Microtubule-associated Rac

描述(申请人提供)：微管相关的RAC调节和急性肺损伤。肺内皮细胞(EC)通透性受损导致肺水肿，这是一种严重的并发症，见于各种肺部疾病，并与高死亡率相关。小分子GTP酶Rac和Rho在EC细胞骨架重塑和细胞连接动力学中扮演着相反的角色，这是内皮屏障调节的关键机制。RAC依赖的外周肌动蛋白、细胞连接复合体和EC屏障保护电位的增强可能是RAC对不同亚细胞亚区小GTP酶活性的精确调控的关键机制。已知，微管(MT)分解增加会导致EC屏障功能障碍。我们已发表的研究表明，促水肿和促炎激动剂凝血酶、TGF2和TNF1通过Rho依赖的MT动力学改变增加EC的通透性，导致MT依赖的肌球蛋白收缩。相反，屏障保护剂对MT之间的串扰、细胞黏附和肌动蛋白细胞骨架的影响尚不清楚，也没有描述MT变化在RAC调节中的作用。我们的新数据表明，MT介导的信号直接参与了肝细胞生长因子(HGF)对EC屏障的保护作用，HGF是一种循环分子，在急性Ling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，即微管可能通过MT相关的信号复合体介导HGF的屏障保护作用，并减轻急性肺内皮细胞功能障碍。我们假设MT相关的接头蛋白APC(腺瘤性息肉病)可以结合并将一种新的RAC特异性的鸟氨酸核苷酸交换因子ASEF沿着MT运送到细胞外围，在那里ASEF将其细胞骨架定位从MT切换到肌动蛋白，并靶向肌动蛋白相关的RAC效应器IGQAP1。这一机制可能导致一种新的范式，即通过靶向传递RAC激活剂ASEF来增强微管依赖的外周肌动蛋白细胞骨架和细胞-细胞连接，从而促进ALI的EC屏障特性和肺修复机制。具体目标#1将研究APC和ASEF在RAC GTP酶激活和HGF诱导的肺内皮细胞屏障增强中的作用。具体目标#2将研究MT在APC/ASEF激活和细胞内定位中的作用。具体目标#3将研究HGF诱导的APC/ASEF与IQGAP1的相互作用，并研究APC/ASEF/IQGAP1复合体在HGF诱导的EC屏障增强中的作用。具体目的#4将使用体内基于siRNA的ASEF基因敲除、ASEF基因敲除小鼠和救援策略来评估ASEF在ALI肺保护机制中的作用。这些研究将表征新的屏障保护机制，并为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确定新的蛋白质靶点。

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.