanism that compensates for M-CSF deficiency [38]. It has been shown that injection of either recombinant human VEGF [39], GM-CSF and/or IL-3 [40] to young mice can similarly induce osteoclast recruitment in op/op mice and correct the osteopetrosis. The receptor for M-CSF (c-Fms) is a single high-affinity transmembrane receptor with a tyrosine kinase domain [41]. In situ hybridization has shown that during embryonic development in mice the expression of c-fms could be detected in both osteoclast precursors and mature osteoclasts [42]. Binding of M-CSF to its receptor in isolated mature osteoclasts stimulates tyrosine phosphorylation in the membrane and cytoplasm followed by oxygen free radical production and associated induction of Ca2 release and bone resorption [43]. Tyrosine (Y) 559 and Y807 in the c-Fms cytoplasmic domain are two residues critical for mediating osteoclast differentiation and also bone resorption by mature osteoclasts. Mutation of each of these tyrosine residues reduces the number of functional osteoclasts. Tyrosine Y697, Y706, and Y721, which are also phosphorylated upon ligand binding, seem to exert no effect on osteoclast formation [44]. Analyzing gene expression patterns induced by M-CSF in mice has indicated that M-CSF alone induces the expression of RANK as well as RANK/NFB pathway components, namely TRAF2, PI3-kinase, MEKK3 and RIPK1 [45]. These findings indicate that M-CSF is not only a survival factor but also a differentiation factor with a synergistic effect with RANKL in osteoclast differentiation. Also, M-CSF induces the expression of both stimulatory and inhibitory factors and receptors, such as interleukin (IL)-1, IL-18 and interferon (IFN)-beta, as well as receptor components for IL11, IL-6 and IFN- in murine bone marrow mononuclear cells [45]. In RA, M-CSF can be involved in migration of monocytes to synovial site and consequently in osteoclast formation. These effects were inhibited in co-cultured system of purified peripheral blood CD14+ monocytes and RA endothelial cells by M-CSF neutralizing antibody [31].

Granulocyte Macrophage- Colony Stimulating Factor GM-CSF is a multi-lineage haematopoietic growth factor. It was first defined by its ability to generate granulocyte and macrophage colonies from precursor cells in vitro [46]. Surprisingly, GM-CSF-deficient mice (GM-CSF-/-) showed no obvious deficiency in the development and numbers of myeloid cells, but exhibit abnormal surfactant accumulation in the lung with numerous large intra-alveolar phagocytic macrophages. These mice frequently suffer from subclinical lung infections involving bacterial or fungal organisms [47]. In human serum, GM-CSF can be found at low levels and is difficult to detect in the circulation, but its level is increased in a variety of pathological conditions such as RA [48-50]. GM-CSF is a

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potent proinflammatory cytokine which produced by synovial cells in rheumatoid arthritis to the synovial fluid of the inflamed joint. It has been shown that treatment of adherent peripheral blood mononuclear cells with MCSF and RANKL significantly induces GM-CSF receptor expression. This suggests that GMCSF might play a direct regulatory role in osteoclast differentiation [51]. Studies examining the effect of GM-CSF on osteoclastogenesis have led to conflicting results. In some studies, GM-CSF has been reported to induce osteoclast-like formation [52-55]. In the study by Kurihara, et al. (1989) murine spleen cells were cultured in semisolid conditions in the presence of IL-3. After one week small colonies consisting of blast cells with little sign of differentiation were lifted and pooled in the presence of GM-CSF plus 1,25 dihydroxyvitamin D3 (1,25[OH]₂D₃); multinuclear TRAP cells appeared from day 14 [54]. Also, when murine marrow cells were first cultured in semisolid conditions in the presence of one of M-CSF, GM-CSF or IL-3 and then the recovered marrow cells from the semisolid cultures were subsequently co-cultured with primary osteoblastic cells in the presence of 1,25[OH] ₂D₃, GM-CSF induced formation of TRAP multinuclear cells [55]. In addition, in osteopetrotic op/op mice, which have a deficiency in active M-CSF production, daily injection of GM-CSF can induce a significant correction of osteopetrosis, expansion of the bone marrow cavity, and haematopoietic recovery [40]. However there is no evidence to suggest that GM-CSF can substitute for M-CSF in osteoclast differentiation in vitro [56-57]. In contrast to studies showing a stimulatory or facilitating effect of GM-CSF on osteoclastogenesis, a number of other studies indicate that GM-CSF inhibits osteoclast formation. In either co-cultures of bone marrow cells with osteoblasts [55, 58], or in cultures of both murine [57] and human [59-60] precursors containing RANKL and M-CSF, an inhibitory effect of GM-CSF on osteoclast formation has been reported. More recently, some explanations for the both the stimulatory and inhibitory effects of GM-CSF on osteoclast formation have been provided. In human monocytes GM-CSF has been shown to be capable of stimulating osteoclast formation indirectly by stimulating the release of M-CSF which maintains survival of osteoclast precursors and is one of the specific osteoclastogenic cytokines [53]. Integrinv3 plays a key role in osteoclast adhesion to the bone [61]. GM-CSF has been shown to induce the expression of v3 in BMM [62]. Moreover, it has been shown that pre-treatment of precursors with GM-CSF in the absence of RANKL induces the number of precursors and consequently stimulates osteoclast formation [57, 59]. In contrast, longer exposure to GM-CSF in co-culture systems of osteoclast precursors, or treatment of cells with GM-CSF in presence of RANKL and M-CSF, inhibits osteoclast differentiation [55, 57-60, 63]. GM-CSF treatment in the presence of M-CSF and RANKL is shown to be associated with reduce expression of c-fos and increased expression of DC surface markers [57, 59]. The expression of c-fos has been suggested to be a key regulator for the differentiation of both osteoclast and DC lineages. Whilst it has been suggested that GMCSF reduces c-fos expression and directs the osteoclast precursors toward DC formation [57, 59], DC maturation is inhibited when c-fos is expressed at an early stage of differentiation [57]. However, we have shown that, GM-CSF can generate a population of adherent macrophage lineage cells from murine bone marrow precursors (GM-BMM) which is also capable of giving rise to OC lineage cells in the presence of M-CSF and RANKL as effectively as BMM. The degree of this differentiation was surprising considering that GM-

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BMM are often referred to as immature dendritic cells and that, for both BMM and the GMBMM, GM-CSF suppressed subsequent OC differentiation governed by M-CSF and RANKL. Unlike for BMM, this GM-CSF-mediated suppression for GM-BMM appeared to be independent of c-fos expression [27]. Additionally, recent gene expression studies show that GM-CSF treatment down-regulates monocyte chemotactic protein 1 (MCP-1). Also, addition of exogenous MCP-1 restored the GM-CSF-mediated suppression of osteoclast formation [60]. It is likely that the microenvironment, in combination with other factors, is another parameter determining the role of GM-CSF on osteoclast formation. It has been shown that in a co-culture system of murine bone marrow-derived precursors with stromal cells, either IL-6 or TNF-α, not only overcame GM-CSF inhibition, but increased osteoclast numbers beyond that seen with either IL-6 or TNF-α alone [64]. In contrast, GM-CSF inhibited direct TNF-induced osteoclast differentiation in mouse M-CSF derived-precursors by down-regulation of mRNA and surface expression of TNFR1 and TNFR2 [65].

In bone TGF- is secreted in a latent complex by osteoblasts, chondrocytes and osteoclasts and deposited in bone matrix. During bone resorption by osteoclasts, acidic conditions and proteases in resorption pits cleave the latent complex and activate TGF-. After activation, TGF-1 can bind to its receptor on the osteoblast membrane and modulates the expression of OPG and RANKL. TGF- can also bind to its receptor on osteoclast precursors and have a direct effect on osteoclast formation and activities Figure 2. Activation and role of TGF-1 in osteoclastogenesis.

Transforming Growth Factor-Beta Transforming growth factor beta (TGF-) is a multifunctional cytokine that is present in large amounts in bone. It is usually secreted as a latent complex. The N-terminus of this TGF complex is noncovalently connected with latency associated peptide (LAP). It is called small latent TGF- and often binds with an associated latent TGF- binding protein (LTBP) resulting in the large latent form (Figure 2). These latent complexes are required for secretion of TGF- from cells and for matrix binding [66]. In bone, TGF-β is secreted by osteoblasts,

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chondrocytes and osteoclasts in the form of a latent complex that is usually deposited in the bone matrix by interacting with a select set of matrix proteins such as latent TGF-betabinding protein-1 (LTBP-1) [67] and LTBP-3 [68]. During bone resorption, secreted lysosomal enzymes in the acidic microenvironment produced by osteoclasts in resorption pits are able to cleave the latent complex and therefore release and activate TGF- (Figure 2) [6971]. Lack of latency proteins in Ltbp-3-/- mice results in a variety of abnormalities, including an increase in bone mass. In this mouse the osteoclast numbers were similar to control mice but the number of osteoblasts was reduced, suggesting lack of Ltbp-3 decreases levels of TGF- in bone, which in turn reduces osteoclast function and bone turnover [68]. Active TGF- has a regulatory effect on both osteoclasts and osteoblasts (Figure 2). Studies investigating the role of TGF-β on osteoclastogenesis have produced contradictory results. These differences may relate to different experimental parameters such as the system used for osteoclast differentiation and the dosage and time of TGF- exposure [72]. Expression of TGF- can be found at elevated levels in the synovial fluids and tissues of RA patients [73-77]. TGF-1 has been shown to be important in bone pathology of RA [72]. Although TGF- has some proinflammatory properties, such as acting as a chemoattractant for human peripheral blood monocytes [78], inhibiting apoptosis of synovial fibroblasts [7980] and inducing expression of IL-1, IL-8 and MMP-1 in synovial fibroblasts [80], it has anti-inflammatory actions as well. For example, TGF-1 knockout mice develop lymphoid infiltrates in heart, lungs, salivary glands, and other organs similar to a systemic autoimmune disease [81]. Also, the number of circulating monocytes is elevated in these animals [82]. TGF-1 inhibits macrophage activation and the production of inflammatory factors such as IL-1, TNF-, scavenger receptors and Matrix metalloproteinases (MMP) in these cells [8387]. It has been shown that blocking the action of endogenous TGF- increases the percentage of responding natural killer (NK) cells as well as the amount of IFN- produced by human NK cells [88-89]. In synovial fibroblasts, TGF-1 treatment inhibits IFN--induced DR antigen expression at both the level of gene and protein expression [90], and its expression has been shown to correlate with the severity of RA disease [91]. Also, carriage of the T allele (T869C gene ) that was associated with decreasing TGFβ1 production, was correlated with increased inflammatory activity, which worsen disease outcome in a study of 208 RA patients. Therefore reduction of TGFβ1 was linked to severity of disease in those patients [92-93]. Furthermore, TGF-1 has been shown to inhibit collagenase production, suppress T-cell and NK-cell proliferation and activation, and block free radical generation [94]. Taken together these finding indicate that TGF- can be beneficial by suppressing the inflammatory processes in bone and joints. It has been shown that, in the presence of M-CSF and RANKL, TGF- has a direct co-stimulatory effect with RANKL and enhances osteoclast differentiation as well as bone resorption [95-97]. Even in the absence of RANKL, TGF- induced differentiation of human monocytes to TRAP and VNR cells which were capable of forming actin rings (ringed structures of F-actin proteins in the sealing zone) and causing lacunar resorption [98]. Addition of osteoprotegerin or antibodies to TNF- and its receptors, as well as antibodies to gp130, did not have any effect on TGF--mediated osteoclastogenesis. Therefore osteoclast formation induced by TGF- would not appear to be due to the production of endogenous RANKL, TNF- or IL-6 by monocytes [98]. Also it has been shown that TGF-β promotes osteoclast survival in part by rapid induction of expression

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of B-cell lymphoma-extra large (Bcl-xl) and myeloid cell leukemia sequence 1 (Mcl-1) (transcription of pro-survival factors) [99] , as well as by induction of leukemia inhibitory factor and suppressor of cytokine signalling 3 (SosteoclastS3) expressions, which are promote osteoclast survival [100]. However, in whole human bone marrow cultures, TGF- exposure suppressed osteoclast generation and bone resorption [101]. As we investigated the effect of TGF-β in proinflammatory and anti-inflammatory macrophage differentiation into osteoclast, interestingly, we have found that TGF- inhibited the differentiation of both bone marrow derive macrophages [27]. Therefore, it appears that the influence of TGF-β on osteoclastogenesis depends on the presence of other stimuli such as RANKL and possibly upon the maturation state of the osteoclast precursors [27]. In a co-culture system of osteoblasts and osteoclast precursors, TGF- inhibited osteoclast differentiation [102-103]. This could be due to induction of OPG expression in osteoblasts by TGF- [102, 104]. It has been reported that, in co-culture systems, the concentration is important for the effect of TGF- on osteoclast differentiation. TGF-1 at low concentrations stimulates both the RANKL/OPG ratio and the level of M-CSF expression in osteoblasts; in contrast, TGF-1 at high concentrations reduced RANKL expression and stimulated expression of OPG by the osteoblasts and consequently suppressed the ratio of RANKL/OPG [104]. However, even in OPG deficient mice, TGF- had an inhibitory effect in a co-culture system of osteoblasts and hemopoietic cells, and the addition of exogenous RANKL only partly rescued osteoclast differentiation, suggesting that there is also an inhibitory mechanism independent of OPG which is partly attributed to reduced RANKL expression [96] (Figure2). In human peripheral blood mononuclear cell cultures, TGF- increased osteoclast formation and resorption only in the presence of a non-adherent lymphocyte population. In this culture system, TGF- enhanced osteoclastogenesis in the lymphocyte-rich environment up to the levels of the lymphocyte-poor environment. Hence it is possible that TGF- downregulates inhibitory factors produced by lymphocytes [71, 105]. The exposure time was also found to be important for the effect of the TGF- on osteoclastogenesis: TGF- stimulated p38 MAPK activity in monocytes thereby promoting osteoclastogenesis, but continuous exposure of the monocytes to TGF- down-regulated RANK expression, thereby reducing the stimulatory effect of RANKL [71]. Studies that have examined the role of TGF- in bone indicate that it has a stimulatory effect on bone formation in vivo and in vitro [106-109]. Consistent with its role in bone formation, local application of TGF- has been shown to improve and accelerate fracture healing [110]. Also, in different types of implants, such as those coated with titanium fibre or calcium phosphate ceramic, local application of TGF- increases bone volume and boneimplant contact [111-113]. In addition to its role in tissue repair, it is also important to consider the role of TGF- in inflammation.

TUMOR NECROSIS FACTOR SUPER FAMILY The tumor necrosis factor (TNF) super family (SF), which consists of 19 members, has a conserved C-terminal domain. This trimeric domain is responsible for receptor binding and

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has 20–30% sequence homology between family members [114]. The receptors for TNF ligands also constitute a super family. A number of TNF family members, including receptor activator of nuclear factor B ligand (RANKL) (SF 11) and TNF- (SF 2), are known to be involved in physiological bone remodelling. They are also associated with the pathogenesis of various bone diseases, such as osteoporosis and bone loss in inflammatory arthritis.

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Receptor Activator of NF-Kappa B Ligand For decades bone biologists knew that the presence of osteoblasts and their direct contact are essential for osteoclast precursor differentiation and activation [2], but the reason was a mystery up to the late 1990s. The discovery of receptor activator of NF-kappa B ligand (RANKL) by two independent groups [115-116] in T cells, and later as an osteoclast differentiation factor [6], solved this mystery. The discovery of RANKL led to the finding that osteoblasts/stromal cells support osteoclast differentiation primarily by production of RANKL [5, 117]. Different lines of evidence indicate the role of RANKL in osteoclastogenesis. RANKL knockout mice show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts. Dendritic cells appeared to be normal in these mice but T and B lymphocytes are defective in early differentiation [118]. The binding of RANKL to its cell-surface receptor (RANK) on the osteoclast precursors in the presence of M-CSF initiates osteoclast differentiation in vitro [119-120]. This differentiation is characterized by the induction of the expression of osteoclast lineage makers such as TRAP, CTR, Cath K and αvβ3 integrins, as well as by the stimulation of fusion and the formation of multinuclear cells. RANKL is not only crucial in osteoclast differentiation, but it is also crucial for their activation and the enhanced survival of mature osteoclasts [121-124]. The absence of osteoclasts in either RANK [125] or RANKL [126] knockout mice confirms the vital role of this system in osteoclastogenesis. In RA, RANKL is expressed by synovial fibroblasts, activated T lymphocytes and by B cells derived from synovial tissues from patients [28, 127]. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion [127]. The discovery of the RANKL/RANK system also helped to explain the regulatory mechanisms of some hormones and cytokines on osteoclast differentiation in pathological conditions. The number of osteoclasts strongly correlates with the ratio of RANKL /OPG in RA. Large number of osteoporotic hormones and cytokines stimulate osteoclast differentiation by induction of this ratio in synovial cells [128]. RANKL also has a role in the immune system. It was first discovered in relation to its ability to enhance the stimulation of naive T-cell proliferation by dendritic cells and to increase the survival of T cells [129]. In RANKL knockout mice the lack of lymph nodes and a deficiency at the early stage of T-cell differentiation indicate the important role of RANKL as an immunoregulator. Dendritic cells are normal in these mice [16]. It has been shown that both systemic and local T-cell activation can stimulate RANKL production and subsequent bone loss [12]. The receptor for RANKL is expressed on cells of the monocyte/macrophage lineages, such as osteoclast progenitors, osteoclasts and dendritic cells. RANK also can be found on T and B cells and fibroblasts [120, 130]. Binding of RANKL initiates RANK activation which activates signalling pathways for osteoclast differentiation, function and survival [116, 120].

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Like other TNF receptor family members, RANK also interacts with TNF-associated factors (TRAF). Activation of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6 has been shown following RANKL binding to RANK [131-134]. Human RANK receptor is a 616-amino acid peptide. In the intracellular C-terminal domain RANK has 383 amino acid residues with a 28-amino acid signal peptide. The intracellular domain does not show any homology to any of the known TNFR family members. RANK has a short transmembrane domain and an N-terminal extracellular domain [116]. RANK activation mediates osteoclast formation and function [135-136]. A vital role of RANK signalling for osteoclast differentiation and activation has been shown. RANK knockout mice have severe osteopetrosis characterized with a deficiency in bone resorption and remodeling. In these mice haematopoiesis is not affected but osteoclasts are absent throughout the skeleton. Also in these mice, lymph node formation is disrupted [125, 137]. Moreover, transgenic expression of soluble RANK-Fc fusion protein leads to induction of severe osteopetrosis, similar to that found in the OPG gene transgenic mouse. Also, addition of polyclonal antibodies against the extracellular domain of RANK in bone marrow cultures of these mice can rescue osteoclastogenesis [120, 138].

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Tumor Necrosis Factor-Alpha Tumor necrosis factor- (TNF-) is a member of the TNF ligand superfamily and a multifunctional cytokine produced mainly by activated macrophages. It regulates diverse cellular reactions such as proliferation, differentiation and apoptosis in various types of cells. TNF- acts via binding with two cell-surface receptors termed TNF- receptor1 (TNFR1, also known as p55 receptor) [139-140] and TNFR2 (also known as p75 receptor) [141]. In bone, TNF- is also secreted by osteoblasts [142] but, since mice lacking TNF- or its receptors do not exhibit any bone defects [143-144], TNF--mediated signalling is not essential for skeletal development and physiological bone remodelling. TNF- is a pivotal factor in the pathogenesis of RA that is produced by many types of cells including activated macrophages and synoviocytes within the inflamed joints [145-146]. The important role of TNF- in bone destruction in RA has been shown in transgenic mice that express human-TNF-α. These mice suffer polyarthritis characterized by significant focal bone erosion and systemic bone loss [15, 17, 147]. Also, blockade of TNF-α activity, with either human anti-ΤNF-α antibodies (infliximab, adalimumab) or recombinant soluble p75TNF receptor, has been shown to efficiently reduce RA symptoms, and is beneficial in slowing the progression of focal bone erosion [148-151]. TNF-enhances the proliferation and differentiation of osteoclast precursor in the bone marrow and also in circulating blood monocytes by up-regulation of c-Fms expression [152]. TNF- alone or in combination with IL-1 contributes to the pathogenesis of these conditions by increasing the number of osteoclasts at sites of bone resorption [153-154]. Also it has been shown that mice deficient in either TNF- or TNFR1 are resistant to ovariectomy-induced bone loss, showing the involvement of TNF- in pathological postmenopaUSl osteoporosis [155]. Although TNF- fails to induce osteoclast formation from more mature osteoclast precursors, such as murine adherent bone marrow derive macrophages [156-157], it has been shown in vitro to directly induce osteoclast differentiation from a variety of osteoclast

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precursors, as well as bone marrow and spleen cells [158]. Moreover, the effect of RANKLmediated osteoclast differentiation is enhanced by the addition of TNF- [159-161]. TNF- treatment increased the expression of CTR, Cath K and RANK mRNA in osteoclast precursors [159, 161] and has been shown to stimulate bone resorption in vitro [162] and in vivo [163]. These effects are inhibited by using an antibody to TNF- [160]. These findings indicate a synergistic effect of TNF- and RANKL. TNF- also induces secretion of RANKL in osteoblastic cells [142]. On the other hand, in co-culture systems of bone marrow cells with osteoblasts, low concentrations of TNF– had a completely opposite effect with inhibition of osteoclast formation, suggesting that TNF- may also induce osteoclastogenesis inhibitory factors [158]. The induction of multinuclear osteoclasts by TNF- was completely inhibited by antip55 antibody and partially blocked by anti-p75 antibody, showing the receptor signals were mediated mostly by TNFR1[162]. It has been shown that the synergistic enhancement of osteoclastogenesis in the presence of TNF- and RANKL is associated with stimulation of osteoclast signalling mediators including c-Src, TRAF2, TRAF6, and MEKK-1. Recruitment of TRAFs and MEKK1 lead to activation of downstream pathways, primarily IB/NF-B, ERKs, and cJun/AP-1. These effects are significantly reduced in TNFR1 knockouts [161]. In human TNF-transgenic mice blockade of p38 MAPK inhibits arthritis caused by overexpression of TNF. This suggests that p38 MAPK is a crucial signalling molecule downstream of TNF [164]. Some groups have proposed that TNF- stimulates osteoclast differentiation by a mechanism independent of RANKL [162, 165]. However, the important role of RANK activation in TNF--induced osteoclastogenesis is indicated by blocking of RANKL-RANK signalling pathways in hTNF tg mice (either by knocking out the c-fos gene or by treatment of mice with RANK.Fc or OPG) [15, 166]; these treatments do not prevent the development of inflammation, but fully protect the mice against bone destruction. This protection correlates with the reduction in the number of mature osteoclasts within the inflamed joints [15, 166]. These results suggest that TNF-α-induced osteoclast differentiation, at least to a certain extent, is mediated through RANKL-RANK interaction [167]. Therefore the concept of the capability of TNF- to compensate for RANKL in osteoclastogenesis is still controversial.

Osteoprotegerin Osteoprotegerin (OPG), which is also known as osteoclastogenesis inhibitory factor (OIF) [168] and TNF receptor like molecule 1 (TR1) [169], is a novel member of the TNF receptor super family [170]. Unlike other TNF receptor family members, which are surface membrane-associated, the lack of transmembrane and cytoplasmic domains in OPG means that it is secreted as a soluble protein [170-171]. OPG is a 401 residue polypeptide with two functional regions. The 21-amino acid propeptide in OPG is cleaved, resulting in a mature protein of 380 amino acids [6, 170]. The N-terminal region (residues 22-185) contains four cysteine-rich repeat sequences with striking homology to the ligand binding domain of other members of the TNF receptor superfamily, and more closely related to TNF receptor-2 and CD40 [172]. The C-terminal region contains

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half of the protein (aa residues 186-401) with no obvious homology to other known proteins and is involved in homodimerization and extracellular matrix binding [6, 170, 172-173]. OPG is expressed in different tissues, including lung, heart, kidney, liver, stomach, intestine, brain, spinal cord, thyroid gland, and bone [6, 170]. In bone it is produced by osteoblasts and bone marrow stromal cells in response to osteotropic factors and cytokines [174]. OPG acts as a soluble decoy receptor for RANKL and competes with RANK for RANKL binding. Binding of RANKL with OPG inhibits all the RANKL effects on osteoclast formation and activation [170, 175-177]. OPG knockout mice suffer from severe osteoporosis due to enhanced osteoclastogenesis. Compared to wild type mice, these mice have destruction of the growth plate, lower bone mineral density, severe trabecular and cortical bone porosity and, as a consequence, a high incidence of fractures [178-179]. Besides the high osteoclast activity in OPG-/- mice, enhanced levels of serum alkaline phosphatase show that osteoblastic bone formation is also elevated [179]. Furthermore, these mice exhibit medial calcification of the aorta and renal arteries, suggesting a role for OPG in calcium homeostasis [179-180]. Overexpression of OPG in transgenic mice leads to osteopetrosis, due to decreased osteoclast differentiation [170, 181]. These data indicate that the OPG/RANKL/RANK connection plays an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population [182].

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INTERFERON FAMILY The term interferon (IFN) was initially proposed for a group of proteins because they were found to interfere with viral replication in previously uninfected tissue culture cells. Interferons are glycoproteins with antiviral, anti-proliferative, and immunomodulatory properties. There are two major classes of interferons: i) type one interferons which comprise IFN-α and IFN-β. They share significant homology with each other and are quite distinct from, ii) type two interferon (also known as IFN-γ). Type one interferons are synthesized by many cell types following infection and can bind to a common cell-surface receptor, known as the Type I IFN receptor (IFNR) [183]. IFNR has a multi-chain structure with two distinct components: IFNR1 (previously called -subunit) and IFNR2 (previously called β-subunit) [184]. Both IFNR1 and IFNR2 are capable of signal transduction and mediate the biological effects of interferons [185]. Interferon -γ primarily is secreted by CD4 TH1 cells, CD8 T cells, and NK cells. A primary action of IFN-γ is activation of macrophages. An IFN-γspecific receptor is composed of two main subunits: IFNGR1 and IFNGR2 [186]. Apart from their roles in the immune system, interferons are also important factors in osteoclastogenesis. Osteoclastic differentiation is strongly inhibited by both type I IFNs [187-188]. However, IFN- was more effective than the IFN-α 2 subtype in suppressing the differentiation of human monocytes into osteoclasts [188]. Mice lacking either IFN- or IFN receptor subunits display an osteoporotic phenotype and have increased osteoclast formation [187]. Recently, it has been shown that IFN- has a role as a natural inhibitor of osteoclast formation. Binding of RANKL with RANK stimulates NF-B and c-fos expression. In turn, these pathways induce osteoclast differentiation.

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After binding of RANKL to its receptor, RANK, the TRAF6 protein becomes recruited and, in turn, activates the NF-B pathway. RANKL also stimulates the expression of c-fos, leading to the transcription of downstream target genes. This pathway is essential for osteoclast differentiation. One the other hand, NF-B and c-fos expression lead to the induction of IFN- which, after secretion and binding with its receptor, suppresses osteoclast differentiation by inhibiting c-fos expression. (adapted from [187, 190]).

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Figure 3. IFN- acts as a negative feedback regulator of osteoclastogenesis.

On the other hand, NF-B and c-fos stimulate another pathway that induces IFN-β expression in osteoclast precursor cells. After secretion, IFN-β binds to its receptor on osteoclasts and selectively down regulates the expression of c-fos. Consequently, IFN-β inhibits osteoclast differentiation, thereby acting as a negative feedback control for osteoclast differentiation (Figure 3) [189-190]. The presence of the IFN-γ receptor on osteoclasts suggests the possibility of a direct effect of this factor on osteoclastic activity [191]. IFN-γ has complex and apparently contradictory actions on osteoclastogenesis and bone resorption, depending on the system and model employed [192]. It suppresses osteoclastogenesis by interfering with the RANKLRANK signaling pathway. IFN-γ induces degradation of TRAF6, which in turn strongly inhibits production of NF-κ B and JNK by RANKL. Also, overexpression of TRAF6 rescues the inhibition of osteoclast differentiation. There is cross-talk between RANKL and IFN-γ [193-194]. The suppressive effect is significantly reduced when osteoclast precursors are preexposed to RANKL. This pre-treatment reprograms osteoclast genes into a state in which they can no longer be suppressed by IFN-γ [194]. A deficiency in IFN-γ receptor significantly induced osteoclast formation and bone destruction [195]. These knockout mice have an increased susceptibility to collagen-induced autoimmune arthritis as compared to wild-type control animals. These observations at least to some extent could be explained by enhanced migration of Mac-1 cells into inflammatory joints which further can differentiate into mature osteoclasts and be involved in joint destruction [195]. On the other hand recently it has been shown that IFN- γ also promotes osteoclast formation through stimulation of antigendependent T cell activation. Under conditions of estrogen deficiency, infection, and direct T cell activation by suppression of TGF- signaling, IFN- γ is inducing bone resorption and bone loss [192].

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INTERLEUKIN FAMILY

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Interleukin 1 Interleukin 1 (IL-1) is a potent pro-inflammatory factor, that produced by macrophages, T cells and other inflammatory cells recruited into sites of immune activation, such as the inflamed joint [196]. Different lines of evidence show that IL-1 plays an important role in the processes of bone loss in rheumatoid arthritis (RA). IL-1 stimulates RANKL expression in stromal cells [142] and acts as a co-stimulator, with RANKL, in the induction, activation and survival of osteoclasts [197-199]. IL-1 promotes the survival of purified osteoclast cells in vitro through the activation of NF-B [200]. This pathway also involves the activation of the downstream signalling of TRAF6, PI 3-kinase/Akt and MEK1/ERK. Inhibition of either of these transcription factors, eliminates the survival effect of IL-1 ,[201] [198]. IL-1 has a synergic effect with TNF- in stimulating osteoclast differentiation [202]. In mice, TNF- stimulates osteoclast differentiation, but these osteoclasts are unable to resorb bone; with the addition of IL-1 to this culture the osteoclasts gained this functional capability [165]. This requirement for IL-1 for functional bone resorption is not seen with other osteoclast differentiation systems. When the CD14 monocyte fraction of human peripheral mononuclear cells was cultured with TNF- and M-CSF, they differentiated to multinucleate cells (MNC) osteoclasts with TRAP, VNR, Cath K expression and bone resorption capacity. In this system, IL-1 did, however, significantly stimulate TNF--induced bone resorption [203]. Studies by Kamolmatyakul et al. (2004) showed that IL-1 up-regulates the expression of Cath K protein in a dose- and time-dependent manner, providing an explanation for IL-1’s effect on bone resorption [204]. Recently, the possibility that IL-1 has a role in RANKLstimulated osteoclastogenesis has shown in one system. RANKL-stimulated osteoclast formation in bone marrow cultures was significantly inhibited by IL-1 receptor antagonist. It also has been shown that immature BMM from IL-1 receptor-deficient (IL-1R) mice had decreased osteoclast formation [205]. The hTNFtg mice develop inflammations and severe bone loss. In IL-1(-/-)hTNFtg mice although still developed inflammation in their joints but completely protected from systemic bone loss. Lack of IL1 reversed increased osteoclast formation and bone resorption in hTNFtg mice. Therefore, IL1 is crucial for TNF-mediated bone loss and is a key mediator of inflammatory osteopenia [206].

Interleukin 6 IL-6 is a multifunctional cytokine which binds with the IL-6 receptor (IL-6R) in the membrane of target cells. Its signal is transduced via glycoprotein 130 (gp130). Circulating levels of IL-6 in physiological conditions are below detection and it does not therefore seem to have an important role under physiological conditions [207]. It is elevated in several pathological and inflammatory conditions and plays a role in the development of specific haematopoietic and immunologic responses. In the bone microenvironment, it is produced by a variety of cells including marrow stromal cells, monocytes/macrophages, osteoclasts, and osteoblasts. IL-6 induces osteoclast formation from osteoclast precursors [208-209] and has a

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co-stimulatory effect with TNF-α and IL-1. Neutralizing antibodies to IL-6 inhibits these stimulatory effects [210]. It has been shown that IL-6 is highly elevated in serum [211] and synovial fluids of patients with RA [212]. The concentration of IL-6 and IL-6R correlated with the degree of joint destruction. The addition of synovial fluid from RA patients to co-cultures murine osteoblastic and bone marrow cells was able to stimulate osteoclast-like cell formation; these effects were significantly inhibited by adding anti-IL-6R antibody [212]. IL-6 seems to plays a crucial role in the bone loss caused by estrogen deficiency, such as that follows loss of ovarian function by ovariectomy [213]. IL-6 deficient mice exhibit a normal amount of trabecular bone but have a higher rate of bone turnover than controls. Estrogen deficiency induced by ovariectomy causes a significant loss of bone mass along with an increase in the rate of bone turnover in wild type animals. Interestingly, ovariectomy does not induce any changes in bone mass or the bone remodelling rate in IL-6 deficient mice [214]. Addition of estrogens to primary human osteoblastic cells, or osteoblastic cell lines, leads to a significant reduction in IL-6 production [215]. Clinical studies of ovariectomized women show that there are significant correlations between serum sIL-6R levels and bone turnover [213]. Recently, it has been shown that inhibition of IL‐6 receptor directly blocks osteoclast formation in vitro and in vivo[216]. It directly inhibits osteoclast differentiation by suppressing receptor activator of NF-κB signaling pathways [217].

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Interleukin 10 Interleukin (IL)-10, also known as cytokine synthesis inhibitory factor (CSIF), is an important immunoregulator with potent anti-inflammatory properties. Macrophages are the major source of IL-10. The direct targets of IL-10 are antigen-presenting cells and lymphocytes where it has major immunological impact such as an inhibitory effect on the differentiation and proliferation of T cells, B cells, natural killer cells, antigen-presenting cells, mast cells, and granulocytes [218]. IL-10 completely inhibited colony formation induced by GM-CSF in rat whole bone marrow cultures and significantly inhibited the formation of osteoclast precursors [219]. In a co-culture system of mouse bone marrow cells and primary osteoblastic cells, IL-10 suppressed osteoclast formation by acting directly on osteoclast precursors [220-221], but it had no effect on bone resorption by mature osteoclasts, either in isolation or when incubated in the presence of osteoblastic cells [220]. IL-10 also strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level [222], which in turn suppressed the effect of these regulatory factors on osteoclast generation and activation.

CONCLUSION Taken together, this review gives a better insight into the complex mechanisms of how inflammatory cytokines may be linked to osteoclastogenesis in inflammatory conditions. Much has been learned in recent years regarding the facilitation of osteoclast differentiation

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and activation by regulatory cytokines, but it is less understood how inflammatory conditions affect osteoclastogenesis in RA and how the inflammatory macrophages react to these factors. So far, the treatment options include pain relievers, non-steroidal inflammatory drugs, disease modifying drugs such as methotrexate, steroids, and also biologicals (such as, antitumour necrosis factor agents). Their mechanism of action is immunosuppression and inhibition of certain cytokines, thus reducing the inflammatory activity. No effective treatment has been developed yet to prevent the bone erosion in RA; therefore a better understanding of the factors that control osteoclast differentiation in inflammatory conditions is essential for the future therapy for this disease. The recent findings of the mechanism of osteoclast differentiation have led to the possibility of the therapeutic use of RANKL blockade or OPG to prevent bone erosion in RA. Although these therapies will be important in the prevention of bone loss in future, they are less effective in reducing of inflammation [15, 166]. Finding factors that prevent inflammation and bone erosion might help to combat the disease in RA patients. Consequently, the studies presented in this chapter support taking a closer look at osteoclast cells as key therapeutic targets in inflammation-induced bone resorption.

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[112] Sumner, D.R., et al. Enhancement of bone ingrowth by transforming growth factorbeta. J. Bone Joint Surg. Am., 1995. 77(8): p. 1135-47. [113] Szivek, J.A., et al. Transforming growth factor-beta1 accelerates bone bonding to a blended calcium phosphate ceramic coating: a dose-response study. J. Biomed. Mater. Res. A, 2004. 68(3): p. 537-43. [114] Schmidmaier, G., et al. Local application of growth factors (insulin-like growth factor1 and transforming growth factor-beta1) from a biodegradable poly(d,l-lactide) coating of osteosynthetic implants accelerates fracture healing in rats. Bone, 2001. 28(4): p. 341-350. [115] Bodmer, J.L., P. Schneider, and J. Tschopp, The molecular architecture of the TNF superfamily. Trends Biochem. Sci., 2002. 27(1): p. 19-26. [116] Wong, B.R., et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem., 1997. 272(40): p. 25190-4. [117] Anderson, D.M., et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature, 1997. 390(6656): p. 175-9. [118] Takahashi, N., N. Udagawa, and T. Suda, A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochemical and Biophysical Research Communications, 1999. 256(3): p. 449-55. [119] Kong, Y.Y., et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature, 1999. 397(6717): p. 315-23. [120] Burgess, T.L., et al. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. The Journal of Cell Biology, 1999. 145(3): p. 527-38. [121] Hsu, H., et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(7): p. 3540-5. [122] Matsuzaki, K., et al. Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures. Biochem. Biophys. Res. Commun., 1998. 246(1): p. 199-204. [123] Nakagawa, N., et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem. Biophys. Res. Commun., 1998. 253(2): p. 395-400. [124] Jimi, E., et al. Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. J. Immunol., 1999. 163(1): p. 434-42. [125] Udagawa, N., et al. Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colonystimulating factor: receptor activator of NF-kappa B ligand. Bone, 1999. 25(5): [126] p. 517-23. [127] Li, J., et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(4): p. 1566-71. [128] Gravallese, E.M., Bone destruction in arthritis. Ann. Rheum. Dis., 2002. 61 Suppl 2: p. ii84-6.

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[217] Miyaura, C., et al. Endogenous bone-resorbing factors in estrogen deficiency: cooperative effects of IL-1 and IL-6. J. Bone Miner Res., 1995. 10(9): p. 1365-73. [218] Cheung, J., et al. Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-beta oestradiol and raloxifene. J. Endocrinol., 2003. 177(3): p. 423-33. [219] Axmann, R., et al. Inhibition of interleukin‐6 receptor directly blocks osteoclast formation in vitro and in vivo. Arthritis and Rheumatism, 2009. 60(9): p. 2747-2756. [220] Yoshitake, F., et al. Interleukin-6 directly inhibits osteoclast differentiation by suppressing receptor activator of NF-κB signaling pathways. Journal of Biological Chemistry, 2008. 283(17): p. 11535. [221] Asadullah, K., W. Sterry, and H.D. Volk, Interleukin-10 therapy-review of a new approach. Pharmacol. Rev., 2003. 55(2): p. 241-269. [222] Xu, L.X., et al. Interleukin-10 selectively inhibits osteoclastogenesis by inhibiting differentiation of osteoclast progenitors into preosteoclast-like cells in rat bone marrow culture system. J. Cell Physiol., 1995. 165(3): p. 624-9. [223] Owens, J.M., A.C. Gallagher, and T.J. Chambers, IL-10 modulates formation of osteoclasts in murine hemopoietic cultures. J. Immunol., 1996. 157(2): p. 936-40. [224] Hong, M.H., et al. The inhibitory effect of interleukin-10 on mouse osteoclast formation involves novel tyrosine-phosphorylated proteins. J. Bone Miner Res., 2000. 15(5): p. 911-8. [225] De Waal Malefyt, R., et al. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med., 1991. 174(5): p. 1209-20.

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In: Osteoclasts Editors: A. J. Brown and J. S. Walker

ISBN 978-1-62081-306-5 © 2012 by Nova Science Publishers, Inc.

Chapter 2

OSTEOCLAST FORMATION AND FUNCTION AND ITS ROLE IN SKELETAL BONE DISEASE Monique Bethel1, Angela Bruzzaniti2,3 , and Melissa A. Kacena1,2, Departments of Orthopaedic Surgery1, Anatomy and Cell Biology2, Indiana University School of Medicine, Indianapolis, Indiana, US Department of Oral Biology3, Indiana University School of Dentistry, Indianapolis, Indiana, US

ABSTRACT Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Osteoclasts are highly specialized cells, derived from the hematopoietic lineage, that resorb mineralized bone matrix. Osteoclast precursors can be recruited to the bone remodeling sites by specific cytokines in the microcellular environment, including some secreted by osteoblasts, fibroblasts, and osteocytes. Once the mononuclear osteoclast precursors converge on the bone surface, they fuse into large, multinucleated cells, a process that results in a highly efficient bone-resorbing cell. Cytoskeletal rearrangement then occurs, polarizing the cell such that the area of the cell directly in contact with the bone surface forms a sealing zone surrounding the ruffled border membrane which provides a large surface area for resorption. In the normal physiologic state, bone resorption and formation are tightly linked. However, several diseases have been identified where dysregulated bone resorption leads to bone of abnormal quality. Overly active osteoclasts have been implicated in the bone loss that occurs in a diverse set of diseases including osteoporosis, cancer, and inflammatory arthritis. Conversely, in osteopetrosis, underactive osteoclasts can lead to abnormally dense bone. In either situation, high or low bone mass, the poor bone quality leads to increased risk of fracture. Here we will review the current knowledge of the cellular events that recruit osteoclasts to sites of bone remodeling, initiate differentiation and fusion, and induce the cytoskeletal rearrangements that prepare the cell for bone resorption. 

Corresponding Author: Melissa Kacena, PhD - Assistant Professor, Indiana University School of Medicine, Department of Orthopaedic Surgery, 1120 South Dr, Fesler Hall 115, Indianapolis, IN 46202. (317) 278-3482 – phone. (317) 278-9568 – fax. [email protected].

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Monique Bethel, Angela Bruzzaniti, and Melissa A. Kacena Furthermore, we will examine how disruptions in any one or several of these processes create abnormalities in osteoclast morphology and function and contribute to skeletal disease in humans.

INTRODUCTION

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Osteoclasts (OCs) are highly specialized cells that can degrade the mineralized matrix of bone. Mononuclear osteoclast precursor cells (OCPs) are recruited to sites of bone resorption, where they fuse with other OCPs to form mature OCs. In addition, the mature OC undergoes many cytoskeletal rearrangements to form the multinucleated cell, also developing several distinct morphological regions as it prepares to resorb bone (Figure 1).

Figure 1A-C. A) Schematic representation of a mature OC, including the specialized areas developed in the mature OC to facilitate bone resorption: the functional secretory domain (FSD), the basolateral membrane (BLM), the sealing zone (SZ), and the ruffled border (RB). The dotted arrows depict the flow of proteolytic enzymes into the resorption lacunae and the flow of degradation products through the FSD. B) Transmission electron micrograph of a mature OC. The numerous invaginations of the RB, as well as the SZ, are seen. C) Confocal image of a mature OC on bone, stained with rhodamine phalloidin to detect actin. The OC binds to bone via the actin ring (shown in white). Scale bar is 10 μm. Micrographs courtesy of Lynn Neff.

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Osteoclast Formation and Function and Its role in Skeletal Bone Disease

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These regions include the sealing zone (SZ) or actin ring, which attaches the OC to the bone; the ruffled border (RB), through which proteinases responsible for solubilizing the bone are secreted; the functional secretory domain, which removes the degradation products; and the basolateral membrane, which plays a role in intracellular communication. These regions of the cell act in concert to efficiently resorb bone and this chapter will discuss how these structures form, how alterations to these structures lead to dysfunction in the OC and contribute to human disease, and how pharmaceuticals can target the OC to prevent disease. Critical to any discussion of OCs are several cytokines that are important to the maturation of OCs: macrophage colony stimulating factor (M-CSF), receptor activator of nuclear κB (RANK), and its ligand (RANKL). M-CSF was discovered during the study of the op/op null mouse, which has severely reduced numbers of OCs and as a result, develops osteopetrosis. In this murine model, normal production of M-CSF is absent and OCP proliferation and maturation are inhibited [1-3]. Similarly, mice deficient in RANK also developed significant osteopetrosis and do not form mature OCs, but have normal development of other myeloid lineage cells such as macrophages and dendritic cells [4]. The ligand for RANK, RANKL, was discovered nearly simultaneously and shown to be produced by osteoblasts (OBs), and interact with RANK expressed on OCs to enhance OC maturation [5-7]. Other studies found that M-CSF and RANKL treatment of murine and human hematopoietic cells resulted in the formation of mature OCs, which finally enabled the study of OCs in isolation, without the need for supporting cells, such as OBs [8]. Recently, other cell types have been shown to express RANKL including activated T-cells, fibroblasts, and osteocytes [9, 10]. Although it is not the only pathway to OC maturation, it is clear that the RANK/RANKL pathway is fundamentally important to OC development and the function of the OC.

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MULTINUCLEATION One of the key morphological features of the mature OC is the presence of multiple nuclei. Generally, the presence of three nuclei distinguish the mature OC from its immature form; however, mature OCs accumulate greater than 20 nuclei [11]. Why do these cells expend the energy to fuse multiple times? Although this is controversial, it is thought that multinucleation increases the resorptive efficiency of OCs. In early studies, several investigators demonstrated that in actively resorbing OCs, all of the nuclei are transcriptionally active; however, with improvements in technology, it was more recently shown that only certain nuclei are active [11-14]. While the significance of which nuclei are active remains unclear, resorptive activity appears to be related to the number of nuclei contained within and the overall size of the OC. In 1992, Piper et al demonstrated that OC size and number of nuclei were significantly related to the amount of OC resorptive activity; however, there was also some evidence suggesting that the largest OCs showed a trend of decreasing resorption capability [15]. Fusion of osteoclastic precursors is a complicated process. Several important mediators have been identified; however, the exact sequence of events and relative contribution of these mediators is currently undergoing further study.

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OCP Recruitment The first step in the fusion process is the recruitment of OCPs to the site of bone resorption. OBs and cells of the bone marrow stroma (BMSC) are believed to play an important role in attracting OCPs. CXCL12 is a chemokine secreted by immature OBs, bone endothelium, and BMSC; its secretion by OB precursors has been shown to promote chemotaxis of OCPs to sites of resorption [16]. Calcium gradients have also been implicated, with higher concentrations of Ca2+ attracting OCPs [17]. Recently, data has indicated that osteocytes play a major role in the recruitment of OCPs to sites of bone remodeling [18]. Specifically, in 2011, al-Dujaili et al showed that apoptotic osteocytes had elevated expression of M-CSF, RANKL and vascular endothelial growth factor. Furthermore, RAW264.7 cells (a murine macrophage/monocyte cell line used as a source of OCPs) cultured in the presence of conditioned media from apoptotic osteocytes induced the formation of significantly greater numbers and larger-sized mature OCs and increased their migratory activity [19]. Emerging in vitro data is challenging the current paradigm of OB/BMSC RANKL production driving osteoclastogenesis and osteoclastic bone resorption. Using an elegant murine model in which RANKL production by osteocytes is diminished, Nakashima et al recently demonstrated that osteocyte RANKL production is a much stronger stimulus for osteoclastogenesis than RANKL produced by OB or BMSC [10]. Another factor implicated in OC migration is membrane associated matrix metalloproteinase, MT1-MMP (also known as MMP-14). MT1-MMP degrades type I collagen and through immunohistochemical staining, has been localized to the lamellipodia and podosomes of OCs, which are used for migration and attachment, respectively [20]. Importantly, MT1-MMP was further localized to the SZ of OCs and not the RB, where other matrix metalloproteinases such as MMP-9 can be found; MT1-MMP was also found to activate other downstream matrix metalloproteinases (MMPs) [21, 22]. It was also demonstrated in vitro that OCPs from MT1-MMP null mouse showed decreased velocity and distance traveled across activated human endothelium in response to RANKL stimulation, a further indication that MT1-MMP may be involved in OC migration to bone remodeling sites [23].

OCP Attachment and Intercellular Contact Once at the site of bone resorption, the OC attaches to the bone matrix. The αvβ3 integrin molecule (or vitronectin receptor) is highly expressed on OCs and binds to the RGD (arginine-glycine-alanine) tripeptide sequence expressed in various proteins that compose the extracellular matrix (ECM), such as vitronectin, fibronectin, fibrinogen and others. Importantly, it can also bind osteopontin and bone sialoprotein [24, 25], two non-collagenous matrix proteins of bone. Another integrin molecule, α1β2, is active in OC adhesion to type I collagen [26]. Blockade of both αvβ3 and α1β2 activity has been shown to significantly decrease OC resorption; however, there was evidence that the decrease in resorption activity due to inhibition of αvβ3 was not from decreased OC adhesion to the bone matrix, but from some other effect on the resorption machinery [27]. αvβ3 plays a multifaceted role in OC function centered around the activation of several intracellular signaling pathways involving

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signaling molecules like c-Src, Pyk-2, p130cas, and Ras-ERK, which ultimately control cell survival, migration and adhesion [28]. Prior to fusion of OCPs, the cells initiate contact with each other. Our current understanding of this process involves a family of proteins called cadherins. Cadherins, along with integrins, are members of the cell adhesion molecules family. However, integrins are involved in cell-matrix adhesion whereas cadherins regulate cell-cell adhesion. The intracellular domain of cadherins interacts with cytosolic proteins, the catenins, which then go on to influence the cytoskeleton (α- and β-catenin) or initiate signaling pathways (β-catenin) [29,30]. E-cadherin is expressed on OCs and inactivating antibodies to this protein were found to inhibit fusion but not proliferation of OCs in vitro [31]. Another superfamily of adhesion molecules that has been identified as important in the OC fusion process are the ADAM (A Disintegrase And Metalloproteinase domain) family of adhesion molecules which function in cell-cell adhesion as well as degradation of ECM [32]. Several full length ADAMs have been identified on OCs and OBs, and their expression varies with the age of the OCs in culture; however, the specific isoform, ADAM 12s, has been found in association with human OCs and not in OBs [33]. Although the exact role of ADAM 12 in OC development is not completely elucidated, it is known that its expression is upregulated during the formation of multinucleated OCs and its expression can also be modulated by MCSF and RANKL, strongly implicating its activity in OC fusion.

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OCP Fusion Tetraspanins are small membrane proteins that span the plasma membrane four times. They associate with integrins and are believed to play a role in the fusion of multiple cell types including OCs [34, 35]. Specifically, the tetraspanin CD9 has been implicated in OCP fusion. In 2006, Ishii et al showed that RAW264.7 cells expressed CD9 and its expression was elevated by exposure to RANKL. Furthermore, antibody or siRNA blockade of CD9 decreased the number of mature OCs that formed from RAW264.7 cells, while CD9 overexpression enhanced OCP fusion [35]. CD44, a cell surface receptor for hyaluronic acid, is also developing as a mediator of the OC fusion process. During macrophage fusion, the intracellular domain of CD44 is cleaved and travels to the nucleus, where it interacts with NFkappaβ, the target of RANK [36]. As OCs share a common cell lineage with macrophages, it is reasonable to suggest that CD44 may act similarly in OCPs and there is evidence that this is so. In 2002, Suzuki et al showed that OCPs from CD44 null mice displayed impaired ability to fuse in vitro [37]. The in vivo effects of CD44 are not clear, as CD44 knockout mice have shown generally normal phenotypes [38, 39]. However, neither of these studies discusses bone phenotypes in detail; therefore, more work is needed to fully elucidate the effects of CD44 in OC maturation. Yet another potential mediator of OCP fusion is the d2 isoform of vacuolar acid ATPase (Atp6v0d2). Vacuolar acid ATPase (V-ATPase, discussed in further detail below), is a proteolytic enzyme critical for solubilizing the hydroxyapatite component of bone and has several cell-specific isoforms. Atp6v0d2 null mice developed a high bone mass phenotype that was subsequently linked with defective OCs, which express TRAP activity but had significantly decreased fusion [40]. Although, the exact role of this protein in OC fusion is

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unknown; V-ATPase may be an attractive target of gene therapy for bone loss disease, as Atp6v0d2 null mice also exhibit increased bone formation [40]. The caspases are generally considered pro-apoptotic proteins and caspase 3 has been shown to be elevated in OCs undergoing apoptosis [41, 42]. Surprisingly, caspase 3 has also been shown to be present in non-apoptotic OCs and necessary for OC fusion through a mechanism involving RANKL. RANKL treatment activates caspase 3 by cleaving procaspase 3 to its active form, which then allows caspase 3 to translocate to the plasma membrane as well as areas within the cytosol containing high concentrations of lipids [43, 43]. When procaspase 3 expression was reduced via siRNA in RAW264.7 cells, RANKL exposure did not induce TRAP activity or fusion of the cells [43]. RANK/RANKL interaction was also shown to be reduced in these procaspase knockdown cells, suggesting that procaspase may feedback to enhance RANK/RANKL activity [43]. Macrophage fusion receptor/signal regulatory protein alpha (MFR/SIRPα) and its receptor, CD47, are members of the immunoglobulin super family and were first studied in the context of the immune system. They were later shown to be involved in macrophage fusion into giant cells [44, 45], and it was thought that it might be involved in OC fusion as well. Further studies have provided data implicating these proteins in OC maturation; however, there is also data contradicting the importance of these proteins in OC fusion. In one mouse model, antibody blockade of CD47 or MFR/SIRPα led to fewer mature OCs in vitro, as did culture of OCPs from CD47 null mice; furthermore, histological examination of femurs from 18-week old male CD47 null showed decreased OC numbers in vivo [46]. While antibody blockade of MRF/SIRPα-CD47 interaction reduced the numbers of OCs formed, there was no affect on OC size or number of nuclei. This finding suggests that these proteins may not have a strong effect on OC fusion; however, the authors did note that with the variety of techniques available for culturing OCs, the effects of MFR/SIRPα and CD47 on OC fusion may not have been evident from the conditions they chose. More study will be needed to determine what, if any, role MRF/SIRPα-CD47 may play in OC fusion. Another emerging mediator of OCP fusion is the CD200 membrane protein, another class of immunoglobulins similar to MRF/SIRPα. In recent studies, the CD200 membrane protein, along with its receptor, CD200R, was more concretely linked with OC maturation than MRF/SIRPα [47]. CD200R expression is limited to cells of the myeloid lineage, while CD200 expression is more widespread [48]. In 2007 Cui et al, while studying the CD200 null mouse, found de novo CD200 expression on fusing macrophages and OCs (CD200R expression remained stable) [47]. In addition, CD200 null mice generated significantly fewer mature OCs than did wild-type controls and subsequently had higher bone mass [47]. Data showed that this ligand-receptor interaction activates RANKL signaling through the JNK pathway, which may explain how these proteins influence OCP fusion [47]. Recently, two proteins with a large influence over OCP fusion and maturation have been discovered. Dendritic-cell specific transmembrane protein (DC-STAMP) was discovered in 2000 [49] and was later found to have a critical role in the immune function of dendritic cells. Specifically DC-STAMP has been implicated in establishing self-tolerance [50], in the fusion process for macrophages and OCs [51, 52], and also in the bone-resorbing function of OCs [53]. DC-STAMP is expressed as a dimer on the cell surface of both human and murine OCPs [54]. Mice lacking DC-STAMP can form TRAP+ mononuclear OCPs with some resorptive capacity; however, these mononuclear OCPs do not fuse, and these mice have a moderate osteosclerotic phenotype [51, 55]. OCPs from DC-STAMP null mice can fuse when cultured

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with wild-type OCPs, suggesting that DC-STAMP is heterogeneously expressed in OCPs [51]. Furthermore, DC-STAMP overexpression has been found to increase the formation of mature OCs [56]. Importantly, although RANKL does not directly bind DC-STAMP (the ligand for DC-STAMP remains unidentified), it modulates the expression of DC-STAMP in OCPs [54, 56]. A complete understanding of the role of RANKL and DC-STAMP in OCP fusion is still being developed but it is now clear that exposure of OCPs to RANKL induces some members of the OCP population to internalize the DC-STAMP protein into the cell cytoplasm [54]. This produces two new populations of OCPs, one with high levels of DCSTAMP on the cell surface (DC-STAMPhi) and another with low levels of DC-STAMP (DCSTAMPlo). In 2010, Mensah et al found that the DC-STAMPlo population of OCPs expressed higher levels of genes associated with fusion. Furthermore, only DC-STAMPlo OCP went on to form TRAP+, multinucleated OCs [54]. Conversely, DC-STAMPhi OCPs did not fuse to form mature OCs [54]. Interestingly, a 10:1 population of DC-STAMPlo: DC-STAMPhi yielded the highest population of TRAP+, multinucleated OCs [54]. This data suggests that the population of OCPs that internalizes the DC-STAMP protein comprises the “master fusogens”, while the group of OCPs in which DC-STAMP remains on the surface act as donor molecules to the final multinucleated cell [54]. A molecule very similar to DC-STAMP, OC-STAMP (osteoclast stimulatory transmembrane protein), has been identified and also plays a role in OCP fusion, although its function is less well understood than DC-STAMP. First identified in 2008, OC-STAMP expression was found to be strongly induced in OCPs exposed to RANKL [57]. Unlike DCSTAMP, it appears to be only found in cells of the OC lineage [57]. In murine OCPs, blockade of OC-STAMP by antibody or siRNA has been found to result in a marked, dosedependent reduction of the formation of mature OCs [57]. Conversely, overexpression of OCSTAMP in these same OCPs using two different constructs yielded significantly greater numbers of mature OCs than OCPs transfected with empty vectors [57]. Like DC-STAMP null mice, mice in which OC-STAMP is blocked still form TRAP+, mononuclear cells. OCSTAMP appears to be less important in OCP fusion, as OC-STAMP does not rescue fusion of OCPs in DC-STAMP knockout mice, which form no multinuclear TRAP+ OCs [57]. The interactions, if any, between OC-STAMP and DC-STAMP have yet to be elucidated.

SEALING ZONE OCs have developed a unique cellular structure to sequester regions of active bone resorption called the SZ. Podosomes, which are cellular structures composed of actin, are involved in cell migration and adhesion. Podosomes are also found in monocytes and dendritic cells, which share a common ancestor with OCs. Several mediators are known to play a role in the formation and stabilization of the SZ. Juric et al described how individual podosomes reorganize into the belt-like actin ring which attaches the cell to target calcified substrate. Early in the life of OCs, clusters of podosome cores containing polymerized F-actin are surrounded by an “actin cloud” which consists of soluble actin, and is localized to the basal area of the cell [58]. As the cell matures, these clusters enlarge to form short-lived rings. Using a complex process whereby actin fibrils polymerize and then depolymerize (also referred to as “treadmilling”); new podosomes are formed at the edge of the actin rings and

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not at the center, allowing expansion of the rings to the cell periphery [58]. Once in place, CD44 and integrin β3 mediate adherence of the actin ring to the surrounding substrate. Both the podosome cores and the actin cloud are thought to participate in the ability of the cell to attach to the ECM and likely complement each other. For example, CD44 has been found associated with the F-actin cores of podosomes, while β3 integrin is expressed in the actin cloud as well as the podosome ring [59,60]. Although CD44 and integrins are both known to be important in cellular attachment to the ECM, the specific role that integrins play in formation, stabilization, or activity of the SZ remains unclear. Other proteins have been identified which play a role in the formation of the actin ring. Protein tyrosine kinase 2 (Pyk2), a signaling protein highly expressed in OCs, appears to be important in the reorganization of podosomes. In 2007, Gil-Hen et al showed that in Pyk-2 null mice, podosome dynamics and microtubule stability were altered; leading to OCs that did not properly form a SZ on calcified substrate and consequently had an osteopetrotic phenotype [61]. The full nature of the SZ is not well understood, many more mediators will likely be identified that play a role in its formation.

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RUFFLED BORDER In order to maintain calcium homeostasis and the structural integrity of the skeleton, bone is constantly remodeled throughout the life of an individual. Bone is composed of both an organic matrix composed primarily of type I collagen and other proteins such as osteocalcin and osteopontin, and the inorganic mineral calcium hydroxyapatite, which provides bone with its strength. OCs have evolved a unique structural component, called the RB, as well as specific proteases to degrade both the organic and inorganic components of bone. The RB is critical for OCs to resorb bone and its presence signals the terminal differentiation of OCs. The cytoskeletal rearrangements necessary to form this structure occur at the same time that the actin ring forms to seal off the resorption lacuna [62]. There is evidence that integrins, particularly αvβ3, play a role not only in the binding of OCs to the substrate, but also contribute to the formation of the RB. In 1996, Nakamura et al showed by immunohistochemical staining that αvβ3 was present in both the RB and the SZ. Furthermore, they demonstrated that blocking integrin binding with synthetic RGD (the amino acid recognition sequence for integrin binding) inhibited in vitro OC resorption and SZ formation dose-dependently [63]. Synthetic RGD did not inhibit OC binding to the dentine substrate in this study, indicating that αvβ3 contributes to OC binding, but is not essential. Along the numerous invaginations of the plasma membrane forming the RB are various cellular mechanisms for producing the chemical environment necessary for bone resorption. Vacuolar (H+) ATPase (V-ATPase) is an energy-dependent proton pump that acidifies the contents of endosomes, lysosomes, Golgi vesicles, secretory vesicles, and the plasma membranes of some cells [64, 65]. V-ATPase resides on the plasma membrane of the RB and is responsible for acidifying the resorption space directly underneath the RB [66]. The protons for the V-ATPase are produced by cytoplasmic carbonic anhydrase II and the excess bicarbonate is removed by a chloride-bicarbonate exchanger that resides on the basolateral membrane. The numerous chloride channels on the RB allow free transfer of chloride ions to maintain electronuetrality [67, 68]. The organic components of bone matrix are degraded by

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another set of enzymes released by the RB including MMPs and cysteine proteinases such as cathepsin K. Although both participate in resorption, it appears that there are differences in the mechanism of action and relative importance between the cathepsins and MMPs. Everts et al has shown that cathepsin K was more important in the OC-mediated resorption of long bones; while in calvariae, MMPs and cathepsins are both important [69]. The current understanding of bone solubilization and eventual resorption ensues as follows: first the bone is decalcified by acid proteases; and then the underlying organic matrix degraded by extracellular MMPs and cathepsins. The degradation products are phagocytosed by the OC and the material ultimately disposed of through the apical side of the cell, called the functional secretory domain (FSD) or released during OC migration. At the bone-OC interface, vesicles from within the cell containing proteolytic enzymes fuse with the RB and deliver the enzymes to the bone surface. It is now known that the RB itself is polarized and this activity is concentrated at the periphery of the RB, while endosomes containing degradation products form near the center of the RB [70]. As the bone is digested by these enzymes, large amounts of calcium and phosphate, as well peptide fragments are released into the resorption lacunae. It is believed that the ions exit the lacunae under the SZ and enter the extracellular milieu [71]. The larger protein fragments cannot escape through the SZ and are taken up by the cell in endocytic vesicles. Whereas cathepsin K and MMP-9 are the proteolytic enzymes active in the resorption lacunae, it has been postulated that TRAP, the enzyme commonly used as an OC marker, possibly participates in the intracellular, lysosomal-mediated degradation of organic bone matrix. Under the right conditions, TRAP was identified in the transcytotic vesicles of actively resorbing OCs and was shown to form reactive oxygen species that have the potential to degrade collagen within these vesicles [72]. The Rab family of small GTPases, particularly Rab7, is believed to play a critical role in the intracellular vesicular trafficking of OCs. Rab proteins regulate vesicular travel throughout the cell via their interaction with actin and microtubule associated proteins [73]. Rab 7 has been shown to localize to the RB during active OC resorption, and localize to the perinuclear zone in inactive OCs where endosomes and lysosomes also accumulate [74]. Furthermore, Sun et al showed that Rab7 colocalizes at the RB with Rac1, another protein known to interact with the actin cytoskeleton [75]. This relationship has been postulated to mediate the fusion of late endosomes in the formation of the RB [75].

BASOLATERAL MEMBRANE This structural component of the OC has received less attention than other areas. Several ion channels are located at the basolateral membrane, including the sodium/potassium pump (NaK+-ATPase), the bicarbonate/chloride exchanger, the sodium/proton exchanger, among others [76]. RANK, the receptor for RANKL and the receptor for M-CSF, c-fms, are also located on the basolateral membrane of the OC. These findings suggest that this area of the OC not only functions in maintaining ionic homeostasis as the OC acidifies the resorption lacunae, it also likely functions in communication with other cells.

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OCS AND CLINICAL IMPLICATIONS Osteopetrosis

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Osteopetrosis is a rare disease of elevated bone mass linked to dysfunctional osteoclastic bone resorption [43, 77]. Although there are several different variants based on the pattern of inheritance and the constellation of signs and symptoms, the incidence is quite small: an estimated 1 out of 20,000 children born every year have the autosomal dominant form of osteopetrosis and 1 out of 250,000 have the autosomal recessive form [78]. Depending on the severity of the manifestations, the disease may be discovered incidentally or result in death very early in a patient’s life. The bones appear dense on radiographs; however, they are more fragile than normal bone and more likely to fracture [79]. The dense bone may not allow normal formation of the bone marrow compartment, and subsequently lead to anemia, leucopenia, and thrombocytopenia. With the normal sites of hematopoietic activity obstructed, these patients often have splenomegaly and hepatomegaly as hematopoiesis moves to these sites [79]. In the autosomal recessive form, death may occur in infancy from the hematopoietic deficiencies which can cause uncontrolled bleeding and infection [79]. In general, cytoskeletal changes in OC morphology are not observed in patients affected with osteopetrosis, and there are normal to increased numbers of OCs; however, the resorption machinery in these cells is ineffective. For example, in the form of osteopetrosis accompanied by renal tubular acidosis, a mutation in the gene encoding carbonic anhydrase II has been discovered, and mutations in V-ATPase and the chloride channel ClC-7 have been identified in other forms of osteopetrosis [80]. Defects in these genes would obstruct the means by which the OC lowers the pH within the resorption lacunae and maintains electronuetrality.

Pycnodysostosis Pycnodysostosis is a disease similar to osteopetrosis first described in children in the 1960’s. It was first thought to be a unique variant of osteopetrosis, which had been described even earlier in the twentieth century. However, the first patient with pycnodysostosis had several distinguishing phenotypical findings not seen in patients with osteopetrosis. In 1963, Shuler gave the first English language description of a male child with a short stature, open fontanelles, short, stubby and wrinkled fingers, hypoplastic jaw with poor dentition and frequent fractures [81]. In studying the child’s family, Shuler found a maternal uncle that displayed similar phenotypic characteristics, and likened it to a disease named pycnodysostosis which had been described previously by two other physicians [82], although their findings were published later. Subsequent studies of families with the disorder revealed that its inheritance pattern was autosomal recessive [83]. The explosion of genetic studies in the 1990’s brought the finding that the pathogenic basis of the disease was a lack cathepsin K, one of the primary enzymes used by OCs to degrade organic bone matrix, in the lysosomes of actively resorbing OCs [84]. Similarly to osteopetrosis, OCs from patients with pycnodysostosis appear to have normal morphology.

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Paget’s Disease In Paget’s disease, patients also have dense, fragile bone; however, unlike osteopetrosis, the increased bone mass has been linked to disordered bone turnover, with dysfunctional OBs and OCs. In 1990, Kukita et.al. showed that OCs cultured from the bone marrow of patients with Paget’s disease were more numerous, had many more nuclei and more resorptive capability than control OCs [85]. Importantly, these pagetic OCs were also found to contain nuclear inclusions [85]. Later studies have demonstrated abnormalities in the expression of several genes that are involved in RANKL signaling, including osteoprotegerin [86]. Ultimately, the pathological basis of Paget’s disease is disordered bone turnover, and to that end, new studies are emerging which also implicate OB abnormalities in this disease. Alkaline phosphatase, osteocalcin and P1NP, all markers of OB activity, can be elevated in patients with Paget’s disease and are used as markers to follow disease activity [87]. As bone resorption and formation are tightly coupled, the abnormal and overly active OC’s in Paget’s disease are believed to also activate OBs; and subsequently, new, yet disordered bone formation [88].

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Osteoporosis Osteoporosis is a bone-loss disease common among post-menopausal women and has become a major public health concern in the U.S. Early studies showed that there are decreased numbers of OCs in the iliac bone of patients with osteoporosis, but their resorptive activity was significantly greater than new bone formation by OBs [89]. Eventually, as bone resorption is favored over bone formation, the bony trabeculae thin, the cortical bone becomes more porous and the bone begins to lose its structural integrity [90, 91]. These changes have been linked to the effects of estrogen on both OCs and OBs. It is known that estrogen increases osteoprotegerin (decoy receptor for RANKL, which competes with RANK for RANKL binding) production from OBs in vitro, and decreases RANKL production from inflammatory cells in vivo [92]. Furthermore, estrogen has been shown to directly inhibit osteoclastogenesis and promote OC apoptosis in vitro [93, 94]. Simultaneously, the lack of estrogen decreases the survival of OBs, therefore, in the basic unit of bone remodeling, the BMU, bone metabolism favors resorption [95]. Several pharmaceuticals now exist that can counteract bone loss associated with osteoporosis and other diseases of bone metabolism. Pharmaceuticals used in the treatment of osteoporosis can be assigned to one of two general categories: anti-resorptives, so named for their affect on osteoclastic bone resorption; and anabolic, which would tend to increase new bone formation. There are several pharmaceutical agents available for the treatment of osteoporosis, most of which have deleterious effects on the activity of OCs. For example, it is known that calcitonin, a hormone important in calcium hemostasis, inhibits osteoclastic bone resorption and has therefore been used as an anti-resorptive therapy for osteoporosis. It affects actively resorbing OCs without reducing their overall numbers [96]. In 2006, Okumura et al demonstrated that treatment of murine OCs cultured on dentine slices with calcitonin completely disrupted the SZ [97]. This same effect was later shown to persist in human OCs [98]. Bisphosphonates (BPs) are a popular group of agents generally considered to belong to the anti-resorptive category and are used to treat a diverse set of metabolic bone diseases

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including, osteoporosis, Paget’s disease, multiple myeloma, and osteogenesis imperfect [99]. BPs bind strongly to hydroxyapatite, and as the OC degrades bone BPs are taken up with the degradation products [100]. While this is likely the primary pathway for BPs to access the OC, there is some evidence that BPs can directly affect OCs without being endocytosed [101]. Within the past decade, the mechanism by which BPs specifically interfere with OC morphology and activity and how BPs affect other cells important to bone metabolism has come to light. In 2002, Alakangas et al showed that one BP, alendronate, had profound effects the resorption capability of OCs [102]. In this experiment, rat OCs exposed to alendronate formed fewer, more rounded and superficial resorption pits on ivory substrate as compared to control OCs. Furthermore, the morphology of RB, SZ, and FSD in these OCs exposed to alendronate was abnormal. Interestingly, this effect was only observed on OCs treated with alendronate for more than 24 hours. Initial OC attachment to the substrate was normal, but as the resorption process continued, detrimental changes to the OC occurred and resorption activity declined. Nitrogen-containing BPs, like alendronate, interfere with the proper prenylation of the small GTPases, like members of the Ras family, which play a role in vesicular trafficking and the organization of the cell’s cytoskeleton as discussed previously [103, 104]. It is thought that the improperly functioning SZ, RB and FSD are mediated by dysfunctional Ras proteins induced by BP treatment [102, 104]. Other experiments have demonstrated further effects of BPs on OCs. Several groups have shown that BPs inhibit the activity of V-ATPases [105, 106]. Mayahara and Sasaki showed that long-term administration of pamidronate, another BP, caused the disappearance of the RB and SZ as well as decreased the expression of V-ATPase in ovarectomized rat OCs [106]. This study also examined OC apoptosis and found no significant difference in the numbers of apoptotic OCs between the experimental and control animals; however, other investigators have reported conflicting data showing that apoptosis is elevated in OCs treated with BP. Escudero et al found that treatment of female rats with olpadronate led to greater numbers of apoptotic OCs within the femur [107]. Estrogen has been linked to OC apoptosis: the animals in this study were not ovarectomized and the presence of estrogen in those animals may have potentiated apoptosis [108, 109]. Histomorphometry revealed that these rats had higher bone volume compared to the controls; however, it also revealed several other interesting findings. The treated OCs were larger and had more nuclei than controls, but they also showed a loss of polarity. This supports the findings of the Alankangas et al study by showing that BPs also affected OC morphology [102]. Furthermore, it is interesting to note that the overall number of OCs was greater in the experimental rats, which may have been due to greater recruitment by the significantly larger numbers of megakaryocytes expressing RANKL also found in the femur. Our laboratory and others have shown that megakaryocytes interact with OBs and OCs. Although further discussion of this subject is beyond the scope of this chapter, it reinforces the multiple affects of BPs and the complicated interworkings of bone metabolism [110-113]. Although perhaps best known for their actions on OCs, BPs also affect other cells involved in bone metabolism. BPs have been shown to prevent glucocorticoid-induced apoptosis of both OBs and osteocytes in mice [114]. The significance of this finding is twofold: greater numbers of OBs would obviously contribute to increased bone formation, and this effect may be amplified by the increased numbers of osteocytes. There is a growing body of evidence showing that osteocyte apoptosis is an initiating event in bone resorption, therefore their survival would tend to inhibit bone resorption [18, 115-117]. Taken together,

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these findings suggest that in addition to their negative effects on OCs, BPs may also have positive effects on OB and osteocyte activity and therefore bone metabolism. Several newer osteoporosis treatments have recently been introduced and which also inhibit the OC resorptive machinery, although they use a different mechanism than BPs. Denosumab is a human monoclonal antibody to RANKL which binds to native RANKL produced by OBs and inhibits the maturation of OCPs; it is currently being used to treat osteoporosis [118]. Odanacatib, a cathepsin K inhibitor, was also recently developed and is being used for the treatment of osteoporosis as well as pycnodysostosis [119].

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Cancer Cancer involving the bone can be the result of a primary tumor, such as in osteosarcoma; or, as in the vast majority of bone tumors, the result of metastases from another primary tumor. Metastatic bone tumors have the potential to appear either lytic or osteosclerotic depending on the balance of bone formation and destruction within the lesion [120]. The RANK/RANKL pathway has been implicated in the lytic lesions of prostate cancer, with RANKL guiding the migration of RANK expressing tumor cells; which then go on to interact with both OBs and OCs to favor osteoclastogenesis [120-122]. A similar mechanism involving RANK/RANKL stimulation of osteoclastogenesis with simultaneous tumor cell induction of OB apoptosis appears to be responsible for lytic disease in breast cancer [123]. Multiple myeloma (MM) is an example of a cancer in which the lytic lesions are unique. Bone lesions in MM have a unique “punched out” appearance on radiography, and compared to metastatic lesions from other cancers, there is a complete absence of new bone formation within these lesions [124]. Although MM cells do not express RANKL, they have been shown to increase expression of RANKL in BMSCs [125]. Other mechanisms appear to be at play as well such as DC-STAMP, which has been implicated in the pathophysiologic bone destruction of MM. In 2011 Silvestris et al found that overexpression of DC-STAMP in peripheral macrophages, coupled with the tendency for both dendritic cells and the malignant plasma cells to fuse into OCs (under specific conditions) and express proteolytic enzymes used in OCs to degrade bone which create an environment highly favoring resorption of bone [126]. BPs and Denosumab have both been used to treat metastatic bone loss disease [127-130], as they limit bone resorption. Both BPs and denosumab can be used to treat hypercalcemia of malignancy; bisphosphates have a much longer history of use for this indication [131, 132]. Interestingly, BPs may have anti-tumor effects above and beyond the treatment of metastatic disease. BPs have been shown to have a myriad of in vitro effects on a variety of different tumor cells, including inhibition of tumor cell adhesion to bone and tumor cell growth, as well as induction of tumor cell apoptosis [130]. These new findings may herald a new indication for BPs in the treatment of cancer.

Inflammatory Arthritis Rheumatoid arthritis (RA) is an autoimmune disorder of disarthrodial joints that affects both adults and children. As the disease progresses, erosions into the cartilage and

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subchondral bone develop as well as periarticular and generalized osteopenia. The generalized osteopenia in these patients is likely multifactorial, with contributions from decreased mobility and long-term steroid use suppressing bone formation and favoring an environment conducive to bone resorption [133]. OCs have been directly implicated in the focal erosions of RA and are thought to be involved in both forms of osteopenia [134,135]. The OCs in RA are morphologically normal, with the exception of their size, as we will address shortly. Contrary to other disease states, in which morphological changes in OCs disrupts the resorption process, the morphological changes seen in RA improve the bone resorbing efficiency of OCs. In 2009, using human peripheral blood mononuclear cells as a source of OCPs, Nozawa et al demonstrated that culturing these progenitors in the presence of connective tissue growth factor (CTGF, which was shown to be elevated in the serum of patients with active RA versus those with inactive disease in this same study), M-CSF, and RANKL led to significantly more and larger mature OCs [136]. As discussed above, while up to a point, larger OCs have increased resorption activity, it is important to note that the aforementioned study did not address number of nuclei, which is also important in the context of larger OCs resorbing more bone [15]. Furthermore, RANKL, known to be critical in several facets of OC maturation, particularly OCP fusion, is elevated in the serum and synovial fluid of RA patients [137]. Activated T cells, also found in the synovium of RA patients, secrete RANKL and various other cytokines that also have stimulatory effects on OCs such as IL-7 and TNF-α [138-140]. It appears that the overall effect of these cytokines is to increase osteoclastogenesis and greatly enhance bone resorption. While it remains unclear exactly how the OCs contribute to both focal bony erosions and the generalized osteopenia of RA, continued research will likely reveal more activating cytokines and other targets for therapeutic intervention in RA. That said, like with osteoporosis and cancer patients, RA patients are now being treated with Denosumab [141].

CONCLUSION OCs are highly specialized cells that resorb bone. They are only one player in the complicated story of bone hemostasis; however, the study of the OC morphology and function has led to insights into how OCs maintain bone and calcium hemostasis, how dysfuction can lead to human disease, and how pharmaceuticals can affect OCs to treat disease. Continued study of the OC will lead to better understanding the role of signaling proteins and proteins that control OC morphology and the development of new drug therapies that will improve the quality of life for patients afflicted with many different diseases.

ACKNOWLEDGMENTS This work was supported by the Indiana - Clinical and Translational Sciences Institute funded, in part by NIH grants NCRR RR025760 and RR025761 (MAK, AB), the Department of Orthopaedic Surgery, Indiana University School of Medicine (MAK), the Department of Oral Biology, Indiana University School of Dentistry (AB), and by NIH/NHLBI grant T32

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HL007910 (MB) and NIH/NIAMS R01 AR060332 (MAK, AB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the supporting agencies.

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[92] Zallone A. Direct and indirect estrogen actions on osteoblasts and osteoclasts. Ann. N. Y. Acad. Sci. 2006;1068:173-9. [93] Kameda T, Mano H, Yuasa T, et al. Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts. J. Exp. Med. 1997;186:489-95. [94] Sorensen MG, Henriksen K, Dziegiel MH, Tanko LB, Karsdal MA. Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts. DNA Cell Biol. 2006;25:475-83. [95] Seeman E. Reduced bone formation and increased bone resorption: Rational targets for the treatment of osteoporosis. Osteoporos Int. 2003;14 Suppl 3:S2-8. [96] Karsdal MA, Henriksen K, Arnold M, Christiansen C. Calcitonin: A drug of the past or for the future? physiologic inhibition of bone resorption while sustaining osteoclast numbers improves bone quality. BioDrugs 2008;22:137-44. [97] Okumura S, Mizoguchi T, Sato N, et al. Coordination of microtubules and the actin cytoskeleton is important in osteoclast function, but calcitonin disrupts sealing zones without affecting microtubule networks. Bone 2006;39:684-93. [98] Yamamoto Y, Yamamoto Y, Udagawa N, et al. Effects of calcitonin on the function of human osteoclast-like cells formed from CD14-positive monocytes. Cell Mol. Biol. (Noisy-le-grand) 2006;52:25-31. [99] Drake MT, Clarke BL, Khosla S. Bisphosphonates: Mechanism of action and role in clinical practice. Mayo Clin. Proc. 2008;83:1032-45. [100] Rodan GA, Fleisch HA. Bisphosphonates: Mechanisms of action. J. Clin. Invest. 1996;97:2692-6. [101] Ito M, Amizuka N, Nakajima T, Ozawa H. Bisphosphonate acts on osteoclasts independent of ruffled borders in osteosclerotic (oc/oc) mice. Bone 2001;28:609-16. [102] Alakangas A, Selander K, Mulari M, et al. Alendronate disturbs vesicular trafficking in osteoclasts. Calcif. Tissue Int. 2002;70:40-7. [103] Green JR. Bisphosphonates: Preclinical review. Oncologist 2004;9 Suppl 4:3-13. [104] Itzstein C, Coxon FP, Rogers MJ. The regulation of osteoclast function and bone resorption by small GTPases. Small Gtpases 2011;2:117-30. [105] David P, Nguyen H, Barbier A, Baron R. The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase. J. Bone Miner. Res. 1996;11:1498507. [106] Mayahara M, Sasaki T. Cellular mechanism of inhibition of osteoclastic resorption of bone and calcified cartilage by long-term pamidronate administration in ovariectomized mature rats. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 2003;274:817-26. [107] Escudero ND, Lacave M, Ubios AM, Mandalunis PM. Effect of monosodium olpadronate on osteoclasts and megakaryocytes: An in vivo study. J. Musculoskelet. Neuronal. Interact. 2009;9:109-20. [108] Krum SA, Miranda-Carboni GA, Hauschka PV, et al. Estrogen protects bone by inducing fas ligand in osteoblasts to regulate osteoclast survival. EMBO J. 2008;27:535-45. [109] Nakamura T, Imai Y, Matsumoto T, et al. Estrogen prevents bone loss via estrogen receptor alpha and induction of fas ligand in osteoclasts. Cell 2007;130:811-23. [110] Kacena MA, Shivdasani RA, Wilson K, et al. Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2. J. Bone Miner. Res. 2004;19:652-60.

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[111] Kacena MA, Gundberg CM, Horowitz MC. A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells. Bone 2006;39:978-84. [112] Taylor AF, Barnes CLT, Horowitz MC, et al. A novel role for thrombopoietin in regulating osteoclast development. JBMR. 2008;23:SU104. [113] Beeton CA, Bord S, Ireland D, Compston JE. Osteoclast formation and bone resorption are inhibited by megakaryocytes. Bone 2006;39:985-90. [114] Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J. Clin. Invest. 1999;104:1363-74. [115] Verborgt O, Gibson GJ, Schaffler MB. Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J. Bone Miner. Res. 2000;15:60-7. [116] Noble BS, Peet N, Stevens HY, et al. Mechanical loading: Biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am. J. Physiol. Cell Physiol. 2003;284:C934-43. [117] Cheung WY, Simmons CA, You L. Osteocyte apoptosis regulates osteoclast precursor adhesion via osteocytic IL-6 secretion and endothelial ICAM-1 expression. Bone 2011. [118] McClung MR, Lewiecki EM, Cohen SB, et al. Denosumab in postmenopausal women with low bone mineral density. N. Engl. J. Med. 2006;354:821-31. [119] Lewiecki EM. Odanacatib, a cathepsin K inhibitor for the treatment of osteoporosis and other skeletal disorders associated with excessive bone remodeling. IDrugs 2009;12:799-809. [120] Galasko CS. Mechanisms of lytic and blastic metastatic disease of bone. Clin. Orthop. Relat. Res. 1982;(169):20-7. [121] Armstrong AP, Miller RE, Jones JC, Zhang J, Keller ET, Dougall WC. RANKL acts directly on RANK-expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes. Prostate 2008;68:92-104. [122] Inoue H, Nishimura K, Oka D, et al. Prostate cancer mediates osteoclastogenesis through two different pathways. Cancer Lett. 2005;223:121-8. [123] Chen YC, Sosnoski DM, Mastro AM. Breast cancer metastasis to the bone: Mechanisms of bone loss. Breast Cancer Res. 2010;12:215. [124] Rothschild BM, Hershkovitz I, Dutour O. Clues potentially distinguishing lytic lesions of multiple myeloma from those of metastatic carcinoma. Am. J. Phys. Anthropol. 1998;105:241-50. [125] Giuliani N, Colla S, Rizzoli V. Update on the pathogenesis of osteolysis in multiple myeloma patients. Acta Biomed. 2004;75:143-52. [126] Silvestris F, Ciavarella S, Strippoli S, Dammacco F. Cell fusion and hyperactive osteoclastogenesis in multiple myeloma. Adv. Exp. Med. Biol. 2011;714:113-28. [127] Fleisch H. Bisphosphonates. pharmacology and use in the treatment of tumour-induced hypercalcaemic and metastatic bone disease. Drugs 1991;42:919-44. [128] Fulfaro F, Casuccio A, Ticozzi C, Ripamonti C. The role of bisphosphonates in the treatment of painful metastatic bone disease: A review of phase III trials. Pain 1998;78:157-69. [129] Diel IJ, Solomayer EF, Costa SD, et al. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N. Engl. J. Med. 1998;339:357-63.

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[130] Neville-Webbe HL, Coleman RE. Bisphosphonates and RANK ligand inhibitors for the treatment and prevention of metastatic bone disease. Eur. J. Cancer. 2010;46:1211-22. [131] Schaiff RA, Hall TG, Bar RS. Medical treatment of hypercalcemia. Clin. Pharm. 1989;8:108-21. [132] Castellano D, Sepulveda JM, Garcia-Escobar I, Rodriguez-Antolin A, Sundlov A, Cortes-Funes H. The role of RANK-ligand inhibition in cancer: The story of denosumab. Oncologist 2011;16:136-45. [133] Vosse D, de Vlam K. Osteoporosis in rheumatoid arthritis and ankylosing spondylitis. Clin. Exp. Rheumatol. 2009;27:S62-7. [134] Gravallese EM. Bone destruction in arthritis. Ann. Rheum. Dis. 2002;61 Suppl 2:ii84-6. [135] Roux S, Orcel P. Bone loss. factors that regulate osteoclast differentiation: An update. Arthritis Res. 2000;2:451-6. [136] Nozawa K, Fujishiro M, Kawasaki M, et al. Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis. Arthritis Res. Ther. 2009;11:R174. [137] Ellabban AS, Kamel SR, Ahmed SS, Osman AM. Receptor activator of nuclear factor kappa B ligand serum and synovial fluid level. A comparative study between rheumatoid arthritis and osteoarthritis. Rheumatol. Int. 2011; [138] Shimoyama Y, Nagafuchi H, Suzuki N, Ochi T, Sakane T. Synovium infiltrating T cells induce excessive synovial cell function through CD28/B7 pathway in patients with rheumatoid arthritis. J. Rheumatol. 1999;26:2094-101. [139] Weitzmann MN, Pacifici R. The role of T lymphocytes in bone metabolism. Immunol. Rev. 2005;208:154-68. [140] Gillespie MT. Impact of cytokines and T lymphocytes upon osteoclast differentiation and function. Arthritis Res. Ther. 2007;9:103. [141] Cohen SB, Dore RK, Lane NE, et al. Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: A twelvemonth, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial. Arthritis Rheum. 2008;58:1299-309.

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Chapter 3

THE HIGH OSTEOCLASTOGENIC POTENTIAL OF HUMAN OSTEOSARCOMA CELLS: RECIPROCAL INTERACTION BETWEEN MG63 OSTEOBLAST-LIKE CELLS AND OSTEOCLAST PRECURSORS J. Costa-Rodrigues and M. H. Fernandes Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Portugal.

ABSTRACT Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Bone is in a continuous remodeling process which involves a complex and coordinated activity of osteoblastic and osteoclasticcells. Unbalances in this equilibrium leads to bonemetabolic pathological conditions, occurring in a variety of clinical situations, including bone primary tumors. Osteosarcoma, the most frequent bone oncologic disease, although with a variable behavior, affects bone metabolic activities, leading to a disturbed bone structure. Osteosarcoma is usually associated to the formation of woven bone, but in the last years it become evident that patients with this pathology frequently display a high bone turnover, with a progressive weakening of its structure. Osteosarcoma cells are osteoblast-like cells that display many osteoblastic features. Due to that, osteosarcoma cell lines are widely used as osteoblastic models for in vitro studies. Among them, MG63 cell line is by far the most used cell line in that context. Although the role of osteoblasts in osteoclastogenesis is well documented, the influence of MG63 osteosarcoma cells on osteoclast development remains poorly elucidated. In the last few years, we have been interested in this issue, aiming to characterize the osteoclastogenic potential of MG63 cells regarding both paracrine and direct cell-to-cell mechanisms. To address this, human osteoclast precursors from peripheral blood mononuclear cells were cultured in the presence of conditioned media from MG63 cell cultures or co-cultured with MG63 cells, in several experimental conditions. The osteoclastogenic response was compared with that achieved by human osteoblastic bone 

Maria Helena Raposo Fernandes, Laboratório de Farmacologia e Biocompatiblidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Rua Dr. Manuel Pereira da Silva, 4200-393 Porto, Portugal; Telephone: +351 220 901 100; Fax: +351 220 901 101; E-mail address: [email protected].

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J. Costa-Rodrigues and M. H. Fernandes marrow cells (hBMC), in similar conditions. The influence of some osteoclastogenic signaling pathways was also addressed. MG63 cell line was able to elicit a high degree of osteoclast differentiation. Regarding paracrine mediated mechanisms, conditioned media from MG63 cell culturesinduced a significant osteoclastogenic response. Also, in co-cultures of osteoclast precursors and MG63 cells, osteoclastogenesis proceeded to a high extent in the presence of a low number of MG63 cells. In both situations, the osteoclastogenic response was higher than that elicited by hBMC, in similar experimental conditions. The intracellular mechanisms involved in the osteoclastogenesismediated by MG63 cells and hBMCrevealed some important differences, namely regarding the relative relevance of MEK and NFkB signaling pathways, and PGE2 production. Also, it was observed that the presence of osteoclastic cells modulate the osteoblastic behavior of MG63 cells. In conclusion, MG63 cell line has a high osteoclastogenic potential and, compared to hBMC, differences were found regarding the pattern and intensity of the osteoclastic response, as well as in the involved intracellular pathways. Results suggest that understanding the molecular details of the osteoclastichyperactivation induced by osteosarcoma cells can open new perspectives for the design of therapeutic approaches for thistype of bone metabolic disorders.

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1. INTRODUCTION Due to its continuous exposure to exogenous stimuli, such as physical stress and pressure, bone tissue presents a high rate of remodeling. This process is continuously occurring, and it is believed that the entire skeleton is renewed several times during life [1]. This process involves the coordinated action of two main cellular types, the osteoblasts and the osteoclasts [2, 3]. The former are responsible for new bone formation, being characterized for their ability to produce an extracellular mineralized matrix [2]. The latter are specialized cells that descend from the monocyte/macrophage lineage [4, 5] and promote bone resorption through the secretion of acid and lytic enzymes (which degrade the inorganic and the organic components of bone tissue, respectively) [2, 6]. Osteoblasts are key players in the process of osteoclastogenesis[7, 8]. They have the ability to modulate it through a complex network of crosstalks. Those communications involve the osteoblastic production of soluble and membrane-bound molecules that can activate or inhibit osteoclast development [8]. Among them are the two main important osteoclastogenesis stimulators, the monocyte-colony stimulation factor (M-CSF) and the receptor activator of nuclear factor-B ligand (RANKL), and the inhibitor osteoprotegerin (OPG) [7, 9]. Nevertheless, osteoblastic cells are also known to produce a variety of other osteoclastogenic modulators, such as IL-1, IL-6 and TNF-, for example [2, 9-11]. Besides osteoblasts, there are also other cell types that regulate osteoclast differentiation, like, for example, fibroblasts, endothelial cells, B cells, T cells and tumor cells [6, 12-17]. Osteoblastic and osteoclastic activities are strictly regulated, in order to avoid imbalances that can led to skeletal disorders, such as those that occur in osteoporosis, bone metastasis or even in primary bone tumors [17, 18]. Osteosarcoma, a malignant tumor that affects mesenchymal cells, is the most common primary solid bone tumor, which occurs mainly in children and young adults [19-21]. It accounts for about 15% of all solid extracranial tumors in those age groups, affecting 1.4 times more the male individuals than the females [21]. Osteosarcoma presents a high general incidence of about 2-3 million of new cases per year,

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but this value raises up to 8-11 million when considering just the 15-19 years old teenage population [21-23]. The main symptoms associated with osteosarcoma involve joint movement problems, local pain and, in some cases, pathological fractures. It affects predominantly the metaphyses of long bones, such as the distal femur, proximal tibia and proximal humerus [19, 24, 25]. Although the relevant improvements on surgery and chemotherapy techniques observed in the last years resulted in a better efficiency of osteosarcoma treatment, the overall rate of survival did not increased substantially over the past two decades [26]. As it happens with all primary or metastatic tumors that develop in bone tissue, osteosarcoma can lead to skeletal integrity problems [21, 27]. Despite the rigid structure of bone, which one might expect to become a problem for the proliferation and development of tumor cells, the fact is that cancer cells may circumvent it through the dysregulation of bone metabolic activities [1, 28]. Osteosarcoma cells might interfere with osteoblastic and/or osteoclastic processes, which can lead to different osteogenic profiles of the cancer [29]. It is usually characterized by an abnormal deposition of non-remodeled osteoid and/or woven bone [21]. Nevertheless, local regions of high bone turnover are also observed [20]. Osteoclast are frequently hyperactivated in the pathophysiology of skeletal cancers [15], and osteosarcoma is not an exception. In fact, in the past few years osteoclastic cells have assumed a relevant role as a potential target for osteosarcoma treatments [26]. Furthermore, osteoclasts are frequently associated with osteosarcoma cells in histological and mRNA analysis (63% and 75% of the cases, respectively) [30]. Osteosarcoma cells are malignant mesenchymal cells, which means that they belong to the same lineage as osteoblasts [21, 26]. Although the reciprocal communication between osteoblastic and osteoclastic cells are well characterized [8], substantially less information is available regarding the crosstalks among osteosarcoma and osteoclastic cells. A detailed understanding of when, how and why osteosarcoma cells affect bone tissue is crucial towards the design of new and effective therapeutic approaches for that pathological condition. Unfortunately, the present knowledge about that issue is still very scarce. Particularly, and since osteosarcoma can also reveal an osteolytic behavior, the crosstalks between osteosarcoma malignant cells and osteoclast (or their precursors) are far from being completely elucidated [20, 31, 32]. Those crosstalks might involve direct cell-to-cell contacts and/or paracrine communications through the secretion of soluble modulators by both cell types. Nowadays, there are many different commercially available human osteosarcoma cell lines. However, their behavior might differ significantly among each other, which significantly difficult the establishment of response patterns. A detailed understanding of the specific properties of each of them, in respect to their effects on bone cells is of the outmost importance. The most used cell line as an osteoblast-like model is MG63, which was established from a 14-years old Caucasian male. Although it displays some osteoblastic features, such as a constitutive low expression of alkaline phosphatase (ALP) that increases following vitamin D3 supplementation [33], for example, it also presents some important differences comparatively to osteoblastic cells, such as the low expression of RANKL [32, 34-37] and the inability to produce an extracellular mineralized matrix [38]. This chapter focuses on the osteoclastogenic potential of MG63 cell line, through different mechanisms, that is, by paracrine-mediated [32] and cell-to-cell contact [31] processes.For the former condition, conditioned media from MG63 cell cultures were used as putative osteoclastogenic modulators on human osteoclast precursors isolated from peripheral

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blood (peripheral blood mononuclear cells, PBMC). To evaluate the role of cell-to-cell contacts between both cell types, on human osteoclastogenesis, the behavior of co-cultures composed by different cellular proportions of MG63 cells and PBMC was analyzed. In both cases, results were compared with those obtained with human bone marrow cells (hBMC), maintained either in the absence or presence of dexamethasone, a well-known osteogenic inducer [39-41]. In addition, the reciprocal interaction, i.e. the effects of osteoclast cells on the osteoblastic behavior of MG63 cell line, was also addressed.

2. CELL CULTURE MODELS In this section, a characterization of the cellular models used in the following parts of the chapter is presented, that is, the osteoblastic behavior of MG63 cell line and the osteoclastic differentiation of peripheral blood mononuclear cells.

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2.1. MG63 Cell Line MG63 cell line (ATCC number CRL-1427TM) was assessed for the expression of several osteoblast-related genes, such as collagen type 1 (COL1), ALP and bone morphogeneticprotein 2 (BMP-2) [2], as well as for the osteoclastogenic activators M-CSF and RANKL and the inhibitor OPG [9]. For comparison, the same analysis was performed in human bone marrow cells (hBMC), cultured in the absence or presence of the classic osteogenic inducer dexamethasone (dex) [39-41], respectively –dex and+dex cultures. Cells were maintained in -Minimal Essential Medium (-MEM), supplemented with 10% foetal bovine serum, 100 IU/mL penicillin,2.5 g/mL streptomycin, 2.5 g/mL amphotericin B and50 g/mL ascorbic acid. After 14 days of culture, RNA was extracted and the RT-PCR analysis was performed. The samples were separated on a 1% agarose gel and subjected to a densitometric analysis. The obtained values were normalized for the corresponding expression of glycerol-phosphate dehydrogenase (GAPDH). MG63 expressed all the analysed genes (Figure 1). COL1 expression levels were slightly higher than those of hBMC cultures performed either in the absence or presence of dexamethasone (-dex and +dex, respectively). On the other hand, ALP and BMP-2 expression levels were relatively low, slightly lower (ALP) and higher (BMP-2) than those observed for –dexhBMC cultures, and significantly lower compared to +dex cultures. These genes are involved in osteoblastic function [42-44], and dexamethasone is known to stimulate their expression in hBMC [41, 45, 46]. Considering the results obtained for MG63 cell line, they are in agreement with the literature, and these cells were often classified as immature osteoblast-like cells [33, 36], that produce collagen and display a low constitutive ALP expression [33]; also, osteosarcoma cells are known to express BMP-2 [47]. Regarding the expression of the osteoclastogenic modulators, MG63 cell line revealed a high response for M-CSF and OPG genes, whose values were higher than, or similar to the ones observed for hBMC cultures, respectively. Their ability to express high levels of such molecules is in line with previous findings [34, 48, 49].

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Figure 1. Characterization of the osteoblastic behavior of MG63 cell line. Cells were assessed by RTPCR for the expression of the osteoblastic markers COL1, ALP and BMP-2, and the osteoclastogenic modulators M-CSF, RANKL and OPG. In parallel, the same analysis was conducted with hBMC maintained in the absence or presence of dexamethasone (-dex and +dex, respectively). Values were normalized for the corresponding GAPDH expression value of each experimental condition. Gel analysis was performed with ImageJ 1.41 software. * - Significantly different from hBMC (-dex) cultures (p < 0.05). # Significantly different from hBMC (+dex) cultures (p < 0.05).

Although, in general, cellular mRNA levels of OPG appear to be higher than RANKL [50], RANKL expression on MG63 was particularly low, being substantially lower than on hBMC cultures. In fact, although it is known that MG63 can express RANKL, there are several published reports that claim that its expression, assessed by RT-PCR and qPCR, is very low [34-37]. Nevertheless, different osteosarcoma cells seem to display different abilities to express RANKL [20, 26, 36]. Taken together, although MG63 cells display several traits from osteoblasts, there are important differences among their gene-expressing profiles. For the majority of the analyzed genes, this cell line reveals a behavior that is more closely related to non-differentiated hBMC (-dex cultures) rather than to osteoblastic cells (+dex cultures).

2.2. Peripheral Blood Mononuclear Cells (PBMC) Osteoclasts are multinucleated cells that descend from the CD14+monocyte lineage [5, 6, 51]. It was observed that their precursor cells are found not only in bone marrow, but also in the blood circulation [5, 6]. In that context, peripheral blood mononuclear cells (PBMC) are widely used as the starting material to establish osteoclastic cell cultures. PBMC per se display a very low osteoclastogenic response [5, 13, 32], but when cultured in the presence of activators of the process, usually they reveal a high osteoclastogenic profile. In that context, there are two growth factors that are known to be sufficient to promote in vitro osteoclastogenesis, namely, M-CSF and RANKL [4, 6, 9, 52]. To validate the suitability of PBMC cultures to generate a high number of mature osteoclasts, the behavior of cell cultures maintained in the absence (base medium) or presence of 20 ng/mL M-CSF and 40 ng/mL RANKL [7, 53] was compared. PBMC were separated from other blood components by a gradient centrifugation with Ficoll-PaqueTM PREMIUM.

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Figure 2. Characterization of the osteoclastic behavior of PBMC cultures, maintained in the absence (base medium) or presence of recombinant M-CSF and RANKL. Cells were assessed for: TRAP activity, determined by pNPP hydrolysis assay (A); number of TRAP positive multinucleated cells (B); expression of the osteoclast-related genes c-myc, c-src, TRAP, CATK, CA2 (C); calcium phosphate resorbing ability (D); presence of multinucleated cells with actin rings and expressing VNR and CTR (E). In addition, the presence of osteoclastic cells was also confirmed by SEM visualization of cell cultures (F). Bars represent 40 m.

Usually, for each 100 mL of processed blood, it was obtained about 70x106 PBMC. Cells, seeded at 1.5x106 cells/cm2, were maintained for 21 days in -MEM, supplemented with 30% human serum (from the same donor from whom the PBMC were collected), 100 IU/mL penicillin,2.5 g/mL streptomycin, 2.5 g/mL amphotericin B and 2 mML-glutamine. Cell cultures were assessed at days 14 and 21 for several osteoclastic traits, Figure 2, namely: quantification of tartarate-resistant acid phosphatase (TRAP) activity – evaluated by pNPP hydrolysis assay, and normalized by total protein content, determined by Bradford’s method [54], as described previously [14, 55] – an enzyme that has been used as an osteoclastic marker, and whose serum levels appear to be correlated with the bone resorbing activity in vivo[6, 56]; presence of TRAP-positive multinucleated cells (MNC), determined by staining the PBMC cultures with the acid phosphatase, leucocyte (TRAP) kit; expression of osteoclast-related genes, namely, factors engaged with osteoclast differentiation and

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activation – c-myc and c-src, respectively, and genes that code for the functional markers TRAP, the lytic enzyme catepsin K (CATK) and the enzyme involved in the production of acid carbonic anhydrase 2 (CA2) [6, 52], normalized with the corresponding expression levels of GAPDH; osteoclast resorbing ability, by culturing PBMC on calcium phosphate coated culture plates (BD BioCoat™ Osteologic™ Bone Cell Culture Plates, BD Biosciences); presence of MNC cells with actin rings and expressing vitronectin and calcitonin receptors (VNR and CTR, respectively), observed by confocal laser scanning microspcopy (CLSM); visualization of cell morphology by scanning electron microscopy (SEM). In base medium, cell cultures displayed a very low TRAP activity (Figure 2A) and number of TRAP+ MNC (Figure 2B), but the presence of recombinant M-CSF and RANKL sharply increased those values. A similar behavior was also observed for the expression of osteoclast-related genes (Figure 2C) and calcium phosphate resorbing ability (Figure 2D). Nevertheless, either in base medium or in the presence of the growth factors, it was possible to observe the presence of osteoclastic cells by CLSM (Figure 2E) and SEM (Figure 2F), although in significantly higher amounts in the latter experimental condition. Thus, as described previously [5, 13, 32], PBMC cultured in the absence of any exogenous osteoclastogenic stimuli reveals only a low osteoclastic behavior. However, the presence of M-CSF and RANKL greatly enhances osteoclastic response, which is in line with the well-established role of these growth factors on osteoclastogenesis[4, 6, 9, 52].

3. OSTEOCLASTOGENIC POTENTIAL OF THE MG63 CELL LINE

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3.1. Paracrine-Mediated Mechanisms In order to analyze the influence of MG63 cells in the differentiation of osteoclast precursors, in the absence of physical contacts between both cell types, PBMC cultures, maintained in base medium, were treated with 10% or 20% of MG63 conditioned media (CM), collected after 48 hours, 7 days and 14 days of culture, and previously normalized for their total protein content. In parallel, CM from hBMC cultures (either –dex or +dex) were also included in the experiments. PBMC cultures were assessed at days 14 and 21 for TRAP activity and number of TRAP+ multinucleated cells (Figure 3). Globally, CM from MG63 cell line elicited a higher TRAP activity than CM from hBMC (either –dex or +dex cultures) in PBMC cultures (Figure 3, upper panels). This difference became more pronounced with the increase in the conditioning period (7 and 14 days). Also, the response was higher when CM was used at 20%, and once again this pattern was more evident and values turned to be statistically significant for CM collected after 7 and 14 days of culture. Regarding the number of TRAP-positive multinucleated cells (Figure 3, lower panels), some differences were observed. First, CM from 48h-MG63 cultures elicited a similar (10%) or lower (20%) response than the corresponding CM from +dex cultures. Second, CM from 7 day-cultures (both MG63 and +dex cultures) revealed an identical osteoclastogenic potential. Nevertheless, CM from 14 day-MG63 cell cultures, used at 20%, caused a higher response than the CM from +dexhBMC cultures.

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Thus, it seems that for lower conditioning periods, and contrarily to that found with the CM from +dex cultures, CM from MG63 cell line may stimulate TRAP activity/expression more than it increases the amount of TRAP-positive multinucleated cells. After determining the overall pattern of response of PBMC cultures in the presence of CM from MG63 cells, PBMC cultures supplemented with CM from 7-day cultures, at 20%, were further characterized for the expression of osteoclast-related genes and the calcium phosphate resorbing ability, in a way similar to that described above. For comparison, results regarding the response observed with 20% of CM from –dex and +dexhBMC cultures, collected after 7 days of culture, were also included in the study. All the tested cell cultures expressed the analyzed genes, c-myc, c-src, TRAP, CATK and CA2 [6, 52] (Figure 4A). The presence of CM from MG63 cell line elicited a significant increase in the expression levels, when compared with those achieved following treatment with CM from –dex or +dexhBMC cultures (Figure 4A). Regarding theosteoclastic resorbing ability, formation of resorption lacunae was observed in all tested conditions (Figure 4B).PBMC cultures supplemented with CM from MG63 cells revealed the highest percentage of resorption, while CM collected from hBMC (-dex) cultures caused the lowest response (Figure 4C). Taken together, CM from MG63 cells displayed a high osteoclastogenic potential, that varies with the concentration and the conditioning period tested. A substantial increase in the osteoclastogenic response was evident between 48h-CM and 7day-CM, but not between the latter and 14 day-CM. Thus, it can be hypothesized that as conditioning period increases, CM may become saturated in the osteoclastogenic inducer molecules; there is also the possibility of an increase in the toxicity of the CM, due to the accumulation of metabolites and cellular wastes. In both cases, the CM ability to promote osteoclast differentiation is attenuated. In a previous report, it was observed that CM from MG63 cells could induce, although slightly, osteoclastogenesis on PBMC cultures, assessed by counting the amount of multinucleated cells positive for TRAP present in the cultures [57]. However, the authors have used 10% of CM collected after 24h of culture, which can account for the low osteoclastogenic potential described. The present data is in line with another work, where it was reported that the presence of CM from MG63 cells, simultaneously with RANKL supplementation (but in the absence of M-CSF), supported the resorbing ability of dentine by human PBMC [57]. Moreover, in another study, it was observed that different osteosarcoma cell lines (HSOS-1, OSRb, G292 and KHOS) were able to differentially induce the formation of TRAP+ multinucleated cells on human monocytes, in the absence of cell-to-cell contacts [20]. In addition, they were also able to increase the expression of c-src on human monocytes and to stimulate the osteoclastic bone-resorbing ability [20]. After characterizing the effects of CM from MG63 cells on osteoclast differentiation and function, the involvement of some important osteoclastogenicrelated signaling pathways on the observed cellular responses was addressed. For that, PBMC cultures, performed in the presence of 20% 7day-CM from MG63 cell line, as well as from – dex and +dexhBMC cultures, were treated with different signaling pathway inhibitors, and characterized for TRAP activity and number of TRAP+ multinucleated cells. MEK pathway is involved in osteoclastogenesis, being activated by several factors (M-CSF and RANKL, for example), particularly those related to the earlier stages of the process, i.e., mainly engaged with osteoclast precursor cell survival [4, 52, 58].

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Figure 3. TRAP activity (upper panels) and TRAP+ multinucleated cells (lower panels) of PBMC cultures treated with CM from hBMC (-dex and + dex) and MG63 cell cultures, collected after 48 hours, 7 days and 14 days of culture. TRAP activity was assessed by pNPP hydrolysis assay and normalized with total protein content of cell cultures, determined by Bradfofd’s method [54]. * - Significantly different from hBMC (-dex) (p < 0.05). # - Significantly different from hBMC (+dex) (p < 0.05).

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Figure 4. RT-PCR analysis (A) and calcium phosphate resorbing ability (B and C) of PBMC cultures treated with CM from hBMC (-dex or +dex) or MG63 cells, collected after 7 days of culture. A – Cells were assessed for the expression of the osteoclast differentiation and activation factors (c-myc and csrc, respectively) and the osteoclastic markers TRAP, CATK and CA2. Values were normalized for the corresponding GAPDH expression value of each experimental condition. Gel analysis was performed with ImageJ 1.41 software. B – Cell cultures were performed on calcium phosphate coated culture plates (BD BioCoat™ Osteologic™ Bone Cell Culture Plates, BD Biosciences). After cell removal, resorption lacunae were identified by phase contrast optical microscopy (B) and total resorbed area was quantified with ImageJ 1.41 software (C).* - Significantly different from hBMC (-dex) (p < 0.05). # Significantly different from hBMC (+dex) (p < 0.05).

U0126, an inhibitor of MEK pathway, was used at 1 M and 10 M, due to the contradictory effects attributed to low concentrations of this molecule in respect to the osteoclast development [14, 59-63]. NFkB pathway is a very important mechanism involved in osteoclast differentiation (it is the main osteoclastogenic pathway activated by RANKL, but it is also activated by M-CSF, among others), leading to the activation of several transcription factors associated to the process [4, 52, 64, 65]. PDTC, aNFkB pathway inhibitor, was tested at 10 M and 100 M, because the lower concentration was described as the IC50 on rat osteoclastogenesis[66]. PGE2 is an eicosanoid molecule involved in the modulation of bone metabolism, enhancing both osteoclastogenesisin vitro and bone resorptionin vivo[67]. Indomethacin was tested at 1 M, due to the established role of this molecule as an inhibitor of PGE2 synthesis [67-70]. Cell cultures were characterized for TRAP activity and number of TRAP+ multinucleated cells. It was observed that U0126 at 1 M caused a partial inhibition of osteoclast development on cultures treated with CM from hBMC (either –dex or +dex) (Table 1). On the other hand,

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its inhibitory effect on PBMC cultures treated with MG63 CM was only residual. However, at 10 M, a full inhibition was achieved on hBMC CM-treated cell cultures, and a substantial decrease on cellular response was observed following MG63 CM supplementation. Thus, although MEK pathway seems to be involved in the osteoclastogenic response induced by the CM from MG63 cells, its relative contribution for the process seemed to be lower than the one observed for cultures treated with CM from hBMC. Treatment with PDTC completely abolished osteoclastogenesis on PBMC cultures treated with CM from hBMC (either –dex or +dex), while in the case of the CM from MG63 cells, the lower concentration caused a significant but not total inhibition. So, NFkB pathway is an important player in osteoclastogenesis mediated by CM from MG63 cell line, but once again it seems to be downregulated in that experimental condition, when compared to the response induced by hBMC CM. Finally, indomethacin did not affect significantly osteoclast development, indicating that PGE2 production might not be involved in the process, in all the three tested conditions. Taken together, these observations suggest that although MEK and NFkB signaling pathways seemed to be involved, there might be other pathways contributing for the high osteoclastogenic response induced by MG63 CM. Moreover, since MG63 cell line display a low expression level of RANKL (Figure 1), it is tempting to suggest that the RANKL-RANK interaction may not represent the main activation event of those signaling pathway, as suggested recently [32]. In this context, and due to the absence of published information about this issue, further studies are required in order to fully characterize the intracellular mechanisms involved in the observed cellular response.

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Table 1. Characterization of the involvement of MEK and NFkB pathways and PGE2 production on the osteoclastogenic response of PBMC cultures treated with CM from hBMC (-dex or +dex) or MG63 cells. CM was collected after 7 days of culture Conditioned media (7days) % of inhibition related to the absence of inhibitors (Day 21) Condition hBMC (-dex) 28.9±3.7 U0126 1M 100 U0126 10M 100 PDTC 10M 100 PDTC 100M 3.7±0.6 Indomethacin 1 M

hBMC (+dex) 30.2±1.5 100 97.5±1.7 100 2.5±0.4

MG63 5.9±0.8 69.9±7.7 68.6±5.1 100 4.6±0.9

3.2. Cell-to-Cell Contact Mechanisms In order to evaluate the role of physical contacts between PBMC and MG63 cells on osteoclast development, the same set of analysis described on the previous section was conducted on co-cultures of PBMC and MG63 cells. For that, PBMC (1.5x106 cells/cm2) were co-cultured with different seeding densities of MG63 cells (102, 103 and 104 cells/cm2). In parallel, PBMC were also co-cultured with the same cellular densities of hBMC (either in the absence or presence of dexamethasone). Co-cultures were maintained in the same experimental conditions described above.

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Figure 5. TRAP activity (upper panels) and TRAP+ multinucleated cells (lower panels) of co-cultures of PBMC and different seeding densities (102, 103 and 104 cells/cm2) of hBMC (-dex or + dex) or MG63 cells. TRAP activity was assessed by pNPP hydrolysis assay and normalized with total protein content of cell cultures, determined by Bradfofd’s method [54]. * - Significantly different from hBMC (-dex) (p < 0.05). # - Significantly different from hBMC (+dex) (p < 0.05).

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Results showed that the lower MG63 seeding density (102 cells/cm2) promoted a high TRAP activity (Figure 5, upper panels), significantly higher than that observed for –dex and +dexhBMC cultures. Nevertheless, this pattern of response was inverted with the increase on their seeding densities. That is, the co-culture performed with the higher number of MG63 cells revealed a lower TRAP activity than the co-cultures performed with the same amount of hBMC. The number of TRAP+ multinucleated cells displayed a similar pattern of results (Figure 5, lower panels) as those observed for TRAP activity. Taken together, it seems evident that MG63 cells have a high osteoclastogenic-inducing ability, justified by the fact that a very low seeding density (102 cells/cm2) can cause a highosteoclastogenic activation. Indeed, at the same cellular density (102 cells/cm2), hBMC cells were able to elicit a significantly lower response, that became similar just when there were 10 times more hBMC (103 cells/cm2). The inversion observed in this pattern of results, as a consequence of the increase on the proportion of MG63 cells or hBMC cells on the co-cultures might be due to the fact that the former cells have a proliferation rate that is higher than the latter [71]. Thus, as the culture period increases, MG63 cells will proliferate and fill the culture plate wells, decreasing the overall efficiency of the osteoclastogenic process, simply as a consequence of a kind of dilution of the osteoclastic cells in culture. For that reason, the evaluation of the expression levels of the osteoclast-related genes and the calcium phosphate resorbing ability of co-cultures, as well as the involvement of some intracellular signaling pathways on the cellular response, was conducted on co-cultures performed with the plating density of 102 cell/cm2 MG63 cells. Results were compared with co-cultures containing 10 times more hBMC (either –dex or +dex cultures), that is, with a plating density of 103 cell/cm2. All tested co-cultures expressed the analyzed genes - c-myc, c-src, TRAP, CATK and CA2 (Figure 6A). Regarding the PBMC + MG63 co-cultures, the expression levels were identical to those observed in the co-cultures performed with 10 times more hBMC (either in the absence or presence of dexamethasone). Formation of resorption lacunae was also observed in all tested conditions (Figure 6B). The percentage of resorbed area was similar in all tested conditions, though MG63 cells were seeded at a 10 times lower density (Figure 6C). The present data is in agreement with previously published reports, although with some differences that can be related to different experimental conditions. Nevertheless, it was observed that both co-cultures of MG63 with CD14+ cells [72], maintained for 7 days in the presence of PTH and fetal calf serum (FCS), or co-cultures of MG63 cells and macrophages [34], performed for 14 days in the presence of FCS, supported osteoclastogenesis, assessed by TRAP activity. Furthermore, in the latter study it was also observed that co-cultures were able express CATK [34]. In addition, other papers have described osteoclastogenesis in cocultures of osteoclast precursors with different osteosarcoma cell lines. For example, cocultures of human monocytes and UMR-106 rat osteosarcoma cell line revealed the presence of multinucleated cells positive for TRAP, which had the ability to resorb dentine slices [73]. Furthermore, when the same osteosarcoma cell line was co-cultured with human umbilical cord mononuclear cells, results showed osteoclasticresorption occurring on bone slices [74]. Also, in co-cultures of PBMC and SaOS-2 cell line, treated with parathyroid hormone (PTH) and, especially, PTH and dexamethasone, authors reported the formation of multinucleated cells TRAP+, as well as increase in the expression of TRAP and CATK genes [75].

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In addition, the same study showed that co-cultures composed by human PBMC or mouse bone marrow cells and SaOS-2 osteosarcoma cells were also able to resorb dentine slices [75]. Finally, co-cultures of human monocytes with the osteosarcoma cell lines HSOS1, OSRb, G292 and KHOS, displayed the presence of different amounts of multinucleated cells positive for TRAP [20]. Co-cultures of PBMC and MG63 cells (plated at 102 cells/cm2) were also characterized for the possible involvement of MEK and NFkB signaling pathways, as well as PGE2 production, on the osteoclastogenic response (TRAP activity and number of TRAP+ multinucleated cells). Once again, results were compared with those achieved for PBMC co-cultured with –dex and +dexhBMC (plated at 103 cells/cm2). It was observed that on co-cultures performed with hBMC, the U0126 molecule caused a significant inhibition at 1 M and a full inhibition at 10 M (Table 2).

Figure 6. RT-PCR analysis (A) and calcium phosphate resorbing ability (B and C) of PBMC cocultured with 103hBMC (-dex or +dex)/cm2 or 102 MG63 cells/cm2.. A – Cells were assessed for the expression of the osteoclast differentiation and activation factors (c-myc and c-src, respectively) and the osteoclastic markers TRAP, CATK and CA2. Values were normalized for the corresponding GAPDH expression value of each experimental condition. Gel analysis was performed with ImageJ 1.41 software. B – Cell cultures were performed on calcium phosphate coated culture plates (BD BioCoat™ Osteologic™ Bone Cell Culture Plates, BD Biosciences). After cell removal, resorption lacunae were identified by phase contrast optical microscopy (B) and total resorbed area was quantified with ImageJ 1.41 software (C).* - Significantly different from hBMC (-dex) (p < 0.05). # - Significantly different from hBMC (+dex) (p < 0.05).

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However, in the case of the co-cultures performed with MG63 cells, the lower concentration did not affect the cellular response, while the higher concentration caused a substantialdecrease, but not a total inhibition of TRAP activity. Thus, MEK pathway seems to be clearly less involved in the osteoclastogenic process activated by the presence of MG63 cells. Regarding the effect of PDTC, a total inhibition was achieved in all the tested cocultures, for the two analyzed concentrations, which suggests a strong involvement of the NFkB pathway in these experimental conditions. On the other hand, indomethacin only caused a partial inhibition on osteoclastic parameters in the tested co-cultures, without significant differences among them. This suggests that PGE2 production may be a contributing mechanism through which MG63 cells stimulate osteoclastogenesis. Since MG63 cells exhibit several osteoblastic traits, this can be explained by the known ability of osteoblasts to produce PGE2 [76, 77]. Table 2. Characterization of the involvement of MEK and NFkB pathways and PGE2 production on the osteoclastogenic response of PBMC co-cultured withhBMC (-dex or +dex) or MG63 cells

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Co-cultures % of inhibition related to the absence of inhibitors (Day 21) Condition hBMC (-dex)* hBMC (+dex)* 41.3±3.4 45.5±4.5 U0126 1M 100 100 U0126 10M 98.8±0.9 91.2±7.9 PDTC 10M 100 100 PDTC 100M 26.1±4.1 16.6±3.6 Indomethacin 1M *103cells/cm2;** 102cells/cm2.

MG63** 0.6±0.1 87.1±5.2 99.2±0.8 99.3±0.7 20.7±2.2

Thus, the presence of MG63 cells seemed to induce osteoclastogenesis in a way dependent on MEK and NFkB pathways, and PGE2 production. Nevertheless, the MEK pathway seemed to have a lower importance, comparing to the response observed on PBMC + hBMC co-cultures. A detailed characterization of the involved intracellular mechanisms is still missing and is required for a full understanding of the osteoclastogenic potential of MG63 cell line by cell-to-cell contacts.

4. MODULATION OF THE MG63 CELL BEHAVIOR BY OSTEOCLASTIC CELLS Being observed that when co-cultured with PBMC, MG63 cells have the ability to stimulate osteoclastogenesis, the existence of a reciprocal communication between both cell types could not be discarded. In this context, the effects of co-cultured PBMC on the expression of the osteoblast-related genes ALP, BMP-2 and COL1 [2] by MG63 cells was analyzed. The study was conducted with co-cultures performed with 1.5 x 106cells/cm2 PBMC and 103 MG63 cells/cm2, and not 102 cells/cm2, as in co-cultures performed with the lower seeding density no osteoblastic RNA was detected, most probably because of the very low amount of MG63 cells present in the co-culture. Results were compared with those

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obtained in co-cultures established with the same seeding density of hBMC (either –dex or +dex cultures), as well as with the values obtained in the corresponding monocultures. Expression of COL 1 was decreased in all co-cultures, compared to the respective monocultures. However, this decrease was significantly higher in the MG63 + PBMC cocultures. Also, the presence of PBMC significantly decreased the expression of ALP by MG63, compared with that verified for both hBMC –dex and + dex co-cultures (in this case, the small decrease observed was not statistically significant) (Figure 7). These results suggest that in the presence of PBMC, MG63 cells might display a lower ability to produce a proper extracellular collagenous matrix and also an impairment in osteoblast functional activity [43, 44]. On the other hand, the expression of BMP-2 was increased in co-cultured conditions, involving both MG63 cells and hBMC. BMP-2 is associated with the modulation of bone metabolic activities [42], inducing not only osteoblastogenesis but also increasing the osteoclastic resorbing activity [78-80], and these results might suggest the possibility of alterations in the bone remodeling events.

Figure 7. RT-PCR analysis of hBMC (-dex or +dex) or MG63 cell cultures (103 cells/cm2) cultured isolated or co-cultured with PBMC. Cells were assessed for the expression of the osteoblastic markers COL1, ALP and BMP-2. Values were normalized for the corresponding GAPDH expression value of each experimental condition. Gel analysis was performed with ImageJ 1.41 software. * - Significantly different from the corresponding isolated culture (p < 0.05).

Despite the fact that the modulation of the behavior of osteosarcoma by osteoclastic cells is yet poorly documented, the present results are in line with a published work where it was observed that CM from chicken osteoclasts, human osteoclastoma cells, and chicken and human multinucleated giant cells, decreased the synthesis of collagen and the activity of ALP on rat UMR-106 osteosarcoma cell line [81].

CONCLUSION Although osteosarcoma is usually associated with an increase of osteoblastic activity in affected bones, frequently leading to the formation of non-remodeled osteoid/woven bone tissue [21], in the last few years the involvement of osteoclastic cells in this pathologic

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condition has gained relevance, suggesting a close relationship between osteoclastic activity and osteosarcoma aggressiveness [26, 30, 82, 83]. The present chapter described the cellular and molecular effects of a widely used human osteosarcoma cell line – MG63 cell line – on the osteoclast differentiation and function, either by paracrine-mediated and cell-to-cell contact mechanisms. It was observed that CM from MG63 cells elicited a high osteoclastogenic response in all tested conditions, which was similar or higher than that observed with CM from hBMC cultures. This behavior was observed not only on osteoclastic cell differentiation but also in the resorbing ability. Regarding the intracellular signaling pathways, MEK and NFkB, although important, presented a lower relevance in the osteoclastogenesis of PBMC observed in the presence of CM from MG63 cell cultures, comparing to that found in the cultures treated with CM from hBMC. PGE2 production seemed to be not involved in the observed osteoclastogenic response in all tested conditions. Regarding PBMC and MG63 co-cultures, it was shown that a very low seeding density of MG63 cell line elicits a high osteoclastogenic response, Moreover, MEK pathway seemed to have a lower contribution to the osteoclastogenic behavior of PBMC + MG63 co-cultures, compared to those performed with hBMC. NFkB appeared to be crucial for the process, while PGE2 production was also involved in observed response. Taken together, it was observed that MG63 cells promote a high stimulation of osteoclastogenesis, either by paracrine- and physical contact-mechanisms, suggesting an elevated osteoclastogenic-triggering ability for this cell line. In fact, its osteoclastogenicpotential in both conditions appeared to be significantly higher than that observed with hBMC cells (maintained in the absence or presence of dexamethasone, an osteogenic inducer), the main in vivo regulator of osteoclast development [2, 8, 18]. Furthermore, the relative contribution of MEK and NFkB pathways, as well as PGE2 synthesis, in theosteoclastogenic process was different, not only between MG63 and hBMC cells, but also in the paracrine- and physical contact-stimulated PBMC cultures. In fact, as the only osteoclastogenic stimuli on the cell cultures was derived from the MG63 cell line, the results reveal that these cells have the ability to modulate osteoclast development either by paracrine and direct contact mechanisms. In addition to the two classic osteoclastogenic enhancers M-CSF and RANKL [4, 6, 52], there are numerous osteoclastogenic molecules that are known to be produced by tumor cells, such as, for example, parathyroid hormone-related peptide, IL-1, IL-3, IL-4, IL-6, IL-11, TNF- and VEGF [20, 84-88]. Since MG63 cells express very low RANKL amounts, as observed in Figure 1 and in previous reports [34-37], it seems obvious that a complex network of crosstalks, mediated by different growth factors, cytokines and other modulators, can be established between osteoclasts and MG63 cells, which account for the observed osteoclastogenic modulation by this osteosarcoma cell line.In addition, it was observed that there are also reciprocal crosstalks between osteoclastic and osteosarcoma cells, that ultimately lead to a modulation of the osteoblastic behavior of MG63 cells. Understanding the molecular details of the reciprocal crosstalks established between osteosarcoma and osteoclastic cells, and particularly the osteoclastichyperactivation induced by osteosarcoma cells, will contribute to the development of new therapeutic approaches for thistype of bone metabolic disorders.

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[64] Roodman GD. Cell biology of the osteoclast. Exp. Hematol. 1999;27: 1229-41. [65] Strait K, Li Y, Dillehay DL, Weitzmann MN. Suppression of NF-kappaB activation blocks osteoclastic bone resorption during estrogen deficiency. Int. J. Mol. Med. 2008;21: 521-5. [66] Hall TJ, Schaeublin M, Jeker H, Fuller K, Chambers TJ. The role of reactive oxygen intermediates in osteoclastic bone resorption. Biochem. Biophys. Res. Commun. 1995;207: 280-7. [67] Kawashima M, Fujikawa Y, Itonaga I, Takita C, Tsumura H. The effect of selective cyclooxygenase-2 inhibitor on human osteoclast precursors to influence osteoclastogenesis in vitro. Mod. Rheumatol. 2009;19: 192-8. [68] Kellinsalmi M, Parikka V, Risteli J, Hentunen T, Leskela HV, Lehtonen S, Selander K, Vaananen K, Lehenkari P. Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro. Eur. J. Pharmacol. 2007;572: 102-10. [69] Okada Y, Pilbeam C, Raisz L, Tanaka Y. Role of cyclooxygenase-2 in bone resorption. J. UOEH 2003;25: 185-95. [70] Blackwell KA, Raisz LG, Pilbeam CC. Prostaglandins in bone: bad cop, good cop? Trends Endocrinol. Metab. 2010;21: 294-301. [71] Harbour ME, Gregory JW, Jenkins HR, Evans BA. Proliferative response of different human osteoblast-like cell models to proinflammatory cytokines. Pediatr. Res. 2000;48: 163-8. [72] Michael H, Harkonen PL, Kangas L, Vaananen HK, Hentunen TA. Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro. Br. J. Pharmacol. 2007;151: 384-95. [73] Quinn JM, Fujikawa Y, McGee JO, Athanasou NA. Rodent osteoblast-like cells support osteoclastic differentiation of human cord blood monocytes in the presence of M-CSF and 1,25 dihydroxyvitamin D3. Int J. Biochem. Cell Biol. 1997;29: 173-9. [74] Buckley KA, Hipskind RA, Gartland A, Bowler WB, Gallagher JA. Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblastexpressed receptor activator of nuclear factor-kappa B ligand. Bone 2002;31: 582-90. [75] Matsuzaki K, Katayama K, Takahashi Y, Nakamura I, Udagawa N, Tsurukai T, Nishinakamura R, Toyama Y, Yabe Y, Hori M, Takahashi N, Suda T. Human osteoclast-like cells are formed from peripheral blood mononuclear cells in a coculture with SaOS-2 cells transfected with the parathyroid hormone (PTH)/PTH-related protein receptor gene. Endocrinology 1999;140: 925-32. [76] Laulederkind SJ, Kirtikara K, Raghow R, Ballou LR. The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent. Exp. Cell Res. 2000;258: 409-16. [77] Le Heron L, Guillaume C, Velard F, Braux J, Touqui L, Moriceau S, Sermet-Gaudelus I, Laurent-Maquin D, Jacquot J. Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone. J. Cyst. Fibros. 2010;9: 69-72. [78] Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, Toyama Y, Yabe Y, Kumegawa M, Hakeda Y. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone 2000;27: 479-86.

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[79] Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine1-phosphate. Proc. Natl. Acad. Sci. U S A 2008;105: 20764-9. [80] Garimella R, Tague SE, Zhang J, Belibi F, Nahar N, Sun BH, Insogna K, Wang J, Anderson HC. Expression and synthesis of bone morphogenetic proteins by osteoclasts: a possible path to anabolic bone remodeling. J. Histochem. Cytochem. 2008;56: 569-77. [81] Galvin RJ, Cullison JW, Avioli LV, Osdoby PA. Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis. J. Bone Miner. Res. 1994;9: 1167-78. [82] Akiyama T, Dass CR, Shinoda Y, Kawano H, Tanaka S, Choong PF. Systemic RANKFc protein therapy is efficacious against primary osteosarcoma growth in a murine model via activity against osteoclasts. J Pharm Pharmacol 2010;62: 470-6. [83] Akiyama T, Choong PF, Dass CR. RANK-Fc inhibits malignancy via inhibiting ERK activation and evoking caspase-3-mediated anoikis in human osteosarcoma cells. Clin. Exp. Metastasis 2010;27: 207-15. [84] Cenni E, Granchi D, Ciapetti G, Stea S, Savarino L, Corradini A. Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements. J. Biomater. Sci. Polym. Ed. 2001;12: 243-53. [85] Kakonen SM, Mundy GR. Mechanisms of osteolytic bone metastases in breast carcinoma. Cancer 2003;97: 834-9. [86] Morrissey C, Vessella RL. The role of tumor microenvironment in prostate cancer bone metastasis. J. Cell Biochem. 2007;101: 873-86. [87] Takenaka K, Yamagishi S, Jinnouchi Y, Nakamura K, Matsui T, Imaizumi T. Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells. Life Sci. 2005;77: 3231-41. [88] Benedikt MB, Mahlum EW, Shogren KL, Subramaniam M, Spelsberg TC, Yaszemski MJ, Maran A. 2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells. J. Cell Biochem. 2010;109: 950-6.

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Chapter 4

OSTEOCLAST BIOLOGY IN PAGET’S DISEASE OF BONE Stephen McManus, Lyne Bissonnette, and Sophie Roux Division of Rheumatology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada

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ABSTRACT Paget’s disease of bone (PDB) is characterized by focal and disorganized increases in bone turnover. Because the initial phase of PDB involves excessive bone resorption, osteoclasts have been identified as the cells primarily affected in PDB. Pagetic osteoclasts are both larger and more numerous than normal osteoclasts. They are overactive and hypersensitive to osteoclastogenic factors, and resistant to apoptosis. Although a viral etiology has been suggested for Paget’s disease, several studies have revealed a marked genetic component. The discovery of mutations of the SQSTM1 (Sequestosome1, p62) gene in numerous patients has identified the protein p62 as an important modulator of bone turnover. p62 mediates several diverse cell functions, including the control of NF-B signaling, protein trafficking and autophagy. Since SQSTM1 mutations do not fully explain the osteoclast phenotype of PDB, the contribution of other osteoclast-related genes, viruses or environmental factors may be involved. Here, we review the most recent advances in osteoclast biology regarding PDB with a particular attention to the impact of the p62 mutations.

PDB is the second most common human skeletal disorder, after osteoporosis, affecting up to 3% of adults over 55 years of age [1]. PDB is characterized by focal and disorganized increases in bone turnover, and osteoclasts have been identified as the cells primarily affected in PDB [2]. As the osteoclasts are abnormally overactive, this disease provides an excellent model for elucidating osteoclast behavior. Both the genetic and viral etiologies have been thoroughly studied in the last few years. The discovery of mutations of the SQSTM1 

Corresponding author: Dr Sophie Roux M.D. Ph.D., 3001, 12th avenue N, Sherbrooke, PQ, Canada, J1H5N4. Phone: 819-564-5261, Fax: 819-564-5265. E-mail: [email protected].

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(Sequestosome1, p62) gene in numerous patients has identified the protein p62 as an important modulator of bone turnover, providing new insights in osteoclast biology and highlighting the major role of p62 in these cells. New loci have recently been associated to PDB implying that genes other than SQSTM1 may contribute to the pathogenesis of PDB. Here is a review of recent advances in our understanding of the regulators of osteoclast phenotype, signaling and functions in pagetic osteoclast.

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OSTEOCLAST PHENOTYPE IN PDB Osteoclasts are multinucleated cells formed by the fusion of mononuclear cells of hematopoietic origin, a process dependent on the dendritic cell-specific transmembrane protein (DC-STAMP) [3, 4]. The osteoclast phenotype differs from that of macrophages and macrophage polykaryons in several important respects; in particular, osteoclasts and their precursors express the calcitonin receptor (CTR) and the Receptor Activator of NF-B (RANK), and present the unique capacity to resorb bone [5, 6]. After exercising their resorption activity, osteoclasts undergo apoptosis, and they have been shown to be sensitive to apoptosis induction by a number of cytokines and factors, including Fas-ligand, TRAIL, and TGFβ [7-9]. Osteoclast differentiation and activation are supported by osteoblasts and stromal cells, and regulated by two signaling pathways, which are activated by M-CSF and RANKL (RANK Ligand) respectively, and a recently described ITAM (immunoreceptor tyrosine-based activation motif)-mediated co-stimulatory signaling [10]. The interactions between RANKL, expressed on osteoblasts, and its receptor RANK, found on mature osteoclasts as well as their progenitor cells, trigger a series of signaling events promoting and regulating differentiation, activity, and survival of the osteoclast affected. Upon stimulation, a trimeric receptor complex recruits and associates with TRAF6, resulting in signaling cascades that ultimately activate transcription factors, particularly NF-B and NFATc1 [10] (Figure 1). In PDB, osteoclastic bone resorption is abnormally increased in localized areas of the skeleton, and remains coupled to bone formation. This characteristic rapid and chaotic bone turnover results in an increase in bone volume that is composed of abnormal matrix with irregular and patchy arrangement of collagen fibers. Pagetic osteoclasts are both larger and more numerous, possess a higher number of nuclei per osteoclast, and cause deeper resorption lacunae [11]. They are resistant to apoptosis, and are overactive [12]. They also are hypersensitive to osteoclastogenic factors, such as RANKL, 1,25-(OH)2D3 [13, 14], and express higher levels of TAFII-17, a VDR binding protein [15]. Gene expression profile studies showed different expression pattern between osteoclast cultures from PDB patients compared to healthy controls. Pagetic osteoclasts displayed downregulation in genes involved in apoptosis (CASP3 and TNFRSF10A), in cell signaling (TNFRSF11A), in the osteoclast bone resorbing function (ACP5 and CTSK) and in the gene coding for Tau protein (MAPT) [16]. Another study found an upregulation of the expression of anti-apoptotic Bcl-2 gene in pagetic osteoclasts [17]. The molecular basis of PDB and the characteristic features of the pagetic osteoclasts are not fully understood, however major advances have been made over the past ten years, particularly in genetics, and have provided important new insights into the pathogenesis of this disease.

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Figure 1. Osteoclast signaling pathways. In both pre-osteoclasts and mature osteoclasts, the interaction between RANKL and RANK results in transduction signals via a signaling complex containing TNFRassociated factors, mainly TRAF6. Several signaling cascades are activated, leading to the expression of specific genes involved in osteoclast differentiation, activation and survival, and include the NF-B, both classical and alternative [106], as well as MAP kinases (JNK, p38, ERK) and Src pathways 5. NFATc1, a transcription factor, is also strongly induced and activated after RANKL stimulation in osteoclasts via c-Fos, NF-B and calcium signaling [107]. RANKL may activate the calcium signals that lead to the induction of NFATc1 through a co-stimulatory pathway that involves Ig-like receptors, such as OSCAR or TREM2, and adaptor molecules, such as FcR and DAP12, that contain an ITA motif, which is critical for activating calcium signaling. The phosphorylation of this motif is stimulated by the RANKL–RANK interaction, and by Ig-like receptors, and results in the activation of PLC and calcium signaling [10]. Non-canonical Wnt proteins (Wnt5a, Wnt11) bind another receptor complex including a frizzled receptor and receptor tyrosine kinase-like orphan receptors (Ror 1/2). This pathway activates the planar cell polarity via JNK signals, as well as Ca2+ signaling via PKC- and calcineurindependent signals [108, 109]. The non-canonical -catenin independent Wnt pathway directly affects osteoclast precursors and stimulates their differentiation.

VIRAL AND GENETIC FACTORS IN PAGET’S DISEASE OF BONE The involvement of a virus infection has been proposed in the pathogeny of PDB since the discovery of inclusion bodies in the nuclei and cytoplasm of osteoclasts which resemble the nucleocapsids of paramyxovirus [18, 19]. This hypothesis has since been supported by studies focusing mainly on Measles virus that have detected mRNA or protein expression of paramyxovirus nucleocapsids in osteoclasts, bone marrow cells or peripheral blood cells from PDB patients. The role of a slow infection in the pathogeny of PDB is still a matter of debate, and other studies failed to detect such an expression [20]. Studies have highlighted the role of paramyxovirus infection in some of the characteristic of pagetic osteoclasts. Transfection of Measles virus nucleocapsid (MVNP) gene in human osteoclast precursors resulted in an increase in osteoclast formation and activity, and increase the sensitivity to 1,25(OH)2D3 [15,

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21]. Transgenic mice expressing MVNP targeted in the osteoclast lineage also had an increase in osteoclast formation from bone marrow cultures with increases in osteoclast formation, activity, as well as increased nuclei number per osteoclast, and in vivo develop lesions similar to those observed in PDB [22]. Although a viral etiology has been suggested for Paget’s disease, numerous studies have also revealed a marked genetic component [23]. Some years ago, a germline mutation in the SQSTM1 gene on 5q35 was identified in a high proportion of PDB patients [24-27]. This gene encodes the ubiquitin-binding protein sequestosome 1, also known as p62. Since this initial discovery, 27 other mutations have also been reported, although the P392L substitution is the most frequent [1, 23]. Mutations of this gene have been detected in about 30% of familial Paget's cases, with a high penetrance of about 80% in patients over 60 years of age, and in about 9% of sporadic cases [1, 23, 27]. A role for somatic acquired SQSTM1 mutations in affected tissues has also been suggested in PDB, but is still controversial [28, 29]. Other strong loci of susceptibility for PDB have also been investigated (5q31, 10p13), although no gene has been identified so far in these loci. In a candidate gene approach, the valosin containing protein (VCP) gene was studied in PDB. The VCP gene is involved in a rare disease in which PDB can occur as part of a disease associated with inclusion body myopathy and frontotemporal dementia. Genetic variants of this gene were not associated with PDB in a British population [30], but such an association was found in a Belgian population [31]. Two recently published genome-wide association studies (GWAS) in PDB patients identified seven significant genetic variants for susceptibility to PDB located at the 1p13 (CSF1), 7q33 (CNOT4, NUP205, SLC13A4), 8q22 (TM7SF4), 10p13 (OPTN), 14q32 (RIN3), 15q24 (PML, GOLGA6A), and 18q21 (TNFRSF11A) loci [32, 33]. The genetic risk for PDB close to the CSF1, OPTN, TM7SF4, and TNFRSF11A genes was confirmed in other populations [34]. The data obtained from GWAS and the replications studies strongly suggest that these 7 genetic variants may predispose to PDB. Interestingly, some candidate genes have been involved in osteoclast formation, activity and survival (CSF1 encoding MCSF; TNFRSF11A encoding RANK), and in osteoclast fusion (TM7SF4 encoding DC-STAMP). This implies that genes other than SQSTM1 may also contribute to the pathogenesis of PDB, although so far p62 is the only gene in which mutations have been identified.

ROLE OF P62 IN OSTEOCLAST SIGNALING The discovery of mutations of the SQSTM1 (Sequestosome1, p62) gene in PDB has led to the identification of protein p62 as an important modulator of bone turnover. Genetic inactivation of p62 in mice leads to impaired RANKL-induced osteoclastogenesis, due to defective activation of NF-B [35], identifying p62 as a key player in osteoclast biology and in RANKL signaling pathway. p62, an adaptor protein with multiple binding domains - p62 or sequestosome 1 (SQSTM1) is a highly conserved protein with multiple and varied functional domains. Involved in cell signaling, receptor internalization, and protein turnover, the protein contains a ubiquitin (Ub)-associated (UBA) domain at its C-terminus, two PEST sequences, between which an LC3-interaction region (LIR) stands, a binding site for the RING finger protein TRAF6, a domain binding p38 as well as LIM-containing proteins, a ZZ finger interacting

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with RIP, a PB1 domain for binding atypical PKCs (aPKCs), but also ERK, NBR1, MAPKK5 (MEK5) and MEKK3, and p62 itself as well, and finally an N-terminus capable of direct interaction with the proteasome subunit component [23, 36, 37]. With such numerous protein-protein interaction motifs, p62 is considered a scaffold, playing an important role in the osteoclast, and serving as the switchboard from which the RANKL activation signal is propagated (Figure 2). TRAF6, p62 and RANKL-induced osteoclast signaling - The p62 scaffolding protein is one of the functional links reported between RANKL and TRAF6-mediated NF-B activation [38]. The most clearly established function of p62 is its role as a scaffold protein for the RANKL-induced activation of NF-B, requiring interactions between TRAF6, p62, the PB1interacting atypical Protein Kinase C (PKC and phosphoinositide-dependent kinase 1 (PDK1) which result in the formation of a multimeric protein complex [12, 35, 39]. The sequence of RANKL-induced activation requires the recruitment of TRAF6, which is responsible for most of the downstream events that lead to osteoclast differentiation and activation [40], and is necessary for RANKL-induced NF-B activation [41]. RANKLinduced TRAF6 recruitment also leads to the activation of the MAP kinases p38, ERK (extracellular-signal regulated kinase), and JNK (c-jun terminal kinase), as well as activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

Figure 2. p62/SQSTM1 linear representation. Multiple interaction motifs located within p62 enable recruitment of specific proteins and regulation of downstream signaling pathways. Thus, the aPKCs and ERK interact with the PB1 domain whereas RIP1 binds to the ZZ domain, and TRAF6 interacts with the TF6-b sequence. The UBA domain binds to polyubiquitin chains. PB1: Phox and Bem1p ; ZZ ZNF: ZZ-type zinc finger; TF6-b: TRAF6 binding sequence; PEST: (P, Proline; E, Glutamate; S, Serine; T, Threonine) rich sequence; LIR: LC3 interacting region; UBA, ubiquitin associated.

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Figure 3. p62 in osteoclast signaling. In both precursors and mature OCLs, the interaction between RANKL and RANK results in signaling cascades that require the recruitment of TNFR-associated factor 6 (TRAF6), and ultimately activate transcription factors, particularly NF-B and NFATc1. The cytosolic p62 protein, encoded by the SQSTM1 gene, is a scaffolding protein that interacts with the RANK signaling complex, and is one of the functional links reported between RANKL and TRAF6mediated NF-B activation. p62 may be involved in the formation of mutimeric protein complexes, because of the interactions between its PB1 domain with other proteins. Due to its ability to bind polyubiquitinated substrates at the C-terminal UBA domain, it may also play a role in protein degradation through its association with the proteasome at its N-terminal ubiquitin-like (UBL) domain, and in autophagy as an LC3-interacting protein.

Another function of TRAF6 in RANK signal propagation is the formation of protein complexes with TGFβ-activated kinase 1 (TAK1) and adaptor proteins TAB1 and TAB2 [42]. When TAK1 is activated, it in turn phosphorylates NF-κB-inducing kinase (NIK), which activates the IκB kinase (IKK) complex, leading to NF-κB pathway activation. As for the MAP kinases, TAK1 will also activate the JNK pathway, while TAB1 recruits and binds p38 to the TRAF6 complex, leading to activation of its pathway [43, 44]. RANKL also activates the Akt/PKB pathway with the aid of TRAF6 and associated signaling proteins [45]. The key to these pathways is the scaffolding protein p62, which is recruited and bound to the TRAF6/RANK complex, and acting as a scaffold regulates these signaling pathways (Figure 3). Once bound to TRAF6, p62 permits the recruitment of atypical protein kinase C (aPKC) proteins [46]. After stimulation by interleukin-1 (IL-1) or nerve growth factor (NGF), these kinases regulate NF-κB activation via the phosphorylation of IκB kinase β (IKKβ) [39, 47].

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The p62 complex also binds the scaffolding receptor interacting protein (RIP) and kinase PDK1, recruiting aPKCs to TNF-α signaling complexes [48, 49]. p62, TRAF6 and CYLD- p62 interactions with TRAF6 stimulate TRAF6 K63-linked autoubiquitination and E3 ligase activity, as well as regulate the synthesis of K63 chains on target substrates. This ubiquitination of TRAF6 is an important mechanism mediating its signaling functions [50, 51]. Activated TRAF6 may then stimulate NF-B activity by activation of the IKK complex, either through aPKC or TAK1-dependent phosphorylation, and requiring NEMO (IKK) ubiquitination for optimal activation [52]. In addition to its well established role in the activation of TRAF6, the role of p62 in regulating a deubiquitinating enzyme (DUB) is a current subject of research. p62 appears to be a molecular adaptor associating deubiquitinase CYLD and TRAF6 [53]. In addition to interacting with TRAF6, CYLD has specificity for Lys63 (K63) chains, and reverses the processing of protein ubiquitination. The decrease in the activity of CYLD leads to the accumulation of Lys63 (K63)-ubiquitinated substrates [53]. By interacting with CYLD at its C-terminal domain, p62 promotes the binding of CYLD to TRAF6 [54]. CYLD thus negatively regulates NF-κB activity by reducing the autoubiquitination of TRAF6 [55, 56]. It therefore negatively regulates the activation of the IKK and JNK pathways, and is markedly upregulated under conditions of RANKL-induced osteoclastogenesis, acting as a natural feedback inhibition regulator [54]. CYLD is a crucial down-regulator of RANK signaling in osteoclasts, and accordingly, it has been shown that CYLD-deficient mice are severely osteoporotic, originating from aberrant osteoclast differentiation, having larger and more numerous osteoclasts which are hypersensitive to RANKL [54].

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P62 AS A SHUTTLING FACTOR UBA and PB1 domain function - It has been suggested that p62 is a shuttling factor in the delivery of poly-ubiquinated substrates to the proteasome for degradation [57]. p62 has been proven to sequester polyubiquitin into aggresomes or sequestosomes, common pathological features of many diseases [58, 59]. However, outside of a few examples, evidence suggests that p62 functions primarily as a scaffolding protein for ubiquitinated proteins in processes not directly related to proteasomal degradation, like NF-κB signaling and macro-autophagy [35, 60]. p62 may be involved in the formation of multimeric protein complexes, due to interactions between its PB1 (Phox and Bem1p) domain with other proteins [61]. Given the ability of p62 to bind polyubiquitinated substrates the C-terminal UBA domain, and its association with the proteasome at its N-terminal ubiquitin-like domain, it likely plays a role in protein turnover as well [62]. The aforementioned N-terminal PB1 permits p62 to act as a shuttling factor, directing for turnover polyubiquitinated proteins that interact with the UBA domain [57]. Proteins containing the PB1 domain include PDK1, Par6, the aPKCs, and many more, and can form specific heterodimers between family members [63]. Therefore, in addition to potentially playing a role in protein turnover via proteasome association, p62 may also participate in the formation of multimeric protein complexes through interactions between PB1 domains. This domain allows flexibility, either favoring this oligomeric function or the formation of self-associating p62 molecules into sequestosomes [62].

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Relatively unusual among ubiquitinating proteins of its type, p62’s UBA is rendered biologically inactive by stabilization of the classical canonical UBA conformation via dimer formation [64, 65]. And by making the active and less stable monomer unavailable, p62 further regulates its own function [66]. Once free in monomeric form, p62 undergoes a conformational change favoring K63-linked polyubiquitination, granting the protein additional functions as a multifunctional scaffold protein, affecting processes of NF-κB, autophagy, and more [62, 66]. Autophagy and LC3 interaction – Whereas proteosomal degradation targets soluble proteins, autophagy (macro-autophagy) is a regulated process that can also eliminate large structures such as aggregates, misfolded macromolecules, organelles or pathogens through the formation of autophagosomes. These structures are large vesicles that wrap up damaged material and fuse with lysosomes to deliver their content for subsequent degradation. Autophagy takes place in all cells, in order to maintain cell homeostasis or in response to stress or starvation, by eliminating damaged components, and providing cells with energy and nutrient resourcing [67, 68]. p62 has been identified as an LC3-interacting protein implicated in autophagy [60, 69, 70]. Studies have shown that p62, along with ubiquitinated proteins, is transported into autophagosomes, suggesting that p62 is a receptor for these proteins that directs them to lysosomes [60, 70]. This is made possible by its LRS (LC3 recognition sequence) located between the zinc finger and UBA domains of p62, where residues interact with the Nterminus and ubiquitin domain of LC3 [71, 72]. LC3 covalently conjugates with phosphatidylethanolamine (PE) through an enzymatic cascade, and is found on the inner and outer membranes of the isolation membrane of the autophagosomes, and is vital to membrane biogenesis as well as closure of the isolation membrane [73-76]. This interaction leads to relocation of p62 to the autophagosome, submitting it to degradation by the autophagylysosome system. However, when autophagy is impaired, p62 is allowed to accumulate, which can result in failure to terminate some of its signaling processes, as well as the formation of p62-positive inclusions [59, 69]. Findings support the hypothesis that ubiquitinated proteins interact with p62, followed by aggregation of the protein complex in a p62 dependent manner, and terminating with degradation of these aggregates by autophagy [77, 78]. The exact mechanisms and impact of this pathway are still being developed, as some details are still unknown; like the quantities of cellular ubiquitinated proteins being transported into autophagosomes by p62, or the signals that are responsible for modulating selective degradation of ubiquitinated protein aggregates by autophagy. Interestingly, knockout of p62 does not appear to markedly affect levels of ubiquitinated proteins in the cell, possibly due to compensatory action by Nbr1 (neighbor of BRCA1 gene 1), which interacts with LC3 in a similar manner [69, 79]. Loss of function of Nbr1 leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis [80]. As previously mentioned, the interaction of p62 with TRAF6 promotes the oligomerization and subsequent K63 polyubiquitination and activation of TRAF6, resulting in NF-κB activation [81]. In the case of cell death, cell-surface death receptors like those of TRAIL trigger apoptosis through signalization after initiator caspase-8 activation by polyubiquitination, via interaction of the death-inducing signaling complex (DISC) with cullin3-based ubiquitin ligase (CUL3) [82]. p62 can promote aggregation of the CUL3modified caspase-8, leading to its full activation and processing, favoring commitment to cell

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death [82]. It has thus been suggested that p62-positive aggregates are signaling hubs that can determine whether cells die by caspase activation or survive through TRAF6-NF-κB pathways [83]. p62 also interacts with Keap1, a ubiquitin ligase for Nrf2 [84]. Nrf2 regulates gene expression of a variety of antioxidant proteins and detoxification enzymes [85]. So, a surplus of p62, either by faulty autophagic processes or overproduction, can lead to competitive binding of Keap1, stabilizing Nrf2 and the activation of its target genes. Therefore, because the level of p62 protein is controlled by autophagy, p62-associated autophagy can regulate not just the NF-κB and other TRAF6-associated activation pathways, but apoptosis and environmental stress response as well [84]. Autophagy has been involved in hypoxia-induced osteoclast differentiation [86], in MCP1 (Monocyte chemotactic protein-1)-induced osteoclast differentiation through MCPIPmediated induction of oxidative stress [87], and p62 may play a role in the starvation-induced autophagy in human osteoclasts [88]. Autophagy proteins have been shown to participate in the polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the osteoclast ruffled membrane [89].

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ROLE OF P62 MUTATIONS IN PAGET’S DISEASE OF BONE Impact of p62P392L in osteoclast phenotype - The significance of the most common p62 mutation, P392L, has been studied in a series of in-vitro experiments, using osteoclast precursors derived from the peripheral blood of PDB patients carrying the p62P392L gene or not, and from bone marrow cells or cord blood monocytes of normal subjects transfected with the p62wt or p62P392L gene. These studies strongly suggest that the p62P392L mutation affects the osteoclast phenotype by inducing overactive osteoclasts that are resistant to apoptosis, have an increased ability to resorb bone, and display some of the characteristics of the Paget osteoclast phenotype including an hyper-responsiveness to osteoclastogenetic factors, such as RANKL and TNF [12, 90, 91]. Impact of p62P392L on osteoclast signaling - Previous findings revealed that in human osteoclasts, RANKL stimulation induced the formation of a multiprotein complex containing not only PKC well [12]. Interestingly, even prior to RANKL stimulation p62 was associated with both activated PDK1 and PKC in PDB osteoclasts, in osteoclasts from healthy donors harboring the p62P392L gene, and in p62P392L transfected osteoclasts [12]. Moreover, the signaling complex formed in response to RANKL stimulation normally results in NF-B activation, which was observed in the non-transfected osteoclasts of CBM cultures. Although both wild-type and mutated p62 over-expression favored the formation of activated protein kinases/p62 complexes, only that of the mutated gene led to an increased basal level of NF-B activation [12]. These findings strongly suggest that p62P392L contributes at least in part to the induction of an activated stage in osteoclasts by stimulating signaling pathways involving PDK1-PKC that could lead to NF-B activation. Similarly, overexpression of the p62P392L, p62K378X or p62E396X mutants in HEK293 or Cos-1 cells increased basal NF-B activation, and overexpression of the p62K378X or p62E396X mutants also increased RANKL-induced NF-B activation more than overexpression of the p62 wild type mutant [91].

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Mutant p62 transfects have been shown to upregulate NFATc1 expression in preosteoclasts, favoring increased osteoclastogenesis and osteoclast activity [92]. Naturally, TRAF6 activity is affected by p62 interaction, affording p62 some measure of indirect control over NFATc1 signaling. However, kinases may also contribute to the nuclear shuttling of this factor, as PKCζ has been shown to interact with NFATc1, and may modulate NFAT-mediated transcription by increasing the activity of its N-terminal transactivation domain [93]. Therefore RANKL stimulation may further contribute to osteoclast activation via formation of the p62-aPKC complex, leading to increased NFATc1 activity. As noted earlier, CYLD is a crucial down-regulator of RANK signaling in osteoclasts. Recent studies have further confirmed that CYLD knockdown significantly increased c-Fos expression in cells transduced to express both wild-type and mutant p62P392L, without necessitating RANKL stimulation [92]. Likewise, mutations to the UBA domain of p62 lead to a reduction in CYLD activity (and thus an increase in osteoclast development and resorption) 92, given that binding between the two takes place at the C-terminal end of the p62 protein. Although the presence of the P392L substitution cannot account for all aspects of the PDB osteoclast phenotype, it clearly affects osteoclast behavior, although the mechanisms involved in such p62-driven misregulation are not fully understood. All these studies demonstrate that the p62P392L mutation affects the osteoclast phenotype, inducing overactive osteoclasts that display several characteristics of the Paget osteoclast phenotype. Experimental models of PDB - In vivo, transgenic mice with targeted expression of the human p62P392L gene in the osteoclast lineage developed osteopenia, with an elevated osteoclast perimeter, although no PDB-related bone lesions were observed. Once again, osteoclast precursors were hyperresponsive to RANKL and TNF, and they demonstrated increased proliferation rates, but no change in either nucleus numbers or apoptosis rates [90]. As p62 is ubiquitously expressed, p62 mutations may also affect cells other than osteoclasts, particularly stromal cells or osteoblasts. This hypothesis has been investigated in a study where p62P394L (the murine equivalent of human p62P392L) knock-in mice were generated. In bone marrow cultures, the expression of p62P394L in stromal cells was associated with increased RANKL expression in response to 1,25(OH)2D3. However, the p62P394L knock-in mice had histologically normal bones up to 18 months of age [94]. This implies that the presence of the p62 mutation in bone cells other than osteoclasts may increase RANKL production in the bone microenvironment, thus indirectly enhancing osteoclast formation and activity, and that additional factors are necessary for the development of PDB in vivo. Transgenic mice coexpressing the measles virus nucleocapsid (MVNP) under the TRAP promoter and knocked-in p62P394L develop PDB-like bone lesions, present in 40% of the mice over 18 months [95]. Finally, in another study, transgenic mice expressing the p62P394L gene develop focal osteolytic lesions, which were present in 95% of homozygous mice by 12 months of age, with some of them resembling PDB lesions [96]. Protein turnover and autophagy in PDB osteoclast and impact of the p62P392L mutation In PDB, virtually all p62-related mutations are clustered in or around the UBA-domain, indicating that ubiquitin-binding alteration may play a role in p62-related bone diseases [97, 98], and the failure of specific polyubiquitinated substrates to interact with the p62 UBA domain may contribute to the pathophysiology of PDB. One attractive hypothesis that could explain aberrant cellular functions is the accumulation of p62-associated proteins, due to

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impaired degradation through proteosomal or autophagic pathways. For instance, p62 is commonly found in protein aggregates described in degenerative diseases [59], and may be linked to increased cell survival [60]. However, ubiquitin-binding activity is not related to PDB occurrence or severity [99], and does not correlate with the UBA-mediated inhibition of proteasomal degradation [100]. Notably, even when their ubiquitin-binding activity has been severely impaired, p62 UBA domains have been shown to still be capable of inhibiting proteasomal degradation. While the mechanism by which this is accomplished is not yet definite, the results suggest that the protective effect of mutant UBA domains on p62 proteasomal degradation may primarily depend on their conformational integrity rather than their ubiquitin-binding capacity [100]. In addition, while some p62 mutants exhibit increased basal NF-κB activity relative to wild-type p62 [91, 98], it has also been observed that mutants in which p62-UBA dimerisation was impaired exhibited reduced NF-κB signaling activity [66]. As p62 is a key player in selective autophagy, and may have a role in the formation of inclusion bodies [69, 101]; because the inclusion bodies found in pagetic osteoclasts resemble those observed in diseases with defective autophagy, a dysregulation of the autophagy process may well be part of the pathogeny in PDB, although so far no direct evidence has been provided for its role in the phenotype of pagetic osteoclast [102]. In addition, the p62P392L mutation did not affect p62-related aggregate formation in human osteoclasts [88]. Interestingly, the p62D335E mutation is located in the LC3-interacting domain (position 321342), although its impact on autophagy has not yet been studied [70, 103]. Apoptosis in PDB osteoclast and impact of the p62P392L mutation - In osteoclast cultures derived from PDB patients, lower rates of apoptosis were induced by the deprivation of survival factors or by death inducers, such as TRAIL, Fas activating antibody or TGF- [12]. An interesting observation is that osteoclasts from healthy carriers of the p62P392L gene had an intermediate phenotype, with greater resistance to apoptosis than osteoclasts from normal controls, but less than that of osteoclasts from PDB patients. In cord blood monocytes transfected with p62wt or p62P392L genes, apoptosis rates were lower in osteoclasts overexpressing either p62wt or p62P392L than in empty-vector transfected osteoclasts [12]. An increase in p62 expression has been observed in PDB osteoclasts [12, 104], and this could contribute to their resistance to apoptosis as, according to studies in other systems, increased expression of p62 could in itself have a protective effect against cell death by preventing the build-up of potentially cell-damaging proteins [60, 105]. In addition, NF-B is known to be involved in apoptosis regulation, and a basal activation of NF-B was observed in osteoclasts transfected with the p62P392L gene, but not those transfected with p62wt. Thus basal and RANKL-induced NF-B activation may influence osteoclast survival in PDB. Finally, gene expression studies have suggested that genes involved in osteoclast apoptosis were significantly downregulated in pagetic osteoclasts (genes encoding caspase-3, TRAIL-R1), with a trend towards a decrease for others (TRAIL-R2, TGF-R1) [16], while the antiapoptotic Bcl-2 gene’s expression was upregulated [17]. So, it seems clear that apoptosis is hindered in PDB osteoclasts, and that the presence of PDB mutations in the p62 gene is involved, at least in part.

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CONCLUSION In PDB, p62/SQSTM1 mutations contribute at least in part to inducing an activated phase in osteoclast by stimulating signaling pathways that can lead to NF-B activation, and to apoptosis resistance. In addition to its role as a scaffold protein in the control of RANKLinduced NF-B signaling, protein p62 mediates several other cell functions including protein trafficking and autophagy, a new research area in osteoclast physiology. However, SQSTM1 mutations do not fully account for the osteoclast phenotype of PDB, and other osteoclastrelated genes may also contribute to the osteoclast phenotype. It is also likely that in addition to genetic factors, environmental factors contribute to the development of the disease.

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[94] Hiruma Y, Kurihara N, Subler MA, Zhou H, Boykin CS, Zhang H, et al. A SQSTM1/p62 mutation linked to Paget's disease increases the osteoclastogenic potential of the bone microenvironment. Hum. Mol. Genet. 2008; 17(23): 3708-19. [95] Kurihara N, Hiruma Y, Yamana K, Michou L, Rousseau C, Morissette J, et al. Contributions of the measles virus nucleocapsid gene and the SQSTM1/p62(P392L) mutation to Paget's disease. Cell Metab. 2011; 13(1): 23-34. [96] Daroszewska A, van 't Hof RJ, Rojas JA, Layfield R, Landao-Basonga E, Rose L, et al. A point mutation in the ubiquitin-associated domain of SQSMT1 is sufficient to cause a Paget's disease-like disorder in mice. Hum. Mol. Genet. 2011; 20(14): 2734-44. [97] Cavey JR, Ralston SH, Sheppard PW, Ciani B, Gallagher TR, Long JE, et al. Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone. Calcif. Tissue Int. 2006; 78(5): 271-7. [98] Najat D, Garner T, Hagen T, Shaw B, Sheppard PW, Falchetti A, et al. Characterization of a non-UBA domain missense mutation of sequestosome 1 (SQSTM1) in Paget's disease of bone. J. Bone Miner. Res. 2009; 24(4): 632-42. [99] Hocking LJ, Lucas GJ, Daroszewska A, Cundy T, Nicholson GC, Donath J, et al. Novel UBA domain mutations of SQSTM1 in Paget's disease of bone: genotype phenotype correlation, functional analysis, and structural consequences. J. Bone Miner. Res. 2004; 19(7): 1122-7. [100] Heinen C, Garner TP, Long J, Bottcher C, Ralston SH, Cavey JR, et al. Mutant p62/SQSTM1 UBA domains linked to Paget's disease of bone differ in their abilities to function as stabilization signals. FEBS Lett. 2010; 584(8): 1585-90. [101] Wooten MW, Hu X, Babu JR, Seibenhener ML, Geetha T, Paine MG, et al. Signaling, Polyubiquitination, Trafficking, and Inclusions: Sequestosome 1/p62's Role in Neurodegenerative Disease. J. Biomed. Biotechnol. 2006; 2006(3): 62079. [102] Helfrich MH, Hocking LJ. Genetics and aetiology of Pagetic disorders of bone. Arch. Biochem. Biophys. 2008; 473(2): 172-82. [103] Falchetti A, Di Stefano M, Marini F, Ortolani S, Ulivieri MF, Bergui S, et al. Genetic epidemiology of Paget's disease of bone in italy: sequestosome1/p62 gene mutational test and haplotype analysis at 5q35 in a large representative series of sporadic and familial Italian cases of Paget's disease of bone. Calcif. Tissue Int. 2009; 84(1): 20-37. [104] Collet C, Michou L, Audran M, Chasseigneaux S, Hilliquin P, Bardin T, et al. Paget's disease of bone in the French population: novel SQSTM1 mutations, functional analysis, and genotype-phenotype correlations. J. Bone Miner. Res. 2007; 22(2): 310-7. [105] Paine MG, Babu JR, Seibenhener ML, Wooten MW. Evidence for p62 aggregate formation: role in cell survival. FEBS Lett. 2005; 579(22): 5029-34. [106] Novack DV, Yin L, Hagen-Stapleton A, Schreiber RD, Goeddel DV, Ross FP, et al. The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis. J. Exp. Med. 2003; 198(5): 771-81. [107] Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell 2002; 3(6): 889-901. [108] Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J. Biol. Chem. 2006; 281(32): 22429-33. [109] Takahashi N, Maeda K, Ishihara A, Uehara S, Kobayashi Y. Regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals. Front Biosci. 2011; 16: 21-30.

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Chapter 5

OSTEOCLASTS: THE MAJOR ACTORS IN BONE RESORPTION Lucia D’Amico and Ilaria Roato* CeRMS, A.O.U. San Giovanni Battista, Turin, Italy

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ABSTRACT The Osteoclast (OC), the exclusive bone resorptive cell, is a member of the monocyte/macrophage family and polykarion that can be generated by cytokine-driven proliferation and differentiation of monocyte precursors, which can circulate within the hematopoietic cell pool or be resident in a number of tissues. The maintenance of adequate bone mass is dependent upon the controlled and timely removal of old, damaged bone. This complex process is performed by the OCs, thus an increase of OC activity is observed in many pathologies characterized by bone loss, such as osteoporosis, hyperparathytoidism, rheumatoid arthritis, bone metastasis, periprosthetic osteolysis in aseptic loosening of arthroplasty and also in pediatric diseases like phenilketonuria and 21-hydroxylase deficiency. A primary mediator of osteoclastogenesis is the RANKRANKL-OPG system, but also other factors may promote OC activation, according to the different pathologies. Anyhow the last result is the bone loss, primarily due to an expansion of the osteoclastic pool only partially or not compensated by a stimulation of bone formation. This review summarizes the main mechanisms promoting osteoclastogenesis in diseases characterized by bone loss, focusing on factors and cytokines involved in this process and on the interaction between OCs and T cells.

INTRODUCTION Bone plays an essential role in the structure, protection and movement of the body. It is a dynamic tissue, constantly remodelled by a coordinated system: an equal amount of bone extracellular matrix resorbed by osteoclasts (OCs) is laid down by osteoblasts (OBs), in order *

Corrisponding author: Ilaria Roato, PhD. CeRMS, A.O.U. San Giovanni Battista, via Santena 5, 10126 Turin, Italy.

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to maintain skeletal mass and calcium balance. This process presents three remarkable features particularly important in terms of physiology. First, resorption and formation of bone occur in a balanced manner in order to maintain bone mass nearly constant during most part of adulthood, establishing bone remodelling as a true homeostatic function. The second point is that the destruction/construction activity characterizing bone remodelling occurs constantly and simultaneously in thousands of sites, thus it require a large and constant supply of energy to bone cells. Lastly, the ability of bone remodelling to repair micro-damages has been essential to preserve mobility until our modern days and thereby this function can be considered as a survival function [1]. Pathological bone loss, regardless the etiology, represents an increase in skeletal degradation due to abnormal OC activity. The OC is a multinucleated cell specialised to carry out lacunar resorption. OCs are not commonly seen in normal adult bone but are often found at sites of osteolysis in diseases affecting bones and joints [2]. This chapter summarizes the main mechanisms promoting osteoclastogenesis and the interactions between OCs and immune system in diseases characterized by bone loss, focusing on factors and cytokines involved in this process.

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Osteoclast Differentiation and Activation The OC is the exclusive bone resorptive cell, is a polykaryon originated by the differentiation of monocyte/macrophage precursor cells at or near the bone surface member [3]. The OC precursors circulate in the peripheral blood, they express the monocyte /macrophage integrins CD11b-c and the lipopolysaccaride receptor antigen CD14 [4-5]. These precursors are only a small percentage of the circulating monocytes, about 2-5%, and undergo rapid changes during OC differentiation process [6-7]. OCs formation requires permissive concentrations of macrophage-colony stimulating factor (M-CSF) and is driven by contact with mesenchymal cells in bone, which express the receptor activator of nuclear factor NF-kB ligand (RANKL). M-CSF contributes to the proliferation, survival, and differentiation of OC precursors, as well as the survival and cytoskeletal rearrangement required for efficient bone resorption. RANKL is a member of the TNF superfamily, its discovery was preceded by the identification of its physiological inhibitor osteoprotegerin (OPG), to which it binds with high affinity [8]. RANKL exists as membrane-bound protein [9], cleaved in a soluble form (sRANKL) by metalloproteinases [10-11]. The membrane RANKL is expressed by OBs/stromal cells, and sRANKL is secreted by activated T cells and represents a crucial link between bone metabolism and the immune system [12-13]. Production of RANKL by activated T cells can directly regulate osteoclastogenesis and bone remodelling, and it explains why autoimmune diseases, cancers, leukemias, asthma and periodontal disease result in systemic and local bone loss. RANKL inhibition through its natural decoy receptor OPG prevents bone loss in postmenopausal osteoporosis and cancer metastasis. Moreover, the RANKL to OPG ratio in serum, rather than the individual protein concentrations, has been suggested to be the critical factor in determining OC activation at bone level, with higher serum RANKL to OPG ratio being a marker for up-regulation of osteoclastogenesis [9]. Alteration in RANKL to OPG axis has been demonstrated in several bone loss associated diseases [14]. OC precursors express RANK and its interaction with RANKL on OBs/stromal cells is essential for OC formation [3, 15]. RANKL can activate

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mature OCs in a dose-dependent manner in vitro in absence of stromal cells [10, 16-18] and can lead rapidly to the resorption of bone in vivo by activating pre-existing OCs [19-20]. The unique osteoclastogenic properties of RANKL allowed the generation of pure populations of OCs in culture and hence the performance of meaningful biochemical and molecular experiments that clarified some molecular mechanisms by which OCs resorb bone. Key to the resorptive event is the capacity of OCs to attach themselves to bone, creating a microenvironment between itself and the underlying bone matrix, a specialized structure called sealing zone [21]. This compartment, which is isolated from the general extracellular space, is acidified by an electrogenic proton pump (H+-ATPase) and a Cl- channel in order to solubilize the mineral component of bone. The acidified milieu mobilizes the mineralised component of bone, exposing its organic matrix, consisting largely of type I collagen, which is subsequently degraded by cathepsin K, a lysosomal enzyme. To facilitate the resorption process, OCs polarize their structure and form the ruffled border, which allows the active transport of H+ ions through the vacuolar proton pump [21]. The critical role that the proton pump, Cl- channel, and cathepsin K play in OC action is underscored by the fact that diminished function of each results in a human disease of excess bone mass, namely osteopetrosis or pyknodysostosis [3, 22]. Imbalance between OCs and OBs activities can arise from different hormonal changes or perturbations of inflammatory and growth factors, resulting in skeletal abnormalities. A significant deviation from a neutral balance between resorption and formation would mean severe accelerated bone loss or bone loss gains, with disastrous consequences in terms of increased fracture risks or compression syndromes. Increased OC activity characterize osteopenic disorders, including postmenopausal osteoporosis, Paget’s disease, lytic bone metastases, rheumatoid arthritis, leading to increased bone resorption and crippling bone damage [23]. Many factors have been described to affect osteoclastogenesis: calciotropic hormones and bone cytokines such as PTH, PTH-related protein (PTHrP), 1,25(OH)2D3, glucocorticoids, IL-1, IL-6, IL-7, IL-11, TNF- and prostaglandin-E2 [24-28]. Many of these factors exert most of their osteoclastogenic activity by inducing RANKL expression on OBs [3, 29-30]. Serum PTH is increased in hyperparathyroidism, whereas PTHrP is secreted by metastatic lung and breast cancer [31]. TNF- and other pro-inflammatory cytokines, such as IL-1 synergizes with RANKL in a unique manner, up-regulating a number of key downstream effector pathways, which lastly activate osteoclastogenic transcription factors [32].

Osteoclasts and Immune System Talk The relationship among bone, hematopoietic and immune systems is an intriguing challenge of the past two decade. These systems are in deep physical contact and share several common pathways. Both OC precursors and the various lymphocyte subsets, such as T, B and NK cells arise from the same stem cell, thus some of the same receptors and ligands that mediate the immune process also rule the maturation of OC precursors and the ability of the mature cell to degrade bone. The relationship between immune system and bone is bi-directional because bone cells express surface molecules which are essential for the expansion of hemopoietic stem cells

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from which all cells of the mammalian immune system derive and at the same time various immunoregulatory cytokines influence the fate of bone cells. OC precursors circulate within the mononuclear fraction of peripheral blood and they act not only as a reservoir for replenishing the pre-OC pool in the bone marrow, but also as a potentially abundant source of pre-OCs that can be recruited into bone or joint tissue in response to reparative or pathological signals. On this basis OCs can be considered as immune cells attracted in bone by stimulatory cytokines, expressed on accessory cells and undergoing specific differentiation. T cells play a key role in estrogen deficiency bone loss [33], but also are important in a range of diseases characterized by systemic and local bone loss, such as rheumatoid arthritis [34], periodontal disease [35-37], bone metastases [27, 38], periprosthetic osteolysis [39] and phenylketonuria [40]. Osteoimmunology focused on OC regulation by T cells, in fact only with the discovery of RANKL and its receptor RANK, the molecular links between the immune system and bone emerged. These molecules were first identified as factors expressed on T cells and dendritic cells (DCs), respectively. RANK and RANKL modulate the immunity through DCs because they increase the ability of DCs to stimulate naïve T cell proliferation and enhance DC survival. They were later identified as the key osteoclastogenic molecules, and now it is clear that a host of immune factors, including costimulatory receptors, cytokines such as IFN and TNF, and T and B lymphocytes play a fundamental role in the regulation of bone cell development and bone turnover, and in pathogenesis of bone disease [41]. IFN has a controversial role in osteoclastogenesis because it has an anti osteoclastogenic effect in vitro [42] and in vivo in animal studies [43], whereas in humans it increases in estrogen deficiency and in rheumatoid arthritis with bone loss [44-45]. IFN influences osteoclastogenesis both directly and indirectly: it targets maturing OC, thus blocking OC formation [46] and it stimulates T cell activation, thus pro-osteoclastogenic factors increase [47]. T cells also produce IL-7, a cytokine with different effects on hematopoietic and immunologic systems. IL-7 support B and T lymphopoiesis [48], but it also plays an important role in bone homeostasis [49-50]. Some studies demonstrated that IL7 promotes osteoclastogenesis by up-regulating T cell-derived cytokines, such as RANKL [27, 51-52] and that its production is increased by estrogen deficiency [33]. Recently investigators focused on the OC modulatory activity of T cells, showing that OCs are able to present antigenic peptides to T cells and to induce FoxP3 expression in CD8+ T cells [53]. In this way, CD8/FoxP3+ cells act as T regulatory, able to rule inappropriate activation of the immune response [53]. The cellular responses in cell-to-cell interactions between T cells and OCs are regulated through reciprocal CD137/CD137L and RANK/RANKL interactions [54]. CD137 is a co-stimulatory member of the TNF receptor induced by T cell receptor activation, it is characterized by the ability to transduce signals in both directions, through the receptor and into the cell that expresses the ligand. Its ligand CD137L is expressed on DCs and OC precursors: in vitro CD137L ligation suppresses osteoclastogenesis through the inhibition of multi-nucleation. On the other hand, RANKL expressed on T cells binds to RANK on OCs, producing a reverse signal in T cells able to enhance apoptosis. B cells differentiate from hematopoietic stem cells in supportive niches found on endosteal bone surface, thus they have a close relationship with bone. In particular, the RANK/RANKL/OPG axis rules also B cell differentiation: loss of OPG increases pro- and

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mature B cells, while the loss of RANK or RANKL decreases this cell population [55]. Activated B cells are involved in the development of inflammatory arthritis as well as periodontal disease [56-57]. B cells can express RANKL, thus they may affect osteoclastogenesis. B cell-related malignancies, such as multiple myeloma (MM) and B cell lymphomas produce factors and cytokines which stimulate OC activity [58-59].

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Osteoclasts in Bone Metastases Bone metastases represent a common cause of morbidity in patients suffering different types of cancer and are commonly classified in osteolytic and osteoblastic. This classification actually represents two extremes of a continuum in which dysregulation of the normal bone remodelling process occurs. Patients can show both osteolytic and osteoblastic metastases or mixed lesions containing both elements [60]. Only in MM do purely lytic bone lesions develop, whereas other osteotropic tumors, such as breast, lung, kidney, prostate cancer may show both the aspects. In the osteolytic lesions the bone destruction is mediated by the OCs, even though the activation of OCs varies depending on the tumors. The affinity of some tumors to growth in bone results from the special microenvironment provided by bone. In fact, activated OCs resorb bone and release growth factors enmeshed in the bone matrix, such as bone morphogenetic proteins, TGF-, insulin-like growth factor, fibroblast growth factor and others that stimulate the growth of metastatic tumor cells [61]. The last, in turn, secrete additional factors that act on bone cells, causing osteolytic and osteoblastic metastases. These local interactions between tumor cells and bone form a vicious cycle, which underlies the development of skeletal metastases [62]. The most prominent cause of bone destruction in metastases is PTHrP, which stimulates OC bone resorption and is secreted by many cancer types [63-65]. Other factors, released by tumor cells, such as IL-6, IL-8, IL-11 and vascular endothelial growth factor (VEGF) can increase OC number, survival and activity, thus osteolytic metastases. Tumor production of most of these factors is stimulated by TGF- [66], which derives from the mineralised bone matrix degradation by OCs. By focusing on peripheral blood mononuclear cells (PBMCs) samples of patients affected by osteotropic solid tumors, an increase in circulating OC precursors was documented in bone metastatic patients, compared with both healthy controls and cancer patients without bone metastases [38]. The PBMCs of these same patients differentiated into mature, multinucleated and bone resorbing OCs in vitro, without adding pro-osteoclastogenic factors, such as M-CSF and RANKL [38]. However, these factors were necessary to generate OCs from PBMCs of healthy donors and non-bone metastatic cancer patients. This spontaneous osteoclastogenesis in vitro was dependent mainly on TNF-, but it has been observed also a synergistic effect with RANKL produced by T cells present in the PBMC cultures. In fact, T cell-depleted PBMCs did not differentiate into OCs without adding M-CSF and RANKL [38]. The production of these pro-osteoclastogenic factors by T cells depends, at least in part, on IL-7 release by tumor cells [27, 67]. IL-7 stimulates T cells to release RANKL, thus osteoclastogenesis is promoted. By dosing IL-7 sera level in patients and healthy controls, it has been shown that the highest IL-7 level is detected in patients with bone metastases compared to non-bone metastatic patients and healthy controls [52, 68-69]. The increase of serum IL-7 seems to depend directly on tumor production, since it has been demonstrated a

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strong IL-7 expression in tumor masses originated in a human-in-mice model of bone metastases and in human bone metastatic biopsies [67]. MM is a B-cell neoplasm with frequent occurrence of lytic bone disease. The reciprocal relationship between MM cells and OCs is known to be critical for the induction of osteoclastogenesis and the activation of bone resorption [70] as well as for the OB inhibition, thus preventing lesion repair [71]. In the pathogenesis of MM bone disease, myeloma cells induce an OPG/RANKL imbalance [72]: RANKL production is increased and OPG is decreased [73]. Several studies suggest that myeloma cells produce RANKL other than stromal cells [74-75], and the up-regulation of RANKL is accompanied by the downregulation of IFN-, which is known to be a strong suppressor of osteoclastogenesis [52]. Moreover, the possible implication of IL-7 in MM osteoclastogenesis has been suggested because IL-7 stimulates RANKL production by T cells and its level in MM patients is high [52]. In culture of PBMCs obtained from myeloma patients, OCs developed without adding exogenous stimulating factors and this osteoclastogenesis was RANKL and T cells-dependent [76]. Furthermore, T cells from MM patients overexpress also OPG and TNF-related apoptosis-inducing ligand (TRAIL), thus the persistence of increased osteoclastogenesis despite the high level of OPG could be explained by the OPG binding to TRAIL, which inhibit the OPG action [77]. Several osteoclastogenic factors have been implicated in the increased activity of OC in myeloma, in particular IL-1 and IL-6, which are potent stimulators of OC formation [78-79]. IL-6 can enhance the effect of PTHrP on the OC formation in vivo [80]. In MM, another potent inducer of OC formation in vitro, independently of RANKL, is macrophage inflammatory protein 1 (MIP-1) [81], whichstimulates both RANKL and IL6-induced OC formation [82]. MIP-1 enhances adhesive interactions between myeloma cells and stromal cells, causing an increased production of IL-6 and RANKL, thus further increasing bone destruction.

OSTEOCLASTS IN RHEUMATOID ARTHRITIS In human arthritis, inflammation of the synovial joints is accompanied by bone and cartilage destruction. In rheumatoid arthritis (RA), bone erosion occurs rapidly and is associated with prolonged, increased inflammation. Synovial cytokines, particularly M-CSF and RANKL, promote OC differentiation and invasion of the periosteal surface adjacent to articular cartilage [83]. An inflammatory milieu induces naïve T cells to differentiate in Th17 cells, a specialized inflammatory subset, capable to produce RANKL, TNF and IL-17. This last cytokine increases RANKL expression by OBs, thus OC activity is further augmented [84]. Synovial tissues of patients with RA produce also other factors regulating bone resorption, such as TNF-, IL-1 and IL-6, which amplify OC differentiation and activation [85]. Examination of the cellular constituents of synovial fluid collected from human arthritis patients revealed that all focal T cells expressed RANKL [86]. Recently, investigators demonstrated that RANKL with M-CSF, can induce trans-differentiation of immature DCs to the OC lineage and that this process is significantly enhanced by RA synovial fluid [87]. DCs are known for being antigen presenting cells and do not seem to play a role in nonpathological conditions, but some data suggest that DCs could act as OC precursors in inflammatory conditions, transforming into DC-derived OC [88]. Moreover, DCs modulate T

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cell activity through RANK/RANKL pathway and other cytokines associated with osteoclastogenesis. Thus DCs could act as an osteo-immune interface, contributing to bone loss in inflammatory diseases [89]. OC are found mainly at the site of focal erosion and are critical for bone destruction, they have the acidic enzymatic machinery necessary to destroy mineralised tissues, including mineralised cartilage and subchondral bone [90]. Destruction of these tissues leads to deep resorption pits, which are filled by inflammatory tissue. There is a rapid erosion of periarticular bone, which is often followed by secondary osteoporosis (osteopenia). Eroded periarticular bone shows little evidence of repair in rheumatoid arthritis, unlike bone in other inflammatory arthropathies. Cytokine-induced mediators, such as Dickkopf 1 (DKK-1) and frizzled-related protein 1, which potently inhibit the differentiation of mesenchymal precursors into chondroblasts and OBs, leading to an insufficient bone forming activity [91]. In addition to the typical pro-inflammatory cytokines, other factors expressed in synovial tissues may rule bone resorption. In this regard, Kotake et al.[92] demonstrated that a peptide derived from T-cell leukemia translocation-associated gene (TCTA) protein, was expressed in synovial tissues from RA patients. This peptide also inhibited pit formation of mature human OCs and suppressed the formation of large OCs in the culture of mature OCs. Furthermore, polyclonal antibodies against TCTA protein suppressed the formation of large OCs in the cultures, preventing OC precursor cellular fusion [92].

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Osteoclasts in Periprosthetic Osteolysis Loosening of total hip arthroplasty (THA) caused by periprosthetic osteolysis is a major clinical problem and make revision surgery essential for the patients. The periprosthetic bonedestruction phenomenon is due to local inflammatory reaction to implant wear debris [93]. Activated periprosthetic cells secrete cytokines and chemokines, which induce the recruitment of inflammatory cells, the formation of osteolytic granulomas and affect the bone remodelling [94]. Wear debris induce phagocytosis by macrophages which activate OC signalling pathway. Studies conducted in animal models and in vitro cell cultures have shown that TNF plays a crucial role as a mediator of particle-induced osteoclastogenic and osteolytic events [95-97]. In this regard polymethylmethacrylate particles fail to elicit aggressive osteolysis in TNF receptor null animals. Likewise animals that lack RANK or RANKL resist induction by particles of osteolysis [97-98]. In periprosthetic osteolysis, OC activity is increased and dependent on RANKL [99-100], but also on other secreted factors [101]. Wang et al. showed that loosened THA patients had higher levels of inflammatory cytokines IL-6, IL-8 and IL-10 in the synovial fluid than primary THA patients [93]. Furthermore, they found positive correlation among the levels of IL-6, IL-8, IL-10 and RANKL in the synovial fluid or RANKL expression on osteoblastic stromal cells in the periprosthetic bone marrow. IL-6 increases bone resorption by stimulating RANKL production in OBs and enhancing RANKL sensitivity in OCs [102]. IL-8 induces RANKL expression in OBs and directly stimulates osteoclastogenesis and bone resorption [103]. Anti-inflammatory cytokine IL-10 inhibits the expression of inflammatory cytokines and protects bone against particulate-induced bone resorption.

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Further downstream signalling by wear debris overlaps with that of TNF and RANKL and activate kinases and molecules involved in OC differentiation and activation [97, 104105]. By studying the PBMC derived from periprosthetic osteolysis patients, in vitro spontaneous osteoclastogenesis was detected and it was inhibited by RANK-Fc addition in cell culture and by T cell depletion [39]. The analysis of periprosthetic tissues showed that T cells were close to OCs, suggesting their interaction. Local CD8+ T cells had a regulatory phenotype, expressing CD25 and FoxP3, whereas CD4+ T cells did not express activation markers. The regulatory phenotype of CD8+ T cells may explain the inhibition of effector CD4+ T cells, which are often found inactive in periprosthetic tissue. Thus, in the pathogenesis of periprosthetic osteolysis T cells initially proliferate and support osteoclatogenesis through RANK/RANKL pathway. Later, OCs may provide a negative feedback on T cells, leading to activation of regulatory CD8+ T cells and subsequent inhibition of CD4+ T cells [39].

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Osteoclasts in Pedriatic Disease In literature pediatric disesases with a bone impairment have been widely documented, using both radiological and ultrasound methods [106-107]. Phenylketonuria (PKU) is an inborn error of amino acid metabolism resulting from deficiency of phenylalanine metabolism. An early protein-restricted diet integrated with phenylalanine free medical foods successfully prevents the irreversible developmental delay characteristic of the natural course of the disease, by maintaining plasma phenylalanine concentrations in non-neuro-toxic range [108]. Despite the recommendation of life-long adherence to treatment, poor compliance to dietary prescriptions is common during adolescence, as the risk of the mental retardation due to hyperphenylalaninemia was historically thought to be insignificant at this age. However, this laxity of dietary restriction has been related to systemic complications of PKU in adulthood. Among these complications, bone impairment was documented with clinical parameters and the possible role of OCs in determining bone damage was studied in children affected by PKU. An increased spontaneous osteoclastogenesis in vitro was documented in a cohort of PKU patients [109], consistent with the increased bone resorption markers in affected patients. Further studies were addressed to clarify the mechanisms of osteoclastogenesis in PKU, considering its potential causes and links with immune system. PKU patients showed increased osteoclastogenesis depending on an increased number of circulating OC precursors. TNF- seems to stimulate and be regulated by OC precursors, whereas OC maturation depends on RANKL and T cells. The level of spontaneous osteoclastogenesis and the T cell activation state correlate with PKU patients’ bone condition. Thus, the finding of a specific sub-population of activated T cells accounting for spontaneous osteoclastogenesis infers a dysfunctional immune system activation in PKU patients. Moreover, a direct correlation between plasma phenylalanine concentration and spontaneous osteoclastogenesis was detected in PKU patients. This is consistent with a possible role of hyperphenylalaninemia in enhancing OC differentiation and consequently promoting bone resorption [40]. Another children pathological condition related to bone impairment is 21-Hydroxylase Deficiency (21-OHD). 21-OHD is the most common cause of congenital adrenal hyperplasia,

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resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21) [110]. This disorder is characterized by accumulation of the precursors immediately proximal to the 21hydroxylation step along the pathway of cortisol synthesis, which are shunted into the androgen pathway. Children with 21-OHD need chronic glucocorticoid therapy as soon as they are diagnosed with the disease, to both replace congenital deficit in cortisol synthesis and reduce androgen secretion by adrenal cortex [111]. Glucocorticoid-induced osteoporosis represents the most common cause of drug-induced osteoporosis, and different mechanisms have been proposed to explain its pathogenesis [112]. Faienza et al. studied a slight reduction in bone mass in 21-OHD patients [113]. A spontaneous formation of numerous, mature, multinucleated, and bone-resorbing OCs in unstimulated and unfractionated PBMC culture was also demonstrated, which was not evident in the same type of cultures derived from controls. This spontaneous osteoclastogenesis seems to correlate directly with the higher number of circulating OC precursors present in 21-OHD patients compared with controls and demonstrate that OC precursors are markedly increased in the circulation of 21-OHD patients [113]. Osteoclastogenesis in 21-OHD patients’ PBMC cultures was dependent on T cells, which produce M-CSF and RANKL. Furthermore, high RANKL levels were measured in media from unfractionated PBMC culture of 21-OHD patients, leading to a RANKL to OPG ratio higher in these patients than in controls. Additionally, RANKL was significantly elevated in the sera from 21-OHD patients and controls, whereas OPG decreased in these patients compared with controls and therefore RANKL to OPG ratio was higher in the sera of 21-OHD patients. Thus, spontaneous osteoclastogenesis, high levels of OC precursors, and high RANK to OPG ratio in 21-OHD patients could explain the slight reduction in the BMD of these subjects.

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Osteoclasts in Osteoporosis Osteoporosis is an emerging medical and socio-economic threat characterised by a systemic impairment of bone mass (BMD), strength and micro-architecture, which increases the propensity of fragility fractures. Osteoporosis includes many skeletal disorders, in fact it has led to use of terms such as postmenopausal, senile osteoporosis and idiopathic osteoporosis. The first two overlap clinically, whereas the third is used for younger men and pre-menopausal women with osteoporotic fragility fractures without identifiable secondary causes. Primary osteoporosis is in itself heterogeneous and presumably involves multiple pathogenetic mechanisms. About 40% of white post-menopausal women are affected by osteoporosis and, with an ageing population, this number is expected to steadily increase in the near future [114-115]. The lifetime fracture risk of a patient with osteoporosis is as high as 40%, and fractures cause subsequent loss of mobility and autonomy often representing a major drop in quality of life. Osteoporosis is a multi-factorial disease: genetic, nutrition and lifestyle determinants have been recognized to cooperate in its pathogenesis. Some polymorphisms can affect fracture risk independent of BMD, such as a polymorphism of the 1 collagen gene [116]. Lifestyle factors can contribute further to the optimal or the inadequate development of the skeleton, as well as to subsequent maintenance of skeletal strength. Calcium, vitamin D, and physical activity are probably the most important determinants of peak bone mass and strength [117]. However, total nutrition, protein intake

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and micronutrients such as vitamins B6, B12, folic acid which may be related to homocysteine and Vitamin K may be important [118-119]. From a biological point of view, in osteoporotic patients there is a decreased bone mass and an increased fragility, caused by an up-regulated OC activity and a reduced bone forming activity [33]. Pro-osteoclastogenic cytokines are produced by both bone itself and bone marrow cells [120] [33]. Nevertheless, in patients with fragility fractures, bone marrow cells are the mainly responsible for the pro-osteoclastogenic M-CSF and RANKL production, whereas bone releases OB inhibitors, such as DKK-1 and Sclerostin (SOST). These two molecules own to the Wnt protein family, which is determinant in maintaining bone mass [121]. DKK-1 and SOST are thought to be involved in the pathogenesis of arthritis [122], glucocorticoid-induced osteoporosis [123], and disuse osteoporosis [124]. In early postmenopause period there is a rapid bone loss, due to the acute phase of estrogen deficiency. The anti-resorptive activity of estrogen is a result of multiple genomic and non-genomic effects on bone marrow and bone cells, which leads to decreased OC formation, increased OC apoptosis and decreased capacity of mature OCs to resorb bone. Stimulation of bone resorption in response to estrogen deficiency is mainly due to cytokine-driven increases in OC formation. Imbalance between RANKL and OPG has been indicated as the pivotal mechanism responsible for bone loss in cases of estrogen deficiency. RANKL expression by bone marrow stromal cells and lymphocytes increases and is associated with enhanced bone loss. Additional inflammatory cytokines are responsible for the up-regulation of OC formation observed in estrogen deficiency. TNF- enhances OC formation by up-regulating stromal cell production of RANKL and M-CSF, and by augmenting the responsiveness of OC precursors to RANKL [125-126]. Studies in mice suggest that activated T cells are the most relevant source of TNF- in conditions of estrogen deficiency [45, 127]. Ovariectomy increases the proliferation of unstimulated T cells and the number of bone marrow T cells, which suggests that in vivo estrogens may regulate the number of TNF--producing T cells, rather than TNF gene expression. The finding that in vitro estrogen treatment of cultured T cells did not blunt the ability of T cells from ovariectomized mice to produce TNF- is consistent with this hypothesis and suggests that estrogen deficiency acts indirectly, perhaps targeting a population of bone marrow cells capable of activating T cell proliferation [127]. Furthermore, the adoptive transfer of wild type T cells restores the capacity of ovariectomy to induce bone loss, while transfer of T cells from TNF null mice does not [127-128]. In post-menopausal women there is a greater production of pro-osteoclastogenic cytokines as compared to pre-menopausal subjects, confirming a relationship between estrogen deficiency and the greater production of pro-inflammatory cytokines [129-130]. Estrogen deficiency does not increase the capacity of OC precursors to differentiate into mature OCs, but rather increases the levels of osteoclastogenic cytokines in the bone microenvironment. In PBMCs derived from women affected by post-menopausal osteoporosis, a significant increase in spontaneous OC formation and bone resorbing activity in vitro have been described [131]. This phenomenon is due to an increase of circulating OC precursors and to an enhanced TNF- and RANKL production in patients compared to healthy controls. In the same patients, the OC number was inversely correlated with bone mineral density and directly with RANKL in culture supernatants [131]. Moreover, in postmenopausal osteoporosis women, T cells showed a more active phenotype, stimulating osteoclastogenesis, compared to pre-menopausal and post-menopausal healthy women [33].

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CONCLUSION Pathological bone loss is mainly due to an increased OC activity and the field of bone biology has matured such that key cellular and molecular mechanisms governing the OC differentiation and activation are largely understood. The RANK/RANKL/OPG system is indispensable for osteoclastogenesis, but also the interactions between OCs and immune system are fundamental to explain the pathogenesis of diseases characterized by unbalanced bone remodelling.

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[32] Zwerina, J., et al., Single and combined inhibition of tumor necrosis factor, interleukin1, and RANKL pathways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, bone erosion, and cartilage destruction. Arthritis Rheum., 2004. 50(1): p. 277-90. [33] D'Amelio, P., et al., Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: a key mechanism in osteoporosis. Bone, 2008. 43(1): p. 92-100. [34] Nakashima, T., T. Wada, and J.M. Penninger, RANKL and RANK as novel therapeutic targets for arthritis. Curr. Opin. Rheumatol., 2003. 15(3): p. 280-7. [35] Brunetti, G., et al., T cells support osteoclastogenesis in an in vitro model derived from human periodontitis patients. J. Periodontol, 2005. 76(10): p. 1675-80. [36] Colucci, S., et al., Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFalpha, and IL-7 in an in vitro model derived from human psoriatic arthritis. J. Pathol., 2007. 212(1): p. 47-55. [37] Taubman, M.A., et al., Immune response: the key to bone resorption in periodontal disease. J. Periodontol, 2005. 76(11 Suppl): p. 2033-41. [38] Roato, I., et al., Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement. FASEB J., 2005. 19(2): p. 228-30. [39] Roato, I., et al., Osteoclastogenesis in peripheral blood mononuclear cell cultures of periprosthetic osteolysis patients and the phenotype of T cells localized in periprosthetic tissues. Biomaterials, 2010. 31(29): p. 7519-25. [40] Roato, I., et al., Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase. PLoS One, 2010. 5(11): p. e14167. [41] Pacifici, R., The immune system and bone. Arch. Biochem. Biophys., 2010. 503(1): p. 41-53. [42] Takayanagi, H., et al., T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature, 2000. 408(6812): p. 600-5. [43] Sato, K., et al., Prolonged decrease of serum calcium concentration by murine gammainterferon in hypercalcemic, human tumor (EC-GI)-bearing nude mice. Cancer Res., 1992. 52(2): p. 444-9. [44] Gao, Y., et al., IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J. Clin. Invest., 2007. 117(1): p. 122-32. [45] Cenci, S., et al., Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator. Proc. Natl. Acad. Sci. U S A, 2003. 100(18): p. 10405-10. [46] Takayanagi, H., S. Kim, and T. Taniguchi, Signaling crosstalk between RANKL and interferons in osteoclast differentiation. Arthritis Res., 2002. 4 Suppl 3: p. S227-32. [47] Pacifici, R., Estrogen deficiency, T cells and bone loss. Cell Immunol., 2008. 252(1-2): p. 68-80. [48] Namen, A.E., et al., Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature, 1988. 333(6173): p. 571-3. [49] Miyaura, C., et al., Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: similarity to estrogen deficiency. Proc. Natl. Acad. Sci. U S A, 1997. 94(17): p. 9360-5.

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[105] Abbas, S. and Y. Abu-Amer, Dominant-negative IkappaB facilitates apoptosis of osteoclasts by tumor necrosis factor-alpha. J. Biol. Chem., 2003. 278(22): p. 20077-82. [106] Allen, J.R., et al., Decreased bone mineral density in children with phenylketonuria. Am. J. Clin. Nutr., 1994. 59(2): p. 419-22. [107] Porta, F., et al., Phalangeal quantitative ultrasound in children with phenylketonuria: a pilot study. Ultrasound. Med. Biol., 2008. 34(7): p. 1049-52. [108] Bickel, H., J. Gerrard, and E.M. Hickmans, Influence of phenylalanine intake on phenylketonuria. Lancet, 1953. 265(6790): p. 812-3. [109] Porta, F., et al., Increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells in phenylketonuria. J. Inherit. Metab. Dis, 2008. 31 Suppl 2: p. S339-42. [110] White, P.C. and P.W. Speiser, Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr. Rev., 2000. 21(3): p. 245-91. [111] Joint LWPES/ESPE CAH, W.g., Consensus statement on 21-hydroxylase deficiency from the lawson Wilkins Pediatric Endocrine Society and the European Society for Pediatric Endocrinology. J. Clin. Endocrinol. Metab., 2002. 87: p. 4048-4053. [112] Mazziotti, G., et al., Glucocorticoid-induced osteoporosis: an update. Trends Endocrinol Metab., 2006. 17(4): p. 144-9. [113] Faienza, M.F., et al., Osteoclastogenesis in children with 21-hydroxylase deficiency on long-term glucocorticoid therapy: the role of receptor activator of nuclear factorkappaB ligand/osteoprotegerin imbalance. J. Clin. Endocrinol. Metab., 2009. 94(7): p. 2269-76. [114] Melton, L.J., 3rd, et al., Perspective. How many women have osteoporosis? J. Bone Miner. Res., 1992. 7(9): p. 1005-10. [115] Ray, N.F., et al., Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995: report from the National Osteoporosis Foundation. J. Bone Miner Res., 1997. 12(1): p. 24-35. [116] Ralston, S.H., Genetics of osteoporosis. Proc. Nutr. Soc., 2007. 66(2): p. 158-65. [117] Bischoff-Ferrari, H.A., How to select the doses of vitamin D in the management of osteoporosis. Osteoporos Int., 2007. 18(4): p. 401-7. [118] Ferrari, S.L., et al., Polymorphisms in the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with variation in vertebral bone mass, vertebral bone size, and stature in whites. Am. J. Hum. Genet., 2004. 74(5): p. 866-75. [119] Bischoff-Ferrari, H.A., et al., Estimation of optimal serum concentrations of 25hydroxyvitamin D for multiple health outcomes. Am. J. Clin. Nutr., 2006. 84(1): p. 1828. [120] Hofbauer, L.C., C.A. Kuhne, and V. Viereck, The OPG/RANKL/RANK system in metabolic bone diseases. J. Musculoskelet Neuronal Interact, 2004. 4(3): p. 268-75. [121] Marques-Pinheiro, A., et al., Novel LRP5 gene mutation in a patient with osteoporosispseudoglioma syndrome. Joint Bone Spine, 2010. 77(2): p. 151-3. [122] Appel, H., et al., Altered skeletal expression of sclerostin and its link to radiographic progression in ankylosing spondylitis. Arthritis Rheum, 2009. 60(11): p. 3257-62. [123] Ohnaka, K., et al., Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem. Biophys. Res. Commun., 2005. 329(1): p. 177-81.

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[124] Lin, C., et al., Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J. Bone. Miner Res., 2009. 24(10): p. 165161. [125] Hotokezaka, H., et al., Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan. J. Cell Biochem., 2007. 101(1): p. 122-34. [126] Eghbali-Fatourechi, G., et al., Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J. Clin. Invest., 2003. 111(8): p. 1221-30. [127] Cenci, S., et al., Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha. J. Clin. Invest., 2000. 106(10): p. 1229-37. [128] Roggia, C., et al., Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo. Proc. Natl. Acad. Sci. U S A, 2001. 98(24): p. 13960-5. [129] Pacifici, R., et al., Effect of surgical menopause and estrogen replacement on cytokine release from human blood mononuclear cells. Proc. Natl. Acad. Sci. U S A, 1991. 88(12): p. 5134-8. [130] Bernard-Poenaru, O., et al., Bone-resorbing cytokines from peripheral blood mononuclear cells after hormone replacement therapy: a longitudinal study. Osteoporos Int., 2001. 12(9): p. 769-76. [131] D'Amelio, P., et al., Spontaneous osteoclast formation from peripheral blood mononuclear cells in postmenopausal osteoporosis. FASEB J., 2005. 19(3): p. 410-2.

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Chapter 6

ROLE OF THE IMMUNO-SKELETAL INTERFACE IN PHYSIOLOGICAL AND PATHOLOGICAL OSTEOCLAST REGULATION M. Neale Weitzmann The Atlanta Department of Veterans Affairs Medical Center, Decatur, Georgia, US and the Division of Endocrinology & Metabolism & Lipids, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, US

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ABSTRACT The immuno-skeletal interface is a centralization of shared cells and cytokine effectors that serve critical functions within both the immune and skeletal systems. The precursors of the osteoclasts, the cells that resorb bone, have long been recognized to derive from monocytes/macrophages, cells central to both innate and adaptive immunity. Furthermore, osteoclast differentiation and function is potently responsive to a host of cytokine mediators secreted by multiple immune-related cell types during physiological immune renewal and pathological immune activation. Among these factors are receptor activator of NF-B ligand (RANKL), the key osteoclastogenic cytokine and osteoprotegerin (OPG) its physiological decoy receptor, as well as a host of inflammatory cytokines that directly and/or indirectly amplify osteoclast formation and activity. As a consequence of this close association between the immune and skeletal systems, responses by the immune system to a wide range of pathological and inflammatory stimuli potently upset physiological bone turnover initiating a wave of inappropriate osteoclastogenesis and an upswing in bone resorption. Such disturbances to the immunoskeletal interface may underlie the bone loss associated with numerous diverse pathological conditions ranging from postmenopausal osteoporosis to autoimmune and/or inflammatory states such as rheumatoid arthritis, inflammatory bowel disease, periodontal infection and in ”inflammaging” the generalized inflammatory state associated with aging. Paradoxically, not only do inflammatory states drive bone loss, but disruptions to basal immune function characteristic of immunodeficiency also alter physiological osteoclastogenesis and may be central to the high rates of skeletal

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M. Neale Weitzmann deterioration associated with HIV-infection. This chapter examines our current understanding of the role of the immuno-skeletal interface in the regulation of osteoclast formation and function in the context of normal and pathological immune responses.

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INTRODUCTION Recent studies have demonstrated an inexplicable interrelationship between the immune system and bone turnover, spawning the field of “osteoimmunology”. Indeed, it has long been recognized that osteoclasts, the cells that degrade (resorb) bone tissue derive from a progenitor of monocytic origin. Monocytes and their progeny the macrophages, play a key role in innate immunity, as phagocytic cells, as well as in adaptive immunity as professional antigen presenting cells (APC). More recently it has been recognized that other cells of the adaptive immune system, in particular lymphocytes, both T cells and B cells, play critical roles in the regulation of basal [1, 2] and stimulated osteoclastogenesis [3]. While immune activation is synonymous with inflammation and it has long been recognized that bone loss is a key feature of inflammatory conditions, it is only in the past decade that we have begun to understand the mechanisms by which inflammatory immune activation leads to pathological osteoclastogenesis and bone loss. Inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor alpha (TNF have long been known to promote bone loss in conditions as diverse as estrogen deficiency and rheumatoid arthritis (RA) [3]. However, the discovery of the RANKL decoy receptor OPG in 1997 [4, 5], followed by identification of the key osteoclastogenic effector, RANKL, in 1998 [6-8] opened the door to the study of the immuno-skeletal interface an enigmatic centralization of immune and skeletal functions centered on common cell types and cytokine effectors. This chapter explores some of the discoveries of the past decade into how the immunoskeletal interface regulates physiological osteoclast renewal, and how disruption of this delicate equilibrium underlies osteoclastic bone loss in conditions as diverse as estrogen deficiency and human immunodeficiency virus-1 (HIV-1) infection.

1. PHYSIOLOGICAL OSTEOCLAST FORMATION AND THE ROLE OF THE RANK/RANKL/OPG AXIS Osteoclast precursors derive from cells that circulate among the monocyte lineage. Among this pool exist a subset of monocytic cells that express a surface molecule referred to as receptor activator of NF-B (RANK). These cells are considered to be early osteoclast precursors and in the presence of permissive concentrations of the survival factor macrophage colony stimulating factor (M-CSF), are able to differentiate into pre-osteoclasts in response to the key osteoclastogenic cytokine RANKL. The pre-osteoclasts ultimately fuse with other pre-osteoclasts to form mature multinucleated osteoclasts with the unique intrinsic capacity to resorb bone [9-12]. Evidence for a central role of RANKL in basal osteoclastogenesis has been definitively provided by animal studies in which genetic ablation of RANKL in mice leads to osteopetrosis, a high bone mass phenotype, caused by a complete absence of osteoclasts [13]. Although several different cell types including lymphocytes are capable of

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RANKL production, under basal conditions the primary source of RANKL has generally been ascribed to cells of the osteoblast lineage [14, 15]. These include bone marrow stromal cells (cells of mesenchymal origin and the progenitors of osteoblasts) as well as mature osteoblasts. Recently, new studies involving conditional deletion of RANKL in osteocytes the terminally differentiated osteoblasts that become embedded in bone matrix [16, 17] and in hypertrophic chondrocytes [16] have provided compelling evidence to suggest that these cell types may be critical sources of RANKL in bone remodeling in vivo [16, 17] and in response to bone mechanical unloading [16]. Under inflammatory conditions, as described in detail later in this chapter, activated T cells are considered a significant source of RANKL. However, T cells do not appear to contribute significantly to bone marrow RANKL concentrations as we have shown that T cell deficient nude mice do not show evidence of diminished RANKL mRNA expression in their bone marrow [1]. RANKL activity is moderated by its physiological decoy receptor OPG, and osteoclast formation and activity is consequently a function of the ratio of RANKL with respect to that of OPG in the bone marrow microenvironment [9, 10]. Overexpression of OPG in mice results in osteopetrosis, driven by low osteoclast number and/or activity [4, 5]. While not required for embryonic bone formation, OPG is essential for the maintenance of postnatal bone mass, as decreased BMD is evident by one month in OPG KO mice [5, 18]. In conditions predisposing to bone loss the levels of RANKL exceed those of OPG leading to elevated osteoclastogenesis and bone resorption. Although many cell types are endowed with the capacity to make OPG [19], the dominant source of this factor in the bone marrow microenvironment has historically been attributed to osteoblasts and/or their precursors, the bone marrow stromal cells [11, 20]. Recently, we challenged this notion by suggesting that cells of the B-lineage may constitute an important additional source of OPG that is critical to the attainment and maintenance of peak BMD [1]. Although immune cells have long been recognized as protagonists of osteoclastic bone destruction our data revealed for the first time a defined role of the immune response in the defense of basal bone homeostasis.

2. ROLE OF THE IMMUNO-SKELETAL INTERFACE IN PHYSIOLOGICAL OSTEOCLAST RENEWAL a) T Cells and B Cells, the Centerpiece of Cell-mediated Immunity T cells play a central role in cell-mediated immunity. Each T cell expresses a unique T cell Receptor (TCR) that recognizes a specific antigen. T cells may be subdivided into two major classes: 1) Helper T cells (effector T cells) which express the CD4 surface glycoprotein and respond to antigens presented by Major Histocompatibility (MHC) class II expressing APCs including B cells, macrophages and dendritic cells; 2) Cytotoxic T cells expressing the CD8 glycoprotein are associated with MHC class I antigenic stimuli [21]. Two signals are required for full T cell activation. The first signal is generated when the TCR engages MHC class II bearing antigens on the surface of B cells and other professional APCs, or MHC class I bearing antigens expressed by most nucleated cells. This signal is insufficient for full T cell activation and on its own simply renders T cells unresponsive to further antigenic stimuli

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(anergy). The second signal, or “costimulatory signal,” involves binding of the CD28 receptor on T cells, with B7 molecules (B7-1 (CD80) and B7-2 (CD86), expressed on B cells and other professional APC. These two signals lead to full T cell activation, cytokine production, clonal expansion, and prevention of anergy [22]. CD28 activation further leads to expression of CD40 ligand (CD40L) by CD4 T cells, which binds to its receptor CD40 on B cells and other APC. CD40 promotes B cell activation, proliferation, survival, and up-regulation of surface molecules involved in antigen presentation, germinal center formation, memory B cell development, immunoglobulin isotype switching, and affinity maturation [23]. These interactions are summarized diagrammatically in Figure 1.

Figure 1. T cell activation and hallmarks of the adaptive immune response. Antigen presenting cells (APC) present antigen (Ag) as a complex with MHCII molecules to T cells (signal I). Recognition of a specific antigen by the T cell Receptor (TCR) complex leads to engagement of B7 (CD80 and CD86) molecules on the APC with T cell expressed CD28 (signal II). This dual signal leads to further expression of costimulatory molecules including CD40L by T cells that engages CD40, its cognate receptor on the APC. These bidirectional signals lead to activation and differentiation of both T cells and APC.

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B cells are furthermore an essential component of the adaptive immune response and play a critical role in humoral (antibody (Ab)-mediated) immune responses, the intensity of which is regulated by T cell costimulation and T cell derived cytokines such as IL-4. The loss of CD28 expression on T cells is correlated with a lack of CD40L expression rendering such T cells incapable of promoting B cell differentiation and immunoglobulin secretion [24]. Activated T cells further express cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a molecule that is highly homologous to CD28 and competes for binding of CD28 to B7-1/B72. CTLA4 thus deprives the T cell of its second costimulatory signal, resulting in inhibition of cytokine production and cell cycle progression arrest, and ultimately terminating immune responses [25].

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b) T Cells and B Cells, Critical Regulators of Basal Osteoclastogenesis T cells have long been associated with inflammatory cytokine production and bone loss [3], but interestingly, depletion of CD4+ and CD8+ T lymphocytes in mice in vivo has been reported to enhance rather than reduce osteoclast formation ex vivo. Surprisingly the mechanism was found to involved, in part, a complete suppression of OPG production [26]. Furthermore, we [27] and others [28] have reported that T cell deficient nude mice have a significantly increased basal osteoclast number and reduced BMD, suggesting that in contrast to their pro-osteoclastogenic activities in inflammatory conditions, at baseline T cells have a capacity to protect bone mass. Similar reports have been made for B cells where in vivo B cell depletion was reported to aggravate bone loss in an animal model of periodontitis, suggesting that B cells may act to limit bone resorption under certain conditions [29]. More recently, enhanced osteoclastogenesis was reported in mice null for the transcription factor O/E-1 (Ebf1) that is essential for B lymphocyte development [30]. Knock out (KO) in mice of Pax5, which plays an important role in B-cell differentiation, leading to arrest in the B cell lineage at the pro-B cell stage, leads to severely reduced bone mass, although this has been attributed to indirect consequences of Pax5 deficiency rather than as a direct consequence of B cell depletion [31]. In addition, severe osteopetrosis, a high bone mass phenotype, has been identified in Brutons tyrosine kinase (Btk) and Tec kinase double-KO mice. These two characteristic B cell non-receptor tyrosine kinases were found to be highly expressed in osteoclasts, and their deletion led to defective osteoclastogenesis and bone resorption [32]. Because of this promiscuous expression of what were originally considered to be B cell specific transcription factors, linking changes in B cell related transcription factors to osteoclastogenesis and bone turnover has turned out to be complex and difficult to interpret, and has failed to provide a clear rationale for how lymphocytes may modulate osteoclastogenesis and bone turnover. Using an in vitro human osteoclastogenesis model system we reported inhibitory activities of peripheral blood B cells on osteoclast formation, mediated in part through secretion of TGFβ [33], a cytokine that induces apoptosis of mature osteoclasts [33, 34] and is reported to stimulate OPG production in osteoblasts [35]. Indeed, purified human B cells have been reported to secrete significant concentrations of OPG, which was further upregulated following ligation of the costimulatory receptor CD40, by activating antibodies [36]. Interestingly, osteoporosis and fractures are common in humans with X-linked hyper-IgM syndrome, an inherited disease caused by mutations in the CD40 ligand (CD40L) gene, which

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codes for the complementary ligand to the CD40 receptor. Patients with X-linked hyper-IgM syndrome have significantly lower BMD than sex- and age-matched control subjects and exhibit elevated levels of N-terminal telopeptides of type I collagen (NTx), a urinary marker that correlates strongly with in vivo osteoclast activity [37]. In addition, polymorphisms in the Kozak sequence of the CD40 gene are reported to be associated with low bone mass in postmenopausal women [38]. In fact, we have reported that in mice the CD40/CD40L pathway mediates critical costimulatory signals between T cells and B cells necessary for the maintenance of basal osteoclastogenesis and bone mass [1]. Genetic ablation of either CD40 or CD40L thus leads to increased bone resorption and a decline in bone mineral density (BMD). This stems from the fact that mature murine B cells contribute 45% of total OPG produced in the bone marrow microenvironment. Furthermore, B cell precursor populations contribute an additional 19% of total OPG. As a consequence, genetic ablation of mature B cells leads to a significant decline in bone marrow OPG concentrations, an imbalance in the RANKL/OPG ratio favoring increased osteoclastogenesis and elevated bone resorption leading to a net loss of BMD. Because T cells are the major physiological source of CD40L, T cell KO mice recapitulate this elevated osteoclastogenic environment as a consequence of diminished B cell OPG production, and display diminished BMD [1]. Recently, blockage of the CD40/CD40L costimulatory system has come under intensive investigation for amelioration of autoimmune diseases such as type I diabetes [39], systemic lupus erythematosus [40] and RA [41] and as an immunosuppressant in transplant patients [42]. Abatacept (CTLA4-Ig), an inhibitor of T cell costimulation and Rituximab, a B cell inhibitor, are now approved therapies for the treatment of adult and juvenile forms of intractable RA [43-46]. The implications for bone turnover and skeletal damage remain unclear and may need to be carefully considered given the existence of the immuno-skeletal interface.

c) Disruption of the Immuno-skeletal Interface and Bone Loss Associated with HIV-Infection Given the important role of T- and B-cells in the regulation of physiological osteoclastogenesis and basal bone homeostasis it is perhaps not surprising that conditions associated with immunodeficiency should be accompanied by bone loss. In fact, it has long been recognized that patients infected with the human immunodeficiency virus (HIV-1), display a high incidence of diminished BMD [47] and current estimates suggest that prevalence of osteoporosis in HIV-infected individuals is more than three times greater compared with HIV-uninfected controls [52]. The cause of this skeletal decline however, has been somewhat enigmatic, and masked, in part, by an unusually high number of confounding factors in HIV patients including traditional risk factors for osteoporosis such as low body mass index, smoking, high rates of alcohol consumption, hypogonadism, vitamin D insufficiency and a range of other sequelae commonly associated with the acquired immunodeficiency syndrome (AIDS) with known impact on bone turnover [48-50]. Furthermore, highly active antiretroviral therapy (HAART or ART) used to repress viral replication and ameliorate disease, has itself been directly implicated in the bone loss observed in AIDS patients [51, 52]. Given the inability to differentiate between a plethora of competing osteoporosis risk factors in human patients, it was only recently, with the

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development of appropriate animal models, that direct effects of viral infection on bone turnover could be examined at a mechanistic level in the absence of other confounding factors. To this end we recently examined bone turnover in HIV-1 transgenic rats, a small animal model of HIV infection in which transgenic overexpression of a replication incompetent HIV provirus leads to an immunological disruption and a constellation of pathologies characteristic of human AIDS [53]. Our data revealed a significant decline in BMD and indices of bone structure and architecture in HIV-1 transgenic rats as a consequence of dramatically elevated osteoclastic bone resorption. We hypothesized that this increase in resorption may result from a disruption in B cell OPG production resulting from direct effects of HIV on B cells and/or as a consequence of HIV-induced perturbations to T cells. B cell OPG production was indeed found to be significant diminished in the face of an elevated B cell RANKL production that led to an inverted RANKL/OPG ratio, permissive for enhanced osteoclast formation and activity. Surprisingly, we further identified a dramatic enhancement in the number of osteoclast precursors, a possible consequence of elevated production of macrophage colony stimulating factor (M-CSF), a survival and differentiation factor for cells of the monocyte/macrophage/osteoclast lineage [54]. Taken together this study demonstrated for the first time the potential for HIV-1-induced disruption of the immunoskeletal interface to perturb basal osteoclastogenesis leading to bone loss.

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d) The immuno-skeletal Interface and Osteoclastic Bone Loss Associated with inflammatory Disease Rheumatoid Arthritis (RA) is an inflammatory autoimmune disease that affects ~ 2 percent of the adult population. It is characterized by chronic inflammation in the synovial membrane of affected joints that ultimately leads to loss of daily function due to chronic pain and fatigue. The majority of patients also have deterioration of cartilage and bone in the affected joints, which leads to permanent disability, and increased mortality [55]. In addition, a systemic bone loss is common. RA not only causes destruction of bone and cartilage by immune cell derived factors but also disrupts the systemic control of bone remodeling [56]. Characteristic of RA is the activation of both T- and B-cells and a dense lymphoid infiltration into the synovial membrane. This process is key for both the initiation and progression of the inflammatory state, as well as driving the bone loss associated with RA [55, 57-60]. In RA activated T- and B-cells are potent inducers of osteoclastic bone resorption because they secrete RANKL, the key osteoclastogenic cytokine. In addition, T cells and B cell are known to secrete TNF, a cytokine that not only promotes RANKL production by osteoblast-lineage cells [61], but also amplifies the activity of RANKL [62, 63]. The importance of TNF in the etiology of RA is demonstrated by the fact that transgenic overexpression of TNF in mice leads to bone and joint destruction that closely mimics that of human RA [64, 65]. Pharmacological TNF ablation by contrast alleviates both inflammation and bone loss in TNF transgenic mice [66] and is now an approved therapy for use in human RA patients [67]. Although RANKL is generally considered the final effector of osteoclastogenesis under basal conditions, over a decade ago we reported novel cytokine-like activities in media from activated T cells that potently induced osteoclast formation independently of RANKL [68].

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While the existence of RANKL-independent cytokines has been considered highly controversial, we recently identified a single novel cytokine in T cell conditioned media possessing the capacity to generate functional osteoclasts in the absence of RANKL and in the presence of OPG. We termed this cytokine secreted osteoclastogenic factor of activated T cells (SOFAT) and determined that SOFAT was derived from an unusual mRNA splicevariant coded by the threonine synthase-like 2 (THNSL2) gene homolog. THNSL2 is a gene remnant highly conserved throughout evolution that codes for threonine synthase, an enzyme previously thought to have no function in mammals [69]. While little is known about SOFAT and its potential role in inflammatory bone loss, given that SOFAT, which like RANKL is potently amplified by TNF[69], the potential exists for a defined role in inflammatory diseases such as RA that are characterized by T cell activation.

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e) Role of the Immuno-skeletal interface in Estrogen Deficiency-induced Bone Loss In contrast to the protective role of B cells and T cells in regulating physiological osteoclastogenesis and bone turnover, activated lymphocytes are well-established mediators of inflammatory osteoclastogenesis and bone loss. In fact, elevated osteoclastic bone resorption in the HIV-transgenic model, as discussed above, was in part a consequence of elevated RANKL production by B cells [54]. Production of RANKL by B cells has been reported in human periodontal lesions and in response to bacterial proteins in animal models of periodontitis [70]. B cells and B cell derived plasma cells in multiple myeloma have been reported to have the potential to support osteoclastogenesis, via direct expression of RANKL [71] and/or as an indirect consequence of the secretion of IL-7 [72] a cytokine that we have reported to potently induce RANKL production by T cells [27, 73, 74]. It has long been recognized that B lymphopoiesis is stimulated during estrogen deficiency [75] while estrogen treatment down-regulates B lymphopoiesis, but upregulates immunoglobulin production [76]. B lineage cells have consequently been suggested to play a role in ovariectomy induced bone loss [77]. However, no cause effect relationship between Blymphopoiesis and bone loss in estrogen deficiency has been forthcoming and to further explore this hypothesis we studied the effect of ovariectomy on B cell KO mice. These studies demonstrated that B cell KO mice in fact undergo ovariectomy-induced bone loss that exactly parallels that of WT mice, suggesting that estrogen deficiency-induced bone loss can occur independently of mature B cells [78]. Our studies were not able to exclude a role of immature B cell populations that are still present in B cell KO mice and interestingly, certain early B cell precursors are pluripotent and capable of trans-differentiating into osteoclasts in vitro [27, 79]. These data suggest that estrogen deficiency may expand the reservoir of osteoclast precursors though the trans-differentiation of early B-lineage cells along the monocytic lineage. Like B cells activated T cells are a source of numerous inflammatory cytokines including RANKL. Indeed, one of the major surprises to emerge over the last decade is the important role of T cells in the osteoclastic bone loss associated with estrogen deficiency. Postmenopausal osteoporosis is the archetypal osteoporotic disease of women and is driven in large measure by a deficiency in production of the sex hormone estrogen, following the

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menopause. Production of inflammatory cytokines including IL-1 and TNF have long been recognized to be elevated by human mononuclear cells in postmenopausal humans [80] as well as in ovariectomized mice [81], a surgically induced animal model of postmenopausal osteoporosis. The source of these cytokines, their mode of operation and the complexity of the response have only recently begun to be delineated. Among the first clues for a role of T cells in ovariectomy-induced bone loss was the finding that T cell deficient nude mice are resistant to bone loss associated with ovariectomy [62]. Further studies revealed that T cells were critical to the bone loss associated with estrogen deficiency because of their TNF production. Although RANKL is the final osteoclastogenic effector cytokine, TNFis also capable of promoting osteoclastogenesis though a number of indirect routes. Firstly, TNF is an inducer of RANKL production by osteoblastic cells, secondly it promotes upregulation of the receptor RANK thus converting monocytes into osteoclast precursors and thirdly, it possesses a unique ability to synergize with RANKL thus amplifying its osteoclastogenic activity [62, 63] at the level of the signal transduction machinery [82]. Although T cells are only one source of TNF, the expansion of TNF producing T cell populations during estrogen deficiency drives up concentrations of TNF to a level capable of amplifying RANKL-induced osteoclastogenesis. The importance of T cell derived TNF was demonstrated in studies where T cells from TNF KO mice were reconstituted by adoptive transfer into T cell deficient nude mice. This chimeric mouse had all sources of TNF except those from T cells and in contrast to mice reconstituted with wild type (WT) T cells was resistant to bone loss following ovariectomy [83]. Interestingly, T cell TNF production is a downstream step in a complex cascade of cytokine-driven immunological events. Among the most upstream actions of estrogen deficiency is an increase in reactive oxygen species (ROS) causing an up-regulation of the costimulatory molecule CD80 on dendritic cells [84]. This occurs in parallel with increased production of IGF-1 and a concurrent decline in the immunosuppressive cytokine TGF. Increased IGF-1 and decreased TGF both promote upregulation of IL-7 [3], a potent lymphopoietic cytokine and a master regulator of T cell differentiation, maturation and activity [85]. IL-7 has several actions that likely contribute to osteoclastogenesis including direct promotion of RANKL production by T-cells [27, 73], promoting bone marrow T cell differentiation de novo, enhancement of thymic function and peripheral expansion of mature T cells [86]. IL-7 is further known to increase the sensitivity of T cells to otherwise weak antigenic stimuli, amplifying the antigenic response and driving up T cell activation. In addition, IL-7 promotes production by T cells of the cytokine IFN which is a potent inducer of macrophage class II transactivator (CIITA) protein, a transcription factor that upregulates MHCII expression on macrophages leading to enhanced APC activity and sustained T cell activation and TNF production [3]. Attesting to the importance of IL-7 in this response, pharmacological IL-7 ablation in mice in vivo completely prevents ovariectomy-induced bone loss [87]. While injection of recombinant IL-7 into mice induces pronounced bone loss, injection into T cell deficient mice is ineffective, demonstrating a unique role for T cells as the target of IL-7 induced osteoclastogenesis and bone destruction [27]. This inflammatory model of estrogen deficiency induced bone loss is summarized diagrammatically in Figure 2. The involvement of the adaptive immune system in ovariectomy-induced bone loss raises an interesting specter. Adaptive immune responses center on the capacity of T cells and/or B cells to respond to antigenic stimuli and an intriguing question thus arising from these studies

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is whether there is a unique antigen generated during estrogen deficiency driving T cell activation and if not, what are the antigens involved and where do they come from? Biologically every T cell clone has a unique TCR recognizing a specific antigen. We investigated the issue of whether a specific antigen is necessary for ovariectomy-induced bone loss using the DO11.10 mouse strain in which every T cell expresses an identical transgenic T cell receptor, responsive only to the antigen ovalbumin. Ovalbumin is not endogenously present in rodents and so in this model basal antigen presentation is inactive unless a source of exogenous antigen (in this case ovalbumin) is provided. Interestingly, these mice were found to be resistant to ovx-induced bone loss ratifying the need for an antigenic stimulus. However, after administration of exogenous ovalbumin robust bone loss again ensued in response to ovariectomy demonstrating a requirement for antigen-presentation in ovariectomy-induced bone loss, but also suggesting that no specific type of antigen is needed and that basically any antigen capable of activating T cells can suffice in this role [88].

Figure 2. Inflammatory cascades leading to bone loss in ovariectomized mice. Estrogen deficiency leads to decreases in TGF-β and increases in IGF-1 production driving up IL-7 and promoting T cell activation and expansion. Activated T cells release IFN-γ, which along with elevated ROS promotes antigen presentation by dendritic cells and macrophages though IFN mediated effects on CIITA that drive up MHC class II expression. The net effect is enhanced T cell activation promoting release of the RANKL and TNF. TNF further stimulates RANKL and M-CSF production by osteoblasts and their precursors driving up osteoclast formation. Bone turnover is further disrupted by a negative feedback loop on bone formation where TNF and IL-7 directly blunt bone formation. Adapted from [3].

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Another issue that then arises is the specific nature of the antigens that support bone loss during estrogen deficiency. In fact, it is now recognized that even in perfectly healthy animals and humans there is always a low grade immunological basal response in place, that is mediated by antigenic stimuli which are present as a consequence of chronic exposure to weak self and foreign antigens [89]. Humans and animals are persistently exposed to foreign antigens derived from multiple sources including antigens inhaled into the lungs, peptide products of digestion, and bacterial antigens absorbed in the gut. In addition, there are T cells expressing T cell receptors that recognize weak self-antigens. These T cells escape deletion during thymic section, and accumulate increasingly with advancing age. Together, these foreign and self-antigens all conspire to sustain a weak basal antigenic activity that is necessary for the maintenance of T cell peripheral pools [89, 90]. Estrogen deficiency however appears increase the sensitivity of the immune system to prevailing antigens though upregulation of MHC class II expression on APCs as a consequence of IL-7 driven IFN production and elevated ROS. A systemic increase in IL-7 production further acts to increase the magnitude of T cell response to antigens, in particular weak antigens that are normally tolerogenic and that don’t usually promote significant immune responses now elicit enhanced T cell activation during estrogen deficiency. While further studies are needed, at present no evidence exists for the generation of a unique antigen in estrogen deficiency, but rather ovariectomy appears to cause a general increased sensitivity to prevailing endogenous antigens. While this complex cytokine cascade has been delineated primarily in animal models of postmenopausal osteoporosis, a recent clinical study has reported that women with postmenopausal osteoporosis have a higher T cell activity and increased TNF and RANKL production than healthy post-menopausal subjects. This suggests that T cells contribute to the bone loss induced by estrogen deficiency in humans as is observed in the mouse model [91]. While further clinical studies are needed to ratify these concepts developed in mice, the result of these studies suggest that estrogen deficiency displays significant similarities to an inflammatory state [3].

CONCLUSION Research over the last decade has uncovered a remarkable integration between the skeletal and immune systems. The existence of the immuno-skeletal interface has deep ranging consequences for physiological osteoclast renewal and the maintenance of bone homeostasis and may underlie a plethora of diverse pathological states leading to bone disease. Perturbations in the immune response involve both inflammatory responses (immune activation) leading to T- and/or B-cell production of RANKL, the key osteoclastogenic cytokine. By contrast, immunodeficiency may disrupt basal osteoclastogenesis through alterations in B cell production of OPG, the key physiological antagonist of RANKL. As inflammation is commonly associated with ageing, leading to a generalized inflammatory state referred to as “inflammaging”, skeletal decline would appear to be an inescapable consequence of age-associated immunological changes. Indeed, we recently suggested that postmenopausal osteoporosis may need to be viewed from the context of an inflammatory

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disease [3]. Accumulating evidence now suggests that in general many if not most forms of osteoporosis may need to be viewed from the perspective of an immune disorder. Future antiosteoporotic therapies may ultimately need to take a broader approach involving careful immune rebalancing rather than, or in addition to, the direct anti-osteoclastogenic strategies that form our current standard of care.

ACKNOWLEDGMENTS We gratefully acknowledge research support from the Biomedical Laboratory Research and Development Service of the VA Office of Research and Development (Grant 5I01BX000105), and NIH grants from the NIAMS (grants AR059364, AR056090 and AR053607) and the NIA (grant AG040013). We further acknowledge research support by the Emory Center for AIDS Research (CFAR - NIH Grant P30 AI050409) and the Atlanta Clinical and Translational Science Institute (ACTSI - NIH Grant MO1RR00039).

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[72] Giuliani N, Colla S, Sala R, Moroni M, Lazzaretti M, La Monica S, et al. Human myeloma cells stimulate the receptor activator of nuclear factor-kappa B ligand (RANKL) in T lymphocytes: a potential role in multiple myeloma bone disease. Blood. 2002;100(13):4615-4621. [73] Weitzmann MN, Cenci S, Rifas L, Brown C, Pacifici R. Interleukin-7 stimulates osteoclast formation by up-regulating the T- cell production of soluble osteoclastogenic cytokines. Blood. 2000;96(5):1873-1878. [74] Robbie-Ryan M, Pacifici R, Weitzmann MN. IL-7 drives T cell-mediated bone loss following ovariectomy. Ann N Y Acad Sci. 2006;1068:348-351. [75] Masuzawa T, Miyaura C, Onoe Y, Kusano K, Ohta H, Nozawa S, et al. Estrogen deficiency stimulates B lymphopoiesis in mouse bone marrow. J Clin Invest. 1994;94(3):1090-1097. [76] Erlandsson MC, Jonsson CA, Islander U, Ohlsson C, Carlsten H. Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice. Immunology. 2003;108(3):346-351. [77] Miyaura C, Onoe Y, Inada M, Maki K, Ikuta K, Ito M, et al. Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: similarity to estrogen deficiency. Proc Natl Acad Sci U S A. 1997;94(17):9360-9365. [78] Li Y, Li A, Yang X, Weitzmann MN. Ovariectomy-induced bone loss occurs independently of B cells. J Cell Biochem. 2006;100(6):1370-1375. [79] Sato T, Shibata T, Ikeda K, Watanabe K. Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency. J Bone Miner Res. 2001;16(12):2215-2221. [80] Pacifici R, Brown C, Puscheck E, Friedrich E, Slatopolsky E, Maggio D, et al. Effect of surgical menopause and estrogen replacement on cytokine release from human blood mononuclear cells. Proc Natl Acad Sci U S A. 1991;88(12):5134-5138. [81] Kitazawa R, Kimble RB, Vannice JL, Kung VT, Pacifici R. Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice. J Clin Invest. 1994;94(6):2397-2406. [82] Zhang YH, Heulsmann A, Tondravi MM, Mukherjee A, Abu-Amer Y. Tumor Necrosis Factor-alpha (TNF) Stimulates RANKL-induced Osteoclastogenesis via Coupling of TNF Type 1 Receptor and RANK Signaling Pathways. J Biol Chem. 2001;276(1):563568. [83] Roggia C, Gao Y, Cenci S, Weitzmann MN, Toraldo G, Isaia G, et al. Up-regulation of TNF-producing T cells in the bone marrow: A key mechanism by which estrogen deficiency induces bone loss in vivo. Proc Natl Acad Sci U S A. 2001;98(24):1396013965. [84] Grassi F, Tell G, Robbie-Ryan M, Gao Y, Terauchi M, Yang X, et al. Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation. Proc Natl Acad Sci U S A. 2007;104(38):15087-15092. [85] Fry TJ, Mackall CL. Interleukin-7: master regulator of peripheral T-cell homeostasis? Trends Immunol. 2001;22(10):564-571.

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[86] Ryan MR, Shepherd R, Leavey JK, Gao Y, Grassi F, Schnell FJ, et al. An IL-7dependent rebound in thymic T cell output contributes to the bone loss induced by estrogen deficiency. Proc Natl Acad Sci U S A. 2005;102(46):16735-16740. [87] Weitzmann MN, Cenci S, Roggia C, Toraldo G, Weitzmann L, Pacifici R. Increased production of IL-7 uncouples bone formation from bone resorption during estrogen deficiency. J Clin Invest. 2002;110(11):1643-1650. [88] Cenci S, Toraldo G, Weitzmann MN, Roggia C, Gao Y, Qian WP, et al. Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator. Proc Natl Acad Sci U S A. 2003; 100(18):10405-10410. [89] Surh CD, Sprent J. Homeostasis of naive and memory T cells. Immunity. 2008;29(6):848-862. [90] Tanchot C, Lemonnier FA, Perarnau B, Freitas AA, Rocha B. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science. 1997; 276(5321):2057-2062. [91] D'Amelio P, Grimaldi A, Di Bella S, Brianza SZ, Cristofaro MA, Tamone C, et al. Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: A key mechanism in osteoporosis. Bone. 2008;43(1):92-100.

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Chapter 7

MULTIPLE FUNCTIONS OF OSTEOCLASTS AND POTENTIAL USEFULNESS OF PHOSPHATIDYLSERINE-CONTAINING LIPOSOMES ON BONE DISEASES Zhou Wu* and Hiroshi Nakanishi† Department of Aging Science and Pharmacology, Faculty of Dental Science, Kyushu University, Fukuoka, Japan

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Osteoclasts (OCs) are well known as the bone resorption cells in normal bone remodeling and pathological bone loss by increasing their number and resorptive activity. They are derived from myeloid osteoclast precursors (OPs) under the influence of the surrounding cells. There is growing evidence that OCs have multiple functions besides bone resorption. OCs secrete mediators to work as functional antigen-presenting cells in immune responses, affect angiogenesis by regulating the function of capillaries’ endothelial cells, and regulate hematopoietic stem cell (HSCs) functions. Furthermore, OCs can phagocytoze apoptotic bone cells. Phagocytosis of apoptotic cells causes phagocytes to secrete anti-inflammatory mediators to control their functions in autocrine and paracrine manners. Posphatidylserine-containing liposomes (PSLs) are known to mimic the effects of apoptotic cells on phagocytes. We have found that phagocytosis of PSLs by OPs can regulate their secretion of anti-inflammatory mediators, including prostaglandin E2 and transforming growth factor-1, to inhibit OCs formation and inflammatory bone loss. In addition, PSLs promote the maturation of osteoblasts. Therefore, PSLs may provide potential pharmacological interventions against bone diseases through the regulation of these multiple functions of OCs.

* †

To whom correspondence should be addressed. E-mail: Zhouw@ dent.kyushu-u.ac.jp. To whom correspondence should be addressed [email protected].

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INTRODUCTION Apoptotic cells provide an “eat me signal” to phagocytes through exposure of phosphatidylserine (PS) from inner to the outer leaflet of the plasma membrane in the early stage of apoptotic process [Krahling, et al., 1999; Verhoven, et al., 1999]. After phagocytosis of apoptotic cells, phagocytes secrete anti-inflammatory mediators to suppress immune and inflammatory responses [Hoffmann, et al., 2001; Huynh, et al., 2002]. Furthermore, PScontaining liposomes (PSLs) have been found to mimic the effects of apoptotic cells on phagocytes. PSLs induce the production and secretion of anti-inflammatory mediators including prostagrandin E2 (PGE2) after phagocytozed by microglia and macrophages [Zhang et al., 2006; Shi et al., 2007; Wu et al., et al., 2010a; Wu & Nakanishi, 2010b]. It has been reported that osteoclasts (OCs), multinucleated cells derived from myeloid osteoclast precursors (OPs), can phagocytoze apoptotic bone cells [Boabaid et al., 2001; Cerri, P et al., 2003]. These observations prompted us to further examine the effects of PSLs on OCs and OPs. We have found that OPs phagocytoze PSLs to suppress OCs formation and inflammatory bone loss through the production and secretion of transforming growth factorβ1 (TGF-β1) and PGE2 [Wu et al., 2010a; Ma et al., 2011; Wu & Nakanishi, 2011]. Although OCs have long been recognized as specialized cells that are involved in both physiological bone remodeling and pathological bone destruction, the growing evidence suggests that OCs have multiple functions besides bone resorption. For example, both OCs and OPs secrete mediators to work as functional antigen-presenting cells (APCs) in immune responses [Li, et al., 2010; Grassi, et al., 2011], directly affect bone angiogenesis by regulating the function of capillaries’ endothelial cells [Pritzker, et al., 2004; McGonigle, et al., 2009: Cackowski, et al., 2010], and regulate hematopoietic stem cell (HSCs) maintenance and mobilization [Kollet, et al., 2006; Lymperi, et al., 2011; Miyamoto, et al., 2011]. Therefore, PSLs may have several broad pharmacological interventions, including the regulation of immune responses, bone angiogenesis and HSCs regulation, besides the inhibition of OCs formation and inflammatory bone loss. This chapter first summarizes recent findings of multiple functions of OCs, and then discusses the possible clinical usefluness of PSLs on bone diseases.

IMMUNE FUCTIONS OF OCS It is becoming clear that OCs can work as immune cells [Takayanagi, 2007; Xing et al., 2005]. Similar to macrophages, OCs can secrete immune regulators, including tumor necrosis factor-α (TNF-α, interleukin (IL)-6, IL-1β, IL-10, TGF-β1 and PGE2 to affect the surrounding cells as well as themselves [Blaine TA, et al., 1996; Li et al., 2010; Wu et al., 2010a; Ma et al., 2011]. Although T cells are known as the critical regulators of OCs through their production of receptor activator for nuclear factor κB ligand (RANKL) production, recent observations have revealed that OCs can also regulate the functions of T cells [Kiesel et al., 2009; Li et al., 2010; Grassi, et al., 2011]. OCs express MHC molecules (HLA-ABC and HLA-DR), CD80, CD86 and CD40 after stimulation with RANKL and macrophagecolony stimulating factor (M-CSF). These findings indicate that OCs can work as the functional APCs to activate T cells [Li et al., 2010; Grassi et al., 2011]. Moreover, OCs can

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secrete high levels of most T cell chemoattractants, such as chemokine C-C ligand (CCL) 2, CCL3, CCL4 and C-X-C chemokine 10 (CXCL10), to effectively retain T cell adhesion. OCs also inhibit proliferation and apoptosis of T cells and suppress their production of TNF-α and interferon--γ (IFN-γ). These observations strongly suggest that OCs have an ability to engage T cells in an antigen-dependent fashion [Grassi et al., 2011]. More interestingly, OCs can secret higher amounts of IL-10 than dendritic cells (DCs) upon stimulation with lipopolysaccharide (LPS) and stimulate T cells to secrete more IL-10 than IFN-γ, suggesting that OCs may promote T-cell immunity and tolerance [Li et al., 2010]. Therefore, OCs can be targeted for the regulation of immune responses.

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ANGIOGENIC FUNCTIONS OF OCS Angiogenesis is important in physiological and pathological processes in bone, including bone development, fracture healing, inflammatory bone loss, and bone tumors [Bakre et al., 2002; Findlay & Haynes, 2005; Ribatti et al., 2006]. Endothelial cells are known to stimulate OCs formation through the expression of RANKL and the recruitment of OPs [Pritzker et al., 2004; McGonigle et al., 2009]. However, recent preclinical reports as well as clinical observations have noted that OCs may also play a role in stimulating angiogenesis. It is likely that OCs affect angiogenesis because OCs localize close proximity to capillaries. OCs form a resorption cavity or cutting cone, into which a capillary then invades [Gartner & Hiatt, 2001]. Clinically, both osteoclastogenesis and angiogenesis are enhanced under pathologic conditions, such as multiple myeloma, bone metastases and rheumatoid arthritis [Findlay & Haynes 2005; Ribatti et al., 2006]. One observation of OCs on angiogenesis in vitro has shown that the conditioned media (CM) from cultured human OCs stimulate angiogenesis and that co-culture of OCs with myeloma cells produced more angiogenesis [Tanaka et al., 2007]. The angiogenic factors gene expression profiles research in OCs further helps to understand their involvement in angiogenesis. Most of angiogenic factors, such as vascular endothelial growth factor (VEGF)-A,VEGF-C, insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor (PDGF)-A are increased with OCs differentiation [Kiesel et al., 2007; Cappellen et al., 2002; Kubota et al., 2003]. However, the bone marrow (BM) cultures contained other cell types besides OPs and OCs, and RANKL, which is added to induce osteoclastogenesis in the cultures, can increase angiogenesis [Kim et al., 2001]. OCs may also stimulate angiogenesis through release of bone matrix-bound growth factors. Bone matrix is a rich source of growth factors, such as PDGF, acidic fibroblast growth factor (FGF), bacis FGF, and TGF-β [Hauschka et al., 1986; Seyedin et al., 1986]. Furthermore, OPs and OCs can produce TGF-β by themselves [Wu et al., 2010a; Li et al., 2010]. The in vivo evidence has shown that the increase of OC formation by both parathyroid hormone related protein (PTHrP) and RANKL stimulates angiogenesis [Bakre, et al., 2002]. Recently, it has been clarified that OCs contribute to angiogenesis through a matrix metalloproteinase-9 (MMP-9)-dependent mechanism. The angiogenic and bone-resorptive effects of PTHrP and RANKL are reduced by MMP-9-deficiency [Cackowski et al., 2010]. Better understanding the functions of OCs in angiogenesis provides a new insight in controlling angiogenesis because angiogenesis is important in physiological and pathological processes in bone.

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HSC REGULATION FUNCTION OF OCS

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In clinically, failure of OCs formation or function leads to the development of osteopetrosis in humans and other mammals is associated with a reduction in the volume of hematopoietic marrow and extramedullary hematopoiesis [Tolar et al., 2004]. OCs are implied as necessary for the proliferation and turnover of HSC [Kollet et al., 2006]. HSC mobilization is associated with the increased numbers of OCs on bone surfaces, RANKLinduced the increase of OCs formation in vivo and the accompaning of HSCs and their progenitors mobilization along with the increase of MMP-9 and cathepsin K in OCs because these enzymes can cleave membrane-bound kit ligand, a growth and adhesion factor for HSCs. Furthermore, RANKL decreases the expression of kit ligand in osteoblasts (OBs), which affects HSCs numbers. Others have shown that reduction of HSCs numbers are parallels with inhibition of OCs function in vivo, and HSCs are detrimental with the inhibition of OCs differentiation, indicating that OCs function is fundamental in the HSC niche [Lymperi, 2011]. These are consistent with the findings obtained from osteopetrotic mice, in which the severe reduction of BM space results in extramedullary hematopoiesis [WiktorJedrzejczak et al., 1982]. Moreover, OCs can remove “old” OBs and recruit “new” active matrix producing OBs for supporting HSCs. However, others have the opposite opinion that OCs are dispensable for HSCs maintenance and mobilization, because HSC progenitors mobilization was comparable in osteopetrotic mice, in which OCs and, by consequence, BM cavities are absent after granulocyte colony-stimulating factor injection. Furthermore, pharmacological ablation of OCs did not inhibit HSC progenitor’s mobilization [Miyamoto, 2011]. OCs are accelerated with age, resulting in decreased bone mass [Teitelbaum, 2007]. Hematopoietic activity also decreases with age (Geiger & Rudolph, 2009; Waterstrat & Van Zant, 2009). Bone aging-caused reduction of bone mass and hematopoiesis implies that OCs may function as negative regulator in the hematopoietic system. Therefore, the precise function of OCs in the niche of the HSC pool needs to be clarified in further studies.

EFFECTS OF PSLS ON OCS PSLs can mimic the effects of apoptotic cells on phagocytes to shift phagocytes from the pro-inflammatory to the anti-inflammatory phenotype [Otsuka et al., 2004; Zhang et al., 2006; Shi et al., 2007; Wu & Nakanishi, 2010b; Ma et al., 2011]. We have found that OPs phagocytoze PSLs to induce the production of TGF-β1 and PGE2 [Wu et al., 2010a]. PGE2 strongly inhibits the human OCs differentiation through activation of EP2 and EP4 receptors [Take et al., 2005]. The CM of human blood CD14+ cells pretreated with PGE2 inhibits RANKL-induced OCs differentiation also in murine culture sysytems. Our findings show that a low concentration of PGE2 (500 pg/ml) significantly inhibits OCs differentiation in rat culture systems. The inhibitory effect of PGE2 on OCs differentiation is significantly suppressed by SQ22536, an adenylate cyclase inhibitor, further indicating the involvement of EP2 and/or EP4 receptors in the inhibitory effect of PGE2 on OCs differentiation [Tanaka et al., 2004; Take et al., 2005]. Both PGE2 and TGF-β1 suppress the expression of RANKL and/or RANK in the bone marrow cells, essential molecules for OCs differentiation in the early stage (Kong et al. 1999). Furthermore, both PGE2 and TGF-β1 suppress the expression

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of ICAM-1 and CD44 in OPs, the important adhesion molecules for OPs fusion [Harada et al., 1998; Tani-Ishii et al., 2002; Nakamura et al., 1995]. Moreover, PSLs significantly inhibit the trabecular bone loss in rat adjuvant arthritic model of rheumatoid arthritis, and the inhibitory effects of PSLs on bone loss are reversed by anti-TGF-β1 antibody as well as SQ22536 [Wu et al., 2010a].

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Figure 1. Schematic of the PSLs inhibitory effects on bone resporption, immune responses, angiogenesis and hematopoietic stem cells (HSCs) regulation through the inhibition of osteoclasts (OCs) differentiation. PSLs can be phagocytozed by osteoclast precursors (OPs), to promote them secrecting soluble factors, including PGE2 and TGF-. PSLs-induced the secretion of PGE2 and TGF down-regulates RANKL/RANK singling and the ICAM-1/CD44 expression for the fusion and differentiation of OPs fusion, in turn to inhibit the OCs maturation.

Our ongoing studies further show that PSLs enhance the expression of alkaline phosphatase and osteocrin to promote mineralization in rat primary cultured OBs. Furthermore, PSLs increase the expression of Runt-related factor-2, one of the master regulators of osteogenesis [Wu et al., unpublished data]. The effects of PSLs on OBs maturation are mediated partly by PGE2, because PSLs increase PGE2 production in whole bone marrow cells as well as OPs [Wu et al., 2010a]. Our findings suggest that PSLs may not only inhibit OCs differentiation, but also facilitate OBs maturation. Therefore, it may be concluded that PSLs can restore the balance between bone formation by OBs and bone resorption by OCs.

EFFECTS OF PSLS ON MACROPHAGES/MIROGLIA Increasing evidence shows that PGE2 negatively regulates the production of proinflammatory mediators including TNF-α by activated macrophages [Rouzer et al., 2004; Chae et al., 2008]. PGE2 also attenuates LPS-induced expression of chemokines including monocyte chemoattractant protein-1, IL-8, CCL3 and CCL4, and selective EP4 receptor antagonist completely reversed PGE2-induced suppression of the chemokine production [Takayama et al., 2002]. Moreover, EP4RAG, a selective EP4 receptor agonist, suppresses the production of pro-inflammatory cytokines and chemokines by inhibiting the activation of NF-κB [Ogawa et al., 2009]. Therefore, an inhibitory effect of PGE2 on the production of proinflammatory mediators by macrophages is mediated through the suppression of NF-κB activation [Takayama et al., 2002; Ogawa et al., 2009]. On the other hand, PGE2 positively

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regulates the production of anti-inflammatory cytokines by activated macrophages. PGE2 enhances the IL-10 production by cultured macrophages in a cAMP-dependent manner [van der Pouw Kraan et al., 1995] and PGE2 is responsible for the increased production of IL-10 by LPS-stimulated peritoneal macrophages in pristane-induced lupus mice [Chae et al., 2008]. PSLs can mimic the effects of apoptotic cells on macrophages. A series of studies has shown that PSLs strongly reduce the release of IL-1β and TNF-α by LPS-activated microglia and macrophages [De et al., 2002]. We have found recently that PSLs can regulate the phenotype of infiltrated macrophages in vivo using the inflammatory bone loss model in adjuvant arthritic (AA) rats. Our observations show that approximately half of the infiltrated macrophages into the ankle joints of AA changed from IL-1β-producing to IL-10-producing cell phenotype after phagocytosis of PSLs [Ma et al., 2011]. At the same time, PGE2 production was increased in serum after phagocytosis of PSLs [Wu et al., 2010a]. PSLs induce the phenotypic change of infiltrated macrophages and are dependent on regulating the activities of p38 MAPK and ERK. The phenotypic changes therefore result in inhibiting inflammatory bone loss [Ma et al., 2011]. In our previous study, microglia rapidly produce PGE2 after phagocytosis of PSLs. Furthermore, high-density lipoprotein, a specific ligand for class B scavenger receptor type I (SR-BI), significantly suppressed both phagocytosis of PSLs and the subsequent PGE2 production by microglia though the COX-1/microsomal prostaglandin E synthase-2 pathway [Zhang et al., 2006].

Figure 2. Schematic of the PSLs regulatory effects in differentiation and functions of myeloid derived phagocytes. PSLs. inhibit the maturation of osteoclasts (OCs) and dendritic cells (DCs). PSLs drive macrophages, microglia and DCs form pro-inflammatory phenotype toward anti-inflammatory phenotype during immune response by regulating their cytokine production. M: macrophages.

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EFFECTS OF PSLS ON DCS DCs play major roles in immune responses and tolerance. Activated DCs can produce large amounts of PGE2 to modulate their functions in an autocrine manner [Harizi et al., 2002]. PGE2 inhibits the release of TNF-α [Vassiliou et al., 2003], CCL3 and CCL4 from LPS-stimulated DCs [Jing et al., 2003]. On the other hand, PGE2 enhances the IL-10 production by BD derived-DCs. Moreover, other report shows that PGE2 released from mesenchymal stem cells (MSCs) inhibit DCs maturation and function [Spaggiari et al. 2009]. PSLs have been shown to decrease the up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD83, as well as the production of IL-12 by LPS-stimulated human DCs. DCs diminish capacity to stimulate allogeneic T cell proliferation and to activate IFN-γproducing CD4+ T cells after exposure to PSLs [Chen et al., 2004]. Recently, in vivo study also demonstrates that PSLs reduce the expression of MHC II by DCs. Furthermore, PSLs inhibit the IL-12 production but increase the IL-10 production by DCs [Harizi, 2002; Shi et al., 2007]. Therefore, the effects of PSLs on DCs may be mediated by the PGE2 production. However, little information is available about the involvement of PGE2 on the effects of PSLs on DCs,

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CONCLUSION As mentioned above, OPs and OCs are the multiple functional cells in regulating T cells and immune response, angiogenesis and HSCs maintenance and mobilization. Because other myeloid derived macrophages and DCs also participate in pathological bone loss, myeloid derived phagocytes including OCs are considerable as the target of bone diseases. PSLs can effectively regulate the maturation of OCs and other myeloid derived phagocytes and shift them from pro- to anti-inflammatory phenotype during immune response. Because PS is a component of mammalian cell membranes, PSLs can be used as potential pharmacological interventions against bone diseases without any deleterious side effects.

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Chapter 8

BONE FORMATION AND OSTEOCLASTIC RESORPTION AFTER IMPLANTATION OF -TRICALCIUM PHOSPHATE (-TCP) Takaaki Tanaka*, Masaaki Chazono, Seiichiro Kitasato, Atsuhito Kakuta, and Keishi Marumo NHO Utsunomiya National Hospital Department of Orthopaedic Surgery, Jikei University School of Medicine, Japan

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The mechanism of bone substitute resorption involves two processes: solutionmediated disintegration and cell-mediated disintegration. An example of the first process is calcium sulfate resorption. In our previous studies, the main -tricalcium phosphate (TCP) resorption process was considered to be cell-mediated disintegration by tartrateresistant acid phosphatase (TRAP)-positive cells. Thus, osteoclast-mediated resorption of -TCP may be important for enabling bone formation. In order to address a role of osteoclast in -TCP resorption deeply, we used two different experimental models. 1. Cylindrical -TCP blocks with 75% porosity were implanted in rabbit cancellous bone defects with or without bisphosphonate treatment. 2. -TCP blocks with or without bisphosphonate treatment were soaked with bone marrow cells obtained from femora of a 6-week-old Fisher rat, and were implanted into 12-week-old Fisher rats subcutaneously. The results showed that local application of bisphosphonate reduced the number of osteoclasts on the surface of -TCP. Inhibition of osteoclast formation resulted in reducing -TCP resorption and bone formation. Thus, these results suggest that osteoclast-mediated resorption plays an important role in -TCP resorption and bone formation.

*

E-mail:[email protected], 2160 Shimo-Okamoto, Utsunomiya city, Tochigi 329-1193, Japan, FAX:+81 28-673-9117.

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INTRODUCTION Zoledronate (ZOL) and alendronate (ALN), nitrogen-containing bisphosphonates, are widely used drugs for diseases associated with bone resorption (1). ZOL is originally used to treat metastatic bone diseases and has recently been used for osteoporosis (2). In addition, it has also been used in many orthopedic fields. Local and systemic application of ZOL or ALN has been shown to enhance screw fixation and prevent prosthetic loosning (3-5) Recently, bone substitute materials have been advocated as alternatives to autografts and allografts. In particular, calcium phosphate compounds have received wide attention because of the close chemical and crystal resemblance of these materials to bone mineral. Hydroxyapatite is used as a bone substitute because of its excellent biocompatibility and osteoconductive properties (6). However, hydroxyapatite has several disadvantages, such as slow biodegradation and no progressive bone formation during bone repair (7).-TCP has recently received considerable attention as a bone graft substitute because of its biocompatibility and biodegradability (8-11). The mechanism of -TCP resorption involves solution-mediated disintegration and cell-mediated disintegration. We previously reported that osteoclasts play a major role in bioresorption of -TCP (8, 12). Thus, resorption of TCP is important for bone formation. The aim of this study was to determine whether local ZOl administration would inhibit osteoclastic resorption of -TCP and promote bone formation.

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MATERIALS AND METHODS The cylindrical -TCP blocks used in this study were provided by Olympus Terumo Biomaterials Co. (Tokyo, Japan). -TCP was synthesized using a mechanochemical method (wet milling). Briefly, CaHPO4/H2O and CaCO3, at a molar ratio of 2:1, were mixed into a slurry with pure water and particles of zirconium in a pot mill for 24 hours and dried at 80°C. Calcium-deficient hydroxyapatite was converted to -TCP by calcination at 750°C for one hour. After sintering of the -TCP powder at 1050°C for one hour, a porous -TCP block, with a mean pore size of 200 μm and a porosity of 75%, was obtained. The block was cut into cylinders (diameter, 4 mm; length, 5 and 10 mm)

Experiment 1 Cylindrical -TCP blocks (4 mm in diameter, 10 mm in length) were immersed in ZOL solutions (Novartis Pharma Co. Tokyo, Japan) at 10-4 M for 2 days. Excess ZOL was then removed with sterilized filter paper. New Zealand White rabbits weighing 3.0 to 3.2 kg were used. Under intravenous pentobarbital and general isoflurane anesthesia, bilateral cylindrical bone defects (4.1 mm in diameter, 10 mm in length) were created by drilling in the lateral aspect of the distal femur. Cancellous bone cavities were filled with ZOL-treated -TCP blocks. Defects filled with a -TCP block without ZOL treatment served as a control (Fig. 1). After surgery, all animals were allowed to move freely in their cages without joint immobile-

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zation. Rabbits were sacrificed at 2 weeks postoperatively, and the distal part of the femur was removed and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS).

Figure 1. A procedure of the experiment 1. A cylindrical β-TCP block with 75% porosity (A) was implanted in the bone defect (B) created in the rabbit femur. The arrow indicates a 4.1 mm in diameter defect.

After decalcification in 0.4M ethylenediaminetetraacetic acid (EDTA) for 2 weeks, serial histological sections were cut to include patellar groove. Decalcified sections were obtained for hematoxylin-eosin (HE) and TRAP staining.

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Experiment 2 Cylindrical -TCP blocks (4 mm in diameter, 5 mm in length) were treated with ZOL in the same manner as the experiment 1. Bone marrow cells were obtained from femora of a 6week-old Fisher rat (Fig. 2).

Figure 2. Bone marrow cells were obtained from femora of a 6-week-old Fisher rat. The arrow indicates marrow cells. Osteoclasts: Morphology, Functions and Clinical Implications : Morphology, Functions and Clinical Implications, edited by Alexander J. Brown, and

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The ZOL-treated -TCP blocks were soaked with the bone marrow cells, and were implanted into 12-week-old Fisher rats subcutaneously. The implanted -TCP blocks were harvested at 6 weeks. Decalcified sections were prepared in the same manner as the experiment 1. The care and use of the animals in this study were in accordance with the guidelines of the Laboratory Animal Facilities of The Jikei University School of Medicine.

RESULTS Experiment 1

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In the control defects without ZOL treatment, newly formed bone was already found and was in direct contact with -TCP at 2 weeks. Osteoblasts were present on the newly formed bone in a monolayer (Fig. 3A). Numerous TRAP-positive cells with multinucleate were found on the surface of -TCP, but some of them were also present on the surface of the newly formed bone (Fig. 3B).

Figure 3. Decalcified histological sections stained with HE 2 weeks after implantation of the β-TCP block (A). The control defect without ZOL treatment showed that new bone formation was found in the whole area of the β-TCP-implanted defect. Decalcified sections stained with TRAP two weeks after implantation of the β-TCP block (B). Numerous TRAP-positive cells were found on the surface of the β-TCP. The arrows indicate TRAP-positive cells.

In contrast, in the ZOL-treated defects, new bone formation was completely inhibited, but soft tissue was invaded and vessel formation was occurred within the -TCP block (Fig. 4). Only a small amount of bone formation was found in the periphery (Fig. 5). The number of TRAP-positive cells was also inhibited by ZOL at concentrations of 10-4 M (Fig. 6).

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Figure 4. HE staining of ZOL-treated β-TCP blocks (A) (Original magnification, x40).

Figure 5. Higher magnification of the boxed area in Fig. 4. A very small amount of new bone was found in the periphery of the defect. The arrows indicate new bone.

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Figure 6. Decalcified sections of ZOL-treated defects stained with TRAP. No TRAP-positive cells were found, but soft tissue and vessel formation was occurred within the β-TCP block

Experiment 2

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 -TCP blocks with allogenic rat bone marrow cells enabled marked bone formation 6 weeks after implantation subcutaneously (Fig.7A). TRAP-positive cells were present on the surface of -TCP and new bone (Fig. 7B).

Figure 7. HE staining of the β-TCP block with rat bone marrow cells 6 weeks after implantation. Marked new bone formation was found within the β-TCP block (A). TRAP staining of serial histological sections (B). TRAP-positive cells were present on the surface of TCP and new bone. The arrows indicate TRAP-positive cells. NB indicates new bone.

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In contrast, no bone formation was detected in the -TCP blocks without implantation of bone marrow cells (data not shown). The inhibitory effects of ZOL on bone formation and TRAP-positive cells were very similar to the experiment 1 (Fig. 8).

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Figure 8. HE staining of ZOL-treated β-TCP blocks with rat bone marrow cells. No bone formation was found but soft tissue was formed within the β-TCP blocks.

DISCUSSION ZOL, a nitrogen-containing third generation bisphosphonate, is widely used in therapy of bone loss associated with osteoclast-mediated bone resorption. Osteoclasts play a crucial role of in the development of osteoporosis, mediated through the osteoprotegrin/RANK/RANKLL signaling system (13). Classic bisphosphonate treatment is by the systemic way of oral administration or intravenous injection. Recently, local administration was attempted. Calcium phosphate ceramics, such as hydroxyapatite and β-TCP, can be a good candidate as a carrier for bisphosphonate delivery. Peter et al. (14) determined that 2 days of treatment with ZOL coating on titanium implants would decrease peri-implant osteolysis. The results showed a positive concentration-dependent effect on the peri-implant bone density. Faucheux et al. (15) reported that ZOL released from a complex of calcium phosphates inhibited osteoclastic resorption without affecting osteoblasts in vitro. The present study showed that the number of osteoclasts was inhibited, but bone formation was also inhibited within the βTCP blocks with ZOL-treatment for 2 days. The mechanism of -TCP resorption has been investigated, and is thought to involve both solution- and cell-mediated disintegration. The former process had been considered as a main process. Zerbo et al. (16) reported that mononucleate and binucleate TRAP-positive cells, but

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no multinucleate TRAP-positive cells, were found at the surface of the -TCP particles in biopsy specimens obtained from 6 months after implantation. The lack of large multinucleate TRAP-positive cells suggested that resorption of the -TCP by osteoclasts played only a minor role in its replacement by bone; thus, chemical dissolution seemed the predominant cause of -TCP degradation. However, our previous electron microscopic study (17) has shown that giant cells with two to five nuclei and ruffled borders were in contact with -TCP. In addition, the present study showed that most of TRAP-positive cells were multinucleated. Thus, the resorption of -TCP occurs mainly through cell-mediated disintegration. The results obtained from the experiment 2 showed that bone marrow cells induced both osteoblast and osteoclast differentiation. These results suggested that a coupling-like phenomenon could be occurring in the -TCP-filled bone defects. Thus, osteoclast-mediated resorption of -TCP is essential for enabling bone formation. The present study also showed that locally administered ZOL reduced the number of osteoclasts on the surface of TCP blocks. A very small amount of new bone was found in the periphery of the defect. This may be due to blood flow from the periphery. Locally administered ZOL inhibited not only TRAP-positive cells, but also new bone formation within the -TCP-TCP block. Inhibition of osteoclast formation resulted in a decrease of TCP replacement by bone. In our previous study (8), the amount of newly formed bone reached a peak at 4 weeks after implantation of -TCP, and was higher than that of the surrounding bone. Excessive new bone was resorbed by osteoclasts similar to the healing process found in fracture healing. TRAP-positive cells were present not only on the surface of -TCP, but also on the surface of new bone. ZOL can affect osteoclasts resorbing -TCP as well as new bone. However, the number of cells on the surface of -TCP was much larger than those on the newly formed bone, which resulted in masking the inhibitory effect of bone resorption.

CONCLUSION We investigated the effects of ZOL on osteoclastic resorption of -TCP and bone formation using two experimental models. The results showed that the inhibitory effects of ZOL on -TCP resorption resulted in bone formation, suggesting that osteoclast-mediated resorption of -TCP is essential for bone formation in the -TCP implanted sites.

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Luckman SP, Hughes DE, Coxon FP, Russell RGG, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 1988; 13: 581589. Lyles KW, Colon-Emeric CS, Magaziner JS, Adachi JD, Pieper CF, Mautalen C, Hyldstrup L, Recknor C, Nordsletten L, Moore KA, Lavecchia C, Zhang J, Mesenbrink P, Hodgson PK, Abrams K, Orloff JJ, Horowitz Z, Eriksen EF, Boonen S. Zoledronic

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acid and clinical fractures and mortality after hip fracture. NEJM 2007; 357: 17991809. Moroni A, Faldini C, Hoang-Kim A, Pegreffi F, Giannini S. Alendronate improves screw fixation in osteoporotic bone. J Bone Jt Surg 2007; 89A: 96-101. Venesmaa PK, Kroeger HP, Miettinen HJ, Jurvelin JS, Suomalainen OT, Alhav EM. Alendronate reduces periprosthetic bone loss after uncemented primary total hip arthroplasty: a prospective randomized study. J Bone Miner Res 2001; 16: 2126-2131. Friedl G, Radl R, Stihsen C, Rehak P, Aigner R, Windhager R. The effect of a single infusion of zoledronic acid on early implant migration in total hip arthroplasty. A randomized, double-blind, controlled trial. J Bone Joint Surg 2009; 91A: 274-281. Kitsugi T, Yamamoto T, Nakamura T, Kotani S, Kokubo T, Takeuchi H. Four calcium phosphate ceramics as bone substitutes for non-weight bearing. Biomaterials 1993; 14: 216-224. Hoogendoorn HA, Renooji W, Akkermans LMA, Visser DDS, Wittebol P. Long-term study of large ceramic implants in dog femora. Clin Orthop 1984; 187: 281-288. Chazono M, Tanaka T, Komaki H, Fujii K. Bone formation and bioresorption after implantation of beta-tricalcium phosphate granules-hyaluronate complex in rabbit bone defects. J Biomed Mater Res 2004; 15: 542-549. Tanaka T, Chazono M, Komaki H. Clinical application of beta-tricalcium phosphate in human bone defects. Jikeikai Med J 2006; 53: 23-31. Komaki H, Tanaka T, Chazono M, Kikuchi T. Repair of segmental bone defects in rabbit tibiae using a complex of beta-tricalcium phosphate, type I collagen, and fibroblast growth factor-2. Biomaterials 2006; 27: 5118-26. Tanaka T, Kumagae Y, Saito M, Chazono M, Komaki H, Kikuchi T, Kitasato S, Marumo K. Bone formation and resorption in patients after implantation of betatricalcium phosphate blocks with 60% and 75% porosity in opening wedge high tibial osteotomy. J Biomed Mater Res Part B: Appl Biomater 2008; 86B:453-9. Tanaka T, Saito M, Chazono M, Kumagae Y, Kikuchi T, Kitasato S, Marumo K. Effects of alendronate on bone formation and osteoclastic resorption of beta-tricalcium phosphate. J Biomed Mater Res 2010; 93A:469-474. Romas E. Bone loss in inflammatory arthritis:mechanism and therapeutic approaches with bisphosphonates. Best Pract Res Clin Rheumatol 2005; 19:1065-1079. Peter B, Pioletti DP, Laib S, Bujoli B, Pilet P, Janvier P, Guicheux J, Zambelli PY, Bouler JM, Gauthier O. Calcium phosphate drug delivery system: influence of local zoledronate release on bone implant osteointegration. Bone 2005; 36:52-60. Faucheux C, Verron E, Soueidan A, Josse S, Arshad MD, Janvier P, Pilet P, Bouler JM, Bujoli B, Guicheux J. Controlled release of bisphosphonate from a calcium phosphate biomaterial inhibits osteoclastic resorption in vitro. J Biomed Mater Res 2009; 89A: 46-56. Zerbo IR, Bronckers ALJJ, De Lange G, Burger EH. Localisation of osteogenic and osteoclastic cells in porous β-tricalcium phosphate particles used for human maxillary sinus floor elevation. Biomaterials 2005; 26:1445-1451. Chazono M, Tanaka T, Kitasato S, Kikuchi T, Marumo K. Electron microscopic study on bone formation and bioresorption after implantation of beta-tricalcium phosphate in rabbit models. J Orthop Sci 2008; 13: 550-555.

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In: Osteoclasts Editors: A. J. Brown and J. S. Walker

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Chapter 9

OSTEOCLASTS OF PATIENTS WITH NEUROFIBROMATOSIS 1 (NF1) Eetu Heervä and Juha Peltonen University of Turku, Department of Cell Biology and Anatomy, Turku, Finland

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ABSTRACT Neurofibromatosis 1 (NF1) is an autosomal dominant neuro-skeletal cutaneous syndrome with an incidence of around 1/3000. Low bone mineral density (BMD) and osteoporosis/osteopenia are often associated with NF1. Bone dynamics include continuous bone formation and bone resorption, and imbalance in bone turnover may lead to low BMD. Bone is resorbed by osteoclasts which have been characterized in NF1 using peripheral blood-derived osteoclast differentiation assays. Peripheral blood mononuclear cells were isolated from patients with NF1, age, and gender-matched controls, and these cells were cultured into mature osteoclasts. The results showed that NF1 osteoclasts are more numerous, resorb larger amounts of bone, and display aberrant morphology compared to controls. NF1 osteoclasts also tolerate apoptotic signals, caused by serum deprivation or bisphosphonates, drugs used to treat osteoporosis. Taken together with the fact that osteoblast-mediated bone formation is impaired in NF1, this chapter provides insight on how mutation in the NF1 gene affects bone health, and these results may partially explain the low BMD in NF1.

SHORT COMMUNICATION Neurofibromatosis type 1 (NF1), also known as von Recklinghausen’s disease, is an autosomal dominant neuro-cutaneous-skeletal syndrome. NF1 is caused by mutations in the NF1 gene, NF1-protein, neurofibromin, and functions such as Ras-GTPase activating proteins or Ras-GAP, thus deactivating Ras [1-3]. The diagnosis of NF1 is based on clinical NIH criteria, and/or on mutation analysis of the NF1 gene [4,5]. The hallmarks of NF1 are benign neurofibroma tumors, café-au-lait macules, axillary freckling, optic gliomas, and typical

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skeletal abnormalities, such as congenital pseudarthrosis of the tibia [6]. Skeletal abnormalities in NF1 are either focal or systemic. Focal bone abnormalities include congenital bowing and pseudarthrosis of long bones, fibrocystic lesions, scoliosis, and sphenoid wing dysplasia. The most common systemic bone abnormalities are short stature and reduced bone mineral density (BMD). Osteoporosis is found in 20-50% of patients with NF1 [7]. Bone turnover is a process of old bone being resorbed by osteoclasts and new bones being formed by osteoblasts. During the bone turnover, both bone formation and resorption products are released into the blood and subsequently into urine. These products are called bone turnover markers, including, for example, serum CTX, a collagen I degradation product [8]. Patients with NF1 have increased levels of serum CTX compared to controls, reflecting increased bone turnover [9]. Osteoclast progenitors can be isolated from peripheral blood samples, and cultured into mature multinuclear osteoclasts in vitro. Receptor activators of nuclear factor kappa-beta ligand (RANKL) and macrophage colony stimulating factors (MCSF) are required for the differentiation of mature osteoclasts, and the cell culture methods have been described in detail [10-12]. Briefly, peripheral blood mononuclear cells are isolated using Ficoll gradient centrifugation, and cultured on bone slices for 10-14 days. Osteoclasts are identified using tartrate resistant acid phosphatase (TRACP) staining, and TRACP positive cells with three or more nuclei are considered as osteoclasts. NF1 osteoclasts have been demonstrated to be hyperactive by us [9,12] and others [11,13]. Specifically, culturing of osteoclast progenitors derived from peripheral blood of NF1 patients resulted in higher amounts of mature osteoclasts compared to controls. Thus, NF1 osteoclasts have increased formation capacity. In addition, the mature NF1 osteoclasts are larger in size and have more nuclei compared to controls, as shown in Figure 1 [11-13]. Moreover, the NF1 osteoclasts have enhanced resorption capacity, since NF1 osteoclasts resorbed higher amounts of bone compared to controls in vitro [11-13]. Bone resorption in vitro can be quantified by counting the resorption pits on the bone slice, or measuring the levels of collagen degradation product CTX in the osteoclast culture medium, which both give comparable results [12]. The increase in the number of NF1 osteoclasts is, however, not sufficient enough to explain the approximately two-fold increase in bone resorption. This leads us to speculate that other factors in addition to osteoclast numbers regulate the total bone resorption by NF1 osteoclasts [12].

Figure 1. A representative comparison of osteoclasts (arrow heads) derived from a NF1 patient and a healthy control person. The black areas represent tartrate resistant acid phosphatase (TRACP) staining. The nuclei appear bright, Hoechst staining. Scale bars 50 micrometers.

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Actin is required for formation of actin rings in osteoclasts, which are essential for normal bone resorption and osteoclast migration [14]. NF1 osteoclasts display an enhanced migration capacity compared to control osteoclasts, and have increased content of actin [11,13]. Actin rings are more frequently found in NF1 osteoclasts, suggesting that a greater proportion of osteoclasts are resorbing in NF1 samples compared to controls [11,13]. Actin abnormalities also suggest that the cytoskeleton in NF1 osteoclasts may be different compared to controls. Since the osteoclast cytoskeleton changes during the different phases of the resorption cycle [14], it may be difficult to assess the cytoskeleton of NF1 osteoclasts. Osteoclast death can be caused by a serum deprivation experiment. NF1 osteoclasts tolerate serum deprivation for up to 24 hours, while the number of control osteoclasts is markedly reduced in the same time [12]. This finding prompted us to evaluate the effects of bisphosphonates in NF1 osteoclasts in vitro. Bisphosphonates are pyrophosphate analogues that bind to bone induce osteoclast apoptosis, and are clinically used to treat osteoporosis [15,16]. Our recent data suggests that NF1 osteoclasts are insensitive to bisphosphonates alendronate, clodronate, and zoledronic acid [9]. Higher numbers and proportions of NF1 osteoclasts survived in vitro treatment with these drugs compared to controls. In addition, in NF1 samples, there was only a slight increase in levels of caspase-3, a marker of apoptotic activity, compared to a marked increase in control samples [9]. However, these in vitro results cannot be extrapolated to the NF1 patients taking these drugs. It has been shown that NF1 osteoclasts display hyperactive Ras signalling pathways, which is in agreement with the fact that neurofibromin functions as a Ras-GAP [13]. In osteoclasts, Ras pathways have been shown to represent anti-apoptotic and proosteoclastogenic pathway s[17]. The results on NF1 osteoclasts described above could thus be explained through hyperactive Ras. Also, the downstream signalling pathways ERK and AKT have been shown to be hyperactive in NF1 osteoclasts, supporting the role of Ras hyperactivation [11,13]. Inhibition of Ras in mature NF1 osteoclasts with farnesyl thiosalisylic acid (FTS) does not affect the number of osteoclasts in vitro. However, the addition of FTS counteracted the NF1-related insensitivity to zoledronic acid-induced apoptosis. Thus, the combination of FTS and zoledronic acid had an equal effect on both NF1 and control osteoclasts. This may suggest that the NF1-related insensitivity to apoptotic signals is mediated through Ras signalling [9]. The studies described in this chapter do not evaluate the osteoblast – osteoclast interaction in NF1, such as the role of RANKL/osteoprotegrin signalling. However, it appears that osteoclasts are affected by mutations in the NF1 gene, instead of being normal osteoclasts in abnormal conditions. Human NF1 osteoblasts derived from NF1 patients display impaired differentiation and osteogenic capacity in vitro compared to osteoblasts derived from control persons [18]. Taken together with findings of NF1 osteoclasts, it is not surprising that patients with NF1 often develop and/or progress to low bone mineral density, regardless of gender and/or age [7]. No correlation between osteoclast formation capacity in vitro, urine bone turnover markers, and bone mineral density was found in young (age 1-25 years) patients with NF1 [13]. Thus, some patients with NF1 have normal BMD and high osteoclast activity in vitro, which is in analogy with our data [9]. However, it may be possible that selected bone turnover markers, such as serum CTX, may correlate with BMD or osteoclast activity in vitro. A positive relationship between levels of serum CTX and the bone resorption activity in vitro

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has been shown in both NF1 patients and healthy controls [9,12]. Our results on serum CTX are promising, but are limited to a small number of patients without prospective data. Increased numbers of both osteoclasts and osteoblasts have been shown in bone biopsies from patients with NF1, suggesting that the findings on NF1 osteoclasts in vitro may also be operative in vivo. These biopsies also show larger amounts of osteoids in NF1 samples compared to controls, suggesting a mineralization disorder in NF1 [19]. Also, in other Rasopathies, rare syndromes of the Ras pathway, increased levels of urine bone turnover markers have been documented, suggesting increased bone turnover. This is in analogy with hyperactive Ras and low BMD in patients with different Rasopathies [20]. One may also speculate about the role of osteoclasts in NF1-related focal skeletal abnormalities. Tissue sections from NF1-related congenital pseudarthrosis sites show multiple multinuclear osteoclasts. These osteoclasts may also reside without direct contact to bone, within the fibrous pseudarthrosis tissue [12,18], and may be able to escape the apoptotic signals of the adjacent microenvironment. In conclusion, NF1 and hyperactive Ras regulate human osteoclastogenesis in vitro. This is in analogy to low bone mineral densities often noted in the NF1 patients. In addition, our preliminary results on bisphosphonate insensitivity in NF1 osteoclasts in vitro suggest that patients with NF1 may clinically respond differently to treatments of osteoporosis. Thus, a study on the effects of bisphosphonates on NF1 patients with osteoporosis is called for. Also, the use of biomarkers to predict low BMD or bone loss in NF1 could provide valuable clinical benefit, if a suitable biomarker could be identified. Therefore, continuing research in both clinical and basic science is fundamental for acquiring better understanding of bone pathology in NF1 and other Rasopathies.

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Riccardi VM. (1992). Neurofibromatosis: phenotype, natural history and pathogenesis. Baltimore, MD: Johns Hopkins University Press. Lammert M, Friedman J, Kluwe L, Mautner V. (2005). Prevalence of neurofibromatosis 1 in German children at elementary school enrollment. Arch. Dermatol, 141(1), 71-74. Jouhilahti EM, Peltonen S, Heape AM, Peltonen J. (2011). The pathoetiology of neurofibromatosis 1. Am. J. Pathol, 178(5), 1932-1939. Stumpf D, Annergers J, Brown S, Conneally P, Housman D, Leppert M, Miller J, Moss M, Pileggi A, Rapin I, Strohman R, Swanson L, Zimmersman A. (1988). Neurofibromatosis. Conference statement. National Institutes of Health Consensus Development Conference. Arch. Neurol 45, 575-578. Huson S. Title: The neurofibromatosis: classification, clinical features and genetic counselling. In: Kaufmann D, editor. Title: Neurofibromatoses. Basel: Karger; 2008; 1– 20.

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Friedman JM, Riccardi VM. Title: Clinical and epidemiological features. In: Gutmann DH, MacCollin M, Riccardi VM, editors. Title: Neurofibromatosis: phenotype, natural history, and pathogenesis. Baltimore, MD: John Hopkins University Press; 2008; 26– 86. Elefteriou F, Kolanczyk M, Schindeler A, Viskochil D, Hock J, Schorry E, Crawford A, Friedman J, Little D, Peltonen J, Carey J, Feldman D, Yu X, Armstrong L, Birch P, Kendler D, Mundlos S, Yang F, Agiostratidou G, Hunter-Schaedle K, Stevenson D. (2009). Skeletal abnormalities in neurofibromatosis type 1: Approaches to therapeutic options. Am. J. Med. Genet. A, 149A(10), 2327-38. Vasikaran S, Eastell R, Bruyère O, Foldes AJ, Garnero P, Griesmacher A, McClung M, Morris HA, Silverman S, Trenti T, Wahl DA, Cooper C, Kanis JA, Group I-IBMSW. (2011). Markers of bone turnover for the prediction of fracture risk and monitoring of osteoporosis treatment: a need for international reference standards. Osteoporos. Int, 22(2), 391-420. Heervä E, Peltonen S, Svedström E, Aro HT, Väänänen K, Peltonen J. (2012). Osteoclasts derived from patients with neurofibromatosis 1 (NF1) display insensitivity to bisphosphonates in vitro. Bone, DOI:10.1016/j.bone.2011.12.011. Shinoda K, Sugiyama E, Taki H, Harada S, Mino T, Maruyama M, Kobayashi M. (2003). Resting T cells negatively regulate osteoclast generation from peripheral blood monocytes. Bone, 33(4), 711-20. Yang F, Chen S, Robling A, Yu X, Nebesio T, Yan J, Morgan T, Li X, Yuan J, Hock J, Ingram D, Clapp D. (2006). Hyperactivation of p21ras and PI3K cooperate to alter murine and human neurofibromatosis type 1-haploinsufficient osteoclast functions. J. Clin. Invest, 116, 2880–91. Heervä E, Alanne MH, Peltonen S, Kuorilehto T, Hentunen T, Väänänen K, Peltonen J. (2010). Osteoclasts in neurofibromatosis type 1 display enhanced resorption capacity, aberrant morphology, and resistance to serum deprivation. Bone, 47, 583-90. Stevenson DA, Yan J, He Y, Li H, Liu Y, Zhang Q, Jing Y, Guo Z, Zhang W, Yang D, Wu X, Hanson H, Li X, Staser K, Viskochil DH, Carey JC, Chen S, Miller L, Roberson K, Moyer-Mileur L, Yu M, Schwarz EL, Pasquali M, Yang FC. (2011). Multiple increased osteoclast functions in individuals with neurofibromatosis type 1. Am. J. Med. Genet. A, 155A(5), 1050-1059. Väänänen HK, Zhao H, Mulari M, Halleen JM. (2000). The cell biology of osteoclast function. J. Cell Sci, 113(Pt 3), 377-81. Reszka AA, Rodan GA. Mechanism of action of bisphosphonates. (2003). Curr. Osteoporos. Rep, 1, 45-52. Rachner TD, Khosla S, Hofbauer LC. (2011). Osteoporosis: now and the future. Lancet, 9;377(9773), 1276-87. Tiedemann K, Hussein O, Sadvakassova G, Guo Y, Siegel PM, Komarova SV. (2009). Breast cancer-derived factors stimulate osteoclastogenesis through the Ca2+/protein kinase C and transforming growth factor-beta/MAPK signaling pathways. J. Biol. Chem, 27;284(48), 33662-70.

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[18] Leskelä H, Kuorilehto T, Risteli J, Koivunen J, Nissinen M, Peltonen S, Kinnunen P, Messiaen L, Lehenkari P, Peltonen J. (2009). Congenital pseudarthrosis of neurofibromatosis type 1: impaired osteoblast differentiation and function and altered NF1 gene expression. Bone, 44, 243-250. [19] Seitz S, Schnabel C, Busse B, Schmidt HU, Beil FT, Friedrich RE, Schinke T, Mautner VF, Amling M. (2010). High bone turnover and accumulation of osteoid in patients with neurofibromatosis 1. Osteoporos. Int, 21(1), 119-27. [20] Stevenson DA, Schwarz EL, Carey JC, Viskochil DH, Hanson H, Bauer S, Cindy Weng HY, Greene T, Reinker K, Swensen J, Chan RJ, Yang FC, Senbanjo L, Yang Z, Mao R, Pasquali M. (2011). Bone resorption in syndromes of the Ras/MAPK pathway. Clin. Genet, 80(6), 566-573.

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INDEX

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A access, 42 accounting, 102 acid, 10, 35, 39, 54, 58, 73, 127, 145, 151, 154, 155 acidic, 6, 7, 101, 133 acidosis, 40 acquired immunodeficiency syndrome, 118 activation state, 102 active transport, 97 adalimumab, 10, 25 adaptive immunity, 114 adenovirus, 27 adhesion, 5, 34, 35, 37, 43, 46, 51, 108, 133, 134, 135 adhesive interaction, 100 adipocyte, 74 adolescents, 71 adrenal hyperplasia, 111 adulthood, 96, 102 adults, 21, 43, 77 aetiology, 89, 94 age, x, 4, 35, 54, 77, 80, 86, 102, 117, 118, 123, 134, 153, 155 ageing population, 103 agencies, 45 aggregation, 84, 91, 93 aggressiveness, 69, 71 agonist, 135, 140 AIDS, 118, 124, 127 alanine, 34 alcohol consumption, 118 amino, 10, 11, 24, 38, 102 amino acid, 10, 11, 24, 38, 102 androgen, 103 anemia, 40 angiogenesis, x, 131, 132, 133, 135, 137, 139, 140 anhydrase, 38, 40, 59

ankylosing spondylitis, 52, 111 antagonism, 21 antibody, 4, 11, 15, 25, 35, 36, 37, 87, 117, 127, 135 antigen, x, 7, 13, 15, 27, 96, 100, 107, 114, 115, 116, 122, 123, 131, 132, 133, 138, 139 antigen-presenting cell, x, 15, 131, 132, 139 antioxidant, 85 antisense, 72 antisense RNA, 72 aorta, 12 APC, 114, 116, 121 APCs, 115, 123, 132 apoptosis, viii, 7, 10, 19, 22, 36, 41, 42, 43, 46, 47, 50, 51, 71, 75, 77, 78, 84, 85, 86, 87, 88, 91, 92, 93, 98, 100, 104, 110, 117, 126, 133, 140, 155 appendicular skeleton, vii, 1, 2 arginine, 34 arteries, 12 arthritis, vii, 1, 2, 11, 13, 17, 20, 21, 23, 25, 27, 43, 52, 98, 100, 104, 106, 107, 109, 127, 128 arthroplasty, ix, 95 articular cartilage, 100 ascorbic acid, 56, 72 aseptic, ix, 95 assessment, 49 asthma, 96 attachment, 34, 38, 42 autoantibodies, 21 autoimmune disease, vii, 1, 2, 7, 96, 118, 119 autoimmune diseases, 96, 118 autosomal dominant, x, 40, 153 autosomal recessive, 40

B base, 57, 58, 59 basic research, 106 benign, 153

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160

Index

bicarbonate, 38, 39 biocompatibility, 72, 144 biodegradability, 144 biological roles, 46 biomarkers, 156 biopsy, 25, 150 biosynthesis, 74 bisphosphonate treatment, x, 143, 149 blood, x, 10, 16, 19, 27, 56, 57, 58, 71, 74, 85, 87, 112, 129, 134, 150, 153, 154 blood circulation, 57 blood monocytes, 10, 16, 71, 74, 85, 87 body mass index, 118 bone biology, 105 bone cells, x, 51, 55, 72, 86, 96, 97, 99, 104, 131, 132, 137, 138 bone form, ix, x, 8, 12, 22, 27, 36, 41, 42, 43, 44, 47, 50, 54, 75, 78, 93, 95, 99, 101, 104, 107, 108, 115, 122, 129, 135, 143, 144, 146, 148, 149, 150, 151, 153, 154 bone marrow, viii, x, 4, 5, 6, 8, 10, 12, 14, 15, 25, 26, 28, 29, 34, 40, 41, 54, 56, 57, 66, 72, 79, 85, 86, 89, 98, 101, 104, 108, 109, 112, 115, 118, 121, 129, 133, 134, 135, 140, 141, 143, 146, 148, 149, 150 bone mass, vii, ix, 7, 15, 22, 23, 31, 35, 36, 40, 41, 47, 95, 96, 97, 103, 104, 111, 114, 115, 117, 118, 124, 126, 134 bone volume, 8, 42, 78 bones, 39, 40, 55, 68, 86, 96, 154 breast cancer, 43, 51, 70, 97, 108 breast carcinoma, 75

C Ca2+, 34, 79, 157 calcification, 12, 27, 125 calcitonin, 41, 45, 50, 51, 59, 78 calcium, x, 8, 12, 23, 38, 39, 41, 44, 46, 58, 59, 60, 62, 65, 66, 79, 96, 107, 143, 144, 149, 151 calvaria, 49 cancer, vii, 31, 43, 44, 51, 52, 55, 70, 96, 99, 107, 108, 157 cancer cells, 55 capillary, 133 carcinoma, 51 cartilage, vii, 1, 2, 43, 50, 100, 101, 107, 119, 128 cascades, 78, 79, 82, 122 caspases, 36 category a, 41 C-C, 133 CD8+, 98, 102, 108, 117, 139 cDNA, 4, 24

cell biology, 48, 70, 157 cell culture, viii, 20, 22, 26, 53, 54, 55, 57, 58, 59, 60, 61, 63, 64, 68, 69, 72, 101, 102, 154 cell culture method, 154 cell death, 84, 87, 91 cell differentiation, 9, 69, 98, 117, 121 cell fusion, 46, 47, 48, 73, 88, 111 cell line, viii, 15, 34, 35, 53, 54, 55, 56, 57, 59, 60, 63, 65, 66, 67, 68, 69, 70, 71, 72, 117 cell surface, 23, 24, 35, 36 ceramic, 8, 23, 151 chemical, 38, 144, 150 chemokine receptor, 46 chemokines, 101, 110, 135, 138 chemotaxis, 34 chemotherapy, 55 chicken, 68 childhood, 71 children, 21, 40, 43, 54, 71, 102, 110, 111, 156 chromatography, 108, 138, 139 circulation, 4, 103 classes, 12, 115 classification, 99, 156 clone, 122 cloning, 24, 49 closure, 84 clusters, 37 coding, 78 collagen, 13, 17, 20, 27, 34, 38, 39, 56, 68, 75, 78, 97, 103, 118, 127, 128, 151, 154 communication, 2, 33, 39, 55, 67, 70 complement, 38 complexity, 106, 121 compliance, 102 complications, 102 compounds, 144 compression, 97 conditioning, 59, 60 conductance, 72, 74 congenital adrenal hyperplasia, 102 connective tissue, 44, 126 Consensus, 111, 156 controversial, 11, 33, 80, 98, 120 correlation, 94, 102, 155 correlations, 15, 94 cortex, 103, 140 cortical bone, 12, 41, 51 costimulatory molecules, 116 costimulatory signal, 115, 117, 118 CSF, 1, 2, 3, 4, 5, 6, 7, 8, 9, 14, 15, 17, 18, 19, 20, 22, 33, 34, 35, 39, 44, 54, 56, 57, 58, 59, 60, 62, 69, 72, 73, 74, 78, 96, 99, 100, 103, 104, 106, 114, 119, 122, 132

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161

Index culture, 5, 6, 8, 11, 12, 14, 15, 16, 18, 29, 35, 36, 56, 59, 60, 61, 62, 63, 65, 66, 67, 68, 72, 97, 100, 101, 103, 104, 133, 134, 154 culture medium, 154 cyclooxygenase, 74 cysteine, 11, 39, 49 cytokines, vii, ix, 2, 3, 5, 9, 12, 16, 21, 31, 33, 44, 52, 69, 74, 78, 95, 96, 97, 98, 99, 100, 101, 104, 105, 106, 110, 112, 113, 114, 117, 120, 128, 135 cytoplasm, 4, 37, 79 cytoplasmic tail, 18 cytoskeletal rearrangements, viii, 31, 32, 38 cytoskeleton, 35, 39, 42, 50, 155

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D damages, 96 defects, x, 10, 143, 144, 146, 148, 150, 151 deficiencies, 40 deficiency, ix, 4, 5, 9, 10, 13, 15, 25, 29, 49, 74, 95, 98, 102, 104, 106, 107, 111, 112, 114, 117, 120, 122, 123, 124, 128, 129, 130, 133 degradation, 13, 32, 33, 35, 39, 42, 49, 82, 83, 84, 87, 91, 92, 93, 96, 99, 150, 154 dendritic cell, 6, 9, 19, 24, 33, 36, 37, 43, 47, 48, 78, 98, 105, 109, 115, 121, 122, 125, 129, 133, 136, 138, 140, 141 deprivation, x, 87, 153, 155, 157 destruction, 2, 3, 10, 11, 12, 13, 17, 23, 26, 43, 51, 52, 96, 99, 100, 101, 107, 109, 115, 119, 121, 127, 132 detection, 14, 72 detoxification, 85 deviation, 97 diabetes, 118, 126, 127 digestion, 123 dimerization, 92 direct action, 106 disability, vii, 1, 2, 119 disease activity, 41 diseases, vii, ix, x, 9, 31, 41, 44, 49, 83, 86, 87, 91, 95, 96, 98, 105, 111, 131, 132, 137, 141, 144 disintegrin, 47 disorder, 40, 43, 77, 94, 103, 124, 156 distribution, 47, 108 diversification, 90 diversity, 125 DNA, 24, 50 DOI, 157 donors, 85, 99 dosage, 7 dosing, 99 down-regulation, 6, 20, 78, 100, 138

drug delivery, 151 drugs, x, 16, 125, 144, 153, 155 dysplasia, 154

E E-cadherin, 35 ECM, 34, 35, 38 editors, 156 electron, 32, 150 electrophoresis, 139 elementary school, 156 encoding, 40, 80, 87, 89 endothelial cells, x, 4, 18, 54, 131, 132, 139 energy, 33, 38, 70, 84, 96, 105 England, 22 enrollment, 156 environment, vii, 8, 31, 38, 43, 44, 109, 118 environmental factors, ix, 77, 88 enzyme, 39, 58, 83, 91, 97, 105, 120 enzymes, 7, 39, 40, 54, 85, 134 epidemiology, 94 epithelium, 75 equilibrium, viii, 53, 114 erosion, vii, 1, 2, 3, 9, 10, 16, 17, 100, 101, 107 estrogen, 13, 15, 25, 29, 41, 42, 47, 50, 74, 98, 104, 107, 112, 114, 120, 122, 123, 129 etiology, viii, 77, 80, 96, 119 evidence, x, 2, 5, 9, 14, 33, 34, 35, 38, 42, 83, 87, 89, 101, 115, 123, 124, 131, 132, 133, 135 evolution, 27, 105, 120 excretion, 26 expenditures, 111 experimental condition, viii, 53, 54, 57, 59, 62, 63, 65, 66, 67, 68 exposure, 5, 7, 8, 35, 36, 37, 54, 123, 132, 137, 141 extracellular matrix, 12, 34, 95 extracts, 72

F families, 27, 40, 106, 125 feedback inhibition, 83 female rat, 42 femur, 42, 55, 144, 145 fibers, 78 fibrinogen, 34 fibroblast growth factor, 99, 133, 151 fibroblasts, vii, 1, 3, 7, 9, 17, 21, 26, 31, 33, 45, 54, 70, 107 fibrosis, 74 Finland, 153

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fixation, 144, 151 flexibility, 83 fluid, 15, 17, 20, 45, 100, 101, 106 folic acid, 104 fractures, vii, 1, 2, 12, 40, 55, 103, 104, 111, 117, 151 fragility, 103, 104 fragments, 39 functional analysis, 94 fusion, viii, 9, 10, 31, 34, 35, 36, 37, 39, 44, 47, 48, 51, 78, 80, 101, 135

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G gel, 56 gene expression, 4, 6, 24, 25, 47, 85, 87, 89, 104, 106, 125, 126, 128, 133, 157 genes, ix, 13, 19, 26, 36, 37, 40, 41, 51, 56, 57, 58, 59, 60, 65, 67, 73, 77, 78, 79, 80, 85, 87, 88, 90 genetic counselling, 156 genetics, 78 genome, 80 genotype, 94 gingival, 70 glucocorticoid, 42, 103, 104, 111 glutamine, 58 glycerol, 56 glycine, 34 glycoproteins, 12 grants, 44, 124 granules, 151 granulomas, 101 growth, 2, 3, 4, 6, 12, 18, 20, 21, 22, 23, 27, 34, 43, 44, 52, 57, 59, 69, 72, 75, 88, 97, 99, 108, 133, 134, 140 growth factor, 2, 3, 4, 6, 18, 20, 21, 22, 23, 27, 34, 44, 52, 57, 59, 69, 88, 97, 99, 108, 133, 140 growth hormone, 22 GTPases, 39, 42, 49, 50 guidelines, 146

H HAART, 118 haplotypes, 88 healing, 8, 22, 23, 133, 150 health, xi, 71, 111, 153 hematopoietic stem cells, 18, 51, 98, 134, 135 hemostasis, 41, 44 hepatomegaly, 40 heterogeneity, 89 highly active antiretroviral therapy, 118

hip arthroplasty, 101, 110, 151 histological examination, 36 history, 43, 156 HIV, ix, 113, 114, 118, 120, 127 HIV/AIDS, 127 HIV-1, 114, 118, 127 HIV-infection, ix, 113 HLA, 21, 132, 137 homeostasis, 12, 27, 38, 39, 45, 84, 98, 115, 118, 123, 124, 125, 129 homocysteine, 104 hormonal control, 26 hormone, 2, 9, 41, 97, 112, 120 host, ix, 98, 113 human immunodeficiency virus, 114, 118, 127 human osteoblastic bone marrow cells (hBMC), viii, 54 Hunter, 157 hybridization, 4 hydrolysis, 58, 61, 64 hydroxyapatite, 35, 38, 42, 144, 149 hypercalcemia, 43, 52, 109 hyperparathyroidism, 97 hypersensitivity, 89 hypogonadism, 118 hypothesis, 26, 79, 84, 86, 104, 120 hypoxia, 73, 85, 93

I ICAM, 51, 135 identification, 19, 24, 72, 80, 88, 96, 114 identity, 140 idiopathic, 103, 127 IFN, 4, 7, 12, 13, 21, 27, 98, 100, 107, 121, 122, 123, 129, 133, 137 IFN-β, 12, 13 IL-8, 7, 15, 17, 99, 100, 101, 135 image, 32 imbalances, 54 immobilization, 145 immune activation, ix, 14, 36, 113, 114, 123 immune response, x, 98, 114, 115, 116, 117, 121, 123, 131, 132, 133, 135, 136, 137, 141 immune system, ix, 9, 12, 36, 96, 97, 98, 102, 105, 107, 113, 114, 121, 123, 125, 139 immunity, ix, 98, 113, 115, 128, 133 immunodeficiency, ix, 113, 118, 123 immunoglobulin, 36, 46, 116, 117, 120, 129 immunomodulatory, 12 immunostimulatory, 140 immunosuppression, 16 immunosuppressive agent, 125

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Index implants, 8, 23, 72, 149, 151 improvements, 33, 55 in situ hybridization, 25 in vivo, 8, 11, 15, 18, 25, 26, 27, 28, 29, 35, 36, 41, 45, 46, 47, 50, 51, 58, 62, 69, 80, 86, 97, 98, 100, 104, 107, 109, 112, 115, 117, 118, 121, 124, 126, 129, 133, 134, 136, 137, 139, 156 incidence, x, 12, 40, 54, 118, 153 individuals, vii, 1, 2, 21, 54, 118, 127, 157 inducer, 56, 60, 69, 100, 121 induction, vii, 1, 2, 4, 7, 8, 9, 10, 11, 13, 14, 20, 21, 27, 28, 43, 50, 73, 78, 79, 85, 100, 101, 107, 110, 126, 128 infancy, 40 infection, ix, 12, 13, 40, 79, 113, 114, 118, 119, 127 inflammation, vii, 1, 2, 3, 8, 11, 14, 16, 25, 100, 107, 109, 114, 119, 123, 138 inflammatory arthritis, vii, 9, 17, 26, 31, 99, 128, 151 inflammatory cells, 14, 41, 101 inflammatory disease, 21, 101, 113, 119, 120, 124 inflammatory mediators, x, 131, 132, 135 infliximab, 10 ingestion, 138 inheritance, 40 inhibition, 6, 11, 13, 15, 16, 19, 20, 21, 34, 43, 50, 52, 62, 63, 66, 67, 73, 75, 87, 96, 98, 100, 102, 106, 117, 126, 132, 134, 135 inhibitor, 12, 26, 27, 43, 50, 51, 54, 56, 62, 74, 96, 105, 118, 134, 137 initiation, 119 injury, 140 innate immunity, 114 insulin, 22, 23, 99, 133 integration, 90, 123 integrin, 34, 38, 48, 73, 108 integrins, 9, 20, 35, 38, 46, 96 integrity, 38, 41, 51, 55, 87 intercellular adhesion molecule, 138 interface, vii, ix, 39, 101, 109, 113, 114, 118, 119, 120, 123, 127 interferon, 4, 12, 21, 27, 107, 133 interferon (IFN), 4, 12 interferon gamma, 27 interferons, 12, 27, 107 internalization, 80 interrelations, 110 intervention, 44 intestine, 12 inversion, 65 ion channels, 39 ions, 38, 39, 97 Iran, 1 Ireland, 51

isolation, 15, 33, 84, 92 isotope, 139 Israel, 91 Italy, 95

J Japan, 131, 143, 144 joint deformities, vii, 1, 2 joint destruction, 13, 15, 17, 109, 119, 127 joint swelling, 2 joints, vii, 1, 2, 3, 7, 10, 11, 13, 14, 43, 96, 100, 119, 136 Jordan, 127 juvenile rheumatoid arthritis, 109

K kidney, 12, 99

L latency, 6 lead, vii, x, 11, 13, 31, 33, 40, 44, 55, 69, 79, 81, 85, 86, 88, 97, 116, 153 lesions, 43, 51, 80, 86, 89, 99, 108, 120, 154 leucocyte, 58 leukemia, 8, 22, 101, 110 LFA, 140 lifetime, 103 light, 42 lipids, 36 liposomes, x, 131, 132, 139, 140, 141 liver, 12 localization, 46, 49 loci, 78, 80, 89, 90 longitudinal study, 112 low-density lipoprotein, 111 lung cancer, 108 lupus, 136, 138 lymph, 9, 10, 23, 24, 45, 125, 139 lymphocytes, 8, 9, 15, 45, 98, 104, 114, 117, 120, 126, 128, 139 lymphoid, 7, 108, 119, 126 lymphoma, 8 lysosome, 84

M machinery, 34, 40, 43, 101, 121 macromolecules, 84

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macrophage inflammatory protein, 100 macrophages, ix, 3, 4, 8, 10, 12, 14, 15, 16, 17, 18, 21, 25, 33, 35, 36, 43, 47, 65, 78, 101, 106, 110, 113, 114, 115, 121, 122, 128, 132, 135, 136, 137, 139, 140 majority, 43, 57, 90, 119 malignancy, 43, 75, 106 mammals, 120, 134 management, 111 mapping, 89 marrow, 4, 5, 8, 11, 15, 22, 28, 46, 49, 54, 80, 104, 109, 115, 118, 134, 145 masking, 150 mass, 15, 17, 96, 103, 104, 117, 134 mast cells, 15 materials, 144 matrix, vii, 6, 20, 31, 32, 34, 35, 38, 39, 40, 46, 47, 49, 54, 55, 68, 72, 78, 97, 99, 108, 115, 133, 134, 138 matrix metalloproteinase, 34, 46, 49, 133 matter, 79 maxillary sinus, 151 MCP, 6, 19, 85 MCP-1, 6, 19, 85 measles, 86, 89, 94 measurement, 17 media, viii, 34, 53, 54, 55, 59, 63, 70, 71, 103, 119, 133 medical, 102, 103 MEK, viii, 22, 54, 60, 62, 63, 66, 67, 69, 73 membranes, 38, 84, 92 memory, 116, 129, 130 menopause, 104, 112, 121, 129 mental retardation, 102 mesenchymal stem cells, 137 messenger RNA, 137 metabolic disorders, viii, 54, 69 metabolism, 17, 23, 26, 41, 42, 52, 62, 70, 91, 92, 96, 102, 108, 124, 127 metabolites, 60 metalloproteinase, 21, 46 metastasis, ix, 51, 54, 70, 75, 95, 96, 106 metastatic disease, 43, 51 MHC, 115, 122, 123, 132, 137 migration, 4, 13, 34, 35, 37, 39, 43, 46, 47, 51, 151, 155 mineralization, 72, 135, 156 MIP, 100 mitogen, 90 MMP, 7, 34, 39, 46, 133, 134 MMP-9, 34, 39, 133, 134 MMPs, 34, 39 model system, 117

models, viii, x, 25, 53, 56, 71, 74, 86, 101, 119, 120, 123, 143, 150, 151 molecules, 35, 37, 46, 54, 56, 60, 69, 79, 83, 97, 98, 102, 104, 116, 132, 134 monoclonal antibody, 25, 43, 72 monocyte chemoattractant protein, 135 monolayer, 146 Moon, 127 morbidity, 99 morphology, vii, viii, x, 4, 32, 40, 42, 44, 59, 153, 157 mortality, vii, 1, 2, 21, 119, 151 mortality rate, vii, 1, 2 motif, 24, 78, 79 mRNA, 6, 11, 21, 22, 24, 45, 55, 57, 79, 115, 120 multiple myeloma, 42, 51, 99, 108, 109, 120, 128, 133, 140 musculoskeletal, 71 mutant, 85, 86, 87 mutation, xi, 3, 18, 40, 80, 85, 86, 87, 88, 89, 93, 94, 111, 153 mutations, vii, ix, 40, 77, 80, 86, 87, 88, 89, 94, 103, 117, 153, 155 myeloid cells, 4 myopathy, 80

N National Academy of Sciences, 16, 18, 22, 23, 25 National Institutes of Health, 156 natural killer (NK) cells, 7, 15, 21 necrosis, 10, 16, 23, 24, 25, 26, 106, 110, 128 negative effects, 43 neoplasm, 100 nerve growth factor, 82, 91 neurofibroma, 153 neutral, 97 New Zealand, 144 nicotine, 72 nitric oxide, 21, 27 nitrogen, 144, 149 NK cells, 7, 12, 97 Nrf2, 85, 93 nucleation, 98 nuclei, 33, 36, 41, 42, 44, 45, 46, 78, 79, 150, 154 nucleus, 35, 86 null, 33, 34, 35, 36, 37, 38, 101, 104, 117 nutrient, 84 nutrition, 103

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Index

O OC activation, ix, 95, 96 oligomerization, 84 opportunities, 108 optic glioma, 153 optical microscopy, 62, 66 organelles, 84 organize, 48 osteoarthritis, 17, 52 osteoclast lineage, 9, 80, 86, 119 osteocyte, 34, 42, 45, 51, 125 osteoporosis, vii, ix, x, 1, 2, 9, 10, 12, 17, 27, 31, 41, 42, 43, 44, 49, 50, 51, 54, 77, 95, 96, 97, 101, 103, 104, 107, 111, 112, 113, 117, 118, 120, 123, 124, 125, 127, 130, 144, 149, 153, 155, 156, 157 osteoprotegerin (OPG), ix, 54, 74, 96, 113, 126 osteotomy, 151 ovariectomy, 10, 15, 28, 104, 120, 121, 123, 128 overproduction, 85 oxidative stress, 85, 93 oxygen, 4

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P pain, 16, 55, 119 parallel, 57, 59, 63, 121 parathyroid, 25, 65, 69, 72, 74, 108, 109, 128, 133 parathyroid hormone, 25, 65, 69, 72, 74, 108, 109, 128, 133 pathogenesis, 3, 9, 10, 17, 25, 27, 49, 51, 70, 78, 80, 90, 98, 100, 102, 103, 104, 105, 108, 110, 156 pathology, viii, 7, 18, 19, 53, 93, 127, 156 pathophysiology, 48, 55, 86, 110 pathways, viii, 9, 11, 12, 22, 27, 28, 49, 51, 54, 63, 67, 69, 79, 82, 83, 85, 87, 91, 94, 97, 106, 128, 155 PBMC, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 99, 102, 103 PCR, 56, 57, 62, 66, 68 penicillin, 56, 58 peptide, 6, 10, 39, 69, 98, 101, 123, 137 periodontal, ix, 70, 96, 98, 99, 106, 107, 108, 113, 120, 126, 128 periodontal disease, 70, 96, 98, 99, 106, 107, 108 periodontitis, 107, 117, 120 peripheral blood, viii, x, 4, 5, 7, 8, 22, 23, 28, 44, 45, 53, 56, 57, 70, 73, 74, 79, 85, 96, 98, 99, 105, 107, 111, 112, 117, 124, 137, 153, 154, 157 peripheral blood mononuclear cell, viii, 5, 8, 23, 44, 53, 56, 57, 70, 73, 74, 99, 107, 111, 112, 124, 154 PGE, 74

165

pH, 40 phagocytic cells, 114 phagocytosis, x, 101, 131, 132, 136, 139, 141 pharmaceuticals, 33, 41, 44 pharmacology, 51 phenotype, ix, 12, 20, 35, 36, 38, 46, 71, 77, 78, 85, 86, 87, 88, 89, 93, 94, 102, 104, 107, 114, 117, 134, 136, 137, 156 phenotypes, 17, 35, 88, 105 phenylalanine, 102, 110 phenylketonuria, 98, 107, 110, 111 phosphate, x, 8, 23, 39, 56, 58, 59, 60, 62, 65, 66, 75, 143, 144, 145, 149, 151 phosphates, 149 phosphatidylethanolamine, 84 phosphatidylserine, 132, 138, 139, 140, 141 phosphorylation, 4, 79, 82, 83 physical activity, 103 physicians, 40 Physiological, v, 113, 114, 115 physiology, 48, 70, 88, 96, 106 PI3K, 73, 81, 157 pilot study, 110 placebo, 25, 52 plasma cells, 43, 120 plasma membrane, 35, 36, 38, 132, 139 playing, 81, 83 PMMA, 110 point mutation, 94 polarity, 42, 79 polarization, 48 polymethylmethacrylate, 101, 110 polymorphisms, 21, 90, 103, 118 polypeptide, 11 pools, 123 population, 5, 8, 12, 37, 55, 80, 94, 99, 102, 104, 119 porosity, x, 12, 143, 144, 145, 151 Portugal, 53 positive correlation, 101 positive relationship, 155 potassium, 39, 72 precursor cells, 4, 13, 32, 46, 57, 70, 73, 96, 110 preservation, 124 president, 143 prevention, 16, 25, 49, 52, 116 primary tumor, viii, 43, 53 progenitor cells, 19, 78, 139, 141 progressive functional disability, vii, 1, 2 pro-inflammatory, 8, 14, 21, 97, 101, 134, 135, 136, 138 project, 71

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proliferation, ix, 3, 7, 9, 10, 15, 18, 25, 33, 35, 45, 55, 65, 71, 86, 95, 96, 98, 104, 107, 108, 116, 129, 130, 133, 134, 137 promoter, 47, 86, 126 propagation, 82 propylene, 22 prostate cancer, 43, 75, 99, 108 prostheses, 110 proteasome, 81, 82, 83, 91 protection, 11, 17, 95 protective role, 120 protein family, 104 protein kinase C, 82, 91, 92, 157 proteins, 7, 12, 24, 26, 29, 34, 35, 36, 38, 39, 42, 44, 47, 49, 72, 75, 79, 80, 81, 82, 83, 84, 85, 86, 87, 91, 92, 93, 99, 120, 139, 150, 153 proteolytic enzyme, 32, 35, 39, 43 protons, 38 proto-oncogene, 72 psoriatic arthritis, 45, 70, 107 public health, 41 pumps, 48 pure water, 144 pyrophosphate, 155

Q

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quality of life, 44, 103 quantification, 58

R Rab, 39 rabbit cancellous bone defects, x, 143 radiography, 43 reactions, 10 reactive oxygen, 19, 39, 49, 74, 121 receptors, 4, 7, 9, 10, 24, 27, 28, 46, 59, 72, 74, 79, 84, 88, 92, 94, 97, 98, 134 reciprocal cross, 69 recognition, 38, 84, 92 recruiting, 83 relevance, viii, 54, 69 remodelling, 9, 10, 15, 96, 99, 101, 105 repair, 8, 96, 100, 101, 140, 144 replication, 12, 118 requirements, 130 residues, 4, 10, 11, 84 resistance, 87, 88, 157 resolution, 138 response, viii, 12, 19, 23, 34, 46, 53, 54, 55, 56, 57, 59, 60, 63, 65, 66, 67, 69, 74, 84, 85, 86, 91, 98,

104, 107, 111, 114, 120, 121, 122, 123, 137, 140, 141 responsiveness, 85, 104 restoration, 4 reticulum, 93 rheumatic diseases, 28, 108 rheumatoid arthritis, ix, 3, 5, 14, 16, 17, 18, 19, 20, 21, 24, 25, 28, 52, 95, 97, 98, 100, 101, 109, 110, 113, 114, 126, 127, 128, 133, 135, 138 Rheumatoid arthritis (RA), vii, 1, 2, 43 rings, 7, 37, 58, 59, 155 risk, vii, 1, 2, 31, 80, 90, 97, 102, 103, 118, 127, 157 rituximab, 127 RNA, 56, 67 rodents, 122 roughness, 73 routes, 121 ruffled border membrane, vii, 31 rules, 98

S salivary glands, 7 scanning electron microscopy, 59 science, 156 scoliosis, 154 scope, 42 secrete, x, 44, 99, 101, 117, 119, 131, 132 secretion, x, 6, 11, 13, 20, 34, 49, 51, 54, 55, 85, 103, 108, 117, 120, 126, 131, 132, 135, 138, 140 seeding, 63, 64, 65, 67, 69 sensing, 46 sensitivity, 20, 79, 101, 121, 123 serum, x, 2, 4, 12, 15, 17, 28, 44, 52, 56, 58, 65, 96, 99, 107, 111, 136, 153, 154, 155, 157 sex, 118, 120 showing, 5, 10, 11, 42, 98 side effects, 137 signal peptide, 10 signal transduction, 12, 24, 121 signaling pathway, viii, 13, 15, 26, 29, 34, 35, 54, 60, 63, 65, 66, 69, 78, 79, 80, 81, 82, 85, 88, 93, 157 signalling, 8, 9, 10, 11, 14, 27, 47, 71, 91, 92, 101, 102, 107, 155 signals, x, 11, 38, 79, 84, 94, 98, 108, 115, 116, 126, 153, 155, 156 signs, 40 sintering, 144 siRNA, 35, 36, 37 skeleton, 10, 38, 54, 78, 103, 108 skin, 70 smoking, 118 sodium, 39

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Index software, 57, 62, 66, 68 solid tumors, 99 solution, x, 143, 144, 149 species, 19, 39, 49, 121 specter, 121 spleen, 5, 11 splenomegaly, 40 SQSTM1 mutations, ix, 77, 80, 88, 89, 94 stability, 38 stabilization, 37, 84, 94 starvation, 84, 85 state, vii, ix, 8, 13, 31, 113, 119, 123 states, ix, 2, 44, 113, 123 stem cells, 97 steroids, 16 stimulation, ix, 9, 11, 13, 22, 25, 26, 28, 34, 43, 54, 69, 72, 74, 78, 79, 82, 85, 86, 93, 95, 106, 110, 128, 132 stimulus, 34, 122 stomach, 12 stress, 54, 84, 93, 129 stroma, 34, 108 stromal cells, 1, 2, 3, 6, 9, 12, 14, 15, 19, 22, 23, 24, 26, 45, 72, 78, 86, 96, 100, 101, 104, 115 structure, viii, 12, 27, 37, 38, 53, 55, 95, 97, 119 subdomains, 49 substitutes, 151 substitution, 80, 86 substrate, 37, 38, 42, 93 substrates, 82, 83, 86, 93 sulfate, x, 143 Sun, 39, 49, 75 supplementation, 55, 60, 63 suppression, 6, 14, 117, 135, 138 surface area, vii, 31, 110 surfactant, 4 surplus, 85 survival, 3, 4, 5, 7, 9, 14, 22, 28, 35, 41, 42, 46, 50, 51, 55, 60, 73, 78, 79, 80, 87, 94, 96, 98, 99, 105, 114, 116, 119, 127, 130, 140 susceptibility, 13, 80, 90 symptoms, 10, 40, 55 syndrome, x, 19, 21, 111, 117, 126, 153 synergistic effect, 4, 11, 99 synovial fluid, 5, 7, 15, 17, 20, 21, 28, 44, 52, 70, 100, 101, 107 synovial membrane, 119 synovial tissue, 9, 20, 101 synthesis, 15, 29, 62, 68, 69, 75, 83, 103, 126, 140 systemic lupus erythematosus, 118, 126

T T lymphocytes, 9, 52, 70, 108, 117, 124, 125, 126, 128, 141 tamoxifen, 74 target, 13, 14, 33, 35, 36, 37, 55, 83, 85, 92, 121, 137 tartrate-resistant acid phosphatase, x, 49, 143 TCC, 56 TCR, 115, 116, 122 techniques, 36, 55 technology, 33 TGF, 3, 6, 7, 8, 14, 17, 20, 21, 22, 44, 87, 88, 99, 108, 121, 122, 126, 132, 133, 134, 135, 138, 140 therapeutic approaches, viii, 54, 55, 69, 151 therapeutic targets, 16, 25, 107 therapeutic use, 16 therapy, 16, 25, 29, 41, 75, 103, 111, 112, 119, 127, 128, 149 threonine, 120 thrombocytopenia, 40 thrombopoietin, 51 thyroid gland, 12 tibia, 55, 154 tissue, 2, 8, 12, 17, 25, 46, 52, 54, 55, 69, 95, 98, 101, 102, 114, 146, 148, 149, 156 titanium, 8, 72, 137, 149 TNF-alpha, 17, 20, 25, 26, 28, 73, 105, 106, 110, 111, 128 TNF-α, 6, 10, 11, 15, 44, 83, 132, 135, 136, 137 tooth, 9 toxicity, 60 trafficking, ix, 39, 42, 49, 50, 77, 88 traits, 57, 58, 67 transcription, 2, 8, 13, 14, 47, 50, 62, 78, 79, 82, 86, 93, 94, 97, 110, 117, 121 transcription factors, 14, 50, 62, 78, 82, 94, 97, 117 transcripts, 18 transducer, 28 transduction, 4, 46, 79 transforming growth factor, x, 20, 21, 22, 23, 126, 131, 132, 140, 157 transgene, 106 translocation, 101, 110 transplant, 118 transplantation, 139 transport, 48 treatment, x, 2, 5, 7, 11, 13, 16, 17, 25, 33, 36, 41, 42, 43, 50, 51, 52, 55, 60, 71, 102, 104, 111, 118, 120, 127, 143, 144, 146, 149, 155, 157 trial, 25, 52, 151 tumor, 8, 17, 19, 20, 23, 24, 25, 26, 27, 28, 43, 45, 47, 51, 54, 55, 69, 75, 88, 90, 91, 99, 105, 106,

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107, 109, 110, 114, 125, 126, 128, 129, 132, 137, 140 tumor cells, 43, 51, 54, 55, 69, 99 tumor necrosis factor, 8, 17, 19, 20, 23, 24, 25, 26, 27, 28, 45, 47, 88, 90, 91, 105, 106, 107, 109, 110, 114, 125, 126, 128, 129, 132, 137, 140 tumor progression, 109 tumorigenesis, 93 tumors, 43, 54, 55, 71, 99, 153 tumours, 71 turnover, viii, ix, x, 2, 7, 15, 22, 41, 52, 53, 55, 77, 78, 80, 83, 86, 98, 113, 114, 117, 118, 120, 122, 128, 134, 140, 153, 154, 155, 156, 157 tyrosine, 4, 28, 29, 38, 78, 79, 117 Tyrosine, 4, 126

U ubiquitin, 80, 81, 82, 83, 84, 86, 90, 91, 92, 94 ultrasound, 21, 102, 110 ultrastructure, 89 umbilical cord, 65 United, 16, 18, 22, 23, 25, 111 United States, 16, 18, 22, 23, 25, 111 urine, 154, 155, 156 USA, 113

V vascular endothelial growth factor (VEGF), 75, 99, 133 vector, 27, 87 velocity, 34 viral infection, 77, 79, 119 visualization, 58, 59 vitamin D, 55, 103, 111, 118 vitamins, 104

W wear, 101, 102 wells, 65 wild type, 12, 15, 85, 104, 121 Wnt signaling, 94

Y yeast, 92 young adults, 54

Z

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zinc, 81, 84 zirconium, 144

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