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

β1整合素（β1 integrin，β 1）是一种重要的粘附分子，可能参与类风湿性关节炎（rheumatoid arthritis，RA）和进行性系统性硬化症（progressive systemic sclerosis，PSS）等自身免疫性炎症性疾病的发病机制。本研究旨在探讨β1整合素及其相关分子Crk相关底物淋巴细胞型（Cas-L）和四跨膜蛋白（tetraspanins）的生物学作用（CD 9和CD 82）。1）我们表明Cas-L通过其C-末端半结构域定位于脂筏中，并且通过shRNAi逆转录病毒载体减少Cas-L表达导致细胞迁移和IL-2产生显著减少，提示Cas-L在脂筏中的定位对细胞迁移和细胞因子的产生是必需的。2）我们发现Cas-L与NF-κB系统中重要的信号分子IKK-α/β和TRAF 6相关。Cas-L与IKK的结合结构域初步确定为HLH结构域。我们进一步分析了RAW 264. 7细胞的破骨细胞分化，发现在RANKL诱导的破骨细胞分化过程中，Cas-L同源物p130 Cas的mRNA表达上调，而Cas-L的mRNA表达下调。我们发现Cas-L和Smad 6，7在细胞质中相互关联和共定位。4）为了进一步人工调控免疫系统，我们构建了针对Cas-L和重组CD 9或CD 82-IgG-Fc融合蛋白胞外环的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.