Nur77和TGF-β交互作用参与MMPs/TIMPs调控的机制研究

Acute respiratory distress syndrome (ARDS) has high mortality and its mechanism is not well known. The prognosis is affected by the remodeling of extracellular matrix (ECM) and its major contributors matrix metalloproteinase/metal matrix protease inhibitors (MMPs/TIMPs). Our previous research results indicated that nuclear receptor subfamily 4 group A member 1 (NR4A1, Nur77) and TGF-β were involved in the pathophysiology of ARDS via PPARγ/TGF-β/p38MAPK/smad2 and Nur77/ p38MAPK pathway repectively. The results indicated that there might be cross-talk between Nur77 and TGF-β in co-regulation and maintaining the balance of MMPs/TIMPs. The experiments are carried to investigate the cross-talk between NUR77 andTGF-β, and its mechanism in regulating balance of MMPs/TIMPs. Gene silencing, site directed mutagenesis, luciferase reporter gene, protein fragment complementation assay, GST pull-down assay, RT-PCR are used to measure the cross-talk activation of Nur77 and TGF-β, and their underline mechanisms of regulation of MMPs/TIMPs. This study is designed to provide a theoretical basis for the treatment of ARDS.

细胞外基质重塑影响ARDS的预后，调控重塑进程的主要介质为金属基质蛋白酶/金属基质蛋白酶抑制物（MMPs/TIMPs）。我们前期研究发现，LPS可分别通过PPARγ/TGF-β/p38MAPK/smad2和Nur77/p38MAPK信号转导通道，调控炎性介质ET-1表达，共同的信号转导通道提示孤儿核受体77（Nur77）和TGF-β之间可能存在交互作用，参与了ARDS的过程。本课题将围绕前期研究的Nur77和TGF-β信号转导通道，在细胞和实验动物模型，综合应用基因沉默技术、基因转染技术、荧光素酶报告基因、蛋白质片段互补分析、GSTpull-down分析、RT-PCR等方法，研究Nur77和TGF-β之间的交互作用对重塑介质MMPs/TIMPs表达的影响，并探索Nur77和TGF-β的交互作用参与MMPs/TIMPs表达的分子调控机制，为减低基质重塑对ARDS预后的影响提供防治思路。

细胞外基质重塑在ARDS发生发展中起重要作用，课题组前期研究发现Nur77通过抑制p38 MAPK和NF-κB信号通路下调LPS诱导的内皮素-1表达，降低肺部炎症与损伤。但Nur77在ARDS肺基质重塑与修复中的作用及分子机制尚不清楚。本项目中，我们通过ARDS的大鼠模型与细胞实验揭示了Nur77通过调控MMP9表达参与ECM重塑；接着发现Nur77可通过TGF-β/Smad信号通路调控MMP9表达，进一步的机制研究表明在TGF-β1刺激下，Nur77可与Smad3结合，敲除Nur77能促进Smad3入核，并上调P-smad3表达；在研究中观察到过表达Nur77的A549细胞MMP9表达低于野生A549细胞，采用ChIP与双荧光素酶报告基因实验证实Nur77能直接与MMP9启动子区结合抑制其转录。此外，我们还发现Cystatin C 通过抑制TGF-β1/Smad3信号通路调控MMP9表达。本项目阐明Nur77与TGF-β1/Smad交互作用调控MMP9表达介导ECM重塑的分子机制，为改善患者预后与提高生存质量提供新靶点与依据。