Kanami Itoh

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis

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 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor

We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.

Transforming Growth Factor β Affects Osteoclast Differentiation via Direct and Indirect Actions

Transforming growth factor β (TGF‐β) 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‐κB 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‐α has also been shown. Therefore, we investigated TGF‐β 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‐α. Both OPG and TGF‐β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF‐β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF‐β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ‐inhibited cocultures of opg−/− cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF‐β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF‐β greatly increased osteoclast formation in recombinant RANKL‐ or TNF‐α‐stimulated cultures of hemopoietic cells or RAW 264.7 macrophage‐like cells to levels several‐fold greater than attainable by maximal stimulation by RANKL or TNF‐α. These data suggest that TGF‐β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF‐α) is essential for osteoclast formation, factors such as TGF‐β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Bone Morphogenetic Protein 2 Stimulates Osteoclast Differentiation and Survival Supported by Receptor Activator of Nuclear Factor-κB Ligand

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

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.

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.

Regulation of osteoclast differentiation and function by receptor activator of NFkB ligand and osteoprotegerin

The differentiation and functions of osteoclasts (OCs) are regulated by osteoblast‐derived factors. Receptor activator of NFkB ligand (RANKL) is one of the key regulatory molecules in OC formation. Osteoprotegerin (OPG) is a novel secreted member of the TNF receptor superfamily that negatively regulates osteoclastogenesis and binds to RANKL. We examined the biological actions of macrophage‐colony‐stimulating factor (M‐CSF), RANKL, and OPG on the differentiation of OCs isolated from cocultures of mouse osteoblastic cells and bone marrow cells. Preosteoclasts (pOCs) and OCs were characterized by their ultrastructure and the expression of OC markers such as tartrate‐resistant acid phosphatase (TRAP) and vacuolar‐type H+‐ATPase. pOCs formed without any additives expressed TRAP, but showed little resorptive activity on cocultured dentine slices. TRAP‐positive pOCs treated with M‐CSF began to fuse with each other, but lacked a ruffled border (RB) and showed almost no resorptive activity. pOCs treated with RANKL became TRAP‐positive multinucleated cells, which expressed intense vacuolar‐type H+‐ATPase along the RB membranes and exhibited prominent resorptive activity. Such effects of RANKL on pOCs were completely inhibited by the addition of OPG. OPG inhibited RB formation in mature OCs and reduced their resorptive activity, and also induced apoptosis of some OCs. These results suggest that 1) RANKL induces differentiation of functional OCs from pOCs, 2) M‐CSF induces macrophage‐like multinucleated cells, but not OCs, 3) OPG inhibits RB formation and resorptive activity in mature OCs, 4) OPG also induces apoptosis of OCs, and 5) RANKL and OPG are, therefore, important regulators of not only the terminal differentiation of OCs but also their resorptive function. Anat Rec 268:137–146, 2002.

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A‐binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1α,25 dihydroxyvitamin D3, prostaglandin E2, and interleukin‐1α. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF‐κB ligand (RANKL). RT‐PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony‐stimulating factor‐dependent bone marrow macrophages (M‐BMMΦs). In addition, phosphorylation of Smad2 was observed in M‐BMMΦs stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A‐enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.

Involvement of vacuolar H-ATPase in incorporation of risedronate into osteoclasts

Although osteoclasts incorporate bisphosphonates during bone resorption, the mechanism of this incorporation by osteoclasts is not known. We previously reported that bisphosphonates disrupt the actin rings (clear zones) formed in normal osteoclasts, but did not disrupt actin rings in osteoclasts derived from osteosclerotic oc/oc mice, which have a defect in the gene encoding vacuolar H(+)-ATPase (V-ATPase). The present study showed that V-ATPase is directly involved in the incorporation of risedronate, a nitrogen containing bisphosphonate, into osteoclasts. Treatment of osteoclasts with risedronate disrupted actin rings and inhibited pit formation by osteoclasts on dentine slices. Bafilomycin A(1), a V-ATPase inhibitor, inhibited the pit-forming activity of osteoclasts but did not disrupt actin rings. Risedronate failed to disrupt actin rings in the presence of bafilomycin A(1). E-64, a lysosomal cysteine proteinase inhibitor, showed no inhibitory effect on the demineralization of dentine by osteoclasts but inhibited the digestion of dentine matrix proteins without disrupting actin rings. Risedronate disrupted actin rings even in the presence of E-64. Treatment of osteoclasts placed on plastic plates with risedronate also disrupted actin rings. Bafilomycin A(1) but not E64 prevented the disruption of actin rings in osteoclasts treated with risedronate on plastic plates. Inhibition of V-ATPase with bafilomycin A(1) also prevented disruption of actin rings by etidronate, a non-nitrogen-containing bisphosphonate. These results suggest that V-ATPase induced acidification beneath the ruffled borders of osteoclasts and subsequent bone demineralization triggers the incorporation of both nitrogen-containing and non-nitrogen-containing bisphosphonates into osteoclasts.