Urokinase-induced smooth muscle cell migration requires PI3-K and Akt activation.
OBJECTIVE
To examine the role of the phospho-inositol-3'-kinase (PI3-K)-akt signaling axis during smooth muscle cell (SMC) migration in response to the aminoterminal fragment of urokinase (ATF).


BACKGROUND
Urokinase (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to urokinase is dependent on ATF. The role of PI3-K/akt signaling during migration in response to the uPA fragments is not understood.


METHODS
Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of ATF with and without the PI3-K inhibitors (Wortmannin, Wn [10 nm] and LY294002, LY [10 microm]) and an akt inhibitor (aktI, [10 microm]). Western blotting was performed for akt, ERK1/2, and GSK3beta phosphorylation after cells were stimulated with ATF in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA.


RESULTS
Both PI3-K and akt inhibitors blocked the migratory response to ATF in both assays. ATF induced time-dependent increases in akt phosphorylation at both S472 and T308 sites and ERK1/2 phosphorylation. Activation of akt and ERK1/2 was inhibited by Wn and LY. Manumycin A, a ras inhibitor, did not inhibit activation of akt but did inhibit ERK1/2 activation. Activation of akt and the dephosphorylation of its downstream kinase GSK3beta were inhibited by the akt inhibitor. Direct inhibition of akt did not influence ERK1/2 activation and inhibition of ERK1/2 did not influence akt activation.


CONCLUSIONS
ATF mediated migration is PI3-K dependent and activates two separate pathways: ERK1/2 and akt. ATF induces akt phosphorylation through a PI3K-mediated but ras-independent mechanism while both ras and PI3K are required for ERK1/2 activation. Defining key signaling pathways is vital to regulate vessel remodeling.
Galaria
Irfan I
II
Division of Vascular Surgery, Department of Surgery, Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642, USA.
Nicholl
Suzanne M
SM
Roztocil
Elisa
E
Davies
Mark G
MG
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one