Purpose: We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs.
Methods: p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer. p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with (32)P-labeled myelin basic protein as a substrate.
Results: NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose- and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase in both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO.
Conclusion: These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.