title: Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate.
abstract: OBJECTIVE
  Overexpression of the inducible nitric oxide synthase (iNOS) gene inhibits neointimal hyperplasia after arterial injury. The purpose of this study was to examine the mechanism by which nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation, specifically focusing on signaling pathways known to be activated by NO, including cyclic guanosine monophosphate (cGMP), p53, and p42/44 mitogen-activated protein kinase (MAPK).
  
  
  RESULTS
  VSMCs that were subjected to iNOS gene transfer demonstrated a reduction in proliferation (80%) that was associated with a marked increase in p21 expression. The antiproliferative and p21 stimulatory effects of NO were not suppressed by the soluble guanylate cyclase inhibitor ODQ, implicating cGMP-independent signaling. The role of p53 in NO-mediated upregulation of p21 and inhibition of proliferation was evaluated using p53 -/- VSMCs. A similar reduction in cellular proliferation and upregulation of p21 expression were achieved with iNOS gene transfer as well as treatment with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrating the p53-independent nature of these NO-mediated pathways. The transfer of the iNOS gene activated the p42/44 MAPK, and inhibition of this MAPK pathway with PD98059 partially blocked the antiproliferative effects of NO and completely inhibited the p21 stimulatory effects of NO. For confirmation that iNOS overexpression upregulated p21 in vivo, injured rat carotid arteries were infected with an adenoviral vector carrying the iNOS gene and demonstrated a marked upregulation of p21 expression at three days. However, the ability of NO to inhibit VSMC proliferation does not solely depend on p21 upregulation since the NO-donor SNAP-inhibited VSMC proliferation in p21 -/- VSMCs.
  
  
  CONCLUSIONS
  Nitric oxide inhibits VSMC proliferation in association with the upregulation of p21; both occur independent of p53 and cGMP while being partially mediated through the p42/44 MAPK signaling cascade. This represents one potential mechanism by which NO inhibits VSMC proliferation.
authors.affiliation: University of Pittsburgh, Department of Surgery, PA 15261, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Kibbe
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: S
authors.initials: S
authors.lastname: Nie
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: A
authors.initials: A
authors.lastname: Lizonova
authors.forename: I
authors.initials: I
authors.lastname: Kovesdi
authors.forename: R L
authors.initials: RL
authors.lastname: Simmons
authors.forename: T R
authors.initials: TR
authors.lastname: Billiar
authors.forename: E
authors.initials: E
authors.lastname: Tzeng
mesh_terms: D000818:Animals; D002339:Carotid Arteries / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000235:genetics; D006152:Cyclic GMP / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D006965:Hyperplasia; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology; D015854:Up-Regulation / Q000235:genetics*
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D016159:Tumor Suppressor Protein p53; D019001:Nitric Oxide Synthase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D006162:Guanylate Cyclase; D010396:Penicillamine; D006152:Cyclic GMP; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one