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.
Reorganization of the endothelial cytoskeleton provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases including ventilator induced lung injury, sepsis, and ARDS. Using multidisciplinary biochemical, cell and molecular approaches as well as animal model of ALI, this application will examine a role of microtubule-associated regulators of Rac signaling in the mediation of protective effects of hepatocyte growth factor on pulmonary endothelial barrier. This translational research proposal will provide mechanistic insights into regulation of EC permeability via novel dynamic interactions between microtubule-associated regulators of Rac GTPase signaling, adherens junction protein complexes, and actin cytoskeleton. This study will link for the first time in vitro analysis of molecular and cellular mechanisms of lung endothelial barrier regulation by microtubule-associated signaling and in vivo models of VILI. We believe that the results of these studies will identify new protein targets involved in microtubule- dependent control of lung permeability, which may be used for drug design and new therapies for prevention of pulmonary vascular barrier dysfunction associated with acute lung inflammation and injury.
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