RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.     

Interaction of Fas ligand and Fas expressed on osteoclast precursors increases osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1beta and TNF-alpha, and the activation of NF-kappaB. Functional blocking of IL-1beta and TNF-alpha using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.     

TLR2-dependent modulation of osteoclastogenesis by Porphyromonas gingivalis through differential induction of NFATc1 and NF-kappaB

Osteolytic diseases, including rheumatoid arthritis, osteomyelitis, and periodontitis, are usually associated with bacterial infections. However, the precise mechanisms by which bacteria induce bone loss still remain unclear. Evidence exists that Toll-like receptor (TLR) signaling regulates both inflammation and bone metabolism and that the receptor activator of NF-κB ligand (RANKL) and its receptor RANK are the key regulators for bone remodeling and for the activation of osteoclasts. Here, we investigate the direct effects of the periodontal pathogen Porphyromonas gingivalis on osteoclast differentiation and show that P. gingivalis differentially modulates RANKL-induced osteoclast formation contingent on the state of differentiation of osteoclast precursors. In addition, although an optimal induction of cytokines by P. gingivalis is dependent on TLR2 and TLR4, as well as myeloid differentiation factor 88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β, P. gingivalis utilizes TLR2/ myeloid differentiation factor 88 in modulating osteoclast differentiation. P. gingivalis modulates RANKL-induced osteoclast formation by differential induction of NFATc1 and c-Fos. More importantly, RANKL-mediated lineage commitment also has an impact on P. gingivalis-induced cytokine production. RANKL inhibits P. gingivalis-induced cytokine production by down-regulation of TLR/NF-κB and up-regulation of NFATc1. Our findings reveal novel aspects of the interactions between TLR and RANK signaling and provide a new model for understanding the mechanism underlying the pathogenesis of bacteria-mediated bone loss.     

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.     

Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation

It has been reported previously that inhibitory kappaB kinase (IKK) supports osteoclastogenesis through NF-kappaB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-kappaB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKbeta/NF-kappaB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKbeta-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKbeta through their inhibition. To accomplish this, we generated mice that lack IKKbeta in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKbeta because reduced IKKbeta mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKbeta-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKbeta in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome.     

1'-Acetoxychavicol acetate inhibits RANKL-induced osteoclastic differentiation of RAW 264.7 monocytic cells by suppressing nuclear factor-kappaB activation

Osteoclastogenesis is commonly associated with various age-related diseases, including cancer. A member of the tumor necrosis factor superfamily, receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL), has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this report, we investigated the effect of 1'-acetoxychavicol acetate (ACA), a component of Alpina galanga, on RANKL signaling and consequent osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and coexposure of the cells to ACA completely suppressed RANKL-induced NF-kappaB activation in a time- and concentration-dependent manner. The suppression of NF-kappaB by ACA was mediated through suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Furthermore, incubation of monocytic cells with RANKL induced osteoclastogenesis, and ACA suppressed it. Inhibition of osteoclastogenesis was maximal when cells were simultaneously exposed to ACA and RANKL and minimum when ACA was added 2 days after RANKL. ACA also inhibited the osteoclastogenesis induced by human breast cancer MCF-7 cells, multiple myeloma MM1 cells, and head and neck squamous cell carcinoma LICR-LON-HN5 cells. These results indicate that ACA is an effective blocker of RANKL-induced NF-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.     

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.     

Tumor necrosis factor-alpha (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNF type 1 receptor and RANK signaling pathways

Tumor necrosis factor-alpha (TNF) and the ligand for receptor activator of NF-kappaB (RANKL) are abundant in sites of inflammatory bone erosion. Because these cytokines are potent osteoclastogenic factors and because their signaling pathways are considerably overlapping, we postulated that under pro-inflammatory conditions RANKL and TNF might synergistically orchestrate enhanced osteoclastogenesis via cooperative mechanisms. We found TNF, via TNF type 1 receptor (TNFr1), prompts robust osteoclastogenesis by osteoclast precursors pretreated with RANKL, and deletion of TNFr1 abrogates this response. Enhanced osteoclastogenesis is associated with high expression of otherwise TNF and RANKL-induced mediators, including c-Src, TRAF2, TRAF6, and MEKK-1, levels of which were notably reduced in TNFr1 knockouts. Recruitment of TRAFs and MEKK1 leads to activation of downstream pathways, primarily I kappa B/NF-kappa B, ERKs, and cJun/AP-1. Consistent with impaired osteoclastogenesis and reduced expression of TRAFs and MEKK1, we found that phosphorylation and activation of I kappa B, NF-kappa B, ERKs, and cJun/AP-1 are severely reduced in RANKL-treated TNFr1-null osteoclast precursors compared with wild type counterparts. Finally, we found that TNF and RANKL synergistically up-regulate RANK expression in wild type precursors, whereas basal and stimulated levels of RANK are significantly lower in TNFr1 knockout cells. Our data suggest that exuberant TNF-induced osteoclastogensis is the result of coupling between RANK and TNFr1 and is dependent upon signals transmitted by the latter receptor.     

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.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

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.     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.