Kindlin-2调控Ras/ERK信号促进肾成纤维细胞的增殖与活化及肾纤维化的机制研究

Fibroblast proliferation and activation play crucial role in renal fibrosis,Ras/ERK signaling is important in this process,whereas the mechanism of Ras/ERK activation is not well elucidated. Kindlin-2 is a novel integrin-binding adaptor protein. The previous research of applicant found that overexpression of Kindlin-2 induced ERK1/2 activation in renal fibroblasts and knockdown of Kindlin-2 inhibited TGF-β1-induced ERK1/2 activation and α-SMA expression. Results of co-immunoprecipitation found Kindlin-2 interacted with Ras. We get a hypothesis that Kindlin-2 may induce renal fibroblast proliferation and activation and promote kidney fibrosis through regulating Ras/ERK signaling. We are going to firstly inverstigate the role of Kindlin-2 in fibroblast proliferation and activation.Then we will research the effect of Kindlin-2 on Ras/ERK activation and the interaction of Kindlin-2 with the key molecule mediating Ras activation，namely SOS, utilizing the method of co-immunoprecipitation, cellular colocalization and RNA interference.What is more,we are going to investigate the role of Kindlin-2 in renal fibrosis using Kindlin-2 knockout mice in the model of renal fibrosis. Our research will offer new clues for elucidating novel mechanism underlying fibroblast proliferation and activation and Ras/ERK signaling, and designing rational strategies for the treatment of chronic kidney diseases.

成纤维细胞的增殖与活化在肾纤维化中起关键作用，Ras/ERK信号在此过程中发挥重要作用，但其机制尚未完全阐明。Kindlin-2是新发现的衔接蛋白，我们前期研究发现，在肾成纤维细胞中过表达Kindlin-2能激活ERK；敲低Kindlin-2明显抑制TGF-β诱导的ERK磷酸化和α-SMA的表达；Kindlin-2能与Ras相互作用。因此，本项目假设Kindlin-2调控Ras/ERK信号调节成纤维细胞的增殖与活化参与肾纤维化。项目将利用培养的肾成纤维细胞，研究Kindlin-2对成纤维细胞的增殖与活化的作用；证实Kindlin-2与Ras及SOS的相互作用；揭示Kindlin-2激活Ras/ERK活化的分子机制；并通过基因敲除肾纤维化动物模型进一步证实Kindlin-2在肾纤维化中的作用，为深入理解成纤维细胞的激活，Ras/ERK信号活化的分子基础提供新信息，为干预肾纤维化提供新靶标。

Kindlin-2是促进肾间质纤维化的一种重要衔接蛋白。上皮-间充质转化（EMT)被认为是肾间质纤维化的关键机制。为了验证Kindlin-2是否介导肾上皮细胞的EMT从而促进肾间质纤维化，我们研究了Kindlin-2对人近端肾小管上皮细胞EMT的调控作用及其机制。本研究中，我们发现过表达Kindlin-2显著抑制上皮标志物E-cadherin的表达，增强纤维连接蛋白FN和激活的成纤维细胞标志物α-SMA的表达。Kindlin-2能有效地激活肾小管上皮细胞的ERK1/2和AKT信号，抑制ERK1/2和AKT的活性显著抑制Kindlin-2诱导的肾小管上皮细胞EMT。我们进一步发现Kindlin-2激活ERK1/2和AKT信号的上游分子Ras，其机制是通过Kindlin-2与小GEF分子Sos-1相互作用，募集Sos-1至Ras，促进Ras的活化。Ras抑制剂显著阻断Kindlin-2诱导的ERK1/2和AKT活化，从而抑制EMT。我们进一步证实敲低Kindlin-2干扰EGF介导的Ras-Sos-1相互作用，抑制EGF诱导的肾小管上皮细胞的EMT。重要地是，在动物试验中发现敲低Kindlin-2能显著减少单侧输尿管结扎小鼠肾中ERK1/2和Akt的活性。本研究证实Kindlin-2通过激活Ras及其下游信号ERK1/2和Akt诱导肾小管上皮细胞的EMT，Kindlin-2是治疗纤维化肾病的潜在靶目标。