Urokinase-induced smooth muscle cell migration requires PI3-K and akt activation

Objective. To examine the role of the phosphoinositol-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 mu m]) and an akt inhibitor (aktI, [10 mu m]). Western blotting was performed for akt, ERK1/2, and GSK3 beta 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 GSK3 beta 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. (c) 2005 Elsevier Inc. All rights reserved.