Biphasic effect of p21Cip1 on smooth muscle cell proliferation: role of PI 3-kinase and Skp2-mediated degradation.

OBJECTIVE Proliferation of vascular smooth muscle cells (VSMC) is an important event in atherogenesis, in-stent restenosis and late vein-graft failure. Cell-cycle progression is positively regulated by cyclin:cdk complexes and negatively regulated by cyclin-dependent kinase inhibitors, including p21Cip1. Here we investigate the mechanisms regulating p21Cip1 levels in VSMCs and its role in controlling VSMC proliferation. METHODS AND RESULTS We studied the S-phase-associated kinase protein-2 (Skp2), an F-box protein implicated in the ubiquitination of p21Cip1. Overexpression of wild-type Skp2 or dominant-negative Skp2 decreased or increased p21Cip1 levels, respectively. Interestingly, levels of endogenous p21Cip1 and Skp2 were both increased in a phosphoinositide PI 3-kinase-dependent manner in late G1 phase. Increased expression of p21Cip1 occurred despite significantly increased Skp2-mediated proteasomal degradation. To determine the role of p21Cip1 in regulating VSMC proliferation, we used adenovirus-mediated overexpression and small-interfering RNA to elevate or silence p21Cip1 expression, respectively. Overexpression of p21Cip1 significantly inhibited VSCM proliferation. p21Cip1 silencing also inhibited proliferation and increased apoptotic cell death. CONCLUSIONS Taken together, this data demonstrates that a balance between PI 3-kinase-driven upregulation and Skp2-mediated degradation controls the level of p21Cip1, which regulates VSMC proliferation in a biphasic manner. Low levels of p21Cip1 are also essential to counter apoptosis during cell-cycle progression.