NF-{kappa}B p50 and p52 Regulate Receptor Activator of NF-{kappa}B Ligand (RANKL) and Tumor Necrosis Factor-induced Osteoclast Precursor Differentiation by Activating c-Fos and NFATc1

Department of Microbiology and Immunology, Keio University, Edo, Tōkyō, Japan
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2007; 282(25):18245-53. DOI: 10.1074/jbc.M610701200
Source: PubMed

ABSTRACT Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-kappaB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-kappaB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-kappaB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-kappaB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-kappaB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-kappaB should prevent RANKL- and TNF-induced bone resorption.

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    • "RANKL stimulates the expression and activation of NFATc1 in osteoclast precursors by regulating other transcription factors. It has been shown that RANKL-induced NFATc1 expression depends on NF-κB and c-Fos, which are activated by RANKL [9, 12]. It was also reported that RANKL suppresses the expression of transcriptional repressors such as inhibitors of differentiation/DNA binding (Ids), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (MafB), and interferon regulatory factor 8 that inhibit NFATc1 expression and osteoclast differentiation [13]. "
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    ABSTRACT: Background Excessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases. There is accumulating evidence that certain herbal extracts have beneficial effects on bone metabolism. The fruits of Alpinia oxyphylla has been traditionally used for the treatment of diarrhea and enuresis. In this study, we investigated the effects of water extract of the fruits of Alpinia oxyphylla (WEAO) on osteoclast differentiation and osteoclast-mediated bone destruction. Methods For osteoclast differentiation assay, mouse bone marrow-derived macrophages (BMMs) were cultured in the presence of RANKL and M-CSF. RANKL signaling pathways and gene expression of transcription factors regulating osteoclast differentiation were investigated by real-time PCR and Western blotting. A constitutively active form of NFATc1 was retrovirally transduced into BMMs. Bone resorbing activity of mature osteoclast was examined on a plate coated with an inorganic crystalline calcium phosphate. The in vivo effect against bone destruction was assessed in a murine model of RANKL-induced osteoporosis by micro-computed tomography and bone metabolism marker analyses. Results WEAO dose-dependently inhibited RANKL-induced osteoclast differentiation from BMMs by targeting the early stages of osteoclast differentiation. WEAO inhibited RANKL-induced expression of NFATc1, the master regulator of osteoclast differentiation. Overexpression of a constitutively active form of NFATc1 blunted the inhibitory effect of WEAO on osteoclast differentiation, suggesting that NFATc1 is a critical target of the inhibitory action of WEAO. WEAO inhibited RANKL-induced expression of c-Fos, an upstream activator of NFATc1, by suppressing the classical NF-κB signaling pathway. WEAO also inhibited RANKL-induced down-regulation of Id2 and MafB, negative regulators of NFATc1. WEAO does not directly affect bone resorbing activity of mature osteoclasts. In accordance with the in vitro results, WEAO attenuated RANKL-induced bone destruction in mice by inhibiting osteoclast differentiation. Conclusions This study demonstrates that WEAO exhibits a protective effect against bone loss by inhibiting RANKL-induced osteoclast differentiation. These findings suggest that WEAO might be useful for the prevention and treatment of bone diseases associated with excessive bone resorption.
    BMC Complementary and Alternative Medicine 09/2014; 14(1):352. DOI:10.1186/1472-6882-14-352 · 2.02 Impact Factor
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    • "Binding of RANKL to RANK activates several transcription factors responsible for promoting osteoclastic gene expression. These are not all activated within the same time frame: early response factors, such as AP-1 (c-Fos/c-Jun dimer), are activated before late-response factors, such as NFATc1 [34]. Furthermore, AP-1 is known to regulate the expression of NFATc1 by binding to the NFATc1 promoter [35]. "
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    ABSTRACT: We recently reported that oral administration of a (+)-vitisin A-enriched product prepared from Vitis thunbergii obviously ameliorated bone loss in ovariectomized mice and (+)-vitisin A was able to inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Here we further clarified the mechanism(s) by which (+)-vitisin A targets osteoclastic differentiation and activity. Osteoclast-characteristic enzyme activity was determined using gel zymography or spectroflurometric-based assay. Expression of signal molecules was analyzed via Western blot or immunoprecipitation. Results showed that (+)-vitisin A suppressed RANKL-induced multinuclear cells (MNCs) formation and bone resorption which was accompanied with reduction in β3 integrin, osteoclast stimulatory transmembrane protein (OC-STAMP), matrix metalloproteinase-9 (MMP-9) and cathepsin K proteins expression. (+)-Vitisin A also down-regulated the proteolytic activities of MMP-9 and cathepsin K via targeting at the late stage function. (+)-Vitisin A prominently abrogated RANKL-triggered nuclear translocations of NF-κB, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream IκB degradation as well as ERK and JNK phosphorylation were also substantially repressed. Transfection with siRNA targeting tumor necrosis factor receptor associated factor 6 (TRAF6) clearly restrained RANKL-induced MNCs formation and NFATc1 induction. Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 was markedly induced by RANKL stimulation. (+)-Vitisin A significantly attenuated this interaction when concomitant treated with RANKL in RAW264.7 cells, but failed to affect c-Src/β3 complex formation when post-cultured with MNCs. Taken together, (+)-vitisin A suppressed bone resorption possibly via interruption of RANKL-induced TRAF6 ubiquitination and associated downstream signaling pathways. Furthermore, action through negative regulation of the proteolytic activity of MMP-9 and cathepsin K might also contribute to the anti-resorption effect of (+)-vitisin A.
    PLoS ONE 02/2014; 9(2):e89159. DOI:10.1371/journal.pone.0089159 · 3.23 Impact Factor
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    • "Genetic studies have demonstrated that NF-κB signaling pathway plays a crucial role in osteoclastogenesis [6]. It has been suggested that NF-κB functions upstream of c-Fos during RANKL-induced osteoclastogenesis [7]. The classic NF-κB signaling pathway involves activation of the IκB kinase (IKK) complex that leads phosphorylation and degradation of IκBα, allowing nuclear translocation of NF-κB complexes containing the p50 and p65 subunits. "
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    ABSTRACT: Background Osteoclasts are primarily responsible for bone resorption. In many pathological bone diseases including osteoporosis and rheumatoid arthritis, osteoclasts are excessively activated. Thus, controlling of osteoclasts would be an effective therapeutic strategy for the treatment of excessive bone loss. The stem of Spatholobus suberectus has been widely used in traditional medicine to treat blood stasis syndrome and arthritis in Asia. In the present study, we investigated the effects and action mechanism of water extract of the stem of Spatholobus suberectus (WESS) on osteoclast differentiation and function. Methods The effect of WESS on osteoclast differentiation was evaluated by counting tartrate resistant acid phosphatase-positive multinucleated cells in bone marrow-derived macrophages system and murine bone marrow cell-osteoblast coculture system. Bone resorption activity of mature osteoclast was examined on a calcium phosphate-coated plate. Actin ring structure of osteoclasts was detected fluorescently by staining for F-actin. Activation of signaling pathways and induction of transcription factors required for osteoclastogenesis were investigated by real-time PCR and Western blotting. Results WESS effectively inhibited osteoclast differentiation from its precursors. The inhibitory effect of WESS on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1 expression, via preventing receptor activator of nuclear factor-κB ligand-induced early signaling pathways and decreasing c-Fos protein level in osteoclast precursors. Furthermore, WESS suppressed bone resorption activity of osteoclasts by disrupting actin ring structure. Conclusions This study demonstrated that WESS inhibits osteoclast differentiation and function. These results suggest that WESS has a potential for treating pathological bone diseases caused by excessive bone resorption.
    BMC Complementary and Alternative Medicine 05/2013; 13(1):112. DOI:10.1186/1472-6882-13-112 · 2.02 Impact Factor
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