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 p21(Cip1). Here we investigate the mechanisms regulating p21(Cip1) 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 p21(Cip1). Overexpression of wild-type Skp2 or dominant-negative Skp2 decreased or increased p21(Cip1) levels, respectively. Interestingly, levels of endogenous p21(Cip1) and Skp2 were both increased in a phosphoinositide PI 3-kinase-dependent manner in late G(1) phase. Increased expression of p21(Cip1) occurred despite significantly increased Skp2-mediated proteasomal degradation. To determine the role of p21(Cip1) in regulating VSMC proliferation, we used adenovirus-mediated overexpression and small-interfering RNA to elevate or silence p21(Cip1) expression, respectively. Overexpression of p21(Cip1) significantly inhibited VSCM proliferation. p21(Cip1) 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 p21(Cip1), which regulates VSMC proliferation in a biphasic manner. Low levels of p21(Cip1) are also essential to counter apoptosis during cell-cycle progression. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.