G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase.
OBJECTIVE
The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs.


RESULTS
S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway.


CONCLUSIONS
These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.
Takashima
Shin-Ichiro
S
Department of Physiology, Graduate School of Medical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan.
Sugimoto
Naotoshi
N
Takuwa
Noriko
N
Okamoto
Yasuo
Y
Yoshioka
Kazuaki
K
Takamura
Masayuki
M
Takata
Shigeo
S
Kaneko
Shuichi
S
Takuwa
Yoh
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D036002:ADP Ribose Transferases / Q000494:pharmacology; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C471998:JTE 013; D008246:Lysophospholipids; D047428:Protein Kinase Inhibitors; D020574:Proto-Oncogene Proteins c-sis; D011720:Pyrazoles; D011725:Pyridines; D044140:Receptor, Angiotensin, Type 1; D049349:Receptors, Lysosphingolipid; D011993:Recombinant Fusion Proteins; C060506:sphingosine 1-phosphate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D001905:Botulinum Toxins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D013110:Sphingosine; C049347:fasudil; D002118:Calcium