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...

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Transforming growth factor ␤ (TGF-␤) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-B ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-␣ has also been shown. Therefore, we investigated TGF-␤ effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-␣. Both OPG and TGF-␤ inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-␤ also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-α, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-κB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1β, TNF-α, and receptor activator of NF-κB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE2 production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE2 in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-κB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE2 is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE2 production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE2 production.