Masamichi Takami

Osteoclast differentiation independent of the TRANCE–RANK–TRAF6 axis

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor κB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE–RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-α in the presence of cofactors such as TGF-β. We provide direct evidence against the current paradigm that the TRANCE–RANK–TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

A novel member of the leukocyte receptor complex regulates osteoclast differentiation.

Osteoclasts (OCs) are multinucleated cells that resorb bone and are essential for bone homeostasis. They develop from hematopoietic cells of the myelomonocytic lineage. OC formation requires cell-to-cell interactions with osteoblasts and can be achieved by coculturing bone marrow precursor cells with osteoblasts/stromal cells. Two of the key factors mediating the osteoblast-induced osteoclastogenesis are macrophage–colony stimulating factor (M-CSF) and the tumor necrosis factor (TNF) family member TNF–related activation-induced cytokine (TRANCE) that are produced by osteoblasts/stromal cells in response to various bone resorbing hormones. In addition, other factors produced by osteoblasts/stromal cells further influence osteoclastogenesis. Here we report the identification and characterization of OC-associated receptor (OSCAR), a novel member of the leukocyte receptor complex (LRC)-encoded family expressed specifically in OCs. Genes in the LRC produce immunoglobulin (Ig)-like surface receptors and play critical roles in the regulation of both innate and adaptive immune responses. Different from the previously characterized members of the LRC complex, OSCAR expression is detected specifically in preosteoclasts or mature OCs. Its putative–ligand (OSCAR-L) is expressed primarily in osteoblasts/stromal cells. Moreover, addition of a soluble form of OSCAR in coculture with osteoblasts inhibits the formation of OCs from bone marrow precursor cells in the presence of bone-resorbing factors, indicating that OSCAR may be an important bone-specific regulator of OC differentiation. In addition, this study suggests that LRC-encoded genes may have evolved to regulate the physiology of cells beyond those of the immune system.

Stimulation by Toll-Like Receptors Inhibits Osteoclast Differentiation

Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-κB activation and up-regulated TNF-α production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone.

Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis

Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis. Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. Irf8−/− osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor-α (TNF-α). IRF-8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated osteoclastogenesis.

Lipopolysaccharide Promotes the Survival of Osteoclasts Via Toll-Like Receptor 4, but Cytokine Production of Osteoclasts in Response to Lipopolysaccharide Is Different from That of Macrophages

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.

Suppression of Osteoprotegerin Expression by Prostaglandin E2 Is Crucially Involved in Lipopolysaccharide-Induced Osteoclast Formation

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.

Cells of Bone: Osteoclast Generation

Publisher Summary This chapter focuses on the generation of osteoclasts in bone. Osteoclasts are multinucleated giant cells that resorb bone and develop from hemopoietic cells of the monocyte-macrophage lineage. Development of osteoclasts proceeds within a local microenvironment of bone and this process can be reproduced ex vivo in a coculture of mouse calvarial osteoblasts and hemopoietic cell. Multinucleated cells formed in the coculture exhibit major characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRAP) activity, expression of calcitonin receptors, c-Src (p60c-src), vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. The discovery of the RANKL–RANK interaction has opened a wide new area in bone biology focused on the investigation of the molecular mechanism of osteoclast development and function. Membrane- or matrix-associated forms of both M-CSF and RANKL expressed by osteoblasts/stromal cells appear to be essential for osteoclast formation. Both RANKL(–/–) mice and RANK(–/–) mice show similar features of osteopetrosis with a complete absence of osteoclasts in bone. Gain-of-function mutations of RANK have been found in patients suffering from familial expansile osteolysis and familial Pagets disease of bone. These findings confirm that the RANKL–RANK interaction is indispensable for osteoclastogenesis not only in mice but also in humans. TRAF2-mediated signals are important for inducing osteoclast differentiation, and TRAF6-mediated signals are indispensable for osteoclast activation. Under physiological conditions, osteoclast formation requires cell-to-cell contact with osteoblasts/stromal cells, which express RANKL as a membrane-bound factor in response to several bone-resorbing factors.Publisher Summary This chapter focuses on the generation of osteoclasts in bone. Osteoclasts are multinucleated giant cells that resorb bone and develop from hemopoietic cells of the monocyte-macrophage lineage. Development of osteoclasts proceeds within a local microenvironment of bone and this process can be reproduced ex vivo in a coculture of mouse calvarial osteoblasts and hemopoietic cell. Multinucleated cells formed in the coculture exhibit major characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRAP) activity, expression of calcitonin receptors, c-Src (p60c-src), vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. The discovery of the RANKL–RANK interaction has opened a wide new area in bone biology focused on the investigation of the molecular mechanism of osteoclast development and function. Membrane- or matrix-associated forms of both M-CSF and RANKL expressed by osteoblasts/stromal cells appear to be essential for osteoclast formation. Both RANKL(–/–) mice and RANK(–/–) mice show similar features of osteopetrosis with a complete absence of osteoclasts in bone. Gain-of-function mutations of RANK have been found in patients suffering from familial expansile osteolysis and familial Pagets disease of bone. These findings confirm that the RANKL–RANK interaction is indispensable for osteoclastogenesis not only in mice but also in humans. TRAF2-mediated signals are important for inducing osteoclast differentiation, and TRAF6-mediated signals are indispensable for osteoclast activation. Under physiological conditions, osteoclast formation requires cell-to-cell contact with osteoblasts/stromal cells, which express RANKL as a membrane-bound factor in response to several bone-resorbing factors.

Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-?, IL-1, and RANKL

Lipopolysaccharide (LPS), a cell component of Gram‐negative bacteria, is a pathogen of inflammatory bone loss. To examine the effects of LPS on the survival and fusion of osteoclasts, mononuclear osteoclasts (preosteoclasts, pOCs) were collected from a mouse co‐culture system and cultured in the presence or absence of LPS. Most pOCs died within 24 h in the absence of any stimulus. LPS as well as receptor activator of NF‐κB ligand (RANKL) supported the survival of pOCs, and induced their fusion to form multinucleated cells (MNCs). Like authentic osteoclasts, MNCs induced by LPS expressed calcitonin receptors, and formed actin rings on culture plates. LPS‐induced MNC formation in pOC cultures was observed even in the presence of osteoprotegerin and interleukin (IL)‐1‐receptor antagonists. MNC formation was also stimulated by LPS in pOC cultures prepared from tumor necrosis factor (TNF)‐receptor‐I or TNF‐receptor‐II deficient mice. LPS induced the degradation of IκB in pOCs within 20 min. Lactacystin, an inhibitor of NF‐κB activation, and wortmannin, an inhibitor of phosphatidylinositol‐3 kinase, strongly inhibited LPS‐induced MNC formation in pOC cultures. LPS induced pit‐forming activity of pOCs in the presence of macrophage‐colony stimulating factor (M‐CSF). These findings suggest that LPS stimulates the survival and fusion of pOCs, independent of RANKL, IL‐1 or TNF‐α action. Activation of NF‐κB and phosphatidylinositol‐3 kinase appeared to be involved in LPS‐induced effects on pOCs. These observations suggest that LPS is involved directly in inflammatory bone loss, and also indirectly through the production of LPS‐induced host factors such as IL‐1 and TNF‐α. J. Cell. Physiol. 190: 101–108, 2002.

The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I

Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis. By using M-GFN-immobilized beads, glyoxalase I (GLO1) was identified as an M-GFN-binding protein. GLO1 knockdown and treatment with an established GLO1 inhibitor in osteoclast progenitor cells interfered with osteoclast generation, suggesting that GLO1 activity is required for osteoclastogenesis. In cells, GLO1 plays a critical role in the detoxification of 2-oxoaldehydes, such as methylglyoxal. M-GFN inhibited the enzymatic activity of GLO1 in vitro and in situ. Furthermore, the cocrystal structure of the GLO1/M-GFN complex revealed the binding mode of M-GFN at the active site of GLO1. These results suggest that M-GFN targets GLO1, resulting in the inhibition of osteoclastogenesis.

Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors.

SaOS‐4/3, a subclone of the human osteosarcoma cell line SaOS‐2, established by transfecting the human parathyroid hormone/parathyroid hormone‐related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS‐4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony‐stimulating factor (M‐CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF‐κB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane‐associated and secreted forms of M‐CSF by SaOS‐4/3 cells was up‐regulated in response to PTH. SaOS‐4/3 cells constitutively expressed OPG mRNA, expression of which was down‐regulated by PTH. To elucidate the mechanism of PTH‐induced osteoclastogenesis, SaOS‐4/3 cells were spot‐cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M‐CSF, osteoclasts were induced exclusively inside the colony of SaOS‐4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS‐4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M‐CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS‐4/3 cells. Adding M‐CSF alone failed to support osteoclast formation in the spot coculture. PTH‐induced osteoclast formation occurring inside the colony of SaOS‐4/3 cells was not affected by the concentration of M‐CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS‐4/3 cells. These findings suggest that the up‐regulation of RANKL/ODF expression is an essential step for PTH‐induced osteoclastogenesis, and membrane‐ or matrix‐associated forms of both M‐CSF and RANKL/ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.