表没食子儿茶素没食子酸酯通过调控类风湿关节炎滑膜成纤维细胞中转化生长因子-β激活激酶的活性：抑制肿瘤坏死因子受体相关因子 6 的 K(63)连接泛素化。
Regulation of Transforming Growth Factor β-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.
机构信息
Washington State University College of Pharmacy, Spokane.
University of Toledo College of Pharmacy and Pharmaceutical Sciences, Toledo, Ohio.
出版信息
Arthritis Rheumatol. 2016 Feb;68(2):347-58. doi: 10.1002/art.39447.
OBJECTIVE
Transforming growth factor β-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1β (IL-1β), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA).
METHODS
The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1β-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA).
RESULTS
Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1β-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination.
CONCLUSION
Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.
目的
转化生长因子β激活激酶 1（TAK1）是白细胞介素-1β（IL-1β）、肿瘤坏死因子（TNF）和 Toll 样受体信号转导中关键的 MAPKKK 家族蛋白。本研究旨在探讨 TAK1 的翻译后修饰及其在类风湿关节炎（RA）中的治疗调控。
方法
在 IL-1β 刺激的人类 RA 滑膜成纤维细胞（RASFs）中，评估 TAK1、IL-1 受体相关激酶 1（IRAK-1）和 TNF 受体相关因子 6（TRAF6）抑制的效果。采用 Western blot、免疫沉淀和 20S 蛋白酶体测定法研究 RASFs 中的泛素化过程。评估表没食子儿茶素没食子酸酯（EGCG），一种有效的抗炎分子，在调节 RASFs 中这些过程的功效。通过分子对接研究 EGCG 与人 TAK1、IRAK-1 和 TRAF6 的相互作用。使用佐剂诱导关节炎（AIA）大鼠模型证实了这些发现。
结果
抑制 TAK1，但不是 IRAK-1 或 TRAF6，完全阻断了 IL-1β 诱导的 RASFs 中 IL-6 和 IL-8 的合成。EGCG 抑制 TAK1 在 Thr(184/187)的磷酸化并占据 ATP 结合位点的 C(174)位置，从而抑制其激酶活性。EGCG 预处理还通过在 TRAF6 的 K(124)位置形成稳定的氢键，抑制了 TRAF6 的 K(63) -连接的自泛素化，这对于 TAK1 的自磷酸化至关重要。此外，EGCG 增强了蛋白酶体相关去泛素化酶的表达，以防止蛋白质被蛋白酶体降解。与正常大鼠相比，AIA 大鼠关节匀浆的 Western blot 分析显示 K(48) -连接的多泛素化、TAK1 磷酸化和 TRAF6 表达显著增加。给予 EGCG（50mg/kg/天）治疗 10 天可通过降低 TAK1 磷酸化和 K(48) -连接的多泛素化来改善 AIA 大鼠的病情。
结论
我们的研究结果为用 EGCG 靶向 TAK1 治疗 RA 提供了依据。
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