P-Rex1与TGFβR2相互作用活化成纤维细胞引发肺纤维化的分子机制研究

Pulmonary fibrosis is a chronic interstitial lung disease that leads to progressive destruction of alveolar structure with a high morbidity rate, however the pathogenesis remains elusive. All forms of PF are characterized by elevated proliferation of interstitial fibroblasts, accompanied by increased synthesis and deposit of extracellular matrix (ECM) proteins. Our original studies indicated that P-Rex1 deficient mice showed attenuated pulmonary fibrosis. The activation of P-Rex1 was increased in mice given bleomycin. P-Rex1 was involved in TGF-β signaling and interacted with TGFβR2. However, the mechanism remains unclear. Based on these results, we hypothesized that P-Rex1 could contribute to the promotion of pulmonary fibrosis via up-regulating fibroblasts activation targeting TGFβR2 molecular. This study intends to clarify the mechanism of P-Rex1 in pulmonary fibrosis from molecular, cellular, tissue and animal levels. The mechanism of the activation of P-Rex1 will be investigated by comparing the activation of primary lung fibroblasts from WT and P-Rex1 knockout mice. Co-immunoprecipitation, site-directed mutagenesis and Mass spectrometry are performed to elucidate a new molecular mechanism for P-Rex1 in TGF-β signal pathway, and reveal the sites. It will provide theoretical and experimental basis for targeted therapies on pulmonary fibrosis.

肺纤维化是以肺泡内成纤维细胞过度增殖、基质蛋白异常堆积、组织结构破坏为特征的间质性疾病，临床发病率高，机制不明确。我们前期研究发现：①P-Rex1基因敲除可显著减弱模型小鼠的肺纤维化程度；②发生纤维化的小鼠肺内P-Rex1蛋白的活化水平升高；③P-Rex1蛋白与TGFβR2发生相互作用并参与TGF-β信号通路，但具体分子机制尚不清楚需深入研究。据此我们提出假说：P-Rex1蛋白可能通过TGFβR2活化成纤维细胞引发肺纤维化。本课题拟从分子、细胞、组织及动物等多层次明确P-Rex1蛋白在肺纤维化中的作用机制；通过比较野生型和P-Rex1基因敲除小鼠原代肺成纤维细胞的活化，阐明引起P-Rex1蛋白活化的作用机制；应用免疫共沉淀、基因定点突变及质谱分析等方法探讨P-Rex1蛋白参与TGF-β信号通路的分子机制，揭示发生相互作用的分子及位点，为肺纤维化疾病的药物开发和临床治疗提供实验依据和新靶点。

肺纤维化可导致肺功能进行性、不可逆的丧失，严重威胁人类健康，病理机制尚不明确。本项目通过构建特异性鸟核苷酸交换因子P-Rex1低/高表达小鼠肺成纤维细胞，揭示P-Rex1蛋白可作为TGF-β1下游信号分子调控肺成纤维细胞迁移；双荧光素酶报告基因和蛋白结合实验证实P-Rex1作用于TGFβR2参与纤维化的形成；临床标本研究发现，纤维化的肺组织内P-Rex1的表达水平显著高于对照正常组织，此外分析肺纤维化患者GEO数据库得出P-Rex1在肺纤维化患者外周血、肺组织和肺成纤维细胞内的表达水平均上调。本项目研究从组织、细胞、分子等多层次阐明P-Rex1分子参与TGF-β1/Smad信号通路促进肺纤维化形成的分子机制，提示P-Rex1可能是肺纤维化治疗的潜在药物靶点。. 本项目在基金委的资助下协助培养硕士研究生2名，青年科研骨干1名；发表SCI收录论文2篇，在投稿SCI收录论文1篇。本项目进展顺利，感谢基金委对本项目的资助。待后续有更多成果，继续向基金委汇报。