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

替代NF-κB途径主要由NF-κ B诱导激酶（NIK）、IκB激酶（IKK）α、p100/p52和RelB组成。替代性NF-κB信号传导的标志是通过NIK将p100加工成p52，从而允许p52和RelB结合。尽管NF-κB的p50和p52亚基的基因靶向已表明NF-κB在破骨细胞分化中发挥关键作用，但骨生物学中替代性NF-κB激活的生理相关性尚未得到很好的理解。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p100到p52的加工由于NIK的非活性形式而不发生。Aly/Aly小鼠表现出轻微的骨硬化症，破骨细胞数量显著减少。aly/aly小鼠骨髓细胞中RANKL诱导的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aly/aly突变则消除了p100至p52的加工。此外，RAN ...更多信息 KL诱导的NFATc 1表达在aly/aly骨髓中受损。组成性活性IKKα或p52的过表达恢复了aly/aly细胞中的破骨细胞生成。将野生型p100、不能加工成p52的p100 GRR或p52转染到NF-κB2缺陷细胞中，随后进行RANKL处理，显示RANKL诱导的破骨细胞数量与p52/p100表达比率之间存在强相关性。为了确定p100本身的作用而不受p52伴随缺乏的影响，我们使用了p100缺陷小鼠，它们特别缺乏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。我们的研究结果表明，NF-κB通路，尤其是p100的抑制作用，在骨吸收的基础和刺激破骨细胞生成中起着关键作用。骨形态发生蛋白（BMPs）不仅在体内诱导骨形成，而且在体外诱导间充质细胞向成骨细胞分化。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。在本研究中，我们发现TNFα抑制MC 3 T3-E1细胞的碱性磷酸酶活性，并显著降低BMP 2和Smad诱导的报告基因活性。TNFα对Smad 1、Smad 5和Smad 8的磷酸化或Smad 1-Smad 4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，NF-κB亚基p65的过表达以剂量依赖性方式抑制BMP 2和Smad诱导的报告基因活性。此外，这种p65介导的BMP 2和Smad反应性启动子活性的抑制在通过显性负性IκBα过表达抑制NF-κB后恢复。虽然TNFα不能影响Smad 1-Smad 4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，特异性NF-κB抑制剂BAY 11 -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结合而抑制BMP信号传导。少

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