Endothelial Cytoprotective Signaling by Activated Protein C/Protease-activated Receptor-1

摘要/摘要 目前用于改善血管疾病中内皮功能障碍的治疗选择有限， 败血症，导致高发病率和死亡率。内皮功能障碍导致内皮细胞活化， 破坏内皮屏障功能和对细胞凋亡的敏感性。该提案的长期目标是 描绘内皮抵抗损伤和破坏以促进推进的途径 治疗发展的新目标。活化蛋白C（APC）是一种有前途的治疗药物， 多种有益作用，包括稳定内皮屏障和抗凋亡活性。蛋白酶- 活化受体-1（PAR 1）是一种G蛋白偶联受体（GPCR），是APC细胞的中心介质， 信号传导，其需要小窝蛋白-1（Cav 1）和小窝中的区室化。我们发现APC- 活化的PAR 1主要通过β-arrestin-2（β-arr 2）信号传导来促进内皮屏障保护， 异源三聚体G蛋白如凝血酶（Th）激活的PAR 1。本提案的总体目标是： 对APC/PAR 1如何产生β-arr 2传感器偏倚以促进内皮细胞增殖的机制的理解 细胞保护我们假设不同的GRK 5决定簇和共受体促进APC/PAR 1诱导的细胞凋亡。 通过Cav 1激活的途径促进内皮细胞保护的EARR-ARR 2换能器偏倚 磷酸化我们提出三个具体目标。目的1：阐明GRK 5的作用机制， 明显调节APC相对于Th诱导的偏置信号传导。GRK 5是APC刺激信号传导所必需的， Th诱导的信号转导的脱敏。然而，使不同GRK 5功能的机制并不 知道的我们将确定不同的GRK 5功能是否通过定位到离散质膜来调节 微区，如小窝，使用人类培养的内皮细胞，一个天然的系统，允许研究 内源性PAR 1和GRK 5以及HEK 293 CRISPR/Cas9敲除细胞的生长。目标2：确定 APC与凝血酶控制β-arrestin传感器偏倚的机制。目前尚不清楚， APC-与Th-激活的PAR 1诱导的转导偏置（信号传导与脱敏），也不是APC/PAR 1 促进两种不同的ARR 2介导的细胞保护信号通路：杂乱2（Dvl 2）-Rac 1对照 内皮屏障保护，而鞘氨醇激酶1（SphK 1）-Akt调节抗凋亡活性。我们 将确定β-arrestin和GPCR共受体的不同决定因素是否控制不同的β-arr 2结合模式 和由APC相对于凝血酶诱导的功能。目的3：确定APC/PAR 1调节的机制 Cav 1的功能是促进细胞保护。APC/PAR 1刺激Cav 1磷酸化，但这如何调节 Cav 1功能和整合到细胞保护途径中尚不清楚，将进行测定。的 拟议的研究是创新的，因为它将测试新的假设，以解释GRK 5，ARR 2和Cav 1 在生理相关的背景下驱动APC/PAR 1内皮细胞保护反应， 作为内皮功能障碍治疗的未来疗法的新靶点的状态。

Summary/Abstract There are currently limited treatment options for improving endothelial dysfunction in vascular diseases such as sepsis, resulting in high morbidity and mortality. Endothelial dysfunction results in endothelial cell activation, disruption of endothelial barrier function and sensitivity to apoptosis. The long-term goal of this proposal is to delineate the pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated protein C (APC) is a promising therapeutic and exhibits multiple beneficial effects including stabilization of endothelial barriers and anti-apoptotic activities. Protease- activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the central mediator of APC cellular signaling, which requires caveolin-1 (Cav1) and compartmentalization in caveolae. We discovered that APC- activated PAR1 signals primarily through -arrestin-2 (-arr2) to promote endothelial barrier protection, and not heterotrimeric G proteins like thrombin (Th)-activated PAR1. The overall objective of this proposal is to develop a mechanistic understanding of how APC/PAR1 generates -arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced -arr2 transducer bias to promote endothelial cytoprotection through pathways enabled by Cav1 phosphorylation. We propose three specific aims. Aim 1: To delineate the mechanisms that enable GRK5 to distinctly regulate APC- vs. Th-induced biased signaling. GRK5 is required for APC-stimulated signaling and desensitization of Th-induced signaling. However, the mechanisms that enable distinct GRK5 functions are not known. We will determine if distinct GRK5 functions are regulated by localization to discrete plasma membrane microdomains such as caveolae using human cultured endothelial cells, a native system that permits the study of endogenous PAR1 and GRK5 and HEK293 CRISPR/Cas9 knockout cells. Aim 2: To determine the mechanisms by which APC vs. thrombin control -arrestin transducer bias. It is not known how -arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct -arr2-mediated cytoprotective signaling pathways: dishevelled2 (Dvl2)-Rac1 controls endothelial barrier protection whereas sphingosine kinase 1 (SphK1)-Akt regulates anti-apoptotic activities. We will determine if distinct determinants of -arrestin and GPCR co-receptors control different -arr2 binding modes and functions induced by APC vs. thrombin. Aim 3: To define the mechanisms by which APC/PAR1 regulates Cav1 function to promote cytoprotection. APC/PAR1 stimulates Cav1 phosphorylation but how this modulates Cav1 function and is integrated into the cytoprotective pathway is not known and will be determined. The proposed research is innovative because it will test novel hypotheses to explain how GRK5, -arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as future therapeutics for the treatment of endothelial dysfunction.