Osteoclasts, macrophages, and the molecular mechanisms of bone resorption

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1 Osteoclasts, macrophages, and the molecular mechanisms of bone resorption Steven L. Teitelbaum, M. Mehrdad Tondravi, and F. Patrick Ross Department of Pathology, Washington University School of Medicine, St. Louis, Missouri Abstract: The osteoclast is a physiological polykaryon and the major if not exclusive resorptive cell of bone. It participates in bone remodeling, repair, and growth and mobilization of mineral to meet homeostatic demands. Most importantly, osteoporosis, a disease endemic in Western society and Asia, is always a reflection of enhanced osteoclastic activity relative to bone formation by osteoblasts. In fact, all forms of antiosteoporosis therapy proven successful involve inhibition of osteoclastic bone resorption. Bone resorption is regulated either by altering recruitment of osteoclast precursors into fully differentiated resorptive polykaryons or modulating the rate at which mahire osteoclasts degrade bone. With this in mind, our laboratory has focused on the molecular mechanisms of osteoclast differentiation and the means by which the cell degrades bone matrix. J. Leukoc. Biol. 61: ; Key Words: integrins. H-ATPase. cytokines. steroid hormones OSTEOCLAST ONTOGENY Before the last decade, little was known regarding osteoclast ontogeny or how the cell resorbs bone. For example, as fundamental an issue as whether the osteoclast degrades both the organic and inorganic phases of bone or mobilizes only the mineral compartment was unresolved. We propose that this paucity of information reflected the lack of meaningful in vitro models useful for evaluating osteoclast differentiation and function. In contrast, recently developed systems permit addressing issues fundamental to osteoclast biology [1]. It is now known that the osteoclast is a member of the monocyte/macrophage family. We believe initial confirmation that the osteoclast is of hematopoietic origin comes from observations, made in conjunction with our colleagues at the University of Minnesota, involving a patient with osteopetrosis [21. This family of sclerotic bone diseases, discussed in this review, reflects failed osteoclastogenesis or the inability of mature osteoclasts to resorb bone. Based on seminal animal studies suggesting that osteoclast precursors are hematopoietic [3, 1, we reasoned that bone marrow transplantation would be curative in circumstances of defective osteoclast function. With this in mind we transplanted an osteopetrotic female infant with marrow of her HLA/MLC-identical brother. Not only was the transplant curative but, by following the Y chromosome, we established that osteoclasts, but not osteoblasts, are donor (i.e. marrow) derived. Ultimate proof that the osteoclast is of myeloid ontogeny came with the capacity to generate these resorptive cells in culture from pure populations of mononuclear phagocytes 151. The osteoclast shares many features with other macrophage polykaryons but is a unique cell. Those characteristics distinguishing the osteoclast are expression of calcitonm receptors, the capacity to degrade bone and in so doing produce resorption lacunae, synthesis of abundant tartrateresistant acid phosphatase and distinctive polarization, the latter eventuating in formation of a unique ruffled membrane at the osteoclast-bone interface. Osteoclast ontogeny predicts that absence of transcription factors governing myeloid differentiation will prompt osteopetrosis. In support of this hypothesis we find that transgenic mice in which the myeloid and B lymphoid transcription factor PU.1 (also called Spi-i or Sfpi-1) is deleted fail to generate macrophages or osteoclasts and develop this sclerotic bone disease 161. We successfully rescue the mutant mice by marrow transplantation, with complete restoration of osteoclast and macrophage differentiation and function. Our observations genetically support the common lineage of osteoclasts and macrophages and demonstrate the PU.i mutation is intrinsic to hematopoietic cells. To date only one other transcription factor, c-fos, has been implicated in osteoclastogenesis. Whereas deletion of c-fos also leads to osteopetrosis, in contrast to the PU.i mouse, the c-fos knockout contains excess marrow macrophages [7]. The abundance of macrophages in the face of absent osteoclasts suggests that c-fos promotes differentiation of a bipotential macrophage/osteoclast precursor toward the osteoclast pathway. Because both osteoclasts and macrophages are absent in mice lacking the PU.1 gene product, PU.1 exerts its effect in osteoclastogenesis Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF-a, tumor necrosis factor a; LPS, lipopolysaccharide; IL-4, interleukin-4. Correspondence: Steven L. Teitelbaum, M.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital, North Campus, 216 South Kingshighway, St. Louis, MO Received November 1, 1996; revised November 27, 1996; accepted December 4, Journal of Leukocyte Biology Volume 61, April

2 L!JLJLJ c-src carbonic Anhydrase H-ATPase II Determination Proliferation, Differentiation Polarization Survival Resorption Fig. 1. PU.! exerts its effect on osteoclast development earlier than other known osteopetrotic mutations. While PU.! and c-fos both affect osteoclastogenesis, PU.1 impacts the developmental pathway earlier. The mutations of c-src, carbonic anhydrase II, and H-ATPase affect function of the mature osteoclast. Absence of M-CSF in the op/op mouse is a mutation targeting stromal cells and osteoblasts that normally express the cytokine and thus indirectly affects osteoclast progenitors. earlier than c-fos. Other examples of the osteopetrotic phenotype include mice bearing the naturally occurring op/op mutations 181, or those in which c-src has been deleted or humans lacking functional H-ATPase [iol or carbonic anhydrase II genes [111. The op/op mutation leads to secretion, by marrow stromal cells, of prematurely terminated, nonfunctional CSF-i 1i2, i31. Although this mutation is not intrinsic to hematopoietic cells, the cytokine appears to be important in macrophage and osteoclast development during early life. Given that PU.1 regulates expression of the CSF-i receptor, c-fms, in myeloid cells 1141, we hypothesize that PU.i acts earlier than CSF-i in the cascade leading to osteoclast formation. In contrast to osteopetrosis arising from a lack of osteoclasts, c-src mutants have abundant multinucleated cells with the osteoclast phenotype However, c-src osteoclasts are incapable of normal bone resorption. A model consistent with the findings summarized above suggests that PU.i represents the earliest mutation in the pathway of osteoclast generation and function (Fig. 1). ION TRANSPORT AND OSTEOCLASTIC BONE RESORPTION Although genetic approaches provide powerful tools to analyze ontogeny, exploration of the biochemical events of bone resorption requires large numbers of functional osteoclasts. With this in mind we developed or adapted systems for the isolation and/or generation, in vitro, of avian or murime osteoclasts 15 i6]. Having techniques in hand for generating osteoclasts or isolating these cells from a variety of animals and maintaining them in culture provided us with the opportunity to explore the molecular mechanisms of bone resorption. Our initial efforts established that osteoclasts degrade both bone mineral and collagen and do so with a temporal asynchrony indicating that the inorganic phase must be removed from collagen bundles prior to collagenolysis [i7j. Bone mineral is removed by acidffication of an isolated compartment, the osteoclast-matrix interface. Following demineralization, the organic phase of bone is degraded by collagenolytic enzymes with a ph optimum approximating 4.5, reflecting that present in the resorptive microenvironment [i7j. The fact that weak base arrests osteoclast activity indicates that acidification of the extracellular microenvironment, at the osteoclast-bone interface, is essential to the resorptive process. In fact, the magnitude of acidification necessary to mobilize bone mineral establishes the osteoclast as the major proton-secreting cell. Thus, we turned to the means by which osteoclasts secrete protons into the resorptive microenvironment. Using avian osteoclasts as our model, we established that these cells contain an abundant vacuolar H-ATPase similar, if not identical, to the proton pump expressed by intercalated cells of the renal tubule [181. Importantly, when in contact with bone, this osteoclast proton pump polarizes to the osteoclast-bone interface where it is needed to acidify the resorptive microenvironment. While there is agreement that the resorptive proton pump of the osteoclast is an electrogenic, vacuolar type H-ATPase, controversy has surrounded the subunit composition of this pump To address this issue, we isolated the functional osteoclast proton pump subunits and reconstituted their protontransporting activity in lipid bilayers. Those subunits essential to osteoclast proton transport are immunologically similar to those within the renal H-ATPase [201. The massive proton transport essential to bone degradation raises the issue as to how the osteoclast maintains intracellular ph in the face of potential accumulation of base equivalents. To address this question we again adapted the paradigm of the renal intercalated cell. We find that osteoclasts express, on their anti-resorptive surface, an anion exchanger similar to band 3 of the erythrocyte and renal tubular cell. This transporter exchanges intracellular HCO3 for extracellular C1 in an energy-independent fashion [21J. Thus, the osteoclast is polarized to acidify the resorptive microenvironment and secrete base equivalents via the anti-resorptive plasma membrane. The model described thus far addresses massive proton transport by osteoclasts and maintenance of intracellular ph. This compendium of events does not account for dissipation of charge due to anion accumulation. Because in- 382 Journal of Leukocyte Biology Volume 61, April 1997

3 ci- Fig. 2. Model for the major steps in osteoclastic bone resorption. The osteoclast attaches to bone, which prompts formation of a convoluted ruffled membrane and a resorptive microenvironment beneath the cell. Hydrochloric acid, the product of a vacuolar-type H + -ATPase and chargecoupled Cl- channel concentrated in the ruffled membrane, is secreted, resulting in mineral dissolution. Vesicles containing acidic collagenolytic enzymes in the form of cathepsins, fuse with the bone-apposed membrane, leading to enzyme release and consequent organic matrix degradation. Intracellular ph balance is maintained by a passive C1/HCO3 exchanger on the contraresorptive surface of the cell. cr H2co3 H cr. :#{149} :#{149} : #{149}. #{149}. #{149} #{149} #{149} : #{149} : #{149} #{149} :: #{149} :#{149} : :#{149} : #{149} #{149} C02-4-.co2H2O Pump \ #{149} #{149} #{149} #{149} : #{149} : #{149} #{149} :#{149} #{149} #{149}. #{149}. Cathepsins ADP + P1 ATP Chloride #{149} #{149}:. #{149}.t Channel #{149} #{149} #{149} #{149}: #{149} #{149} Ruffled. #{149} #{149}: #{149}. #{149} #{149} Membrane. #{149} #{149} #{149}.. H C1 BONE tracellular anion excess in this circumstance would reflect abundance of Cl, the anion exchanged for HC03, we postulated that a mechanism exists in the membrane of the osteoclast juxtaposed to bone, by which C1 passes into the resorptive microenvironment. We find that the osteoclast contains a passive C1 permeability in its resorptive membrane, which is charge coupled to its H-ATPase [221. Thus, the means by which osteoclasts acidify the resorptive microenvironment involves secretion of HC1. We have recently established that this C1 channel is outwardly rectifying and related to the renal microsomal chloride channel p64. Interestingly, expression of the resorptive C1 channel is induced upon contact of osteoclast precursors with bone, a step essential to development of the resorptive phenotype [Schlesinger, P. H., Blair, H. C., Teitelbaum, S. L., Edwards, J. C., unpublished resultsl. In summary, acidification of the osteoclast resorptive microenvironment consists of series of well-defined ion-transport events (Fig. 2). The process begins when, under the influence of carbonic anhydrase II, CO2 is hydrated to H2CO3, which dissociates into protons and bicarbonate ions. Protons are Secreted in an energy-dependent fashion into an isolated microenvironment located at the cell-bone interface and HC03 exchanged for C1 at the cell s anti-resorptive surface. C1 entering the osteoclast passes through a resorptive plasma membrane anion channel charge coupled to the H-ATPase. Bone consists of mineral and an organic phase, 90% of which is type I collagen [23j. Although acidification is sufficient to mobilize bone mineral, organic matrix degradation, which we established is also under the aegis of the osteoclast [i7j, requires proteolytic activity. Given the highly acidic resorptive milieu, we suspected the osteoclast collagenolytic enzyme(s) must enjoy a low ph optimum. We verified that such is the case by demonstrating that osteoclast lysates degrade authentic bone collagen most effectively at ph 4.5 [i7, 241, that extant in the resorptive microenvironment. We established that the avian osteoclast contains a cathepsin B-like acidic protease capable of degrading authentic fibrillar bone collagen [241. Recent studies have identified the presence, in mammalian osteoclasts, of a uniquely expressed cathepsin homolog, designated cathepsin K [25, 261. Absence of this protein leads to failure to resorb bone, attesting to the importance of this family of proteases in osteoclast function [27J. While the detailed pathway for delivery of cathepsins to the osteoclast surface is incompletely understood, transport from the golgi to the resorptive microenvironment involves the mannose- 6-phosphate receptor [28J. This observation indicates yeside movement in the osteoclast contrasts with that of most other cells. Whereas the majority of golgi-derived vesicles are targeted in other cells to lysosomes, in the osteoclast the default pathway of lysosomal enzyme transport, namely targeting to plasma membrane, dominates. OSTEOCLAST INTEGRINS The requirement for an isolated extracellular resorptive microenvironment with a ph distinctly different from the general extracellular space indicates that physical intimacy between the osteoclast and bone matrix is essential to the resorptive process. With this in mind we explored the means by which osteoclasts recognize and attach to matrix. Because of the pivotal role integrins play in cell-matrix attachment we asked if members of this family of heterodimers mediate osteoclast-bone recognition. To identify osteoclast integrins participating in bone binding we coated wells with isolated bone matrix proteins and asked which support attachment of avian osteoclasts. We find that only proteins with the Arg-Gly-Asp (RGD) amino acid motif are bound by osteoclasts, suggesting that the event is mediated Teitelbaum et al. Molecular mechanisms of bone resorption 383

4 through the RGD-recognizing subfamily of a-containing integrins, particularly av33. In fact, a blocking antibody recognizing the external domain of the intact Uv3 heterodimer inhibits, in a dose-dependent fashion, the ability of osteoclasts to attach to and degrade bone [291. Osteoporosis always reflects accelerated osteoclast-mediated bone resorption relative to formation. Thus all successful strategies to prevent or arrest this disease to date, have involved osteoclast inhibition. Our finding that antibody blockade of a433 arrests bone resorption, in vitro, encouraged us to search for an RGD peptide mimetic that recognizes av33 with high affinity. We reasoned that such a molecule would blunt bone resorption in vivo and thus prevent osteoporosis. We have identified a small organic molecule that recognizes isolated av33 in solid-phase assays and prevents osteoclasts from attaching to and resorbing bone in vitro. Most importantly, when administered to rats, this av133 antagonist completely prevents the massive bone loss occurring within 6 weeks of oophorectomy. Thus, av133 inhibition presents itself as a potential form of osteoporosis prophylaxis [30]. Having established that Uv33 plays a central role in osteoclastic bone resorption we turned to regulation of the integrin. We first examined the impact of the osteoclastogenic steroids, vitamin D3 and retinoic acid. We had shown previously that the active metabolite of vitamin D3, namely 1,25 dihydroxyvitamin D3 [1,25(OH)2D3J, is a potent inducer of differentiation of osteoclast precursors and other macrophages [31-33]. We find the steroid, as a component of osteoclast differentiation, enhances av3 expression by marrow macrophages by transcriptional activation of both av and 133 integrin gene [34, 35]. We extended these studies to another osteoclastogenic steroid, retinoic acid, and discovered that it too induces av3 expression. In this circumstance, however, appearance of the heterodimer is regulated by enhanced transcription of the 33 subunit [361. The cascade of events inducing post-menopausal osteoporosis begins with a decline in physiological estrogen, accelerating, in turn, osteoclastic bone resorption. We find that while estrogen alone fails to impact Uv(33 expression by osteoclast precursors, picomolar concentrations of the steroid, namely those circulating in post-menopausal women, enhance the integrin-inductive capacity of 1,25(OH)2D3 [371. In contrast, and in keeping with the anti-resorptive effects of estrogen, the sex steroid in nanomolar amounts, which are present prior to menopause, fail to impact ct433. Similar to retinoic acid induction of av3, the heterodimer is regulated via the n-subunit. We next addressed the mechanisms by which steroid hormones transactivate the avian 33 gene. To this end we cloned the 33 promoter and identffied a classical vitamin D response element [38]. Perhaps of greater interest, we characterized a novel steroid response element consisting of three direct hexameric nucleotide repeats This motif is recognized by both the vitamin D and retinoic acid receptors, each in complex with the RXR receptor. Interestingly, each receptor heterodimer competes for the middie half site, thereby modulating the other s transactivating capacity. We believe this represents the first example of steroid hormone receptors modulating each other s transcriptional activity by competing for the same response element. Having established regulation of av3 expression by osteoclastogenic steroids, we asked if hematopoietic cytokines also alter (i integrin appearance on osteoclast precursors. These experiments required a murine model of osteoclast precursor differentiation. To this end we used pure populations of macrophage colony-stimulating factor-dependent murine marrow macrophages which, when placed in appropriate culture conditions, differentiate into bona fide osteoclasts [5]. Our colleague Roberto Pacifici has demonstrated that human CD34 cells, when cultured in vitro with the proper combination of cytokines, including granulocytemacrophage colony-stimulating factor (GM-CSF), differentiate into osteoclasts [40]. Given the central role of GM-CSF in osteoclast formation, our first efforts were directed at determining if the cytokine impacts a[33. By a combination of Northern analysis and immunoprecipitation studies on murine osteoclast precursors, we demonstrated that GM- CSF induces mrna and surface-expressed Uv33 in a time- and dose-dependent manner. Moreover, transcription is unaltered by GM-CSF, but stability of f33 mrna is substantially enhanced [Inoue, M., Teitelbaum, S. L., Ross, F. P., unpublished results]. Importantly, freshly isolated osteoclast precursors, while failing to express (1433, are still capable of spreading on matrix, an event inhibited by our RGD peptidomimetic. This finding led to us to examine these precursors for the presence of other integrins capable of ligating RGD. Our studies culminated in identification of avs as the integrin mediating matrix attachment of early osteoclast precursors. Further experiments revealed that GM-CSF decreases transcription of the 3s gene, diminishing surface expression of avs. The significance of our findings on the ability of GM-CSF to regulate av integrin expression is underscored by the fact that levels of av3 and av15 change reciprocally during osteoclastogenesis in vitro. Whereas a435 is present before multinucleation and disappears with time, av[3 initially absent, increases during osteoclast formation [Inoue, M., Teitelbaum, S. L., Ross, F. P., unpublished results]. These findings suggest a model for the role of integrins in osteoclast formation in which the osteoclast precursor expresses OtvI-35 and no av3, with the situation reversed in the mature cell (Fig. 3). Thus, av35 may be responsible for attachment of precursors, a prerequisite for their proliferation and differentiation. As av5 disappears avs, the functional integrin of the mature osteoclast, is expressed. TUMOR NECROSIS FACTOR a (TNF-a) Using murine osteoclastogenic cultures, we confirmed earlier reports 141, 42] suggesting that TNF-a, a major secretory product of activated macrophages, is among the most potent of osteoclastogenic cytokines lunpublished data]. 384 Journal of Leukocyte Biology Volume 61, April 1997

5 Fig. 3. Proposed scheme for the role of integrins in osteoclast formation and function. The immature osteoclast precursor, while arising in marrow, circulates in the blood. Attachment to RGD-containing proteins in bone is mediated by the integrin Once adherent, the precursor undergoes differentiation and fusion under the influence of a range of hormones and cytokines whose activities include decreasing expression of while enhancing that of a433, the functional integrin of the mature osteoclast. Mature 7steoclast Differentiation A l4l Multinucleation av) Bone A = RGD-containing protein Bone This finding led us to hypothesize a possible mechanism of implant osteolysis, the most frequent disabling complication following prosthetic replacement of diseased joints. Thus, we suggested that TNF-a secretion, by macrophages that have phagocytosed implant-derived particles, represents a critical first step in the accelerated bone resorption characterizing this important clinical condition. Our initial studies confirmed enhanced transcription of the TNF-a gene by macrophages exposed to implant-derived particles lunpublished data]. We then developed an in vivo model in which either polymethylmethacrylate or polyethylene particles (both found in tissues surrounding failed implants) were placed under the external calvarial periosteum of mice. Within 1-2 weeks an osteolytic, osteoclast-rich lesion develops that is functionally and morphologically indistinguishable from that seen in humans. Resident macrophages contain high levels of TNF-a. Most importantly, mice in which both TNF-a receptors have been deleted are protected from implant particle osteolysis. This finding establishes the central role of TNF-a as an etiological agent in post-implant osteolysis [unpublished data]. Periodontal disease, which is accompanied by the presence of bacterial lipopolysaccharide (LPS)-secreting bacteria, represents a second important clinical situation associated with accelerated bone loss. Cultures containing osteoclast precursors derived from marrow of mice treated in vivo with LPS, yield increased numbers of osteoclasts, an event blocked once again by inhibition of TNF-a function [unpublished data]. In summary, TNF-a-stimulated osteoclast formation represents the mechanism of two separate, clinically important conditions. Because TNF-a regulates osteoclast differentiation we wondered if the cytokine also modulates av integrin expression, a process that parallels generation of bone-resorbing cells. We find that treatment of osteoclast precursors with TNF-a leads to a decline in steady state 35 mrna levels as a result of decreased mrna stability, with the overall result being diminished surface expression of a435. In contrast to GM-CSF, which induces av33, TNFa does not impact this integrin receptor [43]. INTERLEUKIN-4 (ll-4) Regulation of osteoclast differentiation and integrin expression is not limited to hematopoietic cytokines. In this regard, we reported several years ago that a transgenic mouse overexpressing IL-4 develops a form of osteoporosis with decreased osteoblast and osteoclast function [44]. We determined that addition of IL-4 to our in vitro murine osteoclastogenic coculture results in dose-dependent decreased multinucleation [45], a finding correlating with the in vivo result. Furthermore, we find the target cell for IL-4 action is the osteoclast precursor and not the osteoblast/stromal component of the coculture [46] and that IL-4, in addition to decreasing osteoclast formation, blunts the bone-resorbing activity ofthe mature cell [47J. Turning to the action of IL-4 on integrins, we determined that the cytokine increases expression of a433 by stimulating transcription of the 33 subunit, whereas steady state levels of a,, mrna are unaltered [48]. In a finding reminiscent of that for GM-CSF, IL-4 also accelerates disappearance of af35 from the cell Teitelbaum et al. Molecular mechanisms of bone resorption 385

6 surface lunpublished data]. Although the role of IL-4 in regulating integrin expression is of interest, a finding of greater potential significance is that IL-4, by decreasing transcription of the TNF-a gene, blocks secretion of the osteoclastogenic cytokine by activated macrophages [unpublished data]. This observation may explain our earlier report that IL-4 inhibits osteoclast formation [49]. Our studies on the molecular mechanisms whereby IL- 4, GM-CSF, and TNF-a regulate expression of the integrins av33 and av3s reveal a variety of pathways are involved, including both increases and decreases in the rate of transcription and mrna stability. Generally, it is the relevant f3 and not av subunit that mediates heterodimer expression. To understand the molecular basis of these events we have cloned both the murine and 35 promoters [50, 51]. Initial examination reveals the presence of consensus sequences for a number of basal and tissue-specific Iranscription factors. There are also consensus sequences for STAT proteins, cytosol-residing latent transcription factors known to mediate activation of genes following cytokine treatment of cells [52, 53]. Direct proof that specffic sequences in the promoter regions of the and 3s genes are involved in cytokine regulation of a433 and a435 in osteoclast precursors will require experiments in which deletion and/or mutation of the putative active sites is followed by functional analysis. OSTEOCLAST POLARIZATION One of the major remaining unsolved issues in osteoclast biology is the mechanism by which the cell polarizes. Following attachment to bone matrix a characteristic ruffled membrane, containing a number ofcritical proteins, including the vacuolar-type proton pump, is generated. An additional critical event is secretion of one or more cathepsins, whose function is to degrade organic matrix in the acidic bone-adjacent microenvironment. Although the detailed events underlying ruffled membrane formation and regulated exocytosis are unclear, the fact that acidifying vesicles are randomly distributed in the cytoplasm of osteoclasts not in contact with bone, but polarize to the bone-apposed plasma membrane in the substrate-adherent cell, indicates that in the resorptive polykaryon, matrix-derived signals prompt directed vesicular movement. Based on information derived from cells such as neurons, pancreatic beta, mast, and pituitary cells [54-57], a reasonable model suggests that a cell-specific signal (e.g., neuronal membrane depolarization, recognition of glucose by its surface receptor on the islet cell, activation of mast cells) triggers movement of vesicles toward the cell surface, where fusion occurs with the existing membrane, thereby leading to its expansion. The overall process, as revealed by analysis of other cell types, primarily neurons, involves a complex set of events mediated by many proteins, including coatamer I and II complexes, a series of docking-related proteins called NSFs, VAMPS, SNAPs, SNAREs, and multiple members of the small GTPase family (both ARFs and rabs). Although outside the scope of this review, the detailed interactions of the many molecules involved in membrane targeting and fusion of specific vesicles are a matter of intense research, summarized in a number of recent artides [58-60]. There is no documentation, in osteoclasts or their precursors, of any proteins known to regulate exocytosis in other systems. Thus, our finding that murine osteoclast precursors express two isoforms of the rab3 subfamily [61J, proteins that mediate regulated exocytosis in a number of other cell types, is potentially important. In the osteoclast, regulated exocytosis (an event we predict is initiated by recognition of bone matrix by the osteoclast), would result in ruffled membrane generation and secretion of collagenolytic enzymes, critical events in bone resorption. The possible significance of the presence of rab3 proteins in osteoclast precursors is underscored by two additional observations. First, the levels of the same two rab3 family members are increased during osteoclastogenesis in our in vitro murine system. Second, treatment of osteoclast precursors with a range of hematopoietic cytokines, and most notably the potent osteoclastogenic molecule TNF-a, results in enhanced expression of both rab3 isoforms, raising the possibility that GTPases participate in ruffled membrane formation. The above events, although representing a reasonable model for expansion of the ruffled membrane, fail to explain how vesicles are transported to the cell surface where they fuse with the plasmalemma to form the ruffled membrane. The first clue as to how vesicles move in osteoclasts came from studies in which the protooncogene c-src was deleted in mice by targeted recombination [91. The resulting animals exhibit osteopetrosis, whose cellular basis is the inability of the differentiated osteoclasts to resorb bone due to failed formation of a ruffled membrane. Based on the reported localization of c-src in osteoclasts at both the ruffled membrane [62] and within the intravesicular compartment [63J, the protooncogene may play a role in vesicular transport. Because microtubules represent an important pathway by which various proteins and vesicles migrate through cells [64, 65] we hypothesized that proteins destined for the ruffled membrane of the osteoclast associate with intermediate filaments. A series of experiments utilizing a combination of confocal microscopy and co-immunoprecipitation/immunoblot analysis demonstrate that adherence of osteoclast precursors to matrix is followed by co-association of c-src with microtubules and not monomeric tubulin. We extended the study to demonstrate that the H-ATPase, the hallmark and major functional protein complex of the ruffled membrane, also decorates microtubules. Furthermore, the osteoclast proton pump, c-src as well as a rab3 isoform known to mediate cytoplasmic vesicle-plasma membrane fusion, localize to the light golgi fraction of osteoclast precursors [661. Because of the central role played by c-src in osteoclast polarization we asked if osteoclastogenic cytokines regulate the protooncogene. We find that TNF-a is unique in that, via accelerated transcription, it alone stimulates expression of c-src by osteoclast precursors [67]. To our knowledge, 386 Journal of Leukocyte Biology Volume 61, April 1997

7 ACKNOWLEDGMENTS This study was supported in part by National Institutes of Health Grants DE05413, AR32788 (S. L. T.), AR42404, AR42378 (F. P. R.), AR44089 (M. M. T.) and a grant from the Shriners Hospital for Crippled Children, St. Louis Unit, St. Louis, MO (S. L. T.). REFERENCES Fig. 4. Model ofosteoclast polarization. The osteoclast attaches to bone via the integrin a433, resulting in tubulin polymerization. Unidentified signals stimulate movement along microtubules of intracellular vesicles, which bear functional proteins targeted to the bone-apposed plasma membrane. Vesicle generation and targeting is a complex set of events involving a large number of facilitatory proteins. Fusion of vesicles results in production of the characteristic ruffled membrane. this represents the first report of transcriptional regulation of c-src. Taken together, our findings suggest a model in which movement of vesicles containing functionally important proteins destined for the osteoclast ruffled membrane, move along a track comprised of microtubules (Fig. 4). SUMMARY Recognition that the osteoclast is a member ofthe monocyte/ macrophage family has prompted development of meaningful experimental models to study the cellular mechanisms of bone resorption. These efforts have delineated molecular targets to inhibit the resorptive process and thus prevent diseases such as osteoporosis. Many concerns remain to be addressed. Among the most provocative is the means by which the osteoclast polarizes and the impact of matrix recognition and cytokines on this event. The role of tissuespecific transcription factors in osteoclast commitment is also fertile area for investigation. The tools are at hand to address these issues and one may expect progress in understanding osteoclast biology to continue to impact patient care. 1. 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