title: Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways.
abstract: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, 60612, USA.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Usatyuk
authors.forename: Donghong
authors.initials: D
authors.lastname: He
authors.forename: Vytas
authors.initials: V
authors.lastname: Bindokas
authors.forename: Irina A
authors.initials: IA
authors.lastname: Gorshkova
authors.forename: Evgeny V
authors.initials: EV
authors.lastname: Berdyshev
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D051356:Cortactin / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008168:Lung / Q000098:blood supply / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D010755:Organophosphates / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010782:Photolysis; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D000099157:Sphingosine Kinase
chemical_list: D015820:Cadherins; D002118:Calcium; D051356:Cortactin; D020928:Mitogen-Activated Protein Kinases; D010755:Organophosphates; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; D013110:Sphingosine; D051176:beta Catenin; D020830:rac1 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins; D000099157:Sphingosine Kinase; C495868:CTTN protein, human; C101203:IQ motif containing GTPase activating protein 1; C116619:caged sphingosine 1-phosphate