Novel targeted therapeutics for regulating synovial hyperplasia in RA

 描述(申请人提供)：在类风湿性关节炎(RA)中，抗凋亡蛋白髓样细胞白血病-1(Mcl-1)(Bcl2家族成员)的表达增加，导致滑膜增生和RA成纤维样滑膜细胞(RA-FLS)对凋亡的抵抗。MCL-1介导的RA-FLS的凋亡抵抗可能部分归因于缺乏内源性促凋亡的BH3蛋白的表达，该蛋白可以结合Mcl-1等。在仅有BH3的家族中，Noxa因其结合Mcl-1的特异性而脱颖而出。NOXA与Mcl-1的结合促进了结构修饰，使Mcl-泛素连接酶E3(Mule)与泛素化结合，并使‘Prime’Mcl-1与蛋白酶体降解结合。旨在增强NOXA表达或传递BH3模拟物的药物策略尚未在RA中进行测试，可能在调节RA滑膜增生方面具有显著的治疗效果。在这方面，已经确定了一种五环三萜类化合物，熊果酸(UA)， 人RA-FLS中NOXA表达的有效诱导剂。比较信使核糖核酸分析显示RA-FLS患者Mcl-1/Noxa比值是正常人FLS(NL-FLS)的2倍。有趣的是，UA通过诱导Noxa的表达和选择性地抑制Mcl-1而不是Bcl2的表达来诱导RA-FLS的凋亡，从而逆转了这一比例。在WT和Noxa-/-BMK小鼠肾细胞系中，我们发现UA不能诱导Noxa-/-细胞的凋亡。有趣的是，稳定表达NOXA的NOXA-/-BMK细胞能够恢复其诱导细胞凋亡的能力。在UA处理后，RA-FLS中多泛素蛋白的表达也显著高于NL-FLS。此外，蛋白酶体抑制剂(MG132)可抑制UA诱导的Mcl-1降解，证实泛素-蛋白酶体系统(UPS)在RA-FLS凋亡中的作用。UA诱导NOXA表达。这项建议利用了这些新的观察结果，我们资助的中心假设是UA通过诱导NoxA的表达抑制滑膜增生，并使RA-FLS对凋亡敏感。Aim 1中提出的研究将确定UA诱导Noxa上调和UPS激活为Mcl-1以促进蛋白酶体降解和RA-FLS凋亡的分子机制。在目标2中，我们将对NL、OA或RA供者的FLS中Mcl-1的磷酸化位点进行表征和比较，并研究UA在调节它们启动Mcl-1降解和增敏RA-FLS的凋亡中的作用。这些研究的成功将提供诺沙星诱导Mcl-1降解的分子机制，以及仅BH3蛋白诱导剂如UA或其结构类似物在调节RA滑膜增生中的治疗重要性。

 DESCRIPTION (provided by applicant): In rheumatoid arthritis (RA), an increased expression of anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member, contributes to synovial hyperplasia and resistance of RA fibroblast-like synoviocytes (RA- FLS) to apoptosis. Mcl-1 mediated resistance to apoptosis in RA-FLS may partly be attributed to the lack in expression of endogenous pro-apoptotic Bcl-2 homology 3 (BH3)-only proteins that can bind Mcl-1 and others. Among the BH3-only family, Noxa stands out exceptional for its specificity to bind Mcl-1. Noxa's binding to Mcl-1 facilitates structural modification that allows binding of Mcl- Ubiquitin ligase E3 (Mule) to ubiquitinate and `prime' Mcl-1 for proteasomal degradation. Pharmacological strategies aimed at enhancing Noxa expression or delivering BH3 mimetics, which are yet to be tested in RA, may have significant therapeutic impact in regulating synovial hyperplasia in RA. In this regard have identified a pentacyclic triterpenoid, ursolic acid (UA), as a potent inducer of Noxa expression in human RA-FLS. Comparative mRNA analysis showed that Mcl-1/Noxa ratio was >2-fold higher in RA-FLS compared to normal human FLS (NL-FLS). Interestingly, UA treatment flipped this ratio by inducing Noxa expression and selectively inhibiting Mcl-1, not Bcl-2, expression to induce RA-FLS apoptosis. U Bcl-2 proteins to blunt their biological activities sing WT and Noxa-/- BMK mouse kidney cell line, we found that UA was ineffective in inducing apoptosis in Noxa-/- cells. Interestingly, transfection of Noxa-/- BMK cells with stably expressing Noxa restored the apoptosis inducing capabilities of UA. A marked increase in the expression of polyubiqitinated proteins in RA-FLS was also observed compared to the NL-FLS upon UA treatment. In addition, UA-induced Mcl-1 degradation was inhibited by proteasome inhibitor (MG132) validating the role of ubiquitin-proteasome system (UPS) in RA-FLS apoptosis. UA-induced Noxa expression. This proposal capitalizes on these novel observations, and the central hypothesis of our grant is that UA suppresses synovial hyperplasia and sensitizes RA-FLS to apoptosis by inducing Noxa expression. Studies proposed in Aim 1 will determine the molecular mechanisms involved in UA-induced Noxa upregulation and activation of UPS to `prime' Mcl-1 for proteasomal degradation and RA-FLS apoptosis. In Aim 2, we will characterize and compare phosphorylation sites on Mcl-1 in FLS from NL, OA, or RA donors and study the effect of UA in modulating them to initiate Mcl-1 degradation and sensitize RA-FLS to apoptosis. The success of these studies will provide the molecular mechanisms of Noxa-induced Mcl- 1 degradation and the therapeutic importance of BH3-only protein inducers such as UA or its structural analogs in regulating RA synovial hyperplasia.