Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.
The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.
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Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021.
Kashiwabara
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Nathan
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D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004790:Enzyme Induction; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D011759:Pyrrolidines / Q000494:pharmacology; D011994:Recombinant Proteins; D016385:TATA Box; D013859:Thiocarbamates / Q000494:pharmacology
D000975:Antioxidants; D008070:Lipopolysaccharides; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D011759:Pyrrolidines; D011994:Recombinant Proteins; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D007371:Interferon-gamma; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D011505:Protein-Tyrosine Kinases