Xu Hanshi, Liu Peng, Liang Liuqin, Danesh Farhad R, Yang Xiuyan, Ye Yijin, Zhan Zhongping, Yu Xueqing, Peng Hui, Sun Lin
Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, Peoples Republic of China.
Arthritis Rheum. 2006 Nov;54(11):3441-51. doi: 10.1002/art.22169.
Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF-kappaB in tumor necrosis factor alpha (TNFalpha)-stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFalpha-induced activation of RhoA and NF-kappaB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.
Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFalpha and incubated with simvastatin (SMV) (1 muM). RhoA activity was assessed by a pull-down assay. NF-kappaB DNA binding activity and nuclear translocation of NF-kappaB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.
TNFalpha stimulation elicited a robust increase in RhoA activity in a dose-dependent manner, and SMV mitigated this increase. TNFalpha also hastened NF-kappaB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IkappaB, and secretion of interleukin-1beta (IL-1beta) and IL-6. SMV prevented the increase in NF-kappaB activation and rise in IL-1beta and IL-6 levels induced by TNFalpha, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF-kappaB and RhoA. Furthermore, cotransfection with a dominant-negative mutant of RhoA demonstrated that the TNFalpha-induced signaling pathway involved sequential activation of RhoA, leading to NF-kappaB activation and, ultimately, to secretion of cytokines.
This study identifies RhoA as the key regulator of TNFalpha-induced NF-kappaB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.
越来越多的证据表明，RhoA可能在炎症反应中起核心作用。本研究旨在探讨RhoA在介导肿瘤坏死因子α（TNFα）刺激的类风湿性滑膜细胞中核因子κB（NF-κB）激活中的作用，并评估他汀类药物对TNFα诱导的RhoA和NF-κB激活以及类风湿性滑膜细胞促炎细胞因子分泌的调节作用。
用TNFα刺激从活动性类风湿性关节炎患者获得的类风湿性滑膜细胞，并用辛伐他汀（SMV）（1μM）孵育。通过下拉试验评估RhoA活性。分别通过灵敏的多孔比色法和共聚焦荧光显微镜测量NF-κB DNA结合活性和NF-κB的核转位。
TNFα刺激以剂量依赖性方式引起RhoA活性的强烈增加，而SMV减轻了这种增加。TNFα还加速了亚基p65的NF-κB核转位并增加了DNA结合活性、荧光素酶报告基因表达、IκB降解以及白细胞介素-1β（IL-1β）和IL-6的分泌。SMV阻止了TNFα诱导的NF-κB激活增加以及IL-1β和IL-6水平升高，而甲羟戊酸和香叶基香叶基焦磷酸逆转了SMV对NF-κB和RhoA激活的抑制作用。此外，用RhoA的显性负突变体共转染表明，TNFα诱导的信号通路涉及RhoA的顺序激活，导致NF-κB激活，并最终导致细胞因子分泌。
本研究确定RhoA是TNFα诱导的NF-κB激活的关键调节因子，这最终导致类风湿性滑膜细胞中促炎细胞因子的分泌。这些发现为他汀类药物在炎症性关节炎中的抗炎作用提供了新的理论依据。
