title: Post-induction, stimulus-specific regulation of tumor necrosis factor mRNA expression.
abstract: The tumor necrosis factor (TNF) gene is activated by multiple extracellular signals in a stimulus- and cell type-specific fashion. Based on the presence of kappaB-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-kappaB in lipopolysaccharide (LPS)-induced TNF gene transcription in cells of the monocyte/macrophage lineage. However, we have previously demonstrated a general and critical role for a minimal TNF promoter region bearing only one of the kappaB-like motifs, kappa3, which is bound by nuclear factor of activated T cell proteins in lymphocytes and fibroblasts in response to multiple stimuli and Ets proteins in LPS-stimulated macrophages. Here, in an effort to resolve these contrasting findings, we used a combination of site-directed mutagenesis of the TNF promoter, quantitative DNase I footprinting, and analysis of endogenous TNF mRNA production in response to multiple stimuli under conditions that inhibit NF-kappaB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NF-kappaB essential modulator, which is the IkappaB kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-kappaB-independent, but inhibition of NF-kappaB activation attenuates virus- and LPS-induced TNF mRNA levels after initial induction. We conclude that induction of TNF gene transcription by virus or LPS does not depend upon NF-kappaB binding to the proximal promoter; rather, a stimulus-specific post-induction mechanism involving NF-kappaB, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.
authors.affiliation: CBR Institute for Biomedical Research, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Alla V
authors.initials: AV
authors.lastname: Tsytsykova
authors.forename: James V
authors.initials: JV
authors.lastname: Falvo
authors.forename: Marc
authors.initials: M
authors.lastname: Schmidt-Supprian
authors.forename: Gilles
authors.initials: G
authors.lastname: Courtois
authors.forename: Dimitris
authors.initials: D
authors.lastname: Thanos
authors.forename: Anne E
authors.initials: AE
authors.lastname: Goldfeld
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D020816:Amino Acid Motifs; D000818:Animals; D004247:DNA / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D061988:Proteasome Inhibitors; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D007476:Ionophores; D008070:Lipopolysaccharides; D016328:NF-kappa B; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C067713:lactacystin; D004247:DNA; D003850:Deoxyribonuclease I; D000111:Acetylcysteine