Bone remodelling: A signalling system for osteoclast regulation

Dispatch

R679

Bone remodelling: A signalling system for osteoclast regulation Ellen Filvaroff* and Rik Derynck†

Two physiological regulators of osteoclast maturation have recently been identified: the secreted protein osteoprotegerin and the cell-surface ligand to which it binds. These proteins are likely to play an important part in the control of bone resorption, but are also likely to have important roles in other tissues. Addresses: *Department of Endocrinology, Genentech, South San Francisco, California 94080-4990, USA. †Departments of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143, USA. Current Biology 1998, 8:R679–R682 http://biomednet.com/elecref/09609822008R0679 © Current Biology Ltd ISSN 0960-9822

Bone tissue is continuously remodelled through the combined activities of two cell types, osteoblasts and osteoclasts. Osteoblasts, which are derived from mesenchymal cells, deposit bone matrix, whereas osteoclasts, which are derived from hematopoietic precursor cells, resorb bone matrix. To maintain a constant bone mass during adult life, osteoblastic matrix deposition and osteoclastic resorption must be closely coordinated (reviewed in [1,2]). Because bone remodeling is continuous, even a slight perturbation of this balance can have drastic effects on bone mass and structure, and result in progressive, metabolic bone disease. Thus, increased bone resorption relative to bone deposition causes the gradual bone loss associated with osteoporosis, whereas decreased bone resorption relative to bone deposition causes the increased trabecular bone mass that occurs in osteopetrosis. As a disturbance of the balance between osteoblastic bone deposition and osteoclastic bone resorption is responsible for many skeletal diseases, we need to understand how the activities of osteoblasts and osteoclasts are regulated and coordinated. Recent studies have identified factors that regulate osteoclast maturation and control bone resorption. The important role of osteoblasts in the regulation of osteoclast maturation is strikingly illustrated by the almost complete absence of osteoclasts and tissue macrophages in the osteopetrotic op/op mouse [3]. The consequently impaired osteoclastic bone resorption and osteopetrosis, which result from the failure of osteoblasts to produce active colony stimulating factor-1 (CSF-1) can be rescued by injection of CSF-1 (reviewed in [4]). CSF-1 thus plays a critical role in osteoclast development by directly acting on osteoclasts to control their proliferation and differentiation. CSF-1 may also affect the activity of mature osteoclasts, through its ability to induce osteoclast fusion,

survival and chemotaxis (reviewed in [4–6]). The temporal and spatial pattern of expression of CSF-1 during development suggests that it is one of the factors that couples hematopoiesis to osteogenesis [4]. Various other extracellular factors that regulate osteoclast maturation and activity have been identified using osteoblast/stromal cell and osteoclast precursor cell coculture systems (reviewed in [5–7]). In these cell systems, osteoclast formation depends on 1,25-dihydroxyvitamin D3 and CSF-1, but it is inhibited by other factors, such as calcitonin, interleukin-4 (IL-4), IL-13, and interferon-γ [5,6]. Many of the positive regulatory factors in this system act primarily on osteoblasts, stimulating osteoclast maturation indirectly. These factors can be divided into three classes, according to their signal transduction mechanisms [5,7]. One class, represented by 1,25-dihydroxyvitamin D3, acts through nuclear receptors of the steroid receptor family. A second class, including parathyroid hormone (PTH), PTHrelated protein (PTHrP), prostaglandin E2 and IL-1, exerts its effects by activating protein kinase A. And a third class, represented by IL-6, IL-11, oncostatin M and leukocyte inhibitory factor (LIF), stimulates bone resorption through the receptor-associated gp130 protein [5,7]. In vitro studies led to the proposal that activation of any one of these three signal transduction pathways would induce expression of a membrane-associated factor on osteoblasts, and thus stimulate osteoclast progenitors to differentiate. This osteoclast stimulatory factor was named osteoclast-differentiation factor [5] or stromal-cell-derived osteoclast formation activity [8]. The gene encoding this factor, which is expressed by activated osteoblast/stromal cells and induces osteoclast differentiation, has now been cloned [9] and the protein renamed osteoprotegerin ligand (OPGL). OPGL and the soluble protein to which it binds, known as osteoprotegerin (OPG), constitute a ligand–receptor system which directly regulates osteoclast differentiation and hence bone density. From its cDNA sequence, human OPGL is a 317 amino acid, type II transmembrane protein with a short aminoterminal intracellular domain and a large carboxy-terminal extracellular domain [9]. The extracellular region includes a juxtamembrane segment of 87 amino acids and a carboxy-terminal domain of 159 amino acids that shows similarity to the cytokine tumor necrosis factor (TNF) and related proteins. OPGL is identical to the TNF-related protein TRANCE/RANKL, the ligand for a member of the TNF receptor family known as ‘receptor activator of NFκB’ (RANK) [10,11], and closely related to the

R680

Current Biology, Vol 8 No 19

Figure 1

Monocyte Hematopoietic progenitor

Proposed roles for CSF-1, OPGL and OPG in the regulation of osteoclast maturation by osteoblasts. (See text for details.)

Macrophage

Myeloid progenitor

CSF-1

OPGL receptor

Osteoclast precursor

Mononuclear Multinucleated osteoclast osteoclast

OPGL CSF-1

OPGL CSF-1

Active osteoclast

OPGL

OPGL

Bone matrix

OPG CSF-1 receptor CSF-1 Current Biology

apoptosis-inducing, TNF-related ligand TRAIL/Apo-2L [12,13]. In the developing skeleton, OPGL is expressed by the mesenchymal cells surrounding the cartilaginous anlagen and by hypertrophic cartilage, and in areas that undergo ossification and remodelling, such as in trabecular bone. OPGL is also made by stimulated osteoblast/stromal cell lines, which support osteoclastogenesis in vitro, but not by osteoclasts [9,14]. In contrast to most other known cytokines, OPGL stimulates osteoclast differentiation directly, as demonstrated in an in vitro system where osteoclast differentiation can be induced in bone marrow or spleen cells by coculture with osteoblastic/stromal cells. In this system, the stromal cells provide CSF-1, and stimulation with dexamethasone and 1,25-dihydroxyvitamin D3 induces osteoclast differentiation. Addition of a soluble derivative of OPGL, which contains the TNF-related domain, induces osteoclast differentiation in the absence of examethasone and 1,25dihydroxyvitamin D3. This effect cannot be mimicked by the closely related cytokine TRAIL [15]. OPGL and CSF-1 together overcome the need for stromal cells to induce osteoclast maturation [9,14]. These results suggest that osteoblasts and/or stromal cells provide CSF-1 and OPGL, and that contact between osteoblast/stromal cells and differentiating osteoclasts is required to allow interaction of transmembrane OPGL on the stromal cell surface with its receptor on osteoclasts (Figure 1). OPGL also stimulates osteoclast activity [9]. CSF-1 alone allows the generation of small mononuclear cells, which do not resorb bone, whereas OPGL stimulates development of active osteoclasts in a CSF-1-dependent manner [9,14].

Stimulated osteoblastic stromal cell

This synergy of CSF-1 and OPGL may be explained by the ability of CSF-1 to induce OPGL receptor expression on osteoclast precursors. Accordingly, CSF-1-treated cultures show a high percentage of cells that directly bind OPGL. These cells are large and adherent, whereas the non-binding cells are small and non-adherent [9]. OPGL also activates resorption by mature osteoclasts in vitro, but this effect does not depend on CSF-1 [9]. Thus, OPGL can act at multiple points during osteoclast maturation (Figure 1). The nature of the OPGL receptor remains unclear, although, given the identity of OPGL and RANKL, it may turn out to be RANK. The importance of OPGL in osteoclast maturation and bone resorption is underscored by the identification and characterization of OPG [16,17]. OPG is a secreted, glycosylated protein of 401 amino acids, which naturally forms a disulfide-bonded dimer of 110 kDa. The sequence of OPG shows strong similarity to TNF receptor superfamily members in the amino-terminal half, and similarity to TNF receptor ‘death domains’ in two carboxy-terminal segments. OPG lacks a transmembrane region, however, and has been found only in secreted form. OPG is made by a variety of tissues, but is expressed at high levels in cartilaginous primordia of developing bones, consistent with a role in skeletogenesis [16]. OPG inhibits osteoclastic differentiation in the coculture system, presumably as a result of its ability to bind and neutralize OPGL produced by the activated stromal cells [14,17] (Figure 1). OPG production by osteoblast/stromal cells is inhibited by 1,25dihydroxyvitamin D3 [17], reinforcing the osteoclastic differentiation caused by concomitant induction of OPGL production by osteoblasts [17]. OPGL is not the only

Dispatch

ligand for OPG, however, as OPG also binds TRAIL with high affinity and blocks TRAIL-induced apoptosis, just as TRAIL blocks OPG-induced inhibition of osteoclastogenesis in vitro [15]. OPG is therefore not merely an inhibitor of osteoclast maturation and activity. That OPG acts as an inhibitor of osteoclast differentiation is also apparent from the phenotype of OPG-overexpressing transgenic mice. These mice show an increase in trabecular bone density, caused by the decreased bone resorption that results from their relatively low numbers of mature osteoclasts [16]. Unlike other osteopetrotic mouse models, these transgenic mice do not have defects in the relative size or shape of their bones, nor abnormalities in tooth eruption. This difference may reflect OPG’s role in regulating the terminal stages of osteoclast differentiation, consistent with the normal numbers of osteoclast precursors in these mice [16]. In some other osteopetrotic mouse models, such as mice deficient in CSF-1 or NF-κB1 and NF-κB2, osteoclast differentiation is affected at earlier stages (reviewed in [18]). These mutations not only lead to decreased numbers of osteoclasts, but can also influence other cell types derived from hematopoietic or myeloid progenitors, such as monocytes and macrophages [4,18]. In other osteopetrotic mouse mutants, such as microphthalmia or c-Src-deficient mice, the number of osteoclasts is normal but their activity is impaired, presumably because of an intrinsic osteoclast defect [18]. Thus, unlike the defects in other osteopetrotic mice, OPG overexpression induces osteopetrosis by interfering with the ability of osteoblasts to directly stimulate osteoclast differentiation. Further evidence that OPG is a physiological regulator of bone mass comes from the observation that OPG-deficient mice exhibit severe bone porosity and a high incidence of fractures [19], reminiscent of human osteoporosis [20]. The bone porosity in OPG-deficient mice is more severe than in other animal models of osteoporosis, consistent with the direct and potent effect of OPG on osteoclast differentiation. Previous studies have shown that inactivation or overexpression of genes for other secreted regulators of osteoclastic resorption can result in osteoporosis. For example, overexpression of IL-4 [21], G-CSF [22] or TGF-β2 [23] result in osteoporotic phenotypes, albeit with different etiologies. The increased bone resorption in OPG-deficient mice is associated with an increase in osteoclast surface as a proportion of total bone surface [19]. Injection of OPGL into normal mice similarly results in reduced bone volume, but without a change in osteoclast number; rather, the increase in osteoclast activity is accompanied by an increase in size and nuclearity of osteoclasts. This stimulation of osteoclast development and activity by OPGL in vivo is consistent with the finding that OPGL binds to both bone marrow osteoclast progenitors and osteoclasts [9]. OPG-deficient

R681

mice also exhibit an increase in osteoblast surface and higher osteocyte density [19]. These changes are probably caused by the increased osteoclast activity, as bone formation is normally coupled to bone resorption [1,2]. This coupling could be effected by the release of stored growth factors (such as TGF-β) during osteoclastic bone matrix resorption, which would then alter osteoblast differentiation and activity. Osteopetrotic animals similarly show changes in osteoblast function [18]. The current in vitro and in vivo results thus strongly suggest that OPG is a physiological inhibitor of osteoclast differentiation, which plays a role in maintaining the normal balance between osteoblastic bone deposition and osteoclastic bone resorption. OPG is produced by many tissues other than bone, including the intestine, heart, kidney, lung and placenta, and hematopoietic and immune organs [16]. OPG’s function in these tissues remains to be determined, but is most likely not restricted to OPGL neutralization. For example, OPG can inhibit TRAIL-induced apoptosis in vitro [15], which may account for the enlarged spleens in OPG-overexpressing mice [16]. OPG may also be involved in in regulating calcium and phosphate transport and homeostasis, given its expression in the intestine, kidney and bone [2,16]. OPG is also likely to be a physiological suppressor of local calcification in blood vessels, as OPG-deficient mice show calcification of the aorta and renal arteries [19]. This mechanistic connection between bone and blood vessel pathology is consistent with the presence of several bone matrix proteins and minerals in calcified arteries [19], and the association between vascular calcification and osteoporosis in humans [20]. OPGL is also expressed outside the skeleton — high levels occur in lymph nodes, and lower levels in the thymus, spleen, peripheral blood leukocytes, bone marrow, heart, placenta, skeletal muscle, stomach and thyroid gland [9–11]. The OPGL receptor RANK, in contrast, is ubiquitously expressed, with highest levels in thymus, skeletal muscle, liver, colon, small intestine and adrenal gland [10]. These observations suggest that RANK may have several ligands and that OPGL plays a role in cell types other than osteoclasts. In vitro results indeed suggest a role for OPGL in T-cell activation and viability, perhaps through its ability to activate NF-κB [10]. OPGL also increases survival of dendritic cells in vitro and enhances their ability to stimulate naive T cells in a mixed lymphocyte reaction [10,24]. OPGL may therefore help to coordinate the activity of dendritic and T cells [10,11,24]. The seeming similarity between the activities of OPGL in bone and in the immune system is perhaps surprising. In vitro, activation of T cells leads to a rapid induction of OPGL production, which then enhances dendritic cell survival, presumably via the RANK receptor. The survival and activation of antigen-specific T cells and antigen-presenting

R682

Current Biology, Vol 8 No 19

dendritic cells may perhaps be closely linked in this way. This linkage may resemble the coordination of osteoblast and osteoclast activities: OPGL expression is induced following activation of stromal osteoblasts and stimulates osteoclast differentiation. Whereas the OPGL-producing cells in these two systems are of different origin, dendritic cells and osteoclasts are both hematopoietic cell types of the monocyte–macrophage lineage. The transcription factor NF-κB, implicated in the T-cell response to OPGL, may also have a role in OPGL-induced signalling during osteoclast development: mice deficient for NF-κB1 and NF-κB2 develop osteopetrosis because of a defect in osteoclast differentiation [25]. A further characterization of the activities of the OPG/OPGL/RANK system in vitro and in vivo should help elucidate these possible analogies between bone cell biology and immunology. The similarity between OPGL and TRAIL and the ability of OPG to neutralize TRAIL [15] suggest that OPG may be a physiological inhibitor of apoptosis in several contexts, including hematopoietic and lymphoid organs. Furthermore, the likelihood that OPGL acts via RANK, which is related to receptors that are known to induce apoptosis, also suggests a close relationship between osteoclast differentiation and the apoptotic response. Whether RANK activation induces or inhibits apoptosis of osteoclasts or their progenitors remains to be determined. Clearly, the possibility that the OPG/OPGL regulates both differentiation and apoptosis in bone and other organs is interesting and needs to be investigated further if these molecules are to be considered for therapeutic treatment of metabolic bone disorders. Acknowledgements We thank Erwin Wagner and Greg Mundy for their constructive comments on this manuscript.

References 1. Erlebacher A, Filvaroff EH, Gitelman SE, Derynck R: Toward a molecular understanding of skeletal development. Cell 1995, 80:371-378. 2. Reddi AH: Bone morphogenesis and modeling: soluble signals sculpt osteosomes in the solid state. Cell 1997, 89:159-161. 3. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okumura H, Sudo T, Shultz LD, Nishikawa S-I: The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990, 345:442-444. 4. Cecchini MG, Hofstetter W, Halasy J, Wetterwald A, Felix R: Role of CSF-1 in bone and bone marrow development. Mol Repr Dev 1997, 46:75-84. 5. Suda T, Udagawa N, Nakamura I, Miyaura C, Takahashi N: Modulation of osteoclast differentiation by local factors. Bone 1995, 17:87S-91S. 6. Heymann D, Guicheux J, Gouin F, Passuti N, Daculsi G: Cytokines, growth factors and osteoclasts. Cytokine 1998, 10:155-168. 7. Suda T, Nakamura I, Jimi E, Takahashi N: Regulation of osteoclast function. J Bone Min Res 1997, 12:869-879. 8. Chambers TJ, Owens JM, Hattersley G, Jat PS, Noble MD: Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse. Proc Natl Acad Sci USA 1993, 90:5578-5582. 9. Lacey DL, Timms E, Tan H-L, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, et al.: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998, 93:165-176.

10. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L: A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997, 390:175-179. 11. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E, Bartlett FS III, Frankel WN, et al.: TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997, 272:25190-25194. 12. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang C-P, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, Goodwin RG: Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995, 3:673-682. 13. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A: Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996, 271:12687-12690. 14. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S-I, Tomoyasu A, Yano K, Goto M, Murakami A, et al.: Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998, 95:3597-3602. 15. Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R, et al.: Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998, 273:14363-14367. 16. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang M-S, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T: Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997, 89:309-319. 17. Yasuda H, Shima N, Nakagawa N, Mochizuki S-I, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M, et al.: Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998, 139:1329-1337. 18. Felix R, Hofstetter W, Cecchini MG: Recent developments in the understanding of the pathophysiology of osteopetrosis. Eur J Endocrinol 1996, 134:143-156. 19. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, et al.: Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998, 12:1260-1268. 20. Banks LM, Macsweeney JE, Stevenson JC: Effect of degenerative spinal and aortic calcification on bone density measurements in postmenopausal women: links between osteoporosis and cardiovascular disease? Eur J Clin Invest 1994, 24:813-817. 21. Lewis DB, Liggitt HD, Effmann EL Motely ST, Teitelbaum SL, Jepsen KJ, Goldstein SA, Bonadio J, Carpenter J, Perlmutter RM: Osteoporosis induced in mice by overproduction of interleukin 4. Proc Natl Acad Sci USA 1993, 90:11618-11622. 22. Takahashi T, Wada T, Mori M, Kokai Y, Ishii S: Overexpression of the granulocyte colony stimulating factor gene leads to osteoporosis in mice. Lab Invest 1996, 74:827-834. 23. Erlebacher A, Derynck R: Increased expression of TGF-b2 in osteoblasts results in an osteoporosis-like phenotype. J Cell Biol 1996, 132:195-210. 24. Wong BR, Josien R, Lee SY, Sauter B, Li H-L, Steinman RM, Choi Y: TRANCE (tumor necrosis factor (TNF)-related activation induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 1997, 186:2075-2080. 25. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R: Osteopetrosis in mice lacking NF-kB1 and NF-kB2. Nat Med 1997, 3:1285-1289.