G Protein Regulation of Endothelial Barrier Function

G 蛋白对内皮屏障功能的调节

• 批准号: 7312501
• 负责人: TATYANA A VOYNO-YASENETSKAYA
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312501
• 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.

肺血管内皮细胞屏障完整性的丧失导致血管通透性增加和肺泡泛滥，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，诱导肌动蛋白应激纤维形成，从而增加跨内皮细胞的通透性。凝血酶激活内皮细胞中异三聚体G13蛋白的α亚基对 Rho蛋白的激活，以及随后增加的内皮通透性反应。我们已经证明Galpha-13蛋白与肌动蛋白结合蛋白Radixin相互作用，Radixin对焦点粘连和肌动蛋白细丝的组装至关重要。因此，在Galpha-13通过Rho鸟氨酸诱导Rho激活的信号转导过程中，Radix可能是一个重要的效应器 核苷酸解离抑制剂RhoGDI。在项目3中，我们将解决一个新的和潜在的重要信号通路，通过它依赖Galpha-13的Rho激活导致肺内皮细胞通透性增加。我们将确定Rho的上游调控与Radioxin有关，以及介导内皮通透性增加的信号通路。在应对炎症介质如凝血酶时，肌动蛋白细胞骨架的改变和增加 内皮通透性通常在数小时内可逆。反应的可逆性可恢复内皮屏障的完整性；然而，其分子和细胞基础尚不清楚。蛋白激酶A(Protein Kinase A，PKA)是一种与增强内皮屏障功能相关的激酶，通常被环磷酸腺苷激活。我们发现凝血酶和Galpha-13都可以通过两种不需要cAMP的新机制来刺激PKA。一种机制是 依赖于Galpha-13与Radioxin的相互作用，这激活了渗透性反应的逆转。另一种机制依赖于通过丝裂原活化蛋白激酶刺激核因子-kappaB信号通路。最终结果是PKA可能使Galpha-13磷酸化，从而抑制Rho的激活。我们将解决这样一个概念 这是下调Galpha-13活性和逆转通透性反应的关键机制。另一种需要解决的可能性是Galpha-13诱导血管扩张剂刺激的磷酸蛋白Vasp的磷酸化，这可以防止肌动蛋白聚合，从而重建内皮屏障。项目3将检验这一普遍假设，即由PAR-1激活Galpha-13形成的信号复合体发出内皮屏障功能丧失的信号，并随后激活导致内皮屏障恢复的信号，从而逆转通透性增加。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的信号的解体，将有可能找到新的靶点和治疗方法，从而可以控制内皮通透性的不适当增加，减少与ARDS相关的血管“泄漏”。