KLK8通过切割内皮细胞膜蛋白Lu/BCAM参与脓毒症肺血管屏障功能障碍病理发生的机制研究

组织激肽释放酶相关肽酶8(KLK8)是丝氨酸蛋白酶家族成员。我们发现脓毒症时肺组织中KLK8表达增多；气道内感染KLK8腺病毒导致肺组织局部KLK8过表达可促使肺血管内皮屏障功能障碍，并加重脓毒症诱导的急性肺损伤；而KLK8基因敲除则可减轻脓毒症诱导的急性肺损伤症状。结合免疫共沉淀、质谱分析和验证工作，我们发现膜蛋白Lu/BCAM可能是KLK8的作用靶点。细胞实验发现肺血管内皮细胞上Lu/BCAM与Laminin511的选择性结合在肺血管内皮细胞屏障功能维持过程中发挥关键作用，而Rac1、FAK和PP2A可能是与Lu/BCAM相结合的信号分子。在此基础上，本项目将利用肺组织局部KLK8过表达和KLK8基因敲除两种动物模型，首先证实KLK8表达增高在脓毒症肺血管屏障功能障碍病理发生中的关键作用，进而将主要围绕KLK8/Lu/BCAM信号途径深入探讨KLK8促进肺血管屏障功能障碍的分子机制。

The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases. In our preliminary study, we found that pulmonary KLK8 expression was increased in a time-dependent manner during cecal ligation and puncture (CLP)-induced sepsis. In addition, intratracheally instillation of adenovirus particles expressing KLK8 (Ad-KLK8) resulted in pulmonary vascular barrier dysfunction, meanwhile aggravated sepsis-induced acute lung injury. By contrast, gene knockout of KLK8 significantly protected against actue lung injury in the CLP-induced sepsis. Using co-immunoprecipitation and mass spectrometry, we obtained a number of potential target proteins which might be associated with KLK8. We then determined that the membrane protein Lutheran/basal cell adhesion molecule (Lu/BCAM) might associate with KLK8 in pulmonary vascular endothelial cells, as indicated by IP of KLK8, which yielded Lu/BCAM. In the in vitro studies, we found that Lu/BCAM played a critical role in maintaining pulmonary vascular endothelial barrier function via its selective interaction with specific extracellular matrix (ECM) ligand, laminin 511. Using co-immunoprecipitation and mass spectrometry, we obtained a number of potential signaling proteins which might be associated with Lu/BCAM in laminin511-treated pulmonary vascular endothelial cells. We then confirmed that when stimulated with laminin511, Lu/BCAM associated with Rac1, FAK and PP2A in endothelial cells, which might mediate the intracellular signaling transduction of Lu/BCAM. On the basis of these results, the present proposal will firstly verify that upregulation of KLK8 does contribute to the development of pulmonary vascular endothelial barrier dysfunction during sepsis, by using both intratracheally instillation of Ad-KLK8 and KLK8 knockout mice models. Since KLK8 associates with the membrane protein Lu/BCAM in pulmonary vascular endothelial cells, we will then clarify whether upregulation of KLK8 leads to cleavage of Lu/BCAM, thus resulting in inactivation of the intracellular signal transduction pathways mediated by Rac1, FAK and PP2A. Finally, the role of KLK8/Lu/BCAM signaling pathway in sepsis-induced dysfunction of pulmonary vascular barrier will be elucidated. The present study will provide reliable evidence to elucidate the mechanisms involved in the pathogenesis of pulmonary vascular barrier dysfunction during sepsis. Blockade of KLK8/Lu/BCAM signaling pathway may become one of the future treatment options of sepsis-induced acute lung injury.