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The molecular mechanisms by which NF-κB regulates osteoclastogenesis and its application for the treatment of periodontitis

NF-κB调节破骨细胞生成的分子机制及其在牙周炎治疗中的应用

基本信息

批准号：
20390473
负责人：
JIMI Eijiro
金额：
$ 12.4万
依托单位：
Kyushu Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20390473/
关键词：
歯学細胞・組織転写因子 NF-κB 破骨細胞分化 BMP

项目摘要

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 … More 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

另一种 NF-κB 通路主要由 NF-κB 诱导激酶 (NIK)、IκB 激酶 (IKK)α、p100/p52 和 RelB 组成。替代 NF-κB 信号传导的特点是通过 NIK 将 p100 加工成 p52，从而允许 p52 和 RelB 结合。尽管 NF-κB p50 和 p52 亚基的基因靶向表明 NF-κB 在破骨细胞分化中发挥着关键作用，但替代性 NF-κB 激活在骨生物学中的生理相关性尚不清楚。为了解决这个问题，我们分析了淋巴发育不全 (aly/aly) 小鼠，其中由于 NIK 的非活性形式，p100 到 p52 的加工不会发生。 Aly/aly 小鼠表现出轻度骨硬化症，破骨细胞数量显着减少。 RANKL 诱导的 aly/aly 小鼠骨髓细胞破骨细胞生成也受到抑制。 RANKL 仍然诱导 IκBα 降解并激活经典 NF-κB，而 p100 到 p52 的加工被 aly/aly 突变取消。此外，RAN … More KL 诱导的 NFATc1 表达在 aly/aly 骨髓中受损。组成型活性 IKKα 或 p52 的过度表达可恢复 aly/aly 细胞中的破骨细胞生成。将野生型 p100、无法加工成 p52 的 p100 GRR 或 p52 转染到 NF-κB2 缺陷细胞中，然后进行 RANKL 处理，结果表明 RANKL 诱导的破骨细胞数量与 p52 与 p100 表达的比率之间存在很强的相关性。为了确定 p100 本身的作用，而不受同时缺乏 p52 的影响，我们使用了 p100 缺陷小鼠，这些小鼠特别缺乏 p100 抑制剂，但仍然表达 p52。 p100 缺陷小鼠由于破骨细胞增加而具有骨质减少表型。我们的数据表明，替代性 NF-κB 通路，尤其是 p100 的抑制作用，在骨吸收中的基础破骨细胞生成和刺激破骨细胞生成中发挥着关键作用。骨形态发生蛋白 (BMP) 不仅诱导体内骨形成，而且诱导体外间充质细胞的成骨细胞分化。肿瘤坏死因子 (TNF)α 抑制 BMP 诱导的成骨细胞分化和骨形成。然而，这些抑制的分子机制仍然未知。在这项研究中，我们发现 TNFα 抑制碱性磷酸酶活性，并显着降低 MC3T3-E1 细胞中 BMP2 和 Smad 诱导的报告基因活性。 TNFα 对 Smad1、Smad5 和 Smad8 的磷酸化或 Smad1-Smad4 复合物的核转位没有影响。在 p65 缺陷的小鼠胚胎成纤维细胞中，p65（NF-κB 的一个亚基）的过度表达以剂量依赖性方式抑制 BMP2 和 Smad 诱导的报告基因活性。此外，p65 介导的 BMP2 和 Smad 响应启动子活性的抑制在通过显性失活 IκBα 的过表达抑制 NF-κB 后得以恢复。尽管 TNFα 未能影响 Smad1-Smad4 复合物的受体依赖性形成，但 p65 与该复合物相关。染色质免疫沉淀和电泳迁移率变动分析表明，TNFα 抑制 Smad 蛋白与靶基因的 DNA 结合。重要的是，特定的 NF-κB 抑制剂 BAY11-7082 消除了这些现象。这些结果表明，TNFα 通过激活 NF-κB 干扰 Smads 的 DNA 结合，从而抑制 BMP 信号传导。较少的