c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

Abstract

Fu P, Usatyuk PV, Lele A, Harijith A, Gregorio CC, Garcia JG, Salgia R, Natarajan V. c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury. Am J Physiol Lung Cell Mol Physiol 308: L1025-L1038, 2015. First published March 20, 2015 doi:10.1152/ajplung.00306.2014Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.paxillin; c-Abl; lipopolysaccharide; tyrosine kinase; permeability; endothelial dysfunction; cytoskeleton GRAM-NEGATIVE BACTERIA-INDUCED sepsis affects more than 750,000 patients annually in the United States (23). Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), is characterized by endothelial hyperpermeability leading to lung inflammation and injury. Lipopolysaccharide (LPS), the bacterial endotoxin and an outer membrane component, induces proinflammatory cytokines, adhesion molecules, endothelial permeability changes, and apoptosis regulated by TLR-4 signaling via activation of Rho GTPase-dependent signaling cascade, NF-B activation, and rearrangement of cytoskeletal proteins (15, 31). Despite recent advances in understanding the pathogenesis of ALI/ARDS, effective pharmacological therapies are not currently available and the molecular mechanisms regulating endothelial barrier dysfunction in ALI/ARDS are not completely defined.Interactions between focal adhesions, extracellular matrix, and cytoskeletal proteins have been implicated in endothelial cell (EC) remodeling and barrier dysfunction associated with LPS-and ventilator-induced lung injury (15,31,48). Specific posttranslational modifications such as phosphorylation of focal adhesion proteins are also known to play a role in endothelial barrier regulation to agonists...

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Inhibition of connexin 43 attenuates oxidative stress and apoptosis in human umbilical vein endothelial cells

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