Abstract
We have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.
Highlights
Abbreviations adherens junction (AJ) Adherens junction ARDS Acute respiratory distress syndrome BCA Bicinchoninic acid DTT Dithiothreitol electron transport chain (ETC) Electron transport chain FAK Focal adhesion kinase HGF Hepatocyte growth factor HRP Horseradish peroxidase IgG Immunoglobulin gamma IP Immunoprecipitation LD Leucine-rich domain
LPS‐stimulated Mitochondrial ROS (mtROS) production in human lung endothelial cells is attenuated by paxillin Y31F and Y118F mutants
reactive oxygen species (ROS) generated in cells by NADPH Oxidase (NOX) proteins and mitochondrial electron transport contributes to antimicrobial immunity[25,26,27] and to intracellular signaling p athways[28,29]
Summary
Abbreviations AJ Adherens junction ARDS Acute respiratory distress syndrome BCA Bicinchoninic acid DTT Dithiothreitol ETC Electron transport chain FAK Focal adhesion kinase HGF Hepatocyte growth factor HRP Horseradish peroxidase IgG Immunoglobulin gamma IP Immunoprecipitation LD Leucine-rich domain. Phosphorylation of tyrosine residues at the proline-rich SH3 motifs creates new binding sites for SH2-domain containing proteins, while serine/threonine phosphorylation in LIM domains potentiates the localization of paxillin to a dhesions[9]. Three major paxillin N-terminal tyrosine phosphorylation sites, Y31 and Y118, and Y181 have been identified with Y31 and Y118 phosphorylation modulating docking of SH2 domain-containing proteins such as Crk, an adaptor molecule important for regulation of cell motility[8,10]. C-Abl mediated tyrosine phosphorylation of Y31 and Y118 regulates LPS-induced pulmonary vascular permeability and injury[13], and HGF- and S1P-induced reactive oxygen species (ROS) generation, lamellipodia formation and endothelial p ermeability[14]. Our previous study showed that c-Abl mediated paxillin tyrosine phosphorylation at Y31 and Y118 regulates LPS-induced endothelial dysfunction and lung injury[13]; the underlying molecular mechanism(s) by which paxillin regulates mitochondria (mt)-derived ROS dependent endothelial dysfunction is unknown
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