CTNNBIP1调控PPARγ活性的机制及其在糖尿病肾病发病中的意义

peroxisome proliferator-activated receptor γ (PPARγ) and Wnt-β-catenin plays an important role in the regulation of cell differnetiation and proliferation. And it has been notified that PPARγ and Wnt-β-catenin both are the key signal of mesangial cell phenotype transition in the process of diabetic nephropathy ( DN), but the specific mechanism remains unclear. Based on our recent research progress, we demonstrated that both of the expression of β-catenin binding protein (CTNNBIP1) and the activity of PPARγ were inhibited in mesangial cells cocultured with high concentration of glucose and in the glomeruli of db/db mouses. So we put forward a hypothesis that CTNNBIP1 is invovled in the pathogenesis of DN through regulation of PPARγ activity. We designed a project to prove the theory. First, we should examine the binding characteristic between PPARγ, CTNNBIP1 and β-catenin. Second, we should investigate that whether or not CTNNBIP1 and/or β-catenin as coacivators has the ability to regulate the transciptive activity of PPARγ by the method of relative activity of Luciferase Reportor. Third, by using protein binding assay technique such as chromatin immunoprecipitation (CHIP) and super electrophoretic mobility shift assay(super-EMSA), to evaluate that whether or not CTNNBIP1 and β-catenin could combined with PPARγ to form as a transciption complex in vivo under the conditions of db/db with enhancing CTNNBIP1 expression by infection of lentiviral containing CTNNBIP1 gene. Finally, we want to confirm that CTNNBIP1 as a coactivator could influence the activity of PPARγ in the pathogenesis of DN. The results will provide new ideas and contribute to the evaluation of DN mechanism, and provide new candidate drug target for the prevention and treatment of DN.

PPARγ和Wnt-β-catenin都是参与糖尿病肾病（DN）系膜细胞表型转化的重要控制信号，且存在交互作用，但具体机制不清。前期研究发现：高糖时β-catenin结合蛋白（CTNNBIP1）表达下调和PPARγ活性降低。因此，提出“CTNNBIP1调控PPARγ转录活性参与DN发病”的假说。拟以PPARγ活性调节为切入点，首先分析PPARγ、CTNNBIP1、β-catenin三者的结合特性；再利用RNAi和荧光素酶报告基因技术，确认CTNNBIP1、β-catenin均是PPARγ转录复合体的组成辅因子；并通过构建CTNNBIP1超表达的DN小鼠模型，应用CHIP和/或EMSA技术从整体水平分析CTNNBIP1、β-catenin是否与PPARγ形成转录复合体。研究结果将为揭示DN机理、DN防治提供新思路和药物靶标。

糖尿病患者长期的高糖状态是其主要的生理改变，也是其并发症的原因之一，糖尿病肾病（DN）是糖尿病的主要并发症之一。过氧化物酶体增殖物激活受体γ（PPARγ）转录活性的降低已被证实是DN发生发展过程中的机制之一。本课题主要研究在高糖环境下连环蛋白互作蛋白1（ICAT）对PPARγ转录活性的影响，试图从这一途径解释DN发生发展的机制。.我们发现在高糖环境下的系膜细胞中，β连环蛋白（β-catenin）表达升高而ICAT表达下降。高糖环境下过表达ICAT可使PPARγ转录活性的升高，且部分降低了系膜细胞表型转化标志物（α-smooth muscle actin and fibronectin）表达以及细胞的增殖。同时，我们应用生物信息学手段，预测了ICAT与-catenin可以与PPARγ相互结合的可能性。接下来，我们通过体外表达这三个蛋白质，利用蛋白质-蛋白质相互作用实验手段，证实了ICAT与β-catenin可以与PPARγ相互结合为一个复合体。通过以上实验证据，我们发现ICAT--catenin蛋白复合体作为PPARγ的共激活物，对其转录活性的增强有着重要作用。最后，我们利用CHIP实验发现过在高糖环境中培养的系膜细胞中过表达ICAT，有更多的ICAT与β-catenin蛋白结合在PPARγ反应原件上。.这个机制的发现可以让我们更好从Wnt/β-catenin 和 PPARγ两个途径交汇的角度理解DN的发生发展过程，也可能为研发预防DN的新型药物提供新的思路。