Sphingosine-1-phosphate-induced smooth muscle cell migration involves the mammalian target of rapamycin.
BACKGROUND
Vascular smooth muscle cell (SMC) migration is an important component of the development of intimal hyperplasia. Sphingosine-1-phosphate (S-1-P) is a lipid released from activated platelets with numerous cellular effects including the stimulation of SMC migration in vitro. We examined the role of the mammalian target of rapamycin and ribosomal p70S6 kinase (p70S6K) in S-1-P-induced SMC migration .


METHODS
Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P (0.01 to 100 micromol/L) with and without rapamycin (10 nmol/L). Western blotting was performed for phosphorylated and total p70S6K, ERK1/2, and p38(MAPK) after stimulation with S-1-P (0.1 micromol/L), with and without rapamycin pretreatment. Phosphorylation of p70S6K was also assayed after S-1-P treatment in the presence and absence of inhibitors of PI3 kinase (wortmannin, WN, and LY294002, LY), Akt (AktI), p38(MAPK) (SB203580), and MEK1 (PD98059).


RESULTS
S-1-P stimulated migration of SMCs in both linear wound and Boyden chamber assays compared to control (P < .05); these responses were inhibited by rapamycin to below the level of control (P < .05 vs S-1-P alone for both assays) in a dose-dependent manner (inhibitory concentration of 50%, 10 nmol/L). S-1-P stimulated phosphorylation of ERK1/2, p38(MAPK), and p70S6K, which peaked at 5 minutes for ERK1/2 and p38(MAPK) and10 minutes for p70S6K (2-fold increase over control for each, P < .05). Rapamycin prevented the phosphorylation of p70S6K at the Thr 389 site (which correlates with enzyme activity), reduced ERK1/2 phosphorylation, but had no effect on the Thr 421/Ser 424 site or on p38(MAPK) phosphorylation. Wortmannin and LY294002 inhibited phosphorylation of the Thr 389 site of p70S6K. AktI and SB203580 had no effect on p70S6K, whereas PD98059 had a marginal effect.


CONCLUSIONS
S-1-P-induced SMC migration was completely inhibited by rapamycin, indicating that the p70S6K pathway is involved. This mechanism likely involves modulation of the ERK1/2 pathway. S-1-P stimulates phosphorylation of p70S6K in a MEK1-dependent, PI3 kinase-dependent, but Akt-independent manner.
Tanski
William J
WJ
Division of Vascular Surgery, Vascular Biology and Therapeutics Program, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Nicholl
Suzanne M
SM
Kim
Dong
D
Fegley
Allison J
AJ
Roztocil
Elisa
E
Davies
Mark G
MG
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D008246:Lysophospholipids / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism / Q000494:pharmacology; D020123:Sirolimus / Q000494:pharmacology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases / Q000494:pharmacology
D000730:Androstadienes; D002867:Chromones; D005419:Flavonoids; D008246:Lysophospholipids; D009025:Morpholines; C060506:sphingosine 1-phosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048051:p38 Mitogen-Activated Protein Kinases; D013110:Sphingosine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; D000077191:Wortmannin