Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.
Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-kappa B p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, L-arginine methyl ester (L-NAME; 100 microM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 microM), was added. In contrast to that of LPS cells, nuclear extracts from LPS + L-NAME cells demonstrated increased NF-kappa B DNA binding on gel shift analysis. Addition of SNAC to LPS + L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-kappa B p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-kappa B-dependent promoter-reporter constructs demonstrated increased activity in LPS + L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-kappa B p50 and inhibition of NF-kappa B-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-kappa B.
delaTorre
A
A
Department of Surgery, Georgetown University Medical Center, Washington, DC 20007, USA.
Schroeder
R A
RA
Punzalan
C
C
Kuo
P C
PC
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics; D008264:Macrophages / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000276:immunology
D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009569:Nitric Oxide; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse