We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE͞RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Bone-resorbing osteoclasts are of hemopoietic cell origin, probably of the CFU-M-derived monocytemacrophage family (1). Osteoclasts are large multinucleated giant cells that express tartrate-resistant acid phosphatase (TRAP) activity and calcitonin receptors and have the ability to form resorption pits on dentine slices (2-4). In the process of osteoclast differentiation, there is an absolute requirement for cell-cell contact between osteoclast progenitors and bone marrow stromal cells or calvaria-derived osteoblasts (5-8).We developed a mouse coculture system of hemopoietic cells and primary osteoblasts to investigate osteoclast formation in vitro. In this coculture system, several systemic and local factors were capable of inducing osteoclast-like multinucleated cell (OCL) formation (6-9). These boneresorbing factors were classified into 3 categories according to their signal transduction pathways: (a) 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] induced OCL formation via 1α,25(OH) 2 D 3 receptors (VDR) present in the nuclei; (b) parathyroid hormone (PTH), PTH-related protein (PTHrP), prostaglandin E 2 (PGE 2 ), and IL-1 induced OCL formation via the A kinase system; and (c) IL-11, oncostatin M, leukemia inhibitory factor, and IL-6 in the presence of soluble IL-6 receptors, all of which transduce their signals through a signal-transducing gp130 protein, also induced OCL formation in vitro. We reported previously that the target cells of IL-6 are osteoblasts/stromal cells but that they are not osteoclast precursors in inducing osteoclast differentiation (10). Similarly, coculture experiments using VDR knockout mice and PTH/PTHrP receptor knockout mice have indicated that the signals mediated by 1α,25(OH) 2 D 3 and PTH, respectively, are also transduced into osteoblasts/stromal cells, but not into osteoclast precursors, to induce osteoclast formation (11,12). Thus, it is concluded that the signals induced by all bone-resorbing factors are transduced into osteo-blasts/stromal cells to induce osteoclast formation. Our hypothesis proposes that osteoblasts/stromal cells express a critical common mediator named osteoclast differentiation factor (ODF), a membrane-bound factor that promotes differentiation of osteoclast progenitors into osteoclasts in response to various bone-resorbing factors through a mechanism involving cell-cell contact (6, 8). IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 incre...

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

We previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of la,25-dihydroxyvitamin D3 and dexamethasone. In this study, we developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells (103-105 cells per well) obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive mononuclear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to la,25-dihydroxyvitamin D3 and dexamethasone. When hemopoletic cells suspended in a collagengel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. All of the colonies consisted of nonspecific esterase-positive cells. The monocyte-depleted population prepared from peripheral blood failed to form colonies, whereas the monocyte-enriched population produced a large number of TRAPase-positive colonies. In addition, alveolar macrophages formed TRAPase-positive colonies most efficiently on the ST2 cell layers in the presence of the two hormones. Salmon 12'I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. When direct contact between the peripheral blood cells and the ST2 cells was inhibited by a collagen-gel sheet, no TRAPasepositive cells were formed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a sulitable microenvironment is provided by bone marrow-derived stromal cells.Osteoclasts are multinucleated cells responsible for bone resorption. It is evident from chicken-quail chimera experiments (1), parabiosis experiments (2, 3), and marrow transplantation studies in osteopetrotic animals (4, 5) that osteoclasts are derived from circulating mononuclear precursors in hemopoietic tissues. However, the nature and the differentiation process of osteoclast precursors are still not known.We recently reported that osteoclast-like multinucleated cells were formed in response to osteotropic hormones in cocultures of mouse spleen cells with osteoblast-rich cell populations freshly isolated from fetal mouse calvaria (6). These multinucleated cells had the typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase (TRAPase), abundant calcitonin receptors, and the ability to form resorption lacunae on dentine slices (6). Then we reported that the two marrow-derived stromal cell lines, MC3T3-G2/ PA6 and ST2, could be substituted for primary osteoblastrich cell populations in inducing osteoclast-like cells in cocultures with spleen cells...

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

It has been reported that soluble interleukin (IL)-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and human immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates 1L-6 action, its physiological role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant mouse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6R) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In contrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrateresistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation on dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding monoclonal anti-mouse IL-6R antibody (MR16-1). It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gpl3O, even though they may have no or a very small number of IL-6Rs. Factors such as IL-1l, oncostatin M, and leukemia inhibitory factor, which are known to exert their functions through gpl3O (the signal-transducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced sIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanism involving gpl3O. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption.

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.