G Proteins and Endothelial Barrier Regulation

G 蛋白和内皮屏障调节

• 批准号: 6820479
• 负责人: TATYANA A VOYNO-YASENETSKAYA
• 金额: $ 25.83万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820479
• 关键词: G protein; actin binding protein; actins; biological signal transduction; cell surface receptors; cyclic AMP; cytoskeleton; genetically modified animals; guanine nucleotide binding protein; guanine nucleotides; high performance liquid chromatography; laboratory mouse; phospholipase C; phosphorylation; polymerase chain reaction; thrombin; vascular endothelium permeability; western blottings

DESCRIPTION (provided by applicant): Loss of endothelial barrier integrity leads to increased vascular permeability and alveolar flooding, and contributes to morbidity and mortality associated with ARDS. In endothelial cells, thrombin activates G protein-coupled receptors and induces actin stress fibers followed by intercellular gap formation, and resultant increased trans-endothelial permeability to protein and liquid. The activation of the alpha subunit of heterotrimeric G13 protein by thrombin in endothelial cells is critical for the activation of Rho proteins and assembly of actin stress fibers and the subsequent cell retraction and increased endothelial permeability responses. Our Supporting Data suggests that G-alpha13 protein interacts with the actin-binding protein, radixin, implicated in the assembly of focal adhesions and actin filaments. Radixin may be a critical effector in signaling the G-alpha 13-induced Rho activation via the Rho guanine nucleotide dissociation inhibitor, RhoGDI. We will address a new and potentially important signaling pathway for the G-alpha13-dependent Rho activation and the resultant increased lung endothelial permeability. We will define the upstream regulation of Rho involving radixin and the signaling pathways mediating the increase in endothelial permeability. Under physiological conditions, the thrombin-induced cytoskeleton alterations and increased endothelial permeability are short-lived (approximately 2 hr) and reversible. The reversibility of thrombin-induced action is critical in restoring endothelial barrier integrity; however, its molecular and cellular bases are poorly understood. Protein kinase A, PKA, a kinase linked to enhancing integrity of the endothelial barrier is usually activated by cyclic AMP. We have discovered that both thrombin and G-alpha 13 can stimulate PKA via two novel mechanisms that do not require cAMP. One mechanism is dependent on the interaction of G-alpha 13 with radixin. Another mechanism is dependent on stimulation of the NF-kappaB signaling pathway via mitogen-activated protein kinases. The PKA-dependent phosphorylation of G-alpha13 may thereby inhibit Rho activation, thus providing a mechanism for down regulation of G-alpha13 activity and reversal of the permeability response. Also, G-alpha 13 may induce phosphorylation of vasodilator-stimulated phosphoprotein, VASP, which may prevent actin polymerization and thus promote the re-establishment of endothelial junctional barrier. Thus, we will test the hypothesis that signaling complexes formed by PAR-1 activation of G-alpha 13 create the molecular basis for ligand-dependent loss of endothelial barrier function as well as its restoration. This proposal will address the molecular and cellular mechanisms that enable G-alpha 13 to form specific signaling complexes, thereby generating signals that both create either loss and the subsequent restoration of endothelial barrier function. We believe that the proposed studies will generate important and novel information elucidating lung endothelial barrier regulatory mechanisms and will identify novel targets for therapies whereby the inappropriate increase in endothelial permeability can be controlled to reduce the vascular "leak" associated with ARDS.

描述（由申请人提供）：内皮屏障完整性的丧失导致血管通透性增加和肺泡充盈，并导致与 ARDS 相关的发病率和死亡率。在内皮细胞中，凝血酶激活 G 蛋白偶联受体并诱导肌动蛋白应力纤维，随后形成细胞间间隙，从而增加跨内皮细胞对蛋白质和液体的通透性。内皮细胞中凝血酶激活异三聚体 G13 蛋白的 α 亚基对于 Rho 蛋白的激活和肌动蛋白应力纤维的组装以及随后的细胞回缩和增加的内皮通透性反应至关重要。我们的支持数据表明，G-alpha13 蛋白与肌动蛋白结合蛋白 radixin 相互作用，参与粘着斑和肌动蛋白丝的组装。 Radixin 可能是通过 Rho 鸟嘌呤核苷酸解离抑制剂 RhoGDI 发出 G-α 13 诱导的 Rho 激活信号的关键效应器。我们将解决 G-α13 依赖性 Rho 激活和由此导致的肺内皮通透性增加的新的、潜在重要的信号通路。我们将定义涉及根素的 Rho 上游调节以及介导内皮通透性增加的信号通路。在生理条件下，凝血酶诱导的细胞骨架改变和内皮通透性增加是短暂的（大约2小时）并且是可逆的。凝血酶诱导作用的可逆性对于恢复内皮屏障完整性至关重要；然而，人们对它的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完整性相关的激酶，通常由环 AMP 激活。我们发现凝血酶和 G-α 13 都可以通过两种不需要 cAMP 的新机制刺激 PKA。一种机制依赖于 G-α 13 与根素的相互作用。另一种机制依赖于通过丝裂原激活蛋白激酶刺激 NF-kappaB 信号通路。 G-α13 的 PKA 依赖性磷酸化可能会抑制 Rho 激活，从而提供下调 G-α13 活性和逆转通透性反应的机制。此外，G-α 13 可能诱导血管舒张刺激的磷蛋白 VASP 磷酸化，这可能阻止肌动蛋白聚合，从而促进内皮连接屏障的重建。因此，我们将检验以下假设：由 G-α 13 的 PAR-1 激活形成的信号复合物为配体依赖性内皮屏障功能丧失及其恢复奠定了分子基础。该提案将解决使 G-alpha 13 形成特定信号复合物的分子和细胞机制，从而产生既造成内皮屏障功能丧失又随后恢复的信号。我们相信，所提出的研究将产生重要且新颖的信息，阐明肺内皮屏障调节机制，并将确定新的治疗靶标，从而控制内皮通透性的不当增加，以减少与 ARDS 相关的血管“渗漏”。