title: FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction.
abstract: FTY720 phosphate (FTY720-P; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, monodihydrogen phosphate ester) is a nonselective sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P2 receptor subtype. However, we have recently shown that FTY720-P displays significant S1P2 receptor agonist activity in recombinant cells and fibroblasts expressing endogenous S1P2 receptors. To elucidate the S1P2-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and small interfering RNA-based pathway inhibition in recombinant Chinese hamster ovary (CHO)-S1P2 cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P2 cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα12/13 knockdown and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα12/13/Rho/ROCK signaling. S1P similarly activated Gα12/13/Rho/ROCK signaling via S1P2 receptors, whereas the two selective S1P1 receptor agonists (Z,Z)-5-(3-chloro-4-[(2R)-2,3-dihydroxy-propoxy]-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (ponesimond) and 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871) were inactive. In lung myofibroblasts, which mainly expressed the S1P2 receptor subtype, we showed that FTY720-P selectively activated the Gα12/13/Rho/ROCK pathway via the S1P2 receptor. Moreover, the activation of the Gα12/13/Rho/ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or phenotypic assays in native expression systems can uncover activation of additional pathways, such as Gα12/13/Rho/ROCK signaling.
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Katrin
authors.initials: K
authors.lastname: Sobel
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Lucile
authors.initials: L
authors.lastname: Monnier
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Katalin
authors.initials: K
authors.lastname: Menyhart
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bolinger
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Rolf
authors.initials: R
authors.lastname: Studer
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Nayler
authors.affiliation: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, Switzerland john.gatfield@actelion.com.
authors.forename: John
authors.initials: J
authors.lastname: Gatfield
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D017097:Electric Impedance; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008168:Lung / Q000166:cytology; D058628:Myofibroblasts / Q000187:drug effects* / Q000502:physiology; D010755:Organophosphates / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D049349:Receptors, Lysosphingolipid / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C525008:FTY 720P; D010755:Organophosphates; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D013110:Sphingosine; D002118:Calcium