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.

Conclusion: 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.