Tumor suppressor p53 but not cGMP mediates NO- induced expression of p21(Waf1/Cip1/Sdi1) in vascular smooth muscle cells

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascul ar smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-in duced p21 expression, we investigated the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway. The induction of p21 by the NO donor S -nitroso-N-acetylpenicillamine (SNAP) seemed to be due to transactivation b ecause SNAP elevated the activity of p21 promoter but did not stabilize p21 mRNA and protein. Because SNAP did not stimulate the deletion mutant of p2 1 promoter that lacked p53 binding sites, we tested the involvement of p53. The expression level of p53 was down-regulated after mitogenic stimulation , whereas it was sustained in the presence of SNAP. SNAP markedly stimulate d DNA binding activity of p53. Furthermore, SNAP failed to induce p21 in VS MCs obtained from p53-knock out mice and in A431 cells that contained mutat ed p53. The antiproliferative effect of SNAP also was attenuated in these c ells. NO stimulates guanylate cyclase and its product cGMP has been shown t o inhibit VSMC proliferation. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxali n-1-one, a guanylate cyclase inhibitor, did not prevent SNAP-induced p21 ex pression. 8-Bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination did not induce p21. Although 8-bromo-cGMP had a small antiproliferative eff ect, the elevation of cGMP concentration induced by SNAP was little through out the G(1) phase. The antiproliferative effect of SNAP was not attenuated by Rp-8-bromoguanosine-3',5'-monophosphorothioate, an inhibitor of cGMP-de pendent protein kinase. These results suggested that NO induces p21 through a p53-dependent but cGMP-independent pathway.