G Protein Regulation of Endothelial Barrier Function

G 蛋白对内皮屏障功能的调节

• 批准号: 6967981
• 负责人: TATYANA A VOYNO-YASENETSKAYA
• 金额: $ 37.02万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967981
• 关键词: G protein; actin binding protein; adult respiratory distress syndrome; biological signal transduction; clinical research; confocal scanning microscopy; cyclic AMP; enzyme activity; genetically modified animals; guanine nucleotide binding protein; immunoprecipitation; laboratory mouse; mitogen activated protein kinase; nuclear factor kappa beta; phosphorylation; respiratory epithelium; vascular endothelium; vascular endothelium permeability; western blottings

Loss of lung vascular endothelial barrier integrity leads to increased vascular permeability and alveolar flooding, and contributes to the morbidity and mortality associated with ARDS. Thrombin activates G protein-coupled receptor PAR-1 in endothelial cells and induces actin stress fiber formation and the resultant increase in transendothelial permeability. The activation of the alpha subunit of the heterotrimeric G13 protein by thrombin in endothelial cells is critical for the activation of Rho proteins, and the subsequent increased endothelial permeability response. We have shown that Galpha-13 protein interacts with the actin-binding protein, radixin, which is critical for the assembly of focal adhesions and actin filaments. Thus, radixin may be an essential effector in signaling the Galpha-13-induced Rho activation via the Rho guanine nucleotide dissociation inhibitor, RhoGDI. In Project 3, we will address a new and potentially important signaling pathway by which Galpha-13-dependent Rho activation results in increased lung endothelial permeabihty. We will define the upstream regulation of Rho involving radixin and the signaling pathways mediating the increase in endothelial permeability. In response to inflammatory mediators such thrombin, the actin cytoskeleton alterations and the increased endothelial permeability are typically reversible within hours. The reversibility ot the response restores endothelial barrier integrity; however, its molecular and cellular bases are poorly understood. Protein kinase A, PKA, a kinase linked to enhancing endothelial barrier function is usually activated by cyclic AMP. We have discovered that both thrombin and Galpha-13 can stimulate PKA via two novel mechanisms that do not require cAMP. One mechanism is dependent on the interaction of Galpha-13 with radixin, which activates the reversal of the permeability response. Another mechanism is dependent on the stimulation of the NF-kappaB signaling pathway via mitogen-activated protein kinases. The end result is that PKA may phosphorylate Galpha-13, and thereby inhibit Rho activation. We will address the concept that this is a key mechanism for the down-regulation of Galpha-13 activity and the reversal of the permeability response. Another possibility to be addressed is that Galpha-13 induces the phosphorylation of vasodilator-stimulated phosphoprotein, VASP, which can prevent actin polymerization and thus re-establish the endothelial barrier. Project 3 will test the general hypothesis that signaling complexes formed by PAR-1 activation of Galpha-13 signal the loss of endothelial barrier function and sequentially activate signals that lead to endothelial barrier recovery, and the reversal of the permeability-increase. We believe that the proposed studies will generate novel information elucidating the regulation of lung vascular permeability. Moreover, with the unraveling of the signals regulating the reversal of the increase in lung vascular permeability, it will be possible to identify novel targets tor therapies whereby the inappropriate increase in endothelial permeability can be controlled reducing the vascular "leak" associated with ARDS.

肺血管内皮屏障完整性的丧失导致血管通透性增加和肺泡灌流，并导致与ARDS相关的发病率和死亡率。凝血酶激活内皮细胞中的G蛋白偶联受体PAR-1，并诱导肌动蛋白应力纤维形成和由此产生的跨内皮渗透性增加。内皮细胞中凝血酶对异源三聚体G13蛋白α亚基的激活对于内皮细胞的生长至关重要。 Rho蛋白的激活，以及随后增加的内皮渗透性反应。我们已经表明，Galpha-13蛋白与肌动蛋白结合蛋白，radixin，这是关键的黏着斑和肌动蛋白丝的组装相互作用。因此，Radixin可能是通过Rho鸟嘌呤信号传导Ga-13诱导的Rho激活的重要效应物。 核苷酸解离抑制剂。在项目3中，我们将讨论一个新的和潜在的重要信号通路，通过该通路，Ga-13依赖性Rho激活导致肺内皮通透性增加。我们将定义Rho涉及radixin的上游调控和介导内皮通透性增加的信号通路。在对凝血酶等炎症介质的反应中，肌动蛋白细胞骨架改变， 内皮渗透性通常在数小时内可逆。反应的可逆性恢复了内皮屏障的完整性;然而，其分子和细胞基础知之甚少。蛋白激酶A（PKA）是一种与增强内皮屏障功能相关的激酶，通常被环AMP激活。我们已经发现凝血酶和Galalpha-13都可以通过两种不需要cAMP的新机制刺激PKA。一种机制是 依赖于Galpha-13与radixin的相互作用，radixin激活渗透性反应的逆转。另一种机制依赖于通过促分裂原活化蛋白激酶刺激NF-κ B信号通路。最终结果是PKA可以磷酸化Ga-13，从而抑制Rho激活。我们将讨论的概念， 这是下调Ga-13活性和逆转渗透性反应的关键机制。另一种可能性是Galalpha-13诱导血管舒张刺激的磷蛋白VASP的磷酸化，这可以阻止肌动蛋白聚合，从而重建内皮屏障。项目3将检验以下一般假设：由PAR-1激活Ga-13形成的信号复合物发出内皮屏障功能丧失的信号，并依次激活导致内皮屏障恢复的信号，以及渗透性增加的逆转。我们相信，拟议的研究将产生新的信息阐明肺血管通透性的调节。此外，随着调节肺血管通透性增加逆转的信号的解开，将有可能鉴定用于治疗的新靶点，由此可以控制内皮通透性的不适当增加，减少与ARDS相关的血管“渗漏”。