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

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.

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。

组织激肽释放酶相关肽酶(KLKs)家族共有15个家族成员。通过比较15种小鼠KLKs的mRNA表达水平，我们发现KLK8在脓毒症小鼠肺组织和脂多糖(LPS)处理的原代分离小鼠肺血管内皮细胞(MLVECs)中都是上调最为显著的家族成员。肺组织切片免疫荧光染色也证实了内毒素血症时肺组织内皮细胞中KLK8显著上调。采用气道滴注KLK8腺病毒(Ad-KLK8)构建肺组织KLK8高表达小鼠模型，我们首次发现KLK8过表达可导致肺组织中肺血管内皮通透性升高和肺损伤。KLK8过表达可以诱导MLVECs内皮细胞活力降低、通透性升高、增殖能力下降；反之，敲低KLK8表达则可部分逆转脓毒症或LPS诱导的MLVECs细胞损伤和通透性增高。我们发现KLK8基因敲除或使用KLK8中和抗体抑制KLK8，均可显著减轻内毒素血症小鼠的肺内皮屏障高通透性和急性肺损伤，并显著降低小鼠死亡率。对KLK8过表达肺血管内皮细胞进行转录组测序、并进行IPA上游调节因子分析后发现，叉头盒M1 (FOXM1)下调在介导KLK8对肺内皮细胞的促损伤和抗增殖作用中起着核心作用。慢病毒介导FOXM1过表达可以完全逆转Ad-KLK8抑制内皮细胞增殖、促进内皮细胞损伤的作用。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）和细胞粘附分子Lu/BCAM 蛋白可能是KLK8在肺内皮细胞上的作用靶点。一方面，KLK8切断VE-cadherin细胞外结构域，通过灭活VE-cadherin/Akt信号通路抑制FOXM1的表达。另一方面，KLK8切割 Lu/BCAM 蛋白后可以阻断其与 Laminin511的受体-配体结合作用，进而抑制Akt/FOXM1信号通路。KLK8缺失或阻断可挽救VE-cadherin或Laminin511-Lu/BCAM介导的/Akt/FOXM1信号通路，从而促进体内内皮细胞再生。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，但是同时它对于成纤维细胞具有显著的促进其增殖和迁移的作用。对公共数据库中肺组织单细胞测序结果分析表明KLK8主要表达于肺组织成纤维细胞中。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以及它潜在的促进肺纤维化病理发生的可能机制值得我们进行深入研究。