Cell biology of the osteoclast

Experimental Hematology 27 (1999) 1229–1241

Cell biology of the osteoclast G. David Roodman Department of Medicine, Division of Hematology, University of Texas Health Science Center, and the Audie L. Murphy Veterans Administration Hospital, San Antonio, TX (Received 11 March 1999; revised 7 April 1999; accepted 19 April 1999)

The osteoclast is a hematopoietic cell derived from CFU-GM and branches from the monocyte-macrophage lineage early during the differentiation process. The marrow microenvironment appears critical for osteoclast formation due to production of RANK ligand, a recently described osteoclast differentiation factor, by marrow stromal cells in response to a variety of osteotropic factors. In addition, factors such as osteoprotegerin, a newly described inhibitor of osteoclast formation, as well as secretory products produced by the osteoclast itself and other cells in the marrow enhance or inhibit osteoclast formation. The identification of the role of oncogenes such as c-fos and pp60 c-src in osteoclast differentiation and bone resorption have provided important insights in the regulation of normal osteoclast activity. Current research is beginning to delineate the signaling pathways involved in osteoclastic bone resorption and osteoclast formation in response to cytokines and hormones. The recent development of osteoclast cell lines may make it possible for major advances to our understanding of the biology of the osteoclast to be realized in the near future. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Osteoclast—Precursor—Bone resorption

The osteoclast: a cell in the monocyte lineage Origins of the osteoclast The osteoclast (Fig. 1) is hematopoietic in origin and derived from cells in the monocyte-macrophage lineage. Baron and coworkers TRAP [1], using an in vivo model of osteoclast formation, demonstrated that the mononuclear cells that initially attach to the bone surface contained nonspecific esterase and, as they differentiated, expressed tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. These cells eventually lost their nonspecific esterase activity and formed multinucleated osteoclasts. Similarly, Burger et al. [2] showed that when murine fetal bone rudiments that lacked osteoclasts were cocultured Offprint requests to: G. David Roodman, Research Service (151), Audie Murphy Veterans Administration Hospital, 7400 Merton Minter Boulevard, San Antonio, TX 78284. E-mail: [email protected]

in plasma clots with marrow cells and a source of macrophage colony-stimulating factor (M-CSF), osteoclasts formed. These workers identified the precursor for these osteoclasts as a cell in the monocytic lineage. Kurihara et al. [3] showed that osteoclast-like cells formed in human marrow cultures from highly purified populations of colony-forming unit granulocyte-macrophage (CFU-GM), the granulocyte-macrophage progenitor cells. In this culture system, CD341 marrow mononuclear cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 to 10 days, and then the cultures are overlayered with 1,25dihydroxyvitamin D3 for an additional 2 weeks. At the end of the second culture period, four types of colonies were detected in the cultures: (1) granulocytic, (2) macrophage, (3) colonies of mixed hematopoietic lineages, and (4) colonies of cells that had a unique polygonal morphology. These polygonal cells expressed calcitonin receptors and fused to form exclusively osteoclast-like multinucleated cells that resorbed calcified matrices. Multinucleated cells formed from these committed precursors had the phenotypic characteristics of osteoclasts. They contracted in response to calcitonin, reacted strongly with the 23c6 monoclonal antibody that identifies the osteoclast vitronectin receptor, and expressed high levels of TRAP, a marker enzyme for osteoclasts. Further support for osteoclasts being derived from cells in the monocyte-macrophage lineage are recent studies that reported that peripheral blood monocytes, when cultured under the appropriate conditions, formed osteoclast-like cells that expressed the phenotypic characteristics of osteoclasts and resorbed bone [4]. Characteristics of osteoclast precursors Recently, the morphologic and phenotypic characteristics of osteoclast precursors were described. Takahashi (unpublished observation) showed in preliminary studies that the earliest marker for cells in the osteoclast lineage appears to be expression of metalloproteinase-9 (MMP-9), which precedes expression of TRAP in CFU-GM–derived cells treated with 1,25-dihydroxyvitamin D3. Lee and coworkers [5] and Wada et al. [6], using murine marrow cultures, reported that TRAP-positive mononuclear cells initially form in these cultures and then acquire calcitonin receptor ex-

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in vitro. These model systems have been used to identify osteoclast precursors, to characterize their surface phenotype, and to identify and characterize factors that affect osteoclast activity and formation.

Figure 1. Bone biopsy specimen depicting an osteoclast in a resorption lacuna. Note the intimate contact between cells in the bone microenvironment and the osteoclasts (original magnification 3 100).

pression after treatment with 1,25-dihydroxyvitamin D3 for several days. Calcitonin can downregulate the calcitonin receptor messenger RNA in these precursors [7]. Kukita et al. [8] purified the precursors for osteoclast-like multinucleated cells formed in human marrow cultures. They demonstrated that the earliest osteoclast precursor is present in the CD34 population of human marrow cells, and, as it differentiates, it acquires an antigen that is identified by the Kn22 monoclonal antibody. This early precursor is MY91 and CD45RA1 and expresses other myeloid antigens. Wesolowski et al. [9] reported the isolation and characterization of highly purified prefusion murine osteoclast precursors. The cells were mononuclear, and they expressed calcitonin receptors and mRNAs for MMP-9, carbonic anhydrase II, and high levels of pp60c-src protein. These cells were purified by first removing marrow stromal cells with collagenase and then releasing the precursors with echistatin, a protein from snake venom containing an RGD sequence. The precursors could resorb bone only if they were cocultured with 1,25-(OH)2D3 and an osteoblastic cell line. These data suggest that the early mammalian osteoclast precursor expresses TRAP and MMP-9 and is derived from CFU-GM. As these precursors differentiate and become committed to the osteoclast lineage, they express high levels of pp60c-src, carbonic anhydrase, and calcitonin receptor.

Model systems for studying osteoclast formation and bone resorption Progress in understanding the molecular events that occur during osteoclast differentiation and osteoclastic bone resorption has been difficult because osteoclasts are few in number, are fragile when isolated from bone, and are difficult to isolate because they are embedded in a calcified matrix. Until recently, no osteoclast cell lines have been available. To circumvent these problems, a variety of model systems have been developed that form osteoclast-like cells

Mature osteoclasts isolated from bone Several techniques have been developed for isolating mature osteoclasts from long bones. One of the most commonly used sources for osteoclasts has been the endosteal surface of chick long bones. Osdoby and coworkers [10] prepared osteoclasts from embryonic chick tibia by removing the marrow and then releasing the osteoclasts using calcium- and magnesium-free buffers. The cell suspension is sieved through nylon mesh to trap the large multinucleated osteoclasts, and the osteoclasts are enriched by discontinuous density centrifugation. Cell preparations of 50–75% pure osteoclasts can be obtained using these techniques, and the osteoclasts can be cultured for 10 days and retain an osteoclast morphology. However, only a small percentage of these cells are viable and resorb bone. A similar approach was used by ZamboninZallone and coworkers [11] using hypocalcemic egg-laying chickens. With their methods, large numbers of osteoclasts can be isolated, although the viability of these cells is still in question. Osteoclasts can be isolated in large numbers from juvenile rabbit long bones, and they can be highly enriched. These cell preparations have been used to generate osteoclast cDNA expression libraries [12]. However, these systems suffer from two shortcomings: (1) the majority of these cells may not be viable; and (2) the isolated cell populations may be heterogenous, making it difficult to assess whether factors act directly or indirectly on osteoclasts. The majority of studies using isolated osteoclasts have suggested that most osteotropic factors act indirectly on osteoclasts via the osteoblast or stromal cell. Furthermore, there is a large variability in the capacity of these cells to resorb bone, so that large number of bone slices are required to obtain reproducible results. In vivo models of osteoclast formation We reported an in vivo model to examine the mechanism of action of osteotropic factors on various stages of osteoclast formation [13]. In this model system, mice are either injected with a factor or implanted with Chinese hamster ovary cells that have been transfected with the cDNA for the factor of interest and constitutively express the factor at high levels. This system permits us to examine the effects of a factor on three specific stages of osteoclast formation: (1) CFU-GM, the earliest identifiable osteoclast precursor, as assessed by colony assays; (2) more committed osteoclast precursors, as assessed by osteoclast-like cell formation in long-term marrow cultures derived from these animals; and (3) mature osteoclasts as determined by histomorphometry of calvariae from these animals. This in vivo model system helps to determine the site of action of various cytokines involved in normal bone remodeling.

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Bone organ culture systems Bone organ culture systems have provided and continue to be useful bioassays for studying factors that control osteoclastic bone resorption and, in some cases, osteoclast formation. These organ culture systems have been useful for identifying new agents that stimulate, as well as inhibit, bone resorption. Among the most frequently used systems are the fetal rat or mouse calvarial assay and the fetal rat long bone system, in which pregnant rats or mice are injected with calcium-45, and the radius and ulna or the calvaria are dissected from the fetuses or neonatal rodents. These bones are placed on membranes suspended on wire mesh and floated over a chemically defined media, then various osteotropic factors are added to the media. After an appropriate time period, the percent of calcium-45 released from the bone relative to the total amount of calcium-45 in the bone fragment is determined. Bone marrow culture systems Testa and coworkers [14] were the first to modify the classic Dexter long-term marrow culture systems to form osteoclast-like cells. In their original work, multinucleated cells were formed in feline bone marrow cultured in a-minimum essential medium with horse serum in the absence of any osteotropic factors. These multinucleated cells were not well characterized, but they were thought to be osteoclastlike because of their morphology. Ibbotson and coworkers [15] further characterized this culture system. They demonstrated that osteotropic factors could modulate formation of these osteoclast-like cells appropriately and showed that the precursor for these cells was a cell in the monocyte-macrophage lineage. Since those initial studies, marrow culture systems that form osteoclast-like cells have been extended to include culture systems using mouse marrow, in which TRAP multinucleated cells that extensively resorb bone form in 5 to 6 days [16], baboon marrow cultures [17], and human marrow cultures [18–20] in which osteoclast-like cells form in 3 weeks. In a typical human marrow culture, nonadherent marrow mononuclear cells are cultured at 106 cells/mL in the presence of 1,25-dihydroxyvitamin D3 (1028 M) for 3 weeks. Few, if any, multinucleated cells formed in the first week of culture, and the majority of multinucleated cells formed during the second week of culture. Maximum numbers of osteoclast-like multinucleated cells are formed in these cultures after 3 weeks, and by 4 weeks the cells begin to detach from the plastic surface. Multinucleated cells from these cultures fulfill the functional criteria of osteoclasts. Udagawa et al. [21] used mouse spleen cells as a source of osteoclast precursors. When these cells are cocultured with an appropriate bone marrow-derived stromal cell (MC3T3-G2-PA6 or ST2) or with primary mouse calvarial cells in the presence of dexamethasone and 1,25-dihydroxyvitamin D3, multinucleated cells form. Osteoclast-like cells formed in these cultures contain TRAP and have enhanced cyclic AMP production in response to calcitonin. Calcitonin re-

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ceptors also were present on the multinucleated cells produced in these cocultures, as demonstrated by autoradiographic techniques with 125I-labeled calcitonin [22]. Numerous resorption lacunae were formed when spleen cells and ST2 cells were cocultured on sperm whale dentine in the presence of 1,25(OH)2D3 and dexamethasone. Cell-to-cell contact with stromal cells or osteoblasts was absolutely required for osteoclast formation in this culture system. This culture system has been adapted to form large numbers of osteoclast-like cells that can be released by collagenase when the cultures are performed on collagen gel-coated plates [22,23]. Osteoclast precursor cell lines Hentunen et al. [24] were the first to report a cell line that formed large numbers of osteoclasts efficiently. They used the TRAP promoter to target the bcl-XL and/or simian virus 40 large T antigen (Tag) genes to cells in the osteoclasts lineage in transgenic mice as a means of immortalizing osteoclast precursors. Immunocytochemical studies confirmed that they had targeted Bcl-XL and/or Tag to osteoclast, and transformed and mitotic osteoclasts were readily apparent in bones from both Tag and bcl-XL/Tag mice. Osteoclast formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared to those from nontransgenic littermates, and bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than 2 years. These cells form high numbers of bone-resorbing osteoclasts when cultured using standard conditions for inducing osteoclast formation, with approximately 50% of the mononuclear cells incorporated into osteoclasts. These osteoclasts express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of osteoclast precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of osteoclast precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared to Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL Tag mice confirmed that there were increased numbers of osteoclast precursors (TRAP1 mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the osteoclast lineage, we have immortalized osteoclast precursors that form bone-resorbing osteoclasts with an efficiency that is 300–500 times greater than normal marrow. Recently, a second osteoclast precursor cell line has been reported that also forms osteoclasts at high efficiency [25].

Role of the bone microenvironment in osteoclast formation Yudagawa et al. [21] and Takahashi et al. [26] reported that coculture of spleen cells or marrow cells as a source of os-

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teoclast precursors with marrow stromal cells or osteoblasts induced osteoclast formation. However, stromal cell lines developed from mouse bone marrow displayed heterogeneity in their capacity to support osteoclastogenesis [27]. Stromal cells or osteoblasts that induce osteoclast formation must be able to produce M-CSF, because marrow stromal cells or osteoblasts from op/op mice that lack M-CSF do not support osteoclast differentiation [28]. Soluble M-CSF could not replace the requirement for stromal cells in this coculture system, which suggests that membrane-bound M-CSF or other factors produced by marrow stromal cells or osteoblasts was absolutely required for murine osteoclast development from precursors. Recently, a factor produced by marrow stromal cells and osteoblasts was identified that is critical to osteoclast formation and can replace stromal cells in coculture systems to induce osteoclast formation by spleen cells. This factor, RANK ligand, is a new member of the TNF family [29,30]. It is also called TRANCE, osteoclast differentiation inducing factor, or osteoprotegerin (OPG) ligand. RANK ligand activates c-jun terminal kinase and sends signals to NFkB. Yasuda and coworkers [30] and others [31] showed that most osteotropic factors that induce osteoclast formation act indirectly by binding marrow stromal cells, which in turn induce upregulation of RANK ligand expression. RANK ligand then binds the RANK receptor on osteoclast precursors and induces osteoclast formation (Fig. 2). A soluble form of RANK ligand has been engineered that, when added to spleen cells or CFU-GM derived cells in the presence of M-CSF and dexamethasone, induces large numbers of osteoclasts. Furthermore, the importance of RANK ligand has been shown by the techniques of homologous recombination, in which absence of RANK ligand expression in mice results in severe osteopetrosis and absence of osteoclasts [32]. Similarly, overexpression of RANK ligand in transgenic mice induces severe osteoporosis. RANK ligand activity can be blocked by the decoy receptor OPG. OPG, also called osteoclastogenesis inhibitory factor, is a member of the tumor necrosis factor (TNF) receptor superfamily that was identified recently [33,34]. In vitro and in vivo osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. OPG is produced by most cell types and appears to block the fusion/ differentiation stage of osteoclast differentiation, rather than the proliferative phase. It binds to a single class of highaffinity binding sites on the ST2 mouse marrow stromal cell line treated with 1,25-(OH)2D3. Yasuda and coworkers [30] recently demonstrated that RANK ligand, produced by osteoblasts, binds to OPG and blocks the inhibitory actions of OPG on osteoclastogenesis. Workers at both AMGEN and Snow Brand Milk have shown that overexpression of OPG in transgenic mice results in severe osteopetrosis, whereas absence of OPG induces osteopenia [35]. Thus, RANK ligand and OPG are important regulators produced by the marrow microenvi-

Figure 2. Role of RANK ligand in osteoclastogenesis. Osteotropic factors induce upregulation of RANK ligand on marrow stromal cells and osteoblasts. RANK ligand then binds the RANK receptor on osteoclast precursors and induces osteoclast formation.

ronment that regulate osteoclast formation and osteoclast activity.

Soluble factors that enhance osteoclast activity Interleukin 1 Interleukin 1 (IL-1) is a cytokine produced by monocytemacrophages and marrow stromal cells and osteoclasts that can stimulate bone resorption in vitro and in vivo. IL-1 induces bone resorption and osteoclast-like cell formation in murine and human marrow cultures [36,37]. Recently, Uy et al. [13] used an in vivo model of osteoclast formation to examine the systemic effects of IL-1 on the different stages of osteoclast development. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, the earliest identifiable osteoclast precursor. It also increased the number of more committed mononuclear osteoclast precursors and stimulated mature osteoclasts to resorb bone. The data demonstrate that IL-1 affects all stages of osteoclast development and may explain its potent effects on bone turnover in vivo. IL-1 also has been implicated in several pathologic conditions associated with increased bone loss. It is produced by several tumors associated with hypercalcemia, such as squamous cell carcinoma and lymphoma [38,39]. Freshly isolated marrow cells derived from some patients with myeloma produced IL-1b, and the bone-resorbing activity present in culture media from these marrow cell isolates could be neutralized by IL-1b antibodies [40,41]. M-CSF M-CSF appears to play an important role in osteoclast development. Studies in the op/op osteopetrotic mouse, which has a point mutation in the M-CSF gene [42] and has severe osteopetrosis due to an absence of osteoclasts, have clearly shown an important role for M-CSF in murine osteoclast development. Injection of M-CSF into op/op mice improves the skeletal sclerosis in these animals, although some authors have suggested that some residual osteosclerosis remains in these animals [43]. Furthermore, Nilsson and coworkers [44] reported that the osteopetrosis in op/op mice improves with age, which suggests that M-CSF is required

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for early osteoclast development, but that other cytokines can mimic the effects of M-CSF at later times. M-CSF also may affect mature osteoclasts, as well as osteoclast precursors. The c-fms receptor, which is the M-CSF receptor, is expressed on mature osteoclasts [45]. Fuller and coworkers [46] also identified a role for M-CSF in maintaining the survival and chemotactic behavior of mature osteoclasts. In their studies, M-CSF prevented apoptosis of osteoclasts, enhanced osteoclast motility, and inhibited bone resorption. Transforming growth factor alpha Transforming growth factor alpha (TGF-a), a polypeptide that is partially homologous to epidermal growth factor, is produced by several solid tumors associated with the hypercalcemia of malignancy. TGF-a can stimulate osteoclastic bone resorption in murine organ cultures by binding the epidermal growth factor receptor [47]. TGF-a is a proliferative factor that stimulates the growth of early osteoclast precursors, but by itself it has no colony-stimulating factor-like activity [48]. TNFs (TNF-a and TNF-b) Both TNF-a and TNF-b (lymphotoxin) markedly stimulate the formation of osteoclast-like multinucleated cells in human marrow cultures [37]. TNF also can affect the activity of mature osteoclasts. Thomson et al. [49,50] showed that incubating mature osteoclasts cocultured with osteoblastic cells with IL-1 or TNF stimulates bone resorption. TNF potentiates the effects of IL-1 on osteoclast formation [38]. Garrett et al. [51] demonstrated that myeloma cell lines released a bone-resorbing activity into their conditioned media, which was TNF-b, and that neutralizing antibodies to lymphotoxin blocked the bone resorption in bone organ cultures induced by media conditioned by these myeloma cells. However, increased levels of TNF-b have not been found consistently in patients with myeloma or in an in vivo model of human myeloma bone disease [52]. TNFs appear to stimulate both proliferation and differentiation of osteoclast precursors and may be involved in the pathogenesis of hypercalcemia of malignancy. Yoneda and coworkers [53] showed that the hypercalcemia, induced by a squamous cell carcinoma cell line implanted into nude mice, is mediated in part by TNF-a. In this model system, TNF-a was produced by host macrophages in response to the tumor. These data suggest that host factors may further aggravate the hypercalcemia induced by tumor-derived factors. Interleukin 6 Interleukin 6 (IL-6) is produced by many cells in the bone microenvironment, including marrow stromal cells, monocyte-macrophages, osteoclasts, and osteoblasts. It induces osteoclast formation from osteoclast precursors [54,55]. However, its role in osteoclast activity is controversial. It does not appear, by itself, to be a potent osteotropic factor in

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murine systems in vivo [56]. IL-6 potentiates the effect of other hormones, such as parathyroid hormone-related protein (PTHrP), on calcium homeostasis and osteoclastic bone resorption in vivo [56], and soluble IL-6 receptor must be added to murine marrow cultures to induce osteoclast formation in vitro [57]. IL-6 receptors have been demonstrated on human osteoclasts [58], and IL-6 can stimulate osteoclast-like cell formation in human marrow cultures in the absence of added IL-6 receptors [55]. IL-6 also may act as an autocrine/paracrine factor produced by osteoclasts in Paget’s disease of bone [59]. Osteoclast-like multinucleated cells formed in human marrow cultures from patients with Paget’s disease actively express IL-6 transcripts and release IL-6 into their conditioned media; these media stimulated osteoclast-like cell formation in normal marrow cultures. Furthermore, patients with Paget’s disease, but not normals, have elevated levels of IL-6 in their marrow plasma and their peripheral blood. Interleukin 11 Interleukin 11 (IL-11) is produced by marrow stromal cells [60]. Girasole et al. [61] reported that IL-11 induced the formation of osteoclasts in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 showed a high degree of ploidy and formed resorption lacunae on calcified matrices. This study also demonstrated that a neutralizing antibody against IL-11 suppressed osteoclast development induced by either 1,25-(OH)2D3, parathyroid hormone (PTH), IL-1, or TNF. These data also suggest that a variety of osteotropic factors can induce osteoblasts to produce IL-11. The effects of IL-11 on osteoclast differentiation appear to be mediated by inducing RANK ligand expression on osteoblasts and marrow stromal cells [30].

Inhibitory factors Transforming growth factor beta Transforming growth factor beta (TGF-b) has been proposed as one of the key factors involved in coupling bone formation to previous bone resorption [62]. It is secreted by osteoblasts and osteoclasts, and it may act as an autocrine factor stimulating osteoblastic bone formation through enhanced chemotaxis, proliferation, and differentiation of committed osteoblasts [62]. TGF-b is secreted as a dimer composed of 12.5-kDa subunits noncovalently associated with one or more polypeptides to form a higher molecular weight latent complex. Latent TGF-b can be activated experimentally by proteinase treatment, acidification, or denaturation to dissociate the mature factor from the latent complex. Bone contains very high levels of latent TGF-b and is the largest source of TGF-b in the body.

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TGF-b is a potent inhibitor of osteoclastic bone resorption. It modulates both osteoclastic bone resorption, migration, and osteoclast differentiation in bone organ cultures [63]. Chenu et al. [64] showed that TGF-b inhibits both the proliferation and fusion of human osteoclast precursors. Ikeda et al. [65] suggested that TGF-b may regulate normal osteoclast activity, as mRNA levels of TGF-b are decreased in rat bone following ovariectomy. These data suggested that TGF-b may be a “coupling factor” that is released and activated during osteoclastic bone resorption, then inhibits osteoclast formation and activity and induces new bone formation [66]. Interferon gamma Interferon gamma (IFN-g) is a potent inhibitor of bone resorption in vitro [67], and suppresses the formation and maturation of osteoclasts [68]. Tohkin et al. [69] examined the effects of IFN-g on the humoral hypercalcemia of malignancy in nude mice bearing lower jaw tumors, in which PTHrP was responsible for inducing hypercalcemia. Mice were injected with IFN-g for 5 days before the establishment of hypercalcemia, and IFN-g treatment delayed the increase in plasma calcium concentrations. IFN-g treatment also eliminated the formation of multinucleated osteoclastlike cells from bone marrow cells of these mice ex vivo. The data suggest that IFN-g suppresses the formation of osteoclasts in vivo, resulting in decreased plasma calcium concentrations. Interleukin 4 Interleukin 4 (IL-4) is a product of activated T cells that has effects on both immunologic and hematopoietic processes. Shioi et al. [70] reported that IL-4 inhibited the formation of osteoclasts from murine bone marrow cells cocultured with stromal cells. Watanabe et al. [71] and Riancho et al. [71,72] showed that IL-4 inhibited bone resorption in organ cultures. Nakano et al. [73] examined the in vivo effects of IL-4 on spontaneous and PTHrP stimulated mouse osteoclast formation. EC-GI cells, which produce PTHrP, were implanted into nude mice. After the mice became hypercalcemic, they were treated with IL-4. Continuous infusion of IL-4 returned the calcium levels to normal. Histomorphometric analysis revealed that IL-4 inhibited osteoclast formation in these mice as demonstrated by a decrease in osteoclastic surfaces and in the number of osteoclasts per normal bone surface. However, transgenic mice overexpressing interleukin-4 develop an osteopenic syndrome that is similar to osteoporosis [74]. These data suggest that high levels of IL-4 may inhibit bone formation, as well as bone resorption. Nitric oxide Nitric oxide is a potent multifunctional signal molecule that has widespread actions in a variety of tissues, including bone. Brandi and coworkers [75] showed that nitric oxide

produces a rapid contraction and detachment of osteoclasts from cell surfaces and inhibits bone resorption by osteoclasts. Furthermore, inhibition of nitric oxide synthase in rats in vivo is accompanied by increased bone resorption. Nitric oxide synthase activity has been detected in normal rat osteoclasts by both Northern blot and immunocytochemical studies [75]. Lowik and coworkers [76] reported that osteoblast-like cells in fetal mouse long bone explants can produce nitric oxide. IFN-g together with TNF-a and lipopolysaccharide induced nitric oxide synthesis by osteoblast-like UMR106 cells and inhibited bone resorption in fetal mouse bone organ cultures. TGF-b inhibited the stimulatory effects of these cytokines on nitric oxide production. These data suggest that nitric oxide production by osteoblasts may represent an important regulatory mechanism of osteoclast activity, especially in inflammatory conditions associated with cytokine release. Sex steroids Estrogen is one of the major inhibitors of osteoclast formation. With ovariectomy, increased osteoclastic bone resorption and osteoclast formation occur. Osteoblasts contain estrogen receptors. Oursler and coworkers [77,78] demonstrated recently that osteoclasts also contain estrogen receptors. The mechanism responsible for the increased bone turnover with estrogen deficiency is still a point of contention, but IL-6, IL-1, and TNF-a have been implicated in this process. Interleukin 18 Interleukin 18 (IL-18), also called interferon-inducing factor, is a recently described pleotropic cytokine. Horwood and coworkers [79] have shown that IL-18 inhibits osteoclast formation in vitro independent of its capacity to induce gamma-interferon. They showed that inhibition of osteoclast formation by IL-18 was mediated by GM-CSF. GMCSF production was produced in response to IL-18 by T cells. These data further demonstrate an important role for T cells in addition to marrow stromal cells in the control of osteoclast formation. Osteoprotegerin/OPG OPG, also called osteoclastogenesis inhibitory factor, recently was identified as a member of the TNF receptor family. It acts as a decoy receptor for RANK ligand to inhibit osteoclastogenesis. In addition to its capacity to inhibit osteoclastogenesis, Hakeda and coworkers [80] recently presented data that OPG inhibits osteoclastic bone resorption, as well as osteoclast formation. OPG disrupted the formation of F-actin ring. This inhibition was dose dependent and observed as early as 6 hours after addition of OPG to the isolated osteoclasts. Furthermore, mice in which the OPG gene has been disrupted suffer severe osteoporosis due to enhanced osteoclastogenesis [35]. These data demonstrate

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that OPG is a key factor negatively regulating osteoclastogenesis in vivo as well as in vitro.

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nism responsible for this escape phenomenon is unclear, but it may be due to the effects of calcitonin on transcriptional regulation of CTR gene expression and downregulation of CTR on the surface of the osteoclast.

Systemic hormones Calcitriol Metabolites of vitamin D3 are potent stimulators of osteoclastic bone resorption and osteoclast formation. The most active metabolite, 1,25-(OH)2D3, acts as a fusigen for committed osteoclast precursors [3]. It is unknown whether 1,25-(OH)2D3 acts on mature osteoclasts directly, although mature osteoclasts express vitamin D receptors [81]. Yasuda et al. [30] have reported that 1,25-(OH)2D3 can induce RANK ligand expression by osteoblasts, a factor that stimulates osteoclastic bone resorption. Furthermore, 1,25(OH)2D3 enhances osteoclastic bone resorption stimulated by parathyroid hormone. Mice treated with 1,25-(OH)2D3 develop hypercalcemia, and analogs of 1,25-(OH)2D3 have variable effects on osteoclast activity [82]. PTH/PTHrP PTH is a peptide produced by the parathyroid glands that is important in the maintenance of normal calcium homeostasis due to its stimulatory effects on osteoclastic bone resorptive activity and renal reabsorption of calcium [83]. PTHrP is a major mediator of the humoral hypercalcemia of malignancy and is produced by a variety of cells. PTHrP has 70% homology for the first 13 amino acids of PTH. PTHrP binds the PTH receptor and activates cAMP. Uy et al. [84] studied the effects of PTH and PTHrP on osteoclasts and precursors in vivo. Their study demonstrated that neither PTH nor PTHrP had an effect on early osteoclast precursors, but increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts. The primary target cell for PTH appears to be the osteoblast [85]. Isolated osteoclasts do not resorb bone in response to PTH and only do so when osteoblasts or osteoblastic cell lines are added to the cultures [86]. Furthermore, PTH induces RANK ligand expression by marrow stromal cells [30]. Calcitonin Calcitonin is a peptide hormone secreted by the parafollicular cells of the thyroid gland and is a potent inhibitor of osteoclastic bone resorption. Calcitonin receptors (CTR) are expressed on committed osteoclast precursors and mature osteoclasts [87]. Calcitonin downregulates expression of CTR in osteoclast precursors and mature osteoclasts in part by inhibiting CTR mRNA expression [88]. Calcitonin acts on osteoclasts by stimulating adenylcyclase activity and cAMP accumulation, which results in immobilization of the osteoclast and contraction of the osteoclast away from the bone surface [89]. Osteoclasts continuously exposed to calcitonin can escape the effects of calcitonin [90]. The mecha-

Prostaglandins The effect of prostaglandins on osteoclast formation and osteoclastic bone resorption may be dependent on the dose administered and the assay system used. Prostaglandins are stimulators of osteoclastic bone resorption in bone organ culture systems and osteoclast formation in murine marrow cultures [16]. However, prostaglandin (PGE2) inhibits osteoclastic bone resorption and formation in human marrow systems [91]. Quinn and coworkers [92] examined the inhibitory effect of PGE2 on osteoclast differentiation. To identify the cellular mechanisms responsible for this inhibitory effect of PGE2 and to determine whether PGE2 inhibition was dependent on the stromal cells supporting osteoclast differentiation, PGE2 was added to murine monocyte/ rat UMR 106 osteoblastic cells and murine monocyte/ST2 stromal cell cocultures before and during specific phases of differentiation. PGE2 exerts an inhibitory effect on osteoclast differentiation in the monocyte/UMR 106 coculture system, but, in contrast, it stimulated osteoclast formation and bone resorption in monocyte/ST2 cocultures by upregulating RANK ligand expression [30]. These data suggest that prostaglandins strongly influence the differentiation of osteoclast precursors, and this effect is dependent not only on the type and dose of prostaglandin administered but also on the nature of bone-derived stromal cells that support osteoclast formation. Gallwitz et al. [93] showed that other arachidonic acid metabolites such as the peptidoleukotrienes, as well as 5-hydroxyeiocatetraenoic acid, stimulate isolated osteoclasts to resorb bone. Figure 3 shows a model for the regulation of osteoclast formation by cytokines and hormones. Stimulatory and inhibitory factors act at different stages of osteoclast formation.

The osteoclast as a secretory cell Recent evidence has suggested that the osteoclast is a secretory cell that produces factors that can stimulate its own formation and activity. Early studies in our laboratory demonstrated that the osteoclast-like cells formed in marrow cultures from patients with Paget’s disease produce IL-6, and that IL-6 can stimulate osteoclast formation and bone resorption. In addition to producing IL-6, osteoclast-like giant cells from giant cell tumors of bone also produce IL-1. Oursler [94] showed that osteoclasts synthesize and secrete latent TGF-b. These investigators used highly purified avian osteoclasts to examine TGF-b synthesis and demonstrated by Northern blot analysis and metabolic labeling studies that TGF-b was produced. The principal form of TGF-b pro-

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Figure 3. Model for regulation of osteoclast formation. Stimulatory factors (1) such as the colony stimulatory factors (CSF), interleukin-6 (IL-6), transforming growth factor alpha (TGFa), or tumor necrosis factor (TNF) induce proliferation of osteoclast precursors. Factors such as 1,25-(OH)2D3 (1,25D) or parathyroid hormone (PTH) act as fusigens. Inhibitory factors (2) such as transforming growth factor beta (TGFb) act on several stages of osteoclast formation. In contrast, interferon gamma (gIFN) and calcitonin (CT) appear to block the later stages of osteoclast formation.

duced by the osteoclasts was TGF-b2. Furthermore, nearly all the TGF-b that was secreted by the osteoclasts was activated. When presented with exogenous latent TGF-b, these avian osteoclasts could activate TGF-b from a variety of sources, which is consistent with the previous observations of Pfeilschifter and Mundy [66] who showed that latent TGF-b could be activated during osteoclastic bone resorption. Because TGF-b is a negative regulator of osteoclastic bone resorption and osteoclast formation, these data suggest that osteoclasts produced both stimulatory and inhibitory factors that can regulate their own formation and activity. Demonstration that osteoclasts produce autocrine factors represents an important addition to our understanding of the regulation of normal osteoclast formation and activity. Takahashi et al. [95] prepared a mammalian cDNA expression library generated from highly purified human osteoclastlike multinucleated cells formed in bone marrow cultures and screened this library for autocrine factors that enhance osteoclast-like cell formation. In the initial screening, annexin II was identified. Purified recombinant annexin II significantly increased osteoclast-like cell formation in human bone marrow cultures in the absence of 1,25-dihydroxyvitamin D3, and it enhanced the bone resorptive capacity of 1,25-dihydroxyvitamin D3 in bone organ cultures. Annexin II originally was thought to be exclusively an intracellular protein that was an inhibitor of phospholipase A2. We have shown that annexin II is secreted by osteoclasts from giant cell tumors and is present in RNA from pagetic bone. Annexin II can stimulate osteoclast formation in vitro in both human and murine systems. Annexin II appears to be a proliferative factor that stimulates the growth of early osteoclast precursors [96] by inducing GM-CSF production by T cells, rather than by inducing fusion and differentiation of the osteoclast precursors. Nesbitt and Horton [97] reported that annexin II also is expressed on the surface of osteoclasts, and inhibition of annexin II with a specific antibody to annexin II blocked bone resorption by isolated osteoclasts. Expression cloning techniques have been used to identify a variety of other factors produced by osteoclasts. We re-

cently identified a novel osteoclast stimulatory factor, which we named OSF-1, which can stimulate formation of human and murine osteoclast-like cells in vitro [98]. This factor has a novel peptide sequence that is not related to any known cytokine and is currently being characterized. cDNA libraries also have been prepared from rabbit osteoclasts [12] and highly purified giant cells from giant cell tumors of bone. Bartkiewicz and coworkers [99] used these techniques to clone a component of vacuolar proton pump, which plays an important role in hydrogen export to the extracellular space beneath the ruffled border in osteoclasts [100]. We recently identified two novel inhibitors secreted by the osteoclasts; one is a membrane-bound inhibitor that is released from the cell surface and is identical in sequence to human Sca1 [101]. This inhibitor blocks osteoclast formation in both human and murine systems, and it inhibits bone resorption in fetal rat long bone organ cultures. The second inhibitor is identical to human legumain and is a soluble product released by the osteoclasts that inhibits osteoclast formation both in vitro and in vivo [102]. This inhibitor inhibits both osteoclast formation and bone resorption in murine and human marrow cultures as well as in PTHrPtreated mice in vivo.

Proto-oncogenes involved in osteoclast differentiation and bone resorption c-fos c-fos, a proto-oncogene normally associated with osteosarcomas, also appears to be a key regulator of osteoclast differentiation. Using homologous recombination, Grigoriadis and coworkers [103] showed that mice lacking the c-fos proto-oncogene develop osteopetrosis and have normal macrophage differentiation. The fos-deficient mice have a block in differentiation at the branch point between monocyte-macrophages and osteoclasts; they only form macrophages. Furthermore, transfection of c-fos cDNA into avian osteoclast precursors induced a twofold increase in TRAP activity and osteoclastic bone resorption activity in the osteoclasts that formed as compared with controls. These data suggest that prolonged expression of c-fos can enhance osteoclast differentiation. More recently, Wagner et al. [104] identified that all Fos proteins (c-Fos, Fra-1, Fra-2, and FosB), but none of the Jun proteins (c-Jun, Jun-B, and Jun-D) rescued the block in osteoclast differentiation. They also found that the N-terminal portion and the core region of Fos proteins were sufficient for osteoclast differentiation. Recently, Owens and coworkers [105] transduced osteoclast precursors with c-fos or Fra-1 using retroviral constructs. Overexpression of Fra-1, but not c-fos, in an immortalized bipotential osteoclast/macrophage precursor cell line caused a significant increase in the proportion of these precursors that developed calcitonin receptors and subsequent

G.D. Roodman/Experimental Hematology 27 (1999) 1229–1241

bone resorption. These data suggest that Fra-1 may play a role in osteoclast differentiation distinct from that of c-fos. NFkB NFkB appears to be another important transcription factor involved in osteoclast differentiation. NFkB is a family of transcription factors composed of five polypeptide subunits. Mice deficient in both the p50 and p52 subunits of NFkB develop severe osteopetrosis. Franzoso et al. [106] demonstrated that these animals lack normal osteoclast development. Deletion of either the p50 or p52 subunits did not result in a mouse with an abnormal bone phenotype. NFkB plays a critical role in expression of a variety of cytokines involved in osteoclast differentiation, including IL-1, TNF-a, IL-6, GM-CSF, and other growth factors. Deletion of both p50 and p52 may affect the production of growth factors that critical for osteoclast differentiation. c-src c-src, a proto-oncogene, plays a critical role in the activation of quiescent osteoclasts to become bone-resorbing osteoclasts. Osteoclast formation is normal in animals lacking the c-src gene. However, they develop osteopetrosis because the osteoclasts are unable to resorb bone [107]. The osteoclasts cannot form ruffled borders [108]. These animals can be rescued by transplantation of normal hematopoietic precursors from animals expressing c-src [109]. The substrate for src appears to be cortactin, which plays a critical role in attachment of osteoclasts to bone surfaces. However, it is unclear whether the enzyme activity of c-src, which is a non-receptor tyrosine kinase, is required for osteoclast activity. Schwartzberg and coworkers [110] generated transgenic mice that have the wild type or mutated versions of c-src proto-oncogene targeted to the osteoclast using the TRAP gene promoter. They demonstrated that expression of the wild-type transgene in only a limited number of tissues can fully rescue the c-src–deficient phenotype. Interestingly, they reported that expression of kinase-defective mutants of c-src also reduces osteopetrosis in c-src– deficient mice. These data suggest that there are essential kinase-independent functions for c-src in vivo. Using double antibody immunoconfocal microscopy, Abu-Amer et al. [111] recently demonstrated that c-src associated with tubulin only when avian osteoclasts were adherent to bone. This suggests that matrix recognition by osteoclasts induces c-src to associate with microtubules that traffic proteins to the cell surface. Tanaka et al. [112] suggested that the lack of phosphorylation of c-cbl in c-src–deficient mice is the important step in osteoclast activation in c-src–deficient animals.

Mechanisms of osteoclastic bone resorption Osteoclasts resorb bone by secreting proteases that dissolve the matrix and acid that releases bone mineral into the extra-

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cellular space under the ruffled border. Osteoclast secretion of hydrogen ion can be modulated by regulators of osteoclastic bone resorption. For example, PTH and PGE2 increase acid secretion by osteoclasts [113], whereas calcitonin decreases acid secretion. Microelectrode-based pH measurements at the ruffled border have shown pH levels as low as 3 to 4 [114]. Acid secretion by the osteoclast requires a proton pump. At least three different types of proton pumps have been implicated in the acidification process, but the strongest evidence suggests a vacuolar type proton pump, which is similar to the kidney H1-ATPase, is involved in osteoclastic bone resorption [100]. This proton pump appears to transport protons against a concentration gradient. Protons are supplied for the proton pump by the action of several enzymes including carbonic anhydrase II [115]. The critical importance of carbonic anhydrase II in the osteoclast has been shown by studies of patients with a congenital absence of this enzyme and osteopetrosis [116]. Similarly, the carbonic anhydrase inhibitor acetozolamide [117] can inhibit osteoclastic bone resorption. Lysosomal hydrolases that are active at an acid pH resorb the organic matrix. As noted, the osteoclast contains high levels of MMP-9 that may act in concert with collagenase to degrade the collagen matrix. The recent discovery of cathepsin O, a cysteine proteinase that is highly expressed in osteoclasts that can degrade collagen in addition to cathepsin B and L, suggests that the osteoclast secretes enzymes that may directly digest collagen.

Osteoclast apoptosis Although much is known about factors regulating osteoclast formation and osteoclast activity, little information is available about factors involved in osteoclast senescence. Osteoclasts undergo apoptosis, programmed cell death, which is characterized by nuclear and cytoplasmic condensation, and fragmentation of nuclear DNA into nucleosomal-sized units. This fragmentation of DNA can be demonstrated by gel electrophoresis or by in situ labeling of the fragmented DNA through incorporation of labeled deoxyoligonucleotides at the single-stranded ends of these DNAs, using terminal deoxynucleotidal transferase. Hughes and coworkers [118] recently developed an in vitro system for examining apoptosis in osteoclasts. In this system, murine marrow is cultured in the presence of 1,25-dihydroxyvitamin D3 until osteoclast-like cells form. The media then are changed, and the factor of interest is added 24 to 48 hours later. The numbers of osteoclasts released into the supernatant are counted and their morphology examined. Using this culture system, Hughes et al. showed that TGF-b can induce apoptosis of mature osteoclasts. TGF-b also can induce apoptosis in a variety of other tissues, such as liver and hematopoietic cell lines, and it will cause cell death in tumor cell lines derived from prostate, liver, and kidney tumors [119]. More recently, Hughes and coworkers [118] showed that sex steroids, including es-

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trogen and testosterone, can promote osteoclast apoptosis both in vitro and in vivo. Addition of estradiol, tamoxifen, or testosterone to murine marrow cultures increased osteoclast apoptosis two- to threefold compared to controls. Similarly, sex steroids also increased osteoclast apoptosis in vivo in ovariectomized or orchiectomized mice treated with either estradiol or testosterone, respectively. Bisphosphonates, which block bone resorption, also can induce apoptosis in osteoclasts [120], whereas osteoclast stimulatory factors, such as 1,25-dihydroxyvitamin D3 and PTH, inhibit induction of osteoclast apoptosis in vitro. These data suggest that regulation of osteoclast lifespan plays an important role in the normal bone remodeling process to either enhance or inhibit osteoclastic bone resorption. Cytokines that enhance osteoclast activity do so in part by increasing osteoclast lifespan, and factors that inhibit osteoclast activity appear to induce osteoclast apoptosis in addition to blocking osteoclast formation and bone resorption.

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Conclusion The osteoclast is a hematopoietic cell derived from CFUGM and branches from the monocyte-macrophage lineage early during the differentiation process. The marrow microenvironment appears critical for osteoclast formation due to production of RANK ligand by marrow stromal cells in response to a variety of osteotropic factors. Factors such as OPG, a recently described inhibitor of osteoclast formation, as well as secretory products produced by the osteoclast itself and other cells in the marrow, enhance or inhibit osteoclast formation. Identification of the role of oncogenes such as c-fos and pp60 c-src in osteoclast differentiation and bone resorption have provided important insights into the regulation of normal osteoclast activity. Current research is beginning to delineate the signaling pathways involved in osteoclastic bone resorption and osteoclast formation in response to cytokines and hormones. The recent development of osteoclast cell lines may make it possible for major advances to our understanding of the biology of the osteoclast to be realized in the near future.

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