title: Sphingosine 1-phosphate-induced mobilization of intracellular Ca2+ mediates rac activation and adherens junction assembly in endothelial cells.
abstract: Sphingosine 1-phosphate (S1P) ligation of endothelial differentiation gene-1 receptor coupled to the heterotrimeric G protein, Gi, promotes endothelial barrier strengthening via Rac-dependent assembly of adherens junctions (AJs). However, the mechanism of Rac activation induced by S1P stimulation remains unclear. In live endothelial cells expressing GFP-Rac, we observed that S1P induced the translocation of Rac to intercellular junctions, resulting in junctional sealing. We investigated the role of intracellular Ca2+ in signaling Rac activation and the enhancement of endothelial barrier function. We observed that S1P activated the release of Ca2+ from endoplasmic reticulum stores, and subsequent Ca2+ entry via lanthanum-sensitive store-operated Ca2+ channels (SOC) after store depletion. Inhibition of Gi, phospholipase C, or inositol trisphosphate receptor prevented the S1P-activated increase in intracellular Ca2+ as well as Rac activation, AJ assembly, and enhancement of endothelial barrier. Chelation of intracellular Ca2+ with BAPTA blocked S1P-induced Rac activation, indicating the requirement for Ca2+ in the response. Inhibition of SOC by lanthanum or transient receptor potential channel 1 (TRPC1), a SOC constituent, by TRPC1 antibody, failed to prevent S1P-induced Rac translocation to junctions and AJ assembly. Thus, our results demonstrate that S1P promotes endothelial junctional integrity by activating the release of endoplasmic reticulum-Ca2+, which induces Rac activation and promotes AJ annealing.
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA. dmehta@uic.edu
authors.forename: Dolly
authors.initials: D
authors.lastname: Mehta
authors.forename: Maria
authors.initials: M
authors.lastname: Konstantoulaki
authors.forename: Gias U
authors.initials: GU
authors.lastname: Ahmmed
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D017097:Electric Impedance; D004594:Electrophysiology; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D017629:Gap Junctions; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008246:Lysophospholipids / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D002614:Chelating Agents; D004396:Coloring Agents; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008246:Lysophospholipids; D018160:Receptors, Cytoplasmic and Nuclear; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D004533:Egtazic Acid; D010738:Type C Phospholipases; D020744:rac GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013110:Sphingosine; D002118:Calcium