title: Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin.
abstract: Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Singleton
authors.forename: Steven M
authors.initials: SM
authors.lastname: Dudek
authors.forename: Eddie T
authors.initials: ET
authors.lastname: Chiang
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D000185:Actinin / Q000378:metabolism / Q000502:physiology*; D020134:Catalytic Domain; D051242:Caveolin 1 / Q000737:chemistry; D002478:Cells, Cultured; D002784:Cholesterol / Q000737:chemistry; D002867:Chromones / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007249:Inflammation; D008840:Microfilament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism / Q000502:physiology*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011651:Pulmonary Artery / Q000473:pathology; D034741:RNA, Small Interfering / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000502:physiology*; D015398:Signal Transduction; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000502:physiology*
chemical_list: C111453:ACTN4 protein, human; D051242:Caveolin 1; D002867:Chromones; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D008840:Microfilament Proteins; D009025:Morpholines; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; D000185:Actinin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D002784:Cholesterol; D019869:Phosphatidylinositol 3-Kinases; D020830:rac1 GTP-Binding Protein