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

摘要/摘要 目前，改善血管疾病的内皮功能障碍的治疗选择有限，例如 败血症，导致高发病率和死亡率。内皮功能障碍会导致内皮细胞激活， 内皮屏障功能的破坏和对细胞凋亡的敏感性。这项提议的长期目标是 描绘内皮细胞抵抗损伤和破坏以促进进展的途径 治疗发展的新靶点。活化蛋白C(APC)是一种很有前途的治疗方法和展品 多种有益作用，包括稳定内皮屏障和抗细胞凋亡活性。蛋白酶- 活化受体-1(PAR1)是一种G蛋白偶联受体，是APC细胞的中枢介质 信号传递，这需要小窝蛋白-1(Cav1)和小窝中的区划。我们发现APC- 激活PAR1信号主要通过b-arrestin-2(b-arr2)促进内皮屏障保护，而不是 异三聚体G蛋白，如凝血酶(Th)激活的PAR1。这项提议的总体目标是发展 APC/PAR1如何产生b-arr2转导偏向促进内皮细胞生长的机制研究 细胞保护。我们假设不同的GRK5决定簇和辅助受体促进APC/PAR1诱导 B-arr2转导偏向通过Cav1激活的途径促进内皮细胞保护 磷酸化。我们提出了三个具体目标。目标1：描述使GRK5能够 明显调节APC与Th诱导的偏向信号转导。APC刺激的信号转导需要GRK5 Th诱导信号的脱敏。然而，启用不同GRK5功能的机制并不是 为人所知。我们将确定不同的GRK5功能是否通过对离散质膜的定位来调节 使用人类培养的内皮细胞的微域，如小凹，这是一种允许进行研究的本地系统 内源性PAR1、GRK5和HEK293CRISPR/Cas9基因敲除细胞。目标2：确定 Apc与凝血酶控制b-arrestin转导偏置的机制。目前尚不清楚b-arrestin是如何 APC-与Th激活的PAR1或APC/PAR1如何引起转导偏差(信号与脱敏) 促进两个不同的b-arr2介导的细胞保护信号通路：dishevelled2(Dvl2)-rac1对照 内皮屏障保护，而鞘氨醇激酶1(SphK1)-Akt调节抗凋亡活性。我们 将确定b-arrestin和gpr共同受体的不同决定因素是否控制不同的b-arr2结合模式。 APC与凝血酶诱导的功能。目标3：确定APC/PAR1的调节机制 Cav1具有促进细胞保护的功能。APC/PAR1刺激Cav1磷酸化，但这是如何调节的 Cav1的功能和是否整合到细胞保护途径尚不清楚，将被确定。这个 拟议的研究具有创新性，因为它将测试新的假设，以解释GRK5、b-arr2和Cav1如何 在生理相关的背景下推动APC/PAR1内皮细胞保护反应，将有助于推动 作为治疗内皮功能障碍的未来疗法的新靶点的现状。

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 b-arrestin-2 (b-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 b-arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced b-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 is 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 b-arrestin transducer bias. It is not known how b-arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct b-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 b-arrestin and GPCR co-receptors control different b-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, b-arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as a future therapeutics for the treatment of endothelial dysfunction.