Role of p42/p44 mitogen-activated-protein kinase and p21 ^waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

Abstract

The purpose of this study was to determine the involvement of the p42͞p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21 waf1/cip1 in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like ␣-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 M, and DFMO elicited effects at concentrations of 100 M or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1͞2 inhibitors PD 098,059 or U0126. This finding suggests that the p42͞p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42͞p44 MAPK and induction of p21 waf1/cip1 . This effect was prevented by MAPK kinase 1͞2 inhibitors, suggesting that induction of p21 waf1/cip1 depended on activation of p42͞p44. Moreover, activation of p42͞p44 and induction of p21 waf1/cip1 were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42͞p44 MAPK and induction of p21 waf1/cip1 were cGMPindependent. Neither 1H-(1,2,4)oxadiazolo[4,3-␣]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21 waf1/cip1 and consequent inhibition of cell proliferation.polyamines ͉ ornithine decarboxylase ͉ atherosclerosis ͉ MAPK V ascular smooth muscle cells (VSMC) are normally quiescent in the adult, rarely exhibiting mitogenic activity in their contractile state (1). However, vascular injury stimulates VSMC growth by disrupting the physiological balance between inhibition and stimulation. Over-proliferation of VSMC contributes to many clinical vascular pathologies, the causes of which vary from mechanical (restenosis after balloon angioplasty), to immunological (transplant arteriosclerosis), to multifactorial (atherosclerosis). Increased VSMC proliferation has been attributed to dysfunction of or damage to the endothelial lining in the area of intimal thickening and, more specifically, to decreased nitric oxide (NO) production. This effect is made evident by several studies in which NO donor agents or NO synthase gene transfer were shown to inhibit neointimal formation after balloon angioplasty in animal models (2-4). The effect inverse has been shown in studies where animals were subjected to balloon angioplasty and then treated with inhibitors of NO synthase (4). In these studies, intimal formation was enhanced by the inhibition of NO production.The mechanism by whi...

Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats

“…This relationship between nitrite and p21 is suggested in vivo, where p21 was shown to be upregulated within the limited neointima following nitrite treatment. Previously published studies focusing on the inhibition of SMC proliferation by NO have also shown it to be dependent upon p21 (40)(41)(42). Our findings support the hypothesis that the effect of nitrite on SMC proliferation is exerted via NO-mediated downstream signaling.…”

“…Waf1/Cip1 . Others and we have previously illustrated (40)(41)(42) that NO-induced inhibition of proliferation in SMCs is dependent on the upregulation of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 . In order to determine whether sodium nitrite increases p21 expression, we treated SMCs with or without sodium nitrite (0-100 μM) or DETA-NONOate as a positive control (50 μM) and determined p21 protein levels by Western blotting after 24 hours.…”

Interaction between Polyamines and the Mitogen-Activated Protein Kinase Pathway in the Regulation of Cell Cycle Variables in Breast Cancer Cells

“…This finding is in agreement with previous reports indicating that in certain circumstances, stimulation of the MAPK pathway can induce p21 and lead to cell cycle arrest (23,29). Of particular relevance is the report by Bauer et al (37) who also observed an inhibition of DFMO-induced p21 up-regulation with the MEK inhibitors PD98059 and U0126 in rat aortic smooth muscle cells. At variance with our findings, however, these authors observed a reversal of the antiproliferative effects of DFMO with these two compounds.…”

Nitric Oxide Down-regulates Polo-like Kinase 1 through a Proximal Promoter Cell Cycle Gene Homology Region

“…This activity of NO ⅐ has been ascribed to both cGMP-dependent and -independent mechanisms. Experiments in rodents have found, with a few notable exceptions (18,30), that NO ⅐ controls the cell cycle through cGMP. In contrast, the anti-proliferation effects of NO ⅐ in human cells have been more frequently associated with cGMP-independent signaling (29,31).…”