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会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。在内皮细胞中，凝血酶激活G蛋白偶联受体，诱导肌动蛋白应激纤维，随后形成细胞间隙，从而增加对蛋白质和液体的跨内皮通透性。凝血酶激活内皮细胞异源三聚体G13蛋白的α亚基，对于Rho蛋白的激活和肌动蛋白应激纤维的组装，以及随后的细胞收缩和内皮通透性反应的增加至关重要。我们的支持数据表明，G-α13蛋白与肌动蛋白结合蛋白Radixin相互作用，参与局部粘连和肌动蛋白细丝的组装。在通过Rho鸟嘌呤核苷酸解离抑制剂RhoGDI信号转导G-alpha13诱导的Rho激活过程中，Radixin可能是一个关键的效应者。我们将解决一个新的和潜在的重要信号通路的G-α13依赖的Rho激活和由此导致的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(约2小时)和可逆的。凝血酶诱导作用的可逆性是恢复内皮屏障完整性的关键；然而，其分子和细胞基础尚不清楚。蛋白激酶A，PKA，是一种与增强内皮屏障完整性相关的激酶，通常由环磷酸腺苷(CAMP)激活。我们发现凝血酶和G-α13都可以通过两种不需要cAMP的新机制来刺激PKA。一种机制依赖于G-α13与Radioxin的相互作用。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。依赖于PKA的G-α13的磷酸化可能因此抑制Rho的激活，从而提供了一种下调G-α13活性和逆转通透性反应的机制。此外，G-α13可诱导血管扩张剂刺激的磷酸蛋白VASP的磷酸化，从而阻止肌动蛋白聚合，从而促进内皮细胞连接屏障的重建。因此，我们将检验这样的假设，即由PAR-1激活G-Alpha13形成的信号复合体为配体依赖性的内皮屏障功能丧失及其恢复创造了分子基础。这项建议将解决分子和细胞机制，使G-阿尔法13形成特定的信号复合体，从而产生信号，既造成内皮屏障功能的丧失和随后的恢复。我们相信，拟议的研究将产生重要的和新的信息，阐明肺内皮细胞屏障的调节机制，并将确定新的治疗靶点，从而控制内皮通透性的不适当增加，以减少与ARDS相关的血管“泄漏”。