title: Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids.
abstract: Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P(1)), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.
authors.affiliation: Department of Medicine, Division of Biomedical Sciences, Section of Pulmonary and Critical Medicine, University of Chicago, Ill 60637, USA. psinglet@medicine.bsd.uchicago.edu
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Singleton
authors.forename: Santipongse
authors.initials: S
authors.lastname: Chatchavalvanich
authors.forename: Panfeng
authors.initials: P
authors.lastname: Fu
authors.forename: Junjie
authors.initials: J
authors.lastname: Xing
authors.forename: Anna A
authors.initials: AA
authors.lastname: Birukova
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Fortune
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Klibanov
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Birukov
mesh_terms: D000818:Animals; D002199:Capillary Permeability; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000098:blood supply*; D008297:Male; D021962:Membrane Microdomains / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000081025:Sphingosine-1-Phosphate Receptors; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D013997:Time Factors; D055397:Ventilator-Induced Lung Injury / Q000378:metabolism / Q000517:prevention & control; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D020662:Guanine Nucleotide Exchange Factors; D010713:Phosphatidylcholines; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; C477362:S1PR1 protein, human; D000081025:Sphingosine-1-Phosphate Receptors; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; C472349:oxidized-L-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D020830:rac1 GTP-Binding Protein