KLK8促进内皮细胞间质转化在糖尿病心肌病心肌间质纤维化中的作用及其机制研究

The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases. In our preliminary study, we found for the first time that transgenic-mediated KLK8 overexpression led to development of endothelial-to-msenchymal transition (EndMT) and fibrosis in the hearts. It has recently been found that high glucose-induced EndMT plays an important role in the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy. We demonstrated that high glucose led to significant increase of KLK8 expression in human coronary artery endothelial cells (HCAECs). Moreover, KLK8 knockdown partly reversed high glucose-induced EndMT in HCAECs. Using co-immunoprecipitation and mass spectrometry, we obtained a number of potential target proteins which might be associated with KLK8. We then determined that vascular endothelial (VE)-cadherin/catenin complex might associate with KLK8 in endothelial cells, as indicated by IP of KLK8, which yielded VE-cadherin and γ-catenin. On the basis of these results, the present proposal will firstly verify that upregulation of KLK8 does contribute to the development of EndMT and cardiac fibrosis in diabetic cardiomyopathy, by using both KLK8 transgenic and KLK8 knockout rodent models. Since KLK8 associates with VE-cadherin/catenin complex in endothelial cells, we will then clarify whether upregulation of KLK8 leads to degradation of VE-cadherin and disassembly of VE-cadherin/catenin complex, thus resulting in nuclear translocation of β-catenin, γ-catenin and p120 catenin. Finally, the role of KLK8/VE-cadherin/catenin signaling pathway in EndMT and cardiac fibrosis in diabetic cardiomyopathy will be elucidated. The present study will provide reliable evidence to elucidate the mechanisms involved in the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy. Blockade of KLK8/VE-cadherin/catenin signaling pathway may become one of the future treatment options of cardiac fibrosis in diabetic hearts.

组织激肽释放酶相关肽酶8(KLK8)是丝氨酸蛋白酶家族成员。我们采用KLK8转基因大鼠首次发现KLK8过表达可导致心脏组织中发生内皮细胞间质转化（EndMT）和间质纤维化。高糖诱导EndMT已知是糖尿病心肌间质纤维化病理发生的重要机制，而我们发现高糖处理可以促使内皮细胞KLK8表达增加，敲低KLK8表达则可部分逆转高糖诱导的EndMT现象。结合免疫共沉淀、质谱分析和细胞水平验证工作，我们发现血管内皮钙粘蛋白（VE-cadherin）-连环蛋白（catenin）复合物可能是KLK8在内皮细胞上的作用靶点。在此基础上，本项目首先将利用KLK8转基因和KLK8基因敲除两种模式动物，证实KLK8表达增高在糖尿病心肌病心脏组织EndMT和间质纤维化过程中的关键作用，进而将主要围绕KLK8/VE-cadherin/catenin信号途径深入探讨KLK8促进心脏组织EndMT和间质纤维化的分子机制。

组织激肽释放酶相关肽酶8(KLK8)是丝氨酸蛋白酶家族成员。我们发现STZ诱导1型糖尿病小鼠心肌组织中KLK8表达显著上调；细胞实验表明高糖处理可以诱导冠状动脉内皮细胞（HCAECs）KLK8表达增高。KLK8启动子上具有Sp-1结合位点。高糖可以促进Sp-1表达，而Sp-1阻断剂可以阻断高糖诱导的KLK8表达。KLK8启动子荧光素酶报告基因实验证实高糖可以通过Sp-1介导的转录激活作用促进内皮细胞高表达KLK8。采用KLK8转基因大鼠，我们首次发现KLK8过表达可导致心脏组织中发生内皮细胞间质转化（EndMT）和间质纤维化。KLK8过表达可以诱导HCAECs内皮细胞发生损伤，进而发生EndMT；反之，敲低KLK8表达则可部分逆转高糖诱导的HCAECs细胞损伤和EndMT。KLK8基因缺失可以显著减轻糖尿病心肌间质纤维化以及EndMT。心脏超声结果表明KLK8基因缺失可以显著减轻糖尿病小鼠的收缩和舒张功能障碍。以往研究表明KLK8可以通过促进激肽生成、激活激肽受体发挥生物学效应。我们的结果则表明KLK8过表达诱导内皮细胞损伤以及EndMT并非通过激肽B1或B2受体，而是通过其蛋白酶的活性来完成的。结合免疫共沉淀、质谱分析和验证工作，我们发现血管内皮钙粘蛋白（VE-cadherin）-斑珠蛋白（plakoglobin，又名γ-catenin）复合物可能是KLK8的作用靶点。KLK8可以降解内皮细胞膜上的VE-cadherin继而引发γ-catenin发生核转位。γ-catenin参与介导KLK8诱导EndMT，而这一作用是通过与p53相互作用完成的。γ-catenin核转位后可以促使p53与HIF-1α和Smad3相互作用，继而促进TGF-β1/Smad信号通路下游促纤维化靶基因表达。综合上述结果，我们推测高糖促进心肌组织KLK8表达，进而通过诱导EndMT促进心肌间质纤维化、心脏功能紊乱。而这一作用是依赖于KLK8切割VE-cadherin导致γ-catenin发生核转位，进而激活TGF-β信号通路来完成。此外，我们在研究过程中还发现：KLK8虽然具有显著的促进内皮损伤的作用，但是同时它对于心脏成纤维细胞具有显著的促进其增殖和迁移的作用。众所周知，成纤维细胞激活、增殖是器官纤维化病理发生发展的核心机制，KLK8作用于成纤维细胞的分子机制值得我们进行深入研究。