Introduction Oxidized phospholipids appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously shown that oxidized phospholipids exhibit barrier-protective effects on pulmonary endothelial cell (EC) monolayers. However, effects of oxidized phospholipids on EC focal adhesion (FA) and adherence junction (AJ) remodeling have not been yet fully explored.
Goal To study molecular mechanisms of adherens junction and focal adhesion remodeling mediated by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC).
Methods All experiments were performed on human pulmonary artery endothelial cells (HPAEC). Intracellular protein localization was analyzed by immunocytochemistry. Subcellular localization of the proteins of interest was determined using subcellular proteome extraction kit. Protein phosphorylation profile was assessed by Western blot analysis. Protein-protein interactions were analyzed by coimmunoprecipitation assays.
Results Enhancement of EC barrier in response to OxPAPC was accompanied by dynamic remodeling of focal adhesions and adherence junctions. Immunofluorescent analysis of OxPAPC-stimulated EC revealed dramatic translocation and peripheral and enhanced peripheral staining for AJ proteins beta-catenin and VE-cadherin. In addition, OxPAPC treatment increased tyrosine phosphorylation of FA proteins FAK at Tyr-576 and paxillin at Tyr-118, which was associated with peripheral redistribution of FA and AJ complexes. Furthermore, subcellular fractionation analysis showed increase of VE-cadherin, beta-catenin, and GIT2 in membrane fraction after OxPAPC challenge. Remarkably, coimmunoprecipitation studies indicated increased interaction of paxillin with FA components FAK, vinculin and GIT2, and AJ protein beta-catenin upon HPAEC stimulation with OxPAPC. Complementary experiments with immunoprecipitation beta-catenin followed by probing for paxillin confirmed these results.
Conclusions The results of these studies characterize OxPAPC-induced focal adhesion remodeling and determine for the first time the specific interactions between focal adhesion and adherens junction protein complexes in endothelial barrier-protective responses to OxPAPC
Grant support: HL076259, HL075349 for K.G.B., AHA-SDG for A.A.B.
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