Novel targeted therapeutics for regulating synovial hyperplasia in RA

 描述（由申请人提供）：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）（Bcl-2家族成员）的表达增加有助于滑膜增生和RA成纤维细胞样滑膜细胞（RA-FLS）对凋亡的抵抗。Mcl-1介导的RA-FLS细胞凋亡抗性可能部分归因于内源性促凋亡Bcl-2同源3（BH 3）-only蛋白表达的缺乏，该蛋白可以结合Mcl-1和其他蛋白。在仅BH 3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，其允许Mcl-泛素连接酶E3（Mule）与泛素化和“引发”Mcl-1结合以进行蛋白酶体降解。药理学策略，旨在提高Noxa的表达或提供BH 3模拟物，这是尚未在RA中进行测试，可能有显着的治疗影响，在调节滑膜增生RA。在这方面，已经鉴定了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mRNA分析显示，与正常人FLS（NL-FLS）相比，RA-FLS中的Mcl-1/Noxa比率高>2倍。有趣的是，UA处理通过诱导Noxa表达并选择性抑制Mcl-1而不是Bcl-2表达来诱导RA-FLS凋亡，从而翻转了该比率。通过对WT和Noxa-/- BMK小鼠肾细胞系中Bcl-2蛋白的生物学活性的研究，我们发现UA不能诱导Noxa-/-细胞凋亡。有趣的是，用稳定表达的Noxa转染Noxa-/- BMK细胞恢复了UA诱导凋亡的能力。与UA处理后的NL-FLS相比，还观察到RA-FLS中多聚泛素化蛋白表达的显著增加。此外，UA诱导的Mcl-1降解被蛋白酶体抑制剂（MG 132）抑制，验证了泛素-蛋白酶体系统（UPS）在RA-FLS凋亡中的作用。UA诱导的Noxa表达。这个提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表达抑制滑膜增生并使RA-FLS对凋亡敏感。目标1中提出的研究将确定UA诱导的Noxa上调和UPS活化以“引发”Mcl-1进行蛋白酶体降解和RA-FLS凋亡的分子机制。在目标2中，我们将表征和比较来自NL、OA或RA供体的FLS中Mcl-1上的磷酸化位点，并研究UA在调节它们以启动Mcl-1降解和使RA-FLS对凋亡敏感中的作用。这些研究的成功将提供Noxa诱导的Mcl- 1降解的分子机制，以及仅含BH 3的蛋白诱导剂（例如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.