Microtubule-associated Rac

微管相关Rac

• 批准号: 8669123
• 负责人: Anna Birukova
• 金额: $ 37.84万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669123
• 关键词: Actins; Actomyosin; Acute; Acute Lung Injury; Adaptor Signaling Protein; Adenomatous Polyposis Coli; Adherens Junction; Adult Respiratory Distress Syndrome; Agonist; Animal Model; Attenuated; Automobile Driving; Binding; Biochemical; Biological Preservation; Blood Vessels; Cell Adhesion; Cells; Complex; Complication; Cytoskeleton; Data; Development; Disease; Down-Regulation; Drug Design; Elements; Endothelial Cells; Endothelium; Equilibrium; Functional disorder; Future; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hepatocyte Growth Factor; Human; In Vitro; Inflammatory; Injury; Intercellular Junctions; Kinesin; Knockout Mice; Lead; Link; Lung; Lung Inflammation; Lung diseases; Mechanics; Mediating; Mediation; Microtubule Alteration; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Molecular Analysis; Molecular Target; Monomeric GTP-Binding Proteins; Motor; Pathogenesis; Peripheral; Permeability; Pilot Projects; Play; Prevention; Prevention therapy; Property; Protective Agents; Proteins; Publishing; Pulmonary Edema; Regulation; Research Proposals; Role; Sepsis; Signal Pathway; Signal Transduction; Small Interfering RNA; Thrombin; Time; Transducers; Translational Research; Vascular Diseases; Vascular Permeabilities; Ventilator-induced lung injury; base; effective therapy; in vivo; in vivo Model; injured; insight; lung injury; lung preservation; lung repair; monolayer; mortality; multidisciplinary; novel; protective effect; protein complex; restoration; rho; rho GTP-Binding Proteins; targeted delivery

DESCRIPTION (provided by applicant): Microtubule-associated Rac regulation and acute lung injury. Compromised lung endothelial cell (EC) permeability leads to pulmonary edema, a serious complication observed in various lung diseases and associated with high mortality. Small GTPases Rac and Rho play opposing roles in EC cytoskeletal remodeling and cell junction dynamics, the critical mechanisms of endothelial barrier regulation. Precise regulation of small GTPase activities in different subcellular compartments may be a key mechanism of Rac-dependent enhancement of peripheral actin, cell junction complexes and EC barrier protective potential. It is known that increased microtubule (MT) disassembly leads to EC barrier dysfunction. Our published studies show that edemagenic and pro-inflammatory agonists thrombin, TGF2 and TNF1 increase EC permeability via Rho-dependent alteration of MT dynamics leading to MT- dependent actomyosin contraction. In contrast, effects of barrier-protective agents on crosstalk between MT, cell adhesions, and actin cytoskeleton are not clear, nor the role of MT changes in Rac regulation is described. Our novel data show that MT-mediated signaling is directly involved in the EC barrier preservation by hepatocyte growth factor (HGF), a circulating molecule with proven protective effects in the models of acute ling injury (ALI). These results have led us to a hypothesis that microtubules may mediate barrier-protective effects of HGF and attenuate acute pulmonary EC dysfunction via MT-associated signaling complexes. We hypothesize that MT-associated adaptor protein APC (adenomatous polyposis coli) can bind and deliver a novel Rac-specific guanine nucleotide exchange factor Asef along MT to the cell periphery, where Asef switches its cytoskeletal localization from MT to actin and targets actin-associated Rac effector IGQAP1. This mechanism may lead to a novel paradigm of microtubule- dependent enhancement of peripheral actin cytoskeleton and cell-cell junctions via targeted delivery of Rac activator Asef, which promote EC barrier properties and lung repair mechanisms in ALI. Specific Aim #1 will examine the role of APC and Asef in the Rac GTPase activation and HGF-induced barrier enhancement in pulmonary endothelial cells. Specific Aim #2 will study the role of MT in APC/Asef activation and intracellular localization. Specific Aim #3 will examine HGF-induced APC/Asef interactions with IQGAP1 and study the role of APC/Asef/IQGAP1 complex in HGF-induced EC barrier enhancement. Specific Aim #4 will use siRNA-based Asef knockdown in vivo, Asef knockout mice, and rescue strategies to evaluate Asef role in the lung protective mechanisms against ALI. These studies will characterize novel barrier protective mechanisms and identify new protein targets for future therapies aimed at prevention of the pulmonary vascular barrier dysfunction associated with acute lung injury.

描述（由申请方提供）：微管相关Rac调节和急性肺损伤。受损的肺内皮细胞（EC）渗透性导致肺水肿，这是在各种肺部疾病中观察到的严重并发症，并且与高死亡率相关。小GTP酶Rac和Rho在EC细胞骨架重塑和细胞连接动力学中发挥相反的作用，这是内皮屏障调节的关键机制。在不同的亚细胞区室中精确调节小的GT3活性可能是Rac依赖性增强外周肌动蛋白、细胞连接复合物和EC屏障保护电位的关键机制。已知增加的微管（MT）分解导致EC屏障功能障碍。我们发表的研究表明，水肿和促炎激动剂凝血酶，TGF 2和TNF 1增加EC渗透性通过Rho依赖性改变MT动力学，导致MT依赖性肌动球蛋白收缩。相反，屏障保护剂对MT、细胞粘附和肌动蛋白细胞骨架之间的串扰的影响尚不清楚，MT在Rac调节中的变化也没有描述。我们的新数据表明，MT介导的信号转导直接参与了肝细胞生长因子（HGF）对EC屏障的保护，HGF是一种循环分子，在急性肝损伤（ALI）模型中具有保护作用。这些结果使我们提出一个假设，即微管可能介导的屏障保护作用的HGF和减轻急性肺EC功能障碍，通过MT相关的信号复合物。我们假设，MT相关的衔接蛋白APC（腺瘤性息肉病大肠杆菌）可以结合并提供一种新的Rac特异性鸟嘌呤核苷酸交换因子Asef沿着MT到细胞周边，在那里Asef开关其细胞骨架定位从MT肌动蛋白和肌动蛋白相关的Rac效应IGQAP 1。这种机制可能导致通过靶向递送Rac激活剂Asef的微管依赖性增强外周肌动蛋白细胞骨架和细胞-细胞连接的新范例，其促进ALI中的EC屏障特性和肺修复机制。具体目标#1将检查APC和Asef在肺内皮细胞中的Rac GT3活化和HGF诱导的屏障增强中的作用。具体目标#2将研究MT在APC/Asef活化和细胞内定位中的作用。具体目标#3将检查HGF诱导的APC/Asef与IQGAP 1的相互作用，并研究APC/Asef/IQGAP 1复合物在HGF诱导的EC屏障增强中的作用。具体目标#4将使用基于siRNA的体内Asef敲除、Asef敲除小鼠和拯救策略来评估Asef在肺保护机制中对ALI的作用。这些研究将表征新的屏障保护机制，并确定新的蛋白质靶点，用于未来旨在预防与急性肺损伤相关的肺血管屏障功能障碍的治疗。