Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of IkappaB.
The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl-L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappaB activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappaB p65 subunit, and IkappaBalpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of IkappaB. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of IkappaBalpha thereby preventing NF-kappaB activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.
Chang
Kwang
K
Department of Molecular and Cellular Biochemistry, School of Medicine Kangwon National University, Chuncheon, Gangwon-do, Korea.
Lee
Seon-Jin
SJ
Cheong
Ilyoung
I
Billiar
Timothy R
TR
Chung
Hun-Taeg
HT
Han
Jeong-A
JA
Kwon
Young-Guen
YG
Ha
Kwon-Soo
KS
Kim
Young-Myeong
YM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D022781:Hepatocytes / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D051381:Rats
D016207:Cytokines; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; C110881:S-nitro-N-acetylpenicillamine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D010396:Penicillamine; D006152:Cyclic GMP