Abstract 195: Oxidized Phospholipids Mediate Interactions between Adherens Junction and Focal Adhesion Proteins
Oxidized phospholipids appear in the pulmonary circulation during acute lung injury or inflammation. Previous studies have shown that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) enhances basal endothelial cell (EC) barrier properties. Although cellular adhesive structures play a critical role in EC barrier regulation, the effects of oxidized phospholipids on EC focal adhesion (FA) and adherens junction (AJ) remodeling have not been yet explored. Double immunofluorescent staining and subcellular fractionation showed pronounced OxPAPC-induced peripheral accumulation of FA proteins focal adhesion kinase (FAK), vinculin, paxillin, and GIT2, and enhanced peripheral staining for AJ proteins beta-catenin and VE-cadherin. Remarkably, OxPAPC enhanced paxillin association with FA proteins, as well as with beta-catenin, which was detected in series of cross-coimmunoprecipitation assays. The novel interaction between paxillin and beta-catenin was further confirmed by colocalization study using confocal microscopy. Protein depletion of paxillin and beta-catenin using specific siRNA attenuated OxPAPC-induced increases in transendothelial electrical resistance, thus reflecting a critical role for these FA and AJ proteins in the OxPAPC-mediated barrier protection. Importantly, OxPAPC-induced paxillin-beta-catenin interactions were regulated by small GTPases Rac and Cdc42. Depletion of Rac and Cdc42 using specific siRNA or their inhibition by toxin B dramatically reduced paxillin-beta-catenin association, as assessed by confocal microscopy analysis. These results characterize OxPAPC-induced remodeling of cellular contacts and show for the first time the specific interactions between focal adhesion and adherens junction protein complexes, which mediate barrier-protective responses to OxPAPC via Rac/Cdc42-dependent mechanism.