| Budget Amount *help |
¥19,370,000 (Direct Cost: ¥14,900,000、Indirect Cost: ¥4,470,000)
Fiscal Year 2010: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2009: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2008: ¥9,750,000 (Direct Cost: ¥7,500,000、Indirect Cost: ¥2,250,000)
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| Research Abstract |
The alternative NF-κB pathway consists predominantly of NF-κB-inducing kinase (NIK), IκB kinase (IKK)α, p100/p52, and RelB. The hallmark of the alternative NF-κB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. Although gene targeting of the p50 and p52 subunits of NF-κB has shown that NF-κB plays a critical role in osteoclast differentiation, the physiologic relevance of alternative NF-κB activation in bone biology, however, is not well understood. To address this issue, we analyzed alymphoplasia (aly/aly) mice in which the processing of p100 to p52 does not occur owing to an inactive form of NIK. Aly/aly mice showed a mild osteopetrosis with significantly reduced osteoclast numbers. RANKL-induced osteoclastogenesis from bone marrow cells of aly/aly mice also was suppressed. RANKL still induced the degradation of IκBα and activated classical NF-κB, whereas processing of p100 to p52 was abolished by the aly/aly mutation. Moreover, RAN
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KL-induced expression of NFATc1 was impaired in aly/aly bone marrow. Overexpression of constitutively active IKKα or p52 restored osteoclastogenesis in aly/aly cells. The transfection of either wild-type p100, p100 GRR that cannot be processed to p52, or p52 into NF-κB2-deficient cells followed by RANKL treatment revealed a strong correlation between the number of osteoclasts induced by RANKL and the ratio of p52 to p100 expression. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100-deficient mice, which specifically lack the p100 inhibitor but still express p52. p100-deficient mice have an osteopenic phenotype owing to the increased osteoclast. Our data suggest a pivotal role of the alternative NF-κB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis in bone resorption.
Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor (TNF)α inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB. Less
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