Differential regulation of ERK1/2 and p38(MAPK) by components of the Rho signaling pathway during sphingosine-1-phosphate-induced smooth muscle cell migration.
OBJECTIVE
To determine the role of rhosignaling in sphingosine-1-phosphate (S-1-P)-induced smooth muscle cell migration.


BACKGROUND
S-1-P is a bioactive sphingolipid released from activated platelets stimulating migration of smooth muscle cells (SMC) in vitro through Galphai G-proteins and MAPK activation. Rho is one of the key small GTPases required for cytoskeletal reorganization and MAPK activation during migration. We hypothesized that S-1-P-stimulated migration is regulated by the rho-signaling pathway.


METHODS
Rat arterial SMCs were cultured in vitro. Linear wound assays of migration were performed in the presence of S-1-P with and without C3 (a rho antagonist) and Y (Y27632, a Rho kinase inhibitor). Western blotting was performed for MEK1-ERK1/2 and MMK3/MKK6-p38(MAPK) phosphorylation after stimulation with S-1-P with and without pre-incubation with the inhibitors. Statistics were analyzed by one-way ANOVA.


RESULTS
S-1-P stimulated migration of SMCs in a wound assay (2-fold over control; P < 0.01), which was blocked by Rho inhibition (P < 0.05). S-1-P activated rho and induced a time-dependent increase in ERK1/2 and p38(MAPK) activation. In the presence of C3, MEK1 and ERK1/2 phosphorylation were significantly decreased, while MKK3/6 and p38(MAPK) phosphorylation were unchanged. In contrast, when rho kinase was inhibited, there was an increase in ERK1/2 and a decrease in p38(MAPK) phosphorylation. Rho kinase inhibition resulted in a decrease in MEK1/2 and MKK3/6 phosphorylation.


CONCLUSIONS
S-1-P differentially regulates the MAPK pathway through components of the rho pathway. Rho regulates ERK1/2 activation through MEK1/2, while Rho kinase negatively modulates ERK1/2 in a MEK1/2-independent manner and regulates p38(MAPK) through MKK3/6. This is the first description of differential MAPK regulation by a G-protein-coupled receptor through the rho pathway. Understanding signal transduction in SMCs will contribute to the development of molecular therapeutics for intimal hyperplasia.
Galaria
Irfan I
II
Vascular Biology and Therapeutics Program, Division of Vascular Surgery, and Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642, USA.
Fegley
Allison J
AJ
Nicholl
Suzanne M
SM
Roztocil
Elisa
E
Davies
Mark G
MG
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000502:physiology*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020741:rho GTP-Binding Proteins; D013110:Sphingosine