Proteasomal regulation of TNF-mediated local bone loss

描述(申请人提供)：慢性炎症性骨疾病，如类风湿性关节炎，以局部骨丢失为特征，这主要是由于促炎细胞因子、IL-1、IL-6和肿瘤坏死因子的产生增加所致。这些细胞因子可促进破骨细胞的生成，降低成骨细胞的活性和存活率。尽管细胞因子如何刺激局部破骨细胞性骨侵蚀已知很多，但细胞因子抑制成骨细胞功能的机制仍不清楚。在过去的几年里，E3泛素连接酶，Smad泛素调节因子(Smad Ubiquitin Reguling Fine，SMurf1)，被报道促进成骨细胞特异性转录因子Runx2的降解，从而抑制成骨细胞的分化。然而，SMurf的调控及其在炎症诱导的局部骨丢失发病机制中的作用还没有被研究。最近，我们证明了肿瘤坏死因子刺激成骨细胞中SMurf1的表达，导致Runx2的降解。肿瘤坏死因子介导的Runx2降解与泛素化增加有关，泛素化可以被对SMurfl和蛋白酶体抑制剂的抑制RNA所阻断。与此一致的是，类风湿性关节炎患者和转肿瘤坏死因子基因的关节炎小鼠的关节组织中都有较高水平的SMurf1和肿瘤坏死因子的表达。基于这些初步发现，我们假设在炎症条件下，肿瘤坏死因子或/或其他细胞因子通过上调SMurf-蛋白酶体降解途径抑制成骨细胞的功能。在此，我们利用肿瘤坏死因子转基因和SMurf1-/-小鼠，在体外研究了肿瘤坏死因子对成骨细胞SMurf通路的影响，以及在体内SMurf1缺陷对肿瘤坏死因子介导的成骨细胞抑制的影响。这项应用的总体目的是验证蓝精灵-蛋白酶体途径在细胞因子介导的局部骨丢失中的重要性。这项研究的结果将开辟一个新的研究领域，研究SMurf E3连接酶参与炎症介导的成骨细胞抑制在侵蚀骨疾病中的作用。

DESCRIPTION (provided by applicant): Chronic inflammatory bone diseases, such as rheumatoid arthritis, are characterized by local bone loss that is largely due to the effects of increased production of pro-inflammatory cytokines, IL-1, IL-6, and TNF. These cytokines cause enhanced osteoclastogenesis, and reduced osteoblast activity and survival. Although much is known about how cytokines stimulate focal osteoclastic bone erosion, the mechanisms by which cytokines inhibit osteoblast function remain unclear. In the past several years, the E3 ubiquitin ligase, Smad ubiquitin regulatory factor (Smurf1), has been reported to promote degradation of the osteoblast- specific transcription factor, Runx2, thereby inhibiting osteoblast differentiation. However, the regulation of Smurf and its role in the pathogenesis of inflammation-induced local bone loss have not been investigated. Recently, we have demonstrated that TNF stimulates Smurf1 expression in osteoblasts, which result in Runx2 degradation. TNF-mediated Runx2 degradation is associated with increased ubiquitination, which can be blocked by inhibitory RNA to Smurfl and proteasomal inhibitors. Consistent with this, tissues from joints of rheumatoid arthritic patients and TNF transgenic arthritic mice have high levels of Smurf1 and TNF expression. Based on these preliminary findings, we hypothesize that under inflammatory conditions, TNF or/and other cytokines inhibit osteoblast function through the up-regulation of Smurf-proteasomal degradation pathway. Here we propose to determine the effect of TNF on the Smurf pathway in osteoblasts in vitro, and the effect of Smurf1 deficiency on TNF-mediated osteoblast inhibition in vivo using TNF transgenic and Smurf1-/- mice. The overall purpose of this application is to validate the significance of the Smurf-proteasomal pathway in cytokine-mediated local bone loss. Results from this study should open up a new field of investigation into the involvement of Smurf E3 ligases in inflammation-mediated osteoblast inhibition in erosive bone diseases.