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

Cardiovasc Res. 2006 Jan;69(1):198-206. doi: 10.1016/j.cardiores.2005.08.020. Epub 2005 Oct 5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Cell Proliferation
  • Cells, Cultured
  • Cyclin-Dependent Kinase Inhibitor p21 / pharmacology*
  • G1 Phase
  • Male
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / metabolism*
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism*
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism*
  • RNA, Messenger / analysis
  • Rats
  • Rats, Wistar
  • Reverse Transcriptase Polymerase Chain Reaction
  • S-Phase Kinase-Associated Proteins / genetics
  • S-Phase Kinase-Associated Proteins / metabolism*
  • Transduction, Genetic / methods

Substances

  • Cyclin-Dependent Kinase Inhibitor p21
  • RNA, Messenger
  • S-Phase Kinase-Associated Proteins
  • Phosphatidylinositol 3-Kinases