Analysis on β1 integrin and its associating molecules in the pathophysiology of Rheumatoid Arthritis and Progressive Systemic Sclerosis.

β1整合素是可能参与类风湿关节炎（RA）、进行性系统性硬化症（PSS）等自身免疫性炎症发病的关键粘附分子。本研究的目的是探讨β1整合素及其相关分子crk相关底物淋巴细胞型（Cas-L）和四跨蛋白（CD9和CD82）的生物学作用。1)我们发现Cas-L通过其c端半结构域在脂质筏中定位，shRNAi逆转录病毒载体降低Cas-L的表达可显著降低细胞迁移和IL-2的产生，提示Cas-L在脂质筏中的定位是细胞迁移和细胞因子产生的必要条件。2)我们发现Cas-L与NF-κB系统中重要的信号分子IKK-α/β和TRAF6相关。初步确定casl对IKKs的关联域为HLH域。我们进一步利用RAW264.7细胞对破骨细胞分化进行分析，发现在rankl诱导的破骨细胞分化过程中，casl同源物p130Cas mRNA表达上调，而casl mRNA表达下调。3)通过双杂交筛选，我们鉴定出Smad6和7为Cas-L结合分子。我们发现Cas-L和smad6、7在细胞质中相互结合并共定位。进一步对Cas-L的siRNA进行分析，发现Cas-L正调控TGF-β介导的信号通路。4)为了将来对免疫系统进行人工调节，我们为Cas-L和重组CD9或CD82-IgG-Fc融合蛋白的细胞外环制备了高效shRNAi逆转录病毒载体。

β1 integrin is key adhesion molecule possibly involving in pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis (RA) and progressive systemic sclerosis (PSS). The aim of this study is investigating the biological roles of β1 integrin and its associating molecules Crk-associated substrate lymphocyte type (Cas-L) and tetraspanins (CD9 and CD82).1) We showed that Cas-L localize in lipid raft through its c-terminal half domains, and that reduction of Cas-L expression by shRNAi retroviral vector resulted in significantly reduced cellular migration and IL-2 production, suggesting that localization of Cas-L in lipid raft is necessary for cellular migration and cytokine production.2) We showed that Cas-L associated with IKK-α/β and TRAF6 that are important signaling molecules in NF-κB system. The associating domain of Cas-L for IKKs are tentatively determined as HLH domain. We further analyzed osteoclast differentiation using RAW264.7 cells, and find that mRNA of p130Cas, a Cas-L homologue, was up-regulated whereas that of Cas-L was down-regulated during RANKL-induced osteoclast differentiation.3) By Two-Hybrid screening, we identified Smad6 and 7 as Cas-L binding molecules. We showed that Cas-L and Smad 6, 7 associate and co-localize each other in the cytoplasm. Furthermore, analysis on siRNA of Cas-L revealed that Cas-L positively regulates TGF-β-mediated signaling pathway.4) To artificially regulate immune system in future, we generated potent shRNAi retroviral vector for Cas-L and recombinant extracellular loops of CD9 or CD82-IgG-Fc fusion proteins.