UROKINASE‐INDUCED SMOOTH MUSCLE CELL MIGRATION INVOLVES THE MAMMALIAN TARGET OF RAPAMYCIN

Abstract

Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Rapamycin is an antifungal and immunosuppressant that inhibits mTOR, which regulates p70S6 kinase (p70S6K) activity. Purpose: To determine a role for the mammalian target of rapamycin (mTOR) in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of mTOR to influence the response of SMCs to uPA. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1–100 nM) with and without rapamycin (1–10 nM). Additional studies were performed in the presence of the PI3-Kinase inhibitor LY294002 (10μM). Western blotting was performed for phosphorylated and total mTOR and p70S6K after stimulation with uPA (10nM) with and without rapamycin. MMP-2 activity determined by zymography. Results: uPA stimulated migration of SMCs in both the linear wound assay (p<0.01) and in the Boyden chamber (p<0.01); these response were inhibited completely by rapamycin in a concentration dependent manner. uPA stimulated phosphorylation of mTOR and p70S6K (2-fold increase over control for both, p<0.05). These responses were inhibited by the presence of the PI3-K inhibitor (p<0.01). Rapamycin completely inhibited both mTOR and p70S6K phosphorylation. uPA induced expression and activity of MMP-2 was markedly inhibited by the presence of rapamycin Conclusions: uPA-induced SMC migration is mTOR dependent. Activation of mTOR and p70S6K is PI3-K dependent. Inhibition of mTOR and p70S6K resulted in a decrease in MMP-2 expression and activity.