title: TNF-alpha gene expression in macrophages: regulation by NF-kappa B is independent of c-Jun or C/EBP beta.
abstract: The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-kappa B family members interact to regulate the human TNF-alpha gene. A 120-bp TNF-alpha promoter-reporter, possessing binding sites for NF-kappa B (kappa B3), C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-kappa B p65, and c-Jun, but not C/EBP beta, suppressed (p < 0.05-0.001) LPS-induced TNF-alpha secretion in primary human macrophages. Following LPS stimulation, NF-kappa B p50/p65 heterodimers bound to the kappa B3 site and c-Jun to the -103 AP-1 site of the TNF-alpha promoter. By transient transfection, NF-kappa B p65 and p50 synergistically activated the TNF-alpha promoter. In contrast, no synergy was observed between NF-kappa B p65, with or without NF-kappa B p50, and c-Jun or C/EBP beta, even in the presence of the coactivator p300. The contribution of the upstream kappa B binding sites was also examined. Following LPS stimulation, the kappa B1 site bound both NF-kappa B p50/p65 heterodimers and p50 homodimers. The binding by NF-kappa B p50 homodimers to the kappa B1, but not to the kappa 3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent kappa B3 and AP-1 sites in the human TNF-alpha promoter contribute independently to LPS-induced activation. Although both the kappa B1 and kappa B3 sites bound transcriptionally active NF-kappa B p50/p65 heterodimers, only the kappa B1 site contributed to down-regulation by NF-kappa B p50 homodimers.
authors.affiliation: Department of Medicine, Division of Arthritis, Veterans Administration Lakeside Medical Center, and Northwestern University Medical School, Chicago, IL 60611, USA.
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: P
authors.initials: P
authors.lastname: Sidiropoulos
authors.forename: G
authors.initials: G
authors.lastname: Song
authors.forename: L J
authors.initials: LJ
authors.lastname: Pagliari
authors.forename: M J
authors.initials: MJ
authors.lastname: Birrer
authors.forename: B
authors.initials: B
authors.lastname: Stein
authors.forename: J
authors.initials: J
authors.lastname: Anrather
authors.forename: R M
authors.initials: RM
authors.lastname: Pope
mesh_terms: D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D007108:Immune Tolerance / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D052002:NF-kappa B p50 Subunit; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000037:antagonists & inhibitors / Q000502:physiology*; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009687:Nuclear Proteins; D016755:Proto-Oncogene Proteins c-jun; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha