Selective G-Protein Activation Is Required For Urokinase Induced Smooth Muscle Cell Migration

Yanjie Qi, Elisa Roztocil, Suzanne M Nicholl, Mark G Davies.
University of Rochester, Rochester, NY.

OBJECTIVES: Urokinase (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to uPA is dependent on the aminoterminal domain (ATD) of the molecule. The migratory response to ATD is G-protein dependent and involves both PI3K-akt and MEK- ERK1/2 pathways. G-proteins exist in the membrane as trimer of α, β and γ subunits and interact with the uPA receptor to mediate signaling. The role of specific G-proteins during ATD mediated migration is not understood.

METHODS: Murine arterial SMCs were cultured in vitro. Linear wound and transwell assays of migration and western blotting for akt, MEK1/2 - ERK 1/2 and MKK3/6 - p38MAPK phosphorylation were performed in the presence of ATD with and without transfection with siRNA to Gαi(2) and Gαi(3) to down regulate Gαi2 and Gαi(3) respectively or with and without adenoviral transfection with the Gβγ inhibitor βARKCT. siRNA to the G-proteins Gαi(2) and Gαi(3) was developed commercially and cells were transfected by a lipofectamine method with appropriate downregulation of targets. Statistics were analyzed by one-way ANOVA.

RESULTS: VSMC express Gαi(2), Gαi(3) and Gβγ but not Gαi(1). Down regulation of Gαi(3) with siRNA and inhibition of Gβγ with βARKCT inhibited ATD induced migration in both assays. Transfection with siRNA to Gαi(2), scrambled siRNA and empty adenovirus had no inhibitory effect. ATD induced activation of MEK1/2 and ERK1/2 by ATD was inhibited by Gαi(3) siRNA and inhibition of Gβγ with βARKCT. Only siRNA to Gαi(3) but not βARKCT inhibited PI3K-akt activation. ATD induced activation of MKK3/6 and p38MAPK by ATD was unaffected by Gαi(2) and Gαi(3) siRNA and inhibition of Gβγ with βARKCT.

CONCLUSIONS: Urokinase induced smooth muscle cell migration requires activation of Gαi and Gβγ G-proteins in the membrane. Both Gαi(3) and Gβγ are necessary for activation of ERK1/2, while Gαi(3) mediates PI3K-akt activation. Selective subunit activation of G-proteins by ATD allows for differential kinase signaling and is a potential molecular target to treat restenosis.