16 1 CHAPTER^

66. Fuentealba LC, Eivers E, Ikeda A. Hurtado C, Kuroda H, Pera diates cooperative signaling by the transforming -p EM, De Robertis EM 2007 Integrating patterning signals: Wnti and wnt pathways. Proc Natl Acad Sci USA 97:8358-8363. GSK3 regulates the duration of the BMP/Smadl signal. Cell 71. Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai 131 : 980-993. S, Gallea S, Stiot V, Blanchet AM, Courtois B, Baron R, Rawadi 67. Sapkota G, Alarcon C, Spagnoli FM, Brivanlou AH, Massague J G 2001 Opposite effects of bone morphogenetic protein-2 and 2007 Balancing BMP signaling through integrated inputs into the transforming growth factor-pl on osteoblast differentiation. Bone Smadl linker. Mol Cell 25:441454. 29:323-330. M, M, SE, S, 72. Zhao Qiao Harris Chen D, Oyajobi BO, Mundy GR 68. Nakayama K, Tamura Y, Suzawa M, Harada S, Fukumoto Kato 2006 The zinc finger transcription factor Gli2 mediates bone mor- M, Miyazono K, Rodan GA, Takeuchi Y, Fujita T 2003 Receptor phogenetic protein 2 expression in osteoblasts in response to tyrosine inhibit bone morphogenetic protein-Smad re- hedgehog signaling. Mol Cell Biol 26:6197-6208. sponsive promoter activity and differentiation of murine MC3T3- 73. Li Y, Li A, Strait K, Zhang H, Nanes MS, Weitzmann MN 2007 El osteoblast-like cells. J Bone Miner Res 18:827-835. Endogenous TNFalpha lowers maximum peak bone mass and in- 69. Hu MC, Rosenblum ND 2005 Smadl, p-catenin and Tcf4 associate hibits osteoblastic Smad activation through NF-kappaB. J Bone in a molecular complex with the Myc promoter in dysplastic renal Miner Res 22:646-655. tissue and cooperate to control Myc transcription. Development 74. Mukai T, Otsuka F, Otani H, Yamashita M, Takasugi K, Inagaki 132:215-225. K, Yamamura M, Makino H 2007 TNF-alpha inhibits BMP- 70. Labbe E, Letamendia A, Attisano L 2000 Association of Smads induced osteoblast differentiation through activating SAPKiJNK with lymphoid enhancer binding factor 1IT cell-specific factor me- signaling. Biochem Biophys Res Commun 356:1004-1010.

Chapter 3. Osteoclast Biology and Bone Resorption

F. Patrick Ross Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

CELL BIOLOGY OF THE OSTEOCLAST from two major sources: biochemical and genetic.(2) The unique osteoclastogenic properties of RANKL permit genera- Pathological bone loss, regardless of etiology, invariably rep- tion of pure populations of osteoclasts in culture and hence the resents an increase in the rate at which the skeleton is de- graded by osteoclasts relative to its formation by osteoblasts. performance of meaningful biochemical and molecular experi- Thus, prevention of conditions such as osteoporosis requires ments that provide insights into the molecular mechanisms by an understanding of the molecular mechanisms of bone resorp- which osteoclasts resorb bone. Further evidence has come tion. from our capacity to generate mice lacking specific genes, plus The osteoclast, the exclusive bone resorptive cell (Fig. l),is the positional cloning of genetic abnormalities in people with a member of the monocytehacrophage family and a abnormal osteoclast function. Key to the resorptive event is polykaryon that can be generated in vitro from mononuclear the capacity of the osteoclast to form a microenvironment be- phagocyte precursors resident in a number of tissues.(') There tween itself and the underlying bone matrix (Fig. 3A). This is, however, general agreement that the principal physiological compartment, which is isolated from the general extracellular osteoclast precursor is the bone marrow . Two cy- space, is acidified by an electrogenic proton pump (H+- tokines are essential and sufficient for basal osteoclastogenesis, ATPase) and a CI- channel to a pH of -4.5.(6) The acidified the first being RANKL",') and the second being macrophage- milieu mobilizes the mineralized component of bone, exposing colony stimulating factor (M-CSF), also designated CSF-1.(3' its organic matrix, consisting largely of type 1 collagen that is These two proteins, which exist as both membrane-bound and subsequently degraded by the lysosomal enzyme cathepsin K. soluble forms (the former is secreted by activated T cells),(4) The critical role that the proton pump, CI- channel, and ca- are produced by marrow stromal cells and their derivative os- thepsin K play in osteoclast action is underscored by the fact teoblasts, and thus physiological recruitment of osteoclasts that diminished function of each results in a human disease of from their mononuclear precursors requires the presence of excess bone mass, namely osteopetrosis or pyknodysosto- these nonhematopoietic, bone-residing cells."' RANKL, a sis.(2,6) Degraded protein fragments are endocytosed and member of the TNF superfamily, is the key osteoclastogenic transported in undefined vesicles to the basolateral surface of , because osteoclast formation requires its presence or the cell, where they are discharged into the surrounding intra- its priming of precursor cells. M-CSF contributes to the pro- cellular fluid.(',') It is also likely that retraction of an osteoclast liferation, survival, and differentiation of osteoclast precursors, from the resorptive pit results in release of products of diges- as well as the survival and cytoskeletal rearrangement required tion. for efficient bone resorption (Fig. 2; a brief summary of the The above model of bone degradation clearly depends on integrated signaling pathways for each osteoclastic regulator physical intimacy between the osteoclast and bone matrix, a discussed in this review is provided later in this review [Fig. 61). role provided by integrins. Integrins are a@heterodimers with The discovery of RANKL was preceded by identification of its long extracellular and single transmembrane domains.") In physiological inhibitor osteoprotegerin (OPG), to which it most instances, the integrin cytoplasmic region is relatively binds with high affinity.(') In contrast, M-CSF is a moiety long short, consisting of 40-70 amino acids. Integrins are the prin- known to regulate the broader biology of myeloid cells, includ- cipal cellhatrix attachment molecules and they mediate os- ing osteoclasts(') (see Fig. 6). teoclastibone recognition. Members of the pl family of inte- Our understanding of how osteoclasts resorb bone derives grins, which recognize collagen, fibronectin, and laminin, are present on osteoclasts, but avp3 is the principal integrin me- The author states that he has no conflicts of interest. diating bone resorption.('") This heterodimer, like all members

0 2008 American Society for Bone and Mineral Research OSTEOCLASTBIOLOGY AND BONERESORPTION I 17

FIG. 1. The osteoclast as a resorptive cell. Transmis- sion electron microscopy of a multinucleated primary rat osteoclast on bone. Note the extensive ruffled bor- der, close apposition of the cell to bone and the partially degraded matrix between the sealing zones. Courtesy of H. Zhao.

of the (YV integrin family, recognizes the amino acid motif Arg- the osteoclast delivers effector molecules like HC1 and cathep- Gly-Asp (RGD), which is present in a variety of bone-residing sin K into the resorptive microenvironment. Osteoclasts are proteins such as and bone sialoprotein. Thus, os- characterized by a unique cytoskeleton, which mediates the teoclasts attach to and spread on these substrates in an RGD- resorptive process. Specifically, when the cell contacts bone, it dependent manner and, most importantly, competitive ligands generates two polarized structures, which enable it to degrade arrest bone resorption in vivo. Proof of the pivotal role that skeletal tissue. In the first instance, a subset of acidified avp3 has in the resorptive process came with the generation of vesicles containing specific cargo, including cathepsin K and the p3 integrin knockout mouse, which develops a progressive other matrix metalloproteases (MMPs), are transported, prob- increase in bone mass because of osteoclast dysfunction.(33) ably through microtubules and actin, to the bone-apposed Based on a combination of these in vitro and in vivo observa- plasma membrane,(’*’ to which they fuse in a manner not cur- tions, small molecule inhibitors of osteoclast function that tar- rently understood, but which may involve PLEKHM1.(I3’ In- get avp3 have been developed.(”’ sertion of these vesicles into the plasmalemma results in for- Bone resorption also requires a polarization event in which mation of a villous structure, unique to the osteoclast, called the ruffled membrane. This resorptive organelle contains the abundant H’ transporting machinery to create the acidified microenvironment, whereas the accompanying exocytosis serves as the means by which cathepsin K is secreted (Fig. 3B). In addition to inducing ruffled membrane formation, contact with bone also prompts the osteoclast to polarize its fibrillar actin into a circular structure known as the “actin ring.” A separate “sealing zone” surrounds and isolates the acidified resorptive microenvironment in the active cell, but its compo- sition is almost completely unknown. The actin ring, like the ruffled membrane, is a hallmark of the degradative capacity of the osteoclast, because structural abnormalities of either occur in conditions of arrested resorption.(l4) In most cells, such as fibroblasts, matrix attachment prompts formation of stable structures known as focal adhesions that contain both integrins and a host of signaling and cytoskeletal molecules, which me- diate contact and formation of actin stress fibers. In keeping with the substitution of the actin ring for stress fibers in osteo- clasts, these cells form podosomes instead of focal adhesions. Podosomes, which in resorbing osteoclasts are present in the actin ring, consist of an actin core surrounded by avP3 and FIG. 2. Role of , , steroids, and prostaglandins in associated cytoskeletal proteins. osteoclast formation. Under the influence of other cytokines (data not The integrin p3 subunit knockout mouse serves as an im- shown), hematopoietic stem cells (HSCs) commit to the myeloid lin- portant tool for determining the role of avp3 in the capacity of eage, express c-Fms and RANK, the receptors for M-CSF and the osteoclast to resorb bone. Failure to express avp3 results in RANKL, respectively, and differentiate into osteoclasts. Mesenchymal a dramatic osteoclast phenotype, particularly regarding the ac- cells in the marrow respond to a range of stimuli, secreting a mixture tin cytoskeleton. The p3-’- osteoclast forms abnormal ruffled of pro- and anti-osteoclastogenic proteins, the latter primarily OPG. membranes in vivo and, whether generated in vitro or directly Glucocorticoids suppress bone resorption indirectly but possibly also target osteoclasts and/or their precursors. , by a complex isolated from bone, the mutant cells fail to spread when plated mechanism, inhibits activation of T cells, decreasing their secretion of on immobilized RGD ligand or mineralized matrix in physi- RANKL and TNF-a; the sex steroid also inhibits osteoblast and os- ological amounts of RANKL and M-CSF. Confirming their teoclast differentiation and lifespan. A key factor regulating bone re- attenuated resorptive activity, p3-’- osteoclasts generate fewer sorption is the RANKUOPG ratio. and shallower resorptive lacunae on dentin slices than do their

0 2008 American Society for Bone and Mineral Research interest in the cytoplasmic molecules mediating these events in osteoclasts and avp3 signaling in this context is reasonably well understood. The initial signaling evcnt involves the proto- oncogene c-src, which, acting as a and an adaptor pro- tein, regulates formation of lamellipodia and disassembly of podosomes, indicating that c-src controls formation of resorp- tive organelles of the cell, such as the ruffled membrane, and also arrests migration on the bone surface. There is continuing debate surrounding the molecules which link c-src to the cy- toskeleton, one proposal being that the focal adhesion kinase family member Pyk2, acting in concert with c-Cbl, a proto- oncogene and ubiquitin ligase."') A second strong candidate is Syk, a nonreceptor that is recruited to the ac- tive conformation of avP3 in osteoclasts in a c-src-dependent manner,(") where it targets Vav3,"") a member of the large family of guanine nucleotide exchange factors (GEFs) that convert Rho GTPases from their inactive GDP to their active GTP conformation.

SMALL GTPASES The Rho family of GTPases is central to remodeling of the actin cytoskeleton in many cell types,('8) and as such plays a central role in osteoclastic bone resorption. On attachment to bone, Rho and Rac bind GTP and translocate to the cytoskel- eton. Whereas both small GTPases impact the actin cytoskel- eton, Rac and Rho exert distinctive effects. Rho signaling me- diates formation of the actin ring and a constitutively active form of the GTPase stimulates podosome formation, osteo- clast motility, and bone resorption, whereas dominant negative Rho arrests these events.('9) Rac stimulation in osteoclast pre- cursors prompts appearance of lamellipodia, thus forming the migratory front of the cell to which avp3 moves when acti- vated.('()) In sum, it is likely that Rho's effect is principally on cell adhesion, whereas Rac mediates the cytoskeleton's migra- tory machinery. Importantly, absence of Vav3 blunts Rac but not Rho activity in the osteoclast.(21) FIG. 3. Mechanism of osteoclastic bone resorption. (A) The osteo- clast adheres to bone through the integrin ~$3,creating a sealing FACTORS REGULATING OSTEOCLAST zone, into which is secreted hydrochloric acid and acidic proteases such FORMATION AND/OR FUNCTION as cathepsin K, MMP9, and MMP13. The acid is generated by the combined actions of a vacuolar H' ATPase; it coupled chloride chan- Proteins nel and a basolatcral chloride-bicarbonate exchanger. Carbonic anhy- drase converts CO, into H' and HCO'- (data not shown). (B) Integrin In addition to the two key osteoclastogenic cytokines M- engagement results in signals that target acidifying vesicles (+ = pro- CSF and RANKL, a number of other proteins play important ton pump complex) containing specific cargo (black dots) to the bone- roles in osteoclast biology, either in physiological and/or apposed face of thc cell. Fusion of these vesicles with the plasma patho-physiological circumstances. membrane generates a polarized cell capable of secreting the acid and As discussed earlier, OPG, a high-affinity ligand for proteases required for bone resorption. RANKL that acts as a soluble inhibitor of RANKL, is secreted by cells of mesenchymal origin, both basally and in response to other regulatory signals, including cytokines and bone- wildtype counterparts. In keeping with attenuated bone re- targeting steroids.(') Pro-inflammatory cytokines suppress sorption in vivo, p3-'- mice are substantially hypocalcemic.(') OPG expression while simultaneously enhancing that of RANKL, with the net effect being a marked increase in os- INTEGRIN SIGNALING teoclast formation and function. Genetic deletion of OPG in both mice and humans leads to profound osteoporosis,('*) Whereas integrins were viewed initially as merely cell at- whereas overexpression of the molecule under the control of a tachment molecules, it is now apparent that their capacity to hepatic promoter results in severe osteopetrosis.(23)Together, transmit signals to and from the cell interior is equally impor- these observations indicate that skeletal and perhaps circulat- tant, an event that requires that the integrin convert from a ing OPG modulates the bone resorptive activity of RANKL default low affinity state to one in which its capacity to bind and helps to explain the increased bone loss in clinical situa- matrix is significantly enhanced. The process, termed activa- tions accompanied by increased levels of TNF-a, tion, arises from either integrin ligation of their multivalent (1L)-1, PTH, or PTH-related protein (PTHrP). Serum PTH ligands or indirectly by growth factor signaling.('') levels are increased in hyperparathyroidism of whatever etiol- avp3 is absent from osteoclast precursors, but their differ- ogy, whereas PTHrP is secreted by mctastatic lung and breast entiation under the action of RANKL results in marked up- carcin~ma.('~~'~)TNF antibodies or a soluble TNF receptor- regulation of this heterodimer. The capacity of integrins to IgG fusion protein potently suppress the bone loss in disorders transmit intracellular signals to the cytoskeleton heightened of inflammatory osteolysis such as rheumatoid arthritis.(")

0 2008 American Society for Bone and Mineral Research OSTEOCLASTBIOLOGY AND BONERESORPTION I 19

The molecular basis of this observation seems to be that the diates its osteolytic effects are still incompletely understood, synergizes with RANKL in a unique but significant advances have been made over the last decade. manner, most likely because RANKL and TNF each activate The original hypothesis, now considered to only part of the a number of key downstream effector pathways, leading to explanation, is that decreased serum E, led to increased pro- nuclear localization of a range of osteoclastogenic transcrip- duction, by circulating , of osteoclastogenic cyto- tion factors (see Fig. 6). Recent evidence suggests a new para- kines such as IL-6, TNF, and IL-1. These molecules act on digm linking TNF, IL-1, and the natural secreted inhibitor for stromal cells and osteoclast precursors to enhance bone re- the latter cytokine, IL-1 receptor antagonist, which blocks IL-1 sorption by regulating expression of pro- (RANKL, M-CSF) function. Specifically, it seems that, at least in murine osteo- and anti- (OPG) osteoclastogenic cytokines (in the case of clasts and their precursors, many of the effects of TNF are mesenchymal cells) and by synergizing with RANKL itself (in mediated through its stimulation of IL-1, which in turn in- the case of myeloid osteoclast precursors; see Fig. 2). However, creases expression and secretion of IL-lra, a set of events that represent a complex control pathway. The significance of IL the understanding that lymphocytes play a key role in medi- receptor antagonist is shown by the fact an IgG fusion protein ating several aspects of bone biology has led to a growing containing the active component of this molecule has been realization that the cellular and molecular targets for E, are developed and enhances the ability of anti-TNF-c-y antibodies more widespread than previously believed. A model proposes to decrease bone loss in rheumatoid arthriti~.‘~’’ that E, impacts the resorptive component of bone turnover Elegant studies suggest that y (IFNy) is an im- (the steroid has separate effects on osteoblasts), at least in part portant suppressor of osteoclast formation and function.‘2x) by modulating production by T cells of RANKL and TNF.‘”) Nevertheless, these findings seem to be in conflict with other in This effect is itself indirect, with E, suppressing antigen pre- vivo observations, including the report that IFNy treatment of sentation by dendritic cells and macrophages by enhancing children with osteopetrosis ameliorates the disease‘2‘) and the expression by the same cells of TGFP. Antigen presentation fact that a number of in vivo studies indicate that IFNy stimu- activates T cells, thereby enhancing their production of lates bone resorption.(3‘)) This conundrum highlights the im- RANKL and TNF. As discussed previously, the first molecule portance of discriminating between in vitro culture experi- is the key osteoclastogenic cytokine, whereas the second po- ments using single cytokines and results in vivo. Many tentiates RANKL action and stimulates production by stromal additional studies have implicated a range of other cytokines in cells of M-CSF and RANKL. This newly discovered interface the regulation of the osteoclast. These include a range of in- between T cells and bone resorption also clarifies aspects of terleukins, GM-CSF, IFNP, stromal cell-derived factor 1 inflammatory osteolysis. Finally, some studies indicate that E, (SDF-1), macrophage inflammatory protein 1 (MIPc-y), and modulates signaling in pre-osteoclasts and that, acting through monocyte chemoattractant protein 1 (MCP-l),(”-”) but at reactive oxygen species, it increases the lifespan and/or func- this time the results are either contradictory, as for GM-CSF in tion of mature o~teocIasts.(~~) the murine versus human systems, or lack direct proof in hu- Both endogenous glucocorticoids and their synthetic ana- mans. Future studies are likely to clarify the currently confus- logs, which have been and continue to be a major mainstay of ing data set. Finally, interactions between immune receptors immunosuppressive therapy, are members of a third steroid such as DNAX activating protein of 12 kDa (DAP12) and FC family having a major impact on bone biology.(”’ receptor y (FcRy), present on osteoclasts and their precursors, One consequence of their chronic mode of administration is and their ligands on cells of the stromal and myeloid/lymphoid severe osteoporosis arising from decreased bone formation lineages are important for transmission of RANK-derived sig- and resorption with the latter absolutely decreased (low turn- nals.‘’’) IL-17 is a product of Th17 cells, a recently over osteoporosis). The majority of the evidence focuses on identified T-cell subset that is generated from uncommitted pre- the osteoblast as the prime target with the steroid increasing cursors under the influence of TGF-p, IL-23, and 1L-6.(32,31) of these bone-forming cells. However, numerous hu- man studies document a rapid initial decrease in bone resorp- Small Molecules tion, suggesting that the osteoclast and/or its precursors may also be targets. The molecular basis for this latter finding is 1,25-dihydroxyvitamin D has all the characteristics of a ste- unclear. However, because osteoblasts are a requisite part of roid hormone, including a high-affinity nuclear receptor that the resorptive cycle, one consequence of their long-term dimi- binds as a heterodimer with the retinoid X receptor to regulate nution could be decreased osteoclast formation and/or func- transcription of a set of specific target genes. This active form tion secondary to lower levels of RANKL and/or M-CSF pro- of vitamin D, generated by successive hydroxylation in the duction. Alternatively, glucocorticoids have been shown to liver and kidney, is a well-established stimulator of bone re- decrease osteoclast apoptosis.(”) sorption when present at supraphysiological levels. Studies A wide range of clinical information shows that excess pros- over many years have indicated that this steroid hormone in- taglandins stimulate bone loss, but once again, the cellular creases mesenchymal cell transcription of the RANKL gene, basis has not been established. Prostaglandins target stromal whereas diminishing that of OPG.‘” Separately, 1,25- and osteoblastic cells, stimulating expression of RANKL and dihydroxyvitamin D suppresses synthesis of the pro- suppressing that of OPG.“”) This increase in the RANKL/ osteoclastogenic hormone PTH”4’ and enhances calcium up- OPG ratio, seen in a variety of human studies, is sufficient of take from the gut. Taken together, the two latter effects would itself to explain the clinical findings of increased osteoclastic seem to be antiresorptive, but many studies in humans indicate activity. However, highlighting again the dilemma of interpret- the net osteolytic action resulting from high levels of this ste- ing in vitro studies there have been a number of studies in roid hormone, suggesting that its ability to stimulate osteoclast which prostaglandins regulate osteoclastogenesis per se in mu- function overrides any bone anabolic actions. rine cell culture. Loss of estrogen (E2), most often seen in the context of Phosphoinositides play distinct and important roles in orga- menopause, is a major reason for the development of signifi- nization of the osteoclast cyt~skeleton.(~”)Binding of M-CSF cant bone loss in aging. Interestingly, it is now clear that es- or RANKL to their cognate receptors, c-Fms and RANK, or trogen is the main sex steroid regulating bone mass in both activation of olvp3, recruits phosphoinositol-3-kinase (PUK) to men and women.(3s) The mechanisms by which estrogen me- the plasma membrane, where it converts membrane-bound

0 2008 American Society for Bone and Mineral Research 20 / CHAPTEK~ phosphatidylinositol 4,5-bisphosphate into phosphatidylinosi- to1 3,4,5-trisphosphate (Fig. 4). The latter compound is recog- nized by specific motifs in a wide range of cytoskeletally active proteins,‘41) and thus PI3K plays a central role in organizing the cytoskeleton of the osteoclast, including its ruffled mem- brane. Akt is a downstream target of PUK and plays an im- portant role in osteoclast function, particularly by mediating RANKL and/or M-CSF-stimulated proliferation and/or sur- vi~aI.(~”)

Cell-Cell Interactions in Bone Marrow Recent evidence has indicated that a number of additional cell types are important for osteoclast biology in a variety of situations (Fig. 5). First, as discussed previously, T cells play a key role in estrogen deficiency bone loss but also are important FIG. 5. Cell-cell interactions in bone marrow. Hematopoietic stem in a range of inflammatory diseases, most notably rheumatoid cells (H), the precursors of both T cells (T) and osteoclasts (OC), arthritid4*) and periodontal di~ease,‘~’)where the Th17 subset reside in a stem cell niche provided by osteoblasts (OB), which, to- likely secretes TNF and IL-17, a newly described osteoclasto- gether with stromal cells (S), derive from mesenchymal stem cells (M). genic cytokine. Given that both osteoclast precursors and the Bone degradation results in release of matrix-associated growth fac- various lymphocyte subsets, such as T, B, and NK cells, arise tors (thick vertical line), which stimulate mesenchymal cells and thus from the same stem cell, it is not surprising that some of the bone formation. This “coupling” is an essential consequence of osteo- clast activity.(”) After activation, T cells secrete molecules that stimu- same receptors and ligands that mediate the immune process late osteoclastogenesis and function. cells (C) release cytokines also govern the maturation of osteoclast precursors and the ca- that activate bone resorption; in turn, matrix-derived factors stimulate pacity of the mature cell to degrade bone. This interface has given cancer cell proliferation, the so-called “vicious cycle.” rise to the new discipline of osteo-immunology, which prom- ises to provide important and exciting findings in the future. Second, whereas it is well established that mesenchymal and in response to hormones and growth factors, resulting in cells are major mediators of cytokine and prostaglandin action modulation of the capacity of HSCs to become functional os- on osteoclasts, it has become clear recently that cells of the teoclasts. same lineage, residing on cortical and trabecular bone, are the Third, cancer cells facilitate their infiltration into the mar- site of a hematopoietic stem cell (HSC) niche.(44) Specifically, row cavity by stimulating osteoclast formation and function. HSCs reside close to osteoblasts as a result of multiple inter- An initial stimulus is PTHrP generation by lung and breast actions involving receptors and ligands on both cells types.(4s) cancer ell^,(^^^^^^^^) thu s enhancing mesenchymal production Furthermore, the mesenchymally derived cells secrete both of RANKL and M-CSF, whereas decreasing that of OPG and membrane-bound and soluble factors that contribute to sur- possibly chemotactic factors. The resulting increase in matrix vival and proliferation of multipotent osteoclast precursors, as dissolution releases bone-residing cytokines and growth fac- well as molecules that influence osteoclast formation and func- tors that, feeding back on the cancer cells, increase their tion. Both committed osteoblasts and the numerous stromal growth and/or survival. This loop has been termed “the vicious cells in bone marrow produce a range of proteins both basally Multiple myeloma seems to use a different but re- lated strategy, namely secretion of MIPa and MCP-1, both of which are chemotactic and proliferative for osteoclast precur- sor~.(~~.~’)The latter compound has been reported to be se- av03 RTKs creted by osteoclasts in response to RANKL and enhances osteoclast formation.‘” It seems likely further future studies experiments will uncover additional molecules mediating bone loss in metastatic disease. Intracellular Signaling Pathways The discussions above have not described in detail the in- tracellular signals by which osteoclasts are formed or those by which they degrade bone. The final major section of this re- $. Bisphosphonates view lays out the important pathways involved. Briefly, three major protein classes are involved, adaptors, kinases, and tran- $.l scription factors (Fig. 6), with one significant exception, Rho Small GTPases Rho Small GTPases RANKL-induced release of Ca”, a pathway that activates the (inactive) - calmodulin-dependent phosphatase calcineurin. NFATlc is a / (jctive’ major substrate for this enzyme, resulting in its nuclear trans- location and subsequent activation of osteoclast-specific genes. A/ J. Cell viability Actin rearrangement Importantly, the potent immunosuppressive drugs FK506 and cyclosporine inhibit calcineurin activity and therefore may tar- FIG. 4. Regulation and role of small GTPases in osteoclasts. Signals from avP3 andlor receptor tyrosine kinases (RTKs) activate small get the oste~clast.(~”) GTPases of the Rho family in a c-src-dependent manner. Bisphospho- The multiplicity of adaptors that link the various receptors nates, the potent antiresorptive drugs, block addition of hydrophobic to downstream signals precludes providing a meaningful sum- moieties onto the GI‘Pases, preventing their membrane targeting and mary, and so we summarize only the modulatory effects of activation. The active GTPases also regulate cell viability and thus kinases and transcription factors, which together regulate re- bisphosphonates induce osteoclast .(5” ceptor-driven proliferation and/or survival of precursors. Thus,

0 2008 American Society for Bone and Mineral Research OSTEOCLASTBIOLOGY AND BONERESORPTION / 21

c-Fmr RANK 5. Kostenuik PJ, Shalhoub V 2001 Osteoprotegerin: A physiological and pharmacological inhibitor of bone resorption. Curr Pharm Des 7:613435. 6. Teitelbaum SL, Ross FP 2003 Genetic regulation of osteoclast development and function. Nat Rev Genet 4:638-649. 7. Salo J, Lehenkari P, Mulari M, Metsikko K, Vaananen HK 1997 Removal of osteoclast bone resorDtion Droducts bv transcvtosis. Science 276:270-273. 8. Stenbeck G. Horton MA 2004 Endocvtic trafficking- in activelv resorbing osteoclasts. J Cell Sci 117:82?-836. 9. Hynes RO 2002 Integrins: Bidirectional, allosteric signaling ma- chines. Cell 110:673487. 10. Ross FP, Teitelbaum SL 2005 avp3 and macrophage colony- stimulating factor: Partners in osteoclast biology. lmmunol Rev 208:88-10.5. PCS D 0 C SD D D 11. Teitelbaum SL 200.5 Osteoporosis and integrins. J Clin Endocrinol Metab 90:2466-2468. c>=Klmw P = Pmliferation 12. Teitelbaum SL, Abu-Amer Y, Ross FP 199.5 Molecular mecha- C = Cytoskelelal n-afgmhrllon nisms of bone resorption. J Cell Biochem 59:1-10. Tnnscrtptton s = suwlval 01- 13. Van Wesenbeeck L, Odgren PR, Coxon Frattini A, Moens P, Faclor D IDineren(ialion FP, Perdu B, MacKay CA, Van Hul E, Timmermans JP, Vanhoe- FIG. 6. Osteoclast signaling pathways. Summary of the major recep- nacker F, Jacobs R, Peruzzi B, Teti A, Helfrich MH, Rogers MJ, tors, downstream kinases, and effector transcription factors that regu- Villa A, Van Hul W 2007 Involvement of PLEKHMl in osteo- late osteoclast formation and function. Proliferation (P) of precursors clastic vesicular transport and osteopetrosis in incisors absent rats is driven chiefly through ERKs and their downstream cyclin targets and humans. J Clin Invest 117:919-930. and E2F maximal activation of this pathway requires combined signals 14. Vaananen HK, Zhao H, Mulari M, Halleen JM 2000 The cell from c-Fms and the integrin avp3. As expected, the cyloskeleton (C) biology of osteoclast function. J Cell Sci 113:377-381. is independent of nuclear control but depends on a series of kinases IS. Schwartz MA, Ginsberg MH 2002 Networks and crosstalk: Inte- and their cytoskeletal-regulating targets, whereas differentiation (D) is grin signalling spreads. Nat Cell Biol 4:E65-E68. regulated largely by controlling gene expression. The calcium/ 6. Horne WC, Sanjay A, Bruzzaniti A, Baron R 2005 The role(s) of calmodulin (CaM)/calcineurin (CN) axis enhances nuclear transloca- Src kinase and Cbl proteins in the regulation of osteoclast differ- tion of NFATlc, the most distal transcription factor characterized to entiation and function. Immunol Rev 208:106-12.5. date. See Refs. 2, 3, 10, 28, 40, and 53-56 for details. 7. Zou W, Kitaura H, Reeve J, Long F, Tybulewicz VLJ, Shattil SJ, Ginsberg MH, Ross FP, Teitelbaum SL 2007 Syk, c-Src, the avp3 integrin, and ITAM immunoreceptors, in concert, regulate osteo- clastic bone resorption. J Cell Biol 1765377488. 8. Jaffe AB, Hall A 2005 Rho GTPases: Biochemistry and biology. proliferation is mediated by avp3 and c-Fm~,("'~~~))reorgani- Annu Rev Cell Dev Biol 21:247-269. zation of the cytoskeleton by avp3, c-Fms, and RANK,(*.'') 9. Chellaiah MA 2005 Regulation of actin ring formation by rho differentiation of mature osteoclasts from myeloid progenitors GTPases in osteoclasts. J Biol Chem 280:32930-32943. by c-Fms, RANK, TNFRl, and IL-lRl,(2~50~5')and their func- 20. Fukuda A, Hikita A, Wakeyama H, Akiyama T, Oda H, Naka- tion by RANK, TNFRl, and IL-lRl.(52,") Not shown is the mura K, Tanaka S 2005 Regulation of osteoclast apoptosis and fact that multiple other cytokines and growth factors, targeting motility by small GTPase binding protein Racl. J Bone Miner Res the same or other less prominent pathways, or acting indi- 20:224.5-2253. 21. Faccio R, Teitelbaum SL, Fujikawa K, Chappel JC, Zallone A, rectly robably contribute to overall control of bone resorp- tion. Tybulewicz VL, Ross FP, Swat W 2005 Vav3 regulates osteoclast (28 function and bone mass. Nat Med 11:284-290. 22. Whyte MP, Obrecht SE, Finnegan PM, Jones JL, Podgornik MN, McAlister WH, Mumm S 2002 Osteoprotegerin deficiency and Human Genetics juvenile Paget's disease. N Engl J Med 347:175-184. The text above might suggest that numerous mutations in 23. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, many genes linked to the osteoclast are likely to have been DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, discovered in humans. In fact, few such genetic changes have Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D. Pattison been defined, with >50% of those reported being in patients W, Campbell P, Sander S, Van G, Tarpley J, Derby J, Lee R, with osteopetrosis caused by defects in the chloride channel Boyle WJ 1997 Osteoprotegerin: A novel secreted protein in- that modulates osteoclast acid secretion (Fig. 3). Rare reports volved in the regulation of bone density. Cell 89:309-319. link deficiencies in RANK, the proton pump, or carbonic an- 24. Clines GA, Guise TA 200.5 Hypercalcaemia of malignancy and basic research on mechanisms responsible for osteolytic and os- hydrase I1 to osteopetrosis, whereas decreased cathepsin K teoblastic metastasis to bone. Endocr Relat Cancer 12:549-583. function leads to pyknodysostosis. In contrast, RANK activa- 25. Martin TJ 2002 Manipulating the environment of cancer cells in tion manifests as osteolytic bone disease, whereas OPG dcfi- bone: A novel therapeutic approach. J Clin Invest 110:1399-1401. ciency leads to a severe form of high turnover osteoporosis. 26. 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Chapter 4. Osteocytes

Lynda F. Bonewald

Department of Oral Biology, University of Missouri at Kansas City School of Dentistry, Kansas City, Missouri

INTRODUCTION that this is a major function of these cells. Recently, it has been shown that osteocytes have another important function, to In the adult skeleton, osteocytes make up >90-95% of all bone regulate phosphate homeostasis; therefore, the osteocyte net- cells compared with 44% osteoblasts and -1-2% osteoclasts. work may also function as an endocrine gland. Defective os- These cells are regularly dispersed throughout the mineralized teocyte function may play a role in a number of bone diseases, matrix, connected to each other and cells on the bone surface especially glucocorticoid-induced bone fragility and osteopo- through dendritic processes generally radiating toward the rosis in the adult, aging skeleton. bone surface and the blood supply. The dendritic processes travel through the bone in tiny canals called canaliculi (250- OSTEOCYTE ONTOGENY 300 nm), whereas the cell body is encased in a lacuna (15-20 km; Figs. 1 and 2). Osteocytes are thought to function as a Osteoprogenitor cells reside in the bone marrow before dif- network of sensory cells mediating the effects of mechanical ferentiating into plump, polygonal osteoblasts on the bone sur- loading through this extensive lacuno-canalicular network. Not face.".') By an unknown mechanism, some of these cells are only do these cells communicate with each other and with cells destined to become osteocytes, whereas some become lining on the bone surface, but their dendritic processes extend past cells and some undergo programmed cell death known as ap- the bone surface into the bone marrow. Osteocytes have long optosis.(') Osteoblasts, osteoid-osteocytes, and osteocytes may been thought to respond to mechanical strain to send signals of play distinct roles in the initiation and re ulation of mineral- resorption or formation, and evidence is accumulating to show ization of bone matrix, but Bordier et first proposed that osteoid-osteocytes are major regulators of this process. Oste- Dr. Bonewald has received graduate student support from and has consulted for Procter & Gamble. She also holds a patent on MLO cell Key words: osteocytes, mechanical load, phosphate metabolism, lines. apoptosis, bone disease

0 2008 American Society for Bone and Mineral Research