NO suppresses while peroxynitrite sustains NF-kappaB: a paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO.
OBJECTIVE
NO has both cytoprotective and cytotoxic effects. A key cytoprotective action of NO is attributed to inhibition of nuclear factor-kappaB (NF-kappaB)-mediated gene expression; this potentially endows NO with ubiquitous anti-inflammatory activity. Since immunostimulant-induced iNOS gene expression is itself dependent on NF-kappaB, NO is expected to limit its own synthesis. On the other hand, many cytotoxic actions of NO have been attributed to the chemical reactivity of peroxynitrite (ONOO-) formed from NO by near diffusion-limited reaction with O2-. To assess whether ONOO- shares the ability of NO to inhibit NF-kappaB activation and consequent iNOS gene expression, we compared effects of NO donors (NOR3 and SNAP), an ONOO- donor (SIN-1), and pure ONOO- on LPS-induced responses in vascular smooth muscle cells (VSMC).


RESULTS
NO donors, but not ONOO-, suppressed LPS-induced NF-kappaB activation and expression of a murine iNOS promoter/reporter construct. An NO donor also suppressed NF-kappaB activation when induced by IL-1beta or TNFalpha. Northern blot and RT-PCR analyses showed that NO, but not ONOO- or 8-bromo-cGMP, decreases LPS-induced expression of iNOS mRNA. Electrophoretic mobility shift assays (EMSA) and immunocytochemical analyses confirmed that NO but not ONOO- inhibits nuclear translocation of NF-kappaB. Although ONOO- generation from SIN-1 did not inhibit NF-kappaB activation, conversion of SIN-1 to a pure NO donor (by addition of excess superoxide dismutase) resulted in potent inhibition of NF-kappaB activation. Dose-response analyses suggest that the inhibitory effect of NO on iNOS gene transcription results specifically from inhibition of NF-kappaB activation, and is mediated by a G-cyclase-independent mechanism that is unavailable to ONOO-. LPS stimulates IkappaB-alpha phosphorylation by inducing IkappaB kinase (IKK) activity, and NO, but not ONOO-, inhibits LPS-induced IkappaB-alpha phosphorylation and IKK activity.


CONCLUSIONS
We demonstrate that only NO inhibits the activation of NF-kappaB and suppresses iNOS gene expression. This distinction provides a novel paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO.
Hattori
Yoshiyuki
Y
Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Tochigi, 321-0293, Japan. yhattori@dokkyomed.ac.jp
Kasai
Kikuo
K
Gross
Steven S
SS
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D018932:Chemokine CCL2; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse