title: The tumour necrosis factor-alpha-mediated suppression of the CCAAT/enhancer binding protein-alpha gene transcription in hepatocytes involves inhibition of autoregulation.
abstract: Tumour necrosis factor-alpha (TNF-alpha) is a key regulator of the immune and inflammatory responses along with numerous other cellular changes during physiological and pathophysiological conditions. The cellular actions of TNF-alpha are associated with both the activation and the inhibition of gene transcription. In contrast to gene activation, the mechanisms underlying the TNF-alpha-mediated transcriptional inhibition remain largely unclear. We have investigated this aspect using the transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) as a model gene. TNF-alpha decreased the expression of C/EBPalpha mRNA and protein in the human hepatoma Hep3B cell line. The activity of the proximal promoter of both the human and the Xenopus C/EBPalpha genes in transfected Hep3B cells was inhibited by TNF-alpha. Transient transfection assays using various Xenopus C/EBPalpha promoter-luciferase DNA constructs showed that a C/EBP recognition sequence was essential for the TNF-alpha response. Electrophoretic mobility shift assays showed that C/EBPalpha bound to this site and co-transfection assays revealed that it was a major activator of the promoter and its transactivation potential was reduced by TNF-alpha. The potential role of nuclear factor kappaB (NF-kappaB) in the response was also investigated in the light of its pivotal role in TNF-alpha signalling. Inhibition of NF-kappaB using pharmacological agents or by transfection of a plasmid specifying for a superrepressor attenuated the TNF-alpha-inhibited C/EBPalpha promoter activity. In addition, an involvement of NF-kappaB in DNA-protein interactions at the C/EBP recognition sequence was identified.
authors.affiliation: Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK.
authors.forename: Pelagia
authors.initials: P
authors.lastname: Foka
authors.forename: Nishi N
authors.initials: NN
authors.lastname: Singh
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Salter
authors.forename: Dipak P
authors.initials: DP
authors.lastname: Ramji
mesh_terms: D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D022781:Hepatocytes / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000502:physiology; D015854:Up-Regulation; D014982:Xenopus laevis
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha