title: Functional characterization of sphingosine 1-phosphate receptor agonist in human endothelial cells.
abstract: Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N,N-dimethylsphingosine (DMS), FTY720's effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a Gi-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.
authors.affiliation: Novartis Institute for Biomedical Research, Inc., One Health Plaza, Room 2223, Bldg 436, East Hanover, NJ 07936, USA.
authors.forename: Jeannene
authors.initials: J
authors.lastname: Butler
authors.forename: Diana
authors.initials: D
authors.lastname: Lana
authors.forename: Oliver
authors.initials: O
authors.lastname: Round
authors.forename: Kenneth
authors.initials: K
authors.lastname: LaMontagne
mesh_terms: D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D000068876:Fingolimod Hydrochloride; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism* / Q000494:pharmacology; D007536:Isomerism; D008246:Lysophospholipids / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015394:Molecular Structure; D038081:Organogenesis / Q000187:drug effects; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism* / Q000494:pharmacology; D049349:Receptors, Lysosphingolipid / Q000819:agonists* / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D000099157:Sphingosine Kinase
chemical_list: D000068876:Fingolimod Hydrochloride; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D007166:Immunosuppressive Agents; D008246:Lysophospholipids; D020928:Mitogen-Activated Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011409:Propylene Glycols; D049349:Receptors, Lysosphingolipid; D013110:Sphingosine; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate