title: Paxillin-beta-catenin interactions are involved in Rac/Cdc42-mediated endothelial barrier-protective response to oxidized phospholipids.
abstract: Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and coimmunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1, and GIT2, increased association of AJ proteins vascular endothelial-cadherin, p120-catenin, alpha-, beta-, and gamma-catenins, and dramatically enhanced cell junction areas covered by AJ. Coimmunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and beta-catenin association, which was critically dependent on Rac and Cdc42 activities and was abolished by pharmacological or small interfering RNA (siRNA)-mediated inhibition of Rac and Cdc42. Depletion of beta-catenin using the siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated AJ enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.
authors.affiliation: Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Birukova
authors.forename: Irina
authors.initials: I
authors.lastname: Malyukova
authors.forename: Valery
authors.initials: V
authors.lastname: Poroyko
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Birukov
mesh_terms: D022005:Adherens Junctions / Q000187:drug effects; D015824:Blood-Air Barrier / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D051419:Paxillin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D051176:beta Catenin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D051419:Paxillin; D010713:Phosphatidylcholines; D051176:beta Catenin; C472349:oxidized-L-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins