Rho GTPase, Rac1, regulates Skp2 levels, vascular smooth muscle cell proliferation, and intima formation in vitro and in vivo

AIMS: Vascular smooth muscle cell (VSMC) proliferation contributes to intima formation after angioplasty or venous by-pass grafting, and during atherosclerosis. VSMC proliferation requires degradation of p27(Kip1) promoted by S-phase kinase-associated protein-2 (Skp2), an F-box protein component of the Skp-Cullin-F-box(Skp2) ubiquitin-ligase. We investigated the role of Rac(1) in the regulation of Skp2 in rat VSMC. METHODS AND RESULTS: Rat carotid balloon injury increased Rac(1) activity. Rho GTPase inhibition with Clostridium difficile Toxin B or specific Rac(1) inhibition with adenovirus-mediated expression of dominant-negative Rac(1) reduced Skp2 levels, and VSMC proliferation in vitro and intima formation in vivo following carotid balloon injury. Inhibition of Skp2 expression and proliferation by dominant-negative Rac(1) was reversed by exogenous Skp2. Elevation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) with forskolin-inhibited Rac(1) activity, reduced Skp2, increased p27(Kip1) and inhibited VSMC proliferation, effects that were reversed by constitutively active Rac(1). These effects were independent of Rac(1) Cdc42/Rac interactive binding (CRIB)-domain effector proteins but associated with Rac(1)-dependent actin polymerization. CONCLUSION: Rac(1) activity regulates VSMC proliferation by controlling Skp2 levels. Activation of Rac(1) induced by balloon injury in vivo increases Skp2 levels, which promotes VSMC proliferation and intima formation. Inhibition of this novel pathway underlies the negative effects of cAMP on VSMC proliferation.