The tumour necrosis factor-α-mediated suppression of the CCAAT/enhancer binding protein-α gene transcription in hepatocytes involves inhibition of autoregulation

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Tumour necrosis factor-α (TNF-α) 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-α are associated with both the activation and the inhibition of gene transcription. In contrast to gene activation, the mechanisms underlying the TNF-α-mediated transcriptional inhibition remain largely unclear. We have investigated this aspect using the transcription factor CCAAT/enhancer binding protein-α (C/EBPα) as a model gene. TNF-α decreased the expression of C/EBPα mRNA and protein in the human hepatoma Hep3B cell line. The activity of the proximal promoter of both the human and the Xenopus C/EBPα genes in transfected Hep3B cells was inhibited by TNF-α. Transient transfection assays using various Xenopus C/EBPα promoter–luciferase DNA constructs showed that a C/EBP recognition sequence was essential for the TNF-α response. Electrophoretic mobility shift assays showed that C/EBPα 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-α. The potential role of nuclear factor κB (NF-κB) in the response was also investigated in the light of its pivotal role in TNF-α signalling. Inhibition of NF-κB using pharmacological agents or by transfection of a plasmid specifying for a superrepressor attenuated the TNF-α-inhibited C/EBPα promoter activity. In addition, an involvement of NF-κB in DNA–protein interactions at the C/EBP recognition sequence was identified.