Inhibition of histone deacetylase suppresses osteoclastogenesis and bone destruction by inducing IFN-beta production

Osteoclasts are bone-resorptive multinucleated cells that are differentiated from hemopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-kappaB ligand (RANKL) and M-CSF. Downstream signaling molecules of the receptor of RANKL, RANK, modulate the differentiation and the activation of osteoclasts. We recently found that histone deacetylase inhibitors (HDIs), known as anticancer agents, selectively suppressed osteoclastogenesis in vitro. However, the molecular mechanism underlying inhibitory action of HDIs in osteoclastogenesis and the effect of HDIs on pathological bone destruction are still not remained to be elucidated. In this study, we show that a depsipeptide, FR901228, inhibited osteoclast differentiation by not only suppressing RANKL-induced nuclear translocation of NFATc1 but also increasing the mRNA level of IFN-beta, an inhibitor of osteoclastogenesis. The inhibition of osteoclast formation by FR901228 was abrogated by the addition of IFN-beta-neutralizing Ab. In addition, treatment of adjuvant-induced arthritis in rats revealed that FR901228 inhibited not only disease development in a prophylactic model but also bone destruction in a therapeutic model. Furthermore, immunostaining of the joints of therapeutically treated rats revealed significant production of IFN-beta in synovial cells. Taken together, these data suggest that a HDI inhibits osteoclastogenesis and bone destruction by a novel action to induce the expression of osteoclast inhibitory protein, IFN-beta.     

SLAT negatively regulates RANKL-induced osteoclast differentiation

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein, we investigated the role of SWAP-70-like adapter of T cells (SLAT) in RANKL-induced osteoclastogenesis. Expression levels of SLAT were reduced during RANKL-induced osteoclastogenesis. Overexpression of SLAT in BMMs inhibited TRAP-positive multinuclear osteoclast formation and attenuated the expression of NFATc1, which is an important modulator in osteoclastogenesis. Furthermore, silencing of SLAT by RNA interference enhanced osteoclast formation as well as NFATc1 expression. In addition, SLAT was involved in RANKL-induced JNK activation in osteoclasts. Taken together, our data suggest that SLAT acts as a negative modulator of RANKL-induced osteoclastogenesis.     

Connection between B lymphocyte and osteoclast differentiation pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.     

Stimulation by TLR5 modulates osteoclast differentiation through STAT1/IFN-beta

Osteoclasts are bone-resorbing cells that are differentiated from hemopoietic precursors of the monocyte-macrophage lineage. Stimulation of TLRs has been shown to positively or negatively modulate osteoclast differentiation, depending on the experimental condition. However, the molecular mechanism by which this modulation takes place remains unclear. In the present study, we examined the effects of flagellin, a specific microbial ligand of TLR5, on the receptor activator of NF-kappaB ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and a constitutively active form of NFATc1 reversed the flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-beta production. Flagellin stimulated IFN-beta expression and release in bone marrow-derived macrophages, and IFN-beta-neutralizing Ab prevented the flagellin-induced c-Fos down-regulation and the anti-osteoclastogenic effect. IFN-beta gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1 deficiency or the JAK2 inhibitor AG490 dramatically prevented IFN-beta induction in response to flagellin or RANKL. In addition, STAT1 deficiency abolished the anti-osteoclastogenic effect induced by flagellin or LPS. In contrast, flagellin stimulated osteoclast differentiation in cocultures of osteoblasts and bone marrow cells without inducing IFN-beta. Thus, IFN-beta acts as a critical modulator of osteoclastogenesis in response to TLR5 activation.     

The transmembrane adaptor protein, linker for activation of T cells (LAT), regulates RANKL-induced osteoclast differentiation

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein we investigate the role of the transmembrane adaptor proteins in RANKL-induced osteoclastogenesis. LAT positively regulates osteoclast differentiation and is up-regulated by RANKL via c-Fos and NFATc1, whereas LAB and LIME act as negative modulators of osteoclastogenesis. In addition, silencing of LAT by RNA interference or overexpression of a LAT dominant negative in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Furthermore, LAT is involved in RANKL-induced PLC(γ) activation and NFATc1 induction. Thus, our data suggest that LAT acts as a positive regulator of RANKL-induced osteoclastogenesis.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Regulation of receptor activator of NF-kappa B ligand-induced osteoclastogenesis by endogenous interferon-beta (INF-beta ) and suppressors of cytokine signaling (SOCS). The possible counteracting role of SOCSs- in IFN-beta-inhibited osteoclast formation

Bone resorption and the immune system are correlated with each other, and both are controlled by a variety of common cytokines produced in the bone microenvironments. Among these immune mediators, the involvement of type I interferons (IFNs) in osteoclastic bone resorption remains unknown. In this study, we investigated the participation of IFN-beta and suppressors of cytokine signaling (SOCS)-1 and -3 in osteoclastogenesis. Addition of exogenous IFN-beta to osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation toward osteoclasts induced by the receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor with/without transforming growth factor-beta, where inhibition was associated with down-regulation of the gene expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the expression of IFN-beta; furthermore, neutralizing antibody against type I IFNs accelerated the osteoclast formation, indicating type I IFNs as potential intrinsic inhibitors. On the other hand, RANKL also induced the expression of SOCS-1 and -3, suppressors of the IFN signaling. Pretreatment with RANKL for a sufficient time for the induction of SOCSs attenuated phosphorylation of STAT-1 in response to IFN-beta in osteoclast progenitors, causing a decrease in the binding activity of nuclear extracts toward the interferon-stimulated response element. mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor-3gamma, comprising IFN-stimulated gene factor-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-beta is produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the induction of signaling suppressors such as SOCSs.     

Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL)

The receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38alpha MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells.     

Signaling crosstalk between RANKL and interferons in osteoclast differentiation

Regulation of osteoclast differentiation is an aspect central to the understanding of the pathogenesis and the treatment of bone diseases such as autoimmune arthritis and osteoporosis. In fact, excessive signaling by RANKL (receptor activator of nuclear factor kappaB ligand), a member of the tumor necrosis factor (TNF) family essential for osteoclastogenesis, may contribute to such pathological conditions. Here we summarize our current work on the negative regulation of osteoclastogenesis by unique signaling crosstalk between RANKL and interferons (IFNs). First, activated T cells maintain bone homeostasis by counterbalancing the action of RANKL through production of IFN-gamma. This cytokine induces rapid degradation of the RANK (receptor activator of nuclear factor kappaB) adapter protein TRAF6 (TNF-receptor-associated factor 6), resulting in strong inhibition of the RANKL-induced activation of NF-kappaB and JNK (c-Jun N-terminal kinase). Second, RANKL induces the IFN-beta gene but not IFN-alpha genes, in osteoclast precursor cells, and that IFN-beta strongly inhibits the osteoclast differentiation by interfering with the RANKL-induced expression of c-Fos. The series of in vivo experiments revealed that these two distinct IFN-mediated regulatory mechanisms are both important to maintain homeostasis of bone resorption. Collectively, these studies revealed novel aspects of the two types of IFN, beyond their original roles in the immune response, and may offer a molecular basis for the treatment of bone diseases.     

A role for alphaV integrin subunit in TGF-beta-stimulated osteoclastogenesis

TGF-beta increases bone resorption in vivo and greatly increases osteoclast formation stimulated by receptor activator of NF-kappaB ligand (RANKL) in vitro. TGF-beta does not independently affect the differentiation state of RAW264.7 preosteoclasts, but increases cell attachment to vitronectin. This effect is mediated by increased expression of alphaV integrin subunit mRNA and protein. Concomitant with induction of osteoclast differentiation, RANKL causes relocation of alphaV to focal sites in the cell. This effect is potentiated by TGF-beta. Integrin blockade disrupts both attachment to vitronectin and RANKL-induced osteoclast formation, but culture on vitronectin has little effect. Ectopic expression of alphaV stimulates multinucleation of RAW264.7 cells and increases the number of osteoclasts formed in the presence of RANKL. These data suggest that TGF-beta potentiates RANKL-induced osteoclast formation, in part by increased expression of the alphaV integrin subunit, which may contribute to cell fusion.     

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 PGE(2) 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 PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB 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 PGE(2) 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 PGE(2) 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 PGE(2) production.     

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.     

Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.     

RANKing intracellular signaling in osteoclasts

RANKL plays a pivotal role in the differentiation, function and survival of osteoclasts, the principal bone-resorbing cells. RANKL exerts the effects by binding RANK, the receptor activator of NF-kappaB, in osteoclasts and its precursors. Upon binding RANKL, RANK activates six major signaling pathways: NFATc1, NF-kappaB, Akt/PKB, JNK, ERK and p38, which play distinct roles in osteoclast differentiation, function and survival. Recent studies have not only provided more insights into RANK signaling but have also revealed that several factors, including INF-gamma, IFN-beta, and ITAM-activated costimulatory signals, regulate osteoclastogenesis via direct crosstalk with RANK signaling. It was recently shown that RANK contains three functional motifs capable of mediating osteoclastogenesis. Moreover, although both IFN-gamma and IFN-beta inhibit osteoclastogenesis, they exert the inhibitory effects by distinct mechanisms. Whereas IFN-gamma has been shown to block osteoclastogenesis by promoting degradation of TRAF6, IFN-beta inhibits osteoclastogenesis by down-regulating c-fos expression. In contrast, the ITAM-activated costimulatory signals positively regulate osteoclastogenesis by mediating the activation of NFATc1 through two ITAM-harboring adaptors: FcRgamma and DAP12. This review is focused on discussing the current understanding of RANK signaling and signaling crosstalk between RANK and the various factors in osteoclasts.