Tumor Necrosis Factor α Promotes Nuclear Localization of Cytokine-inducible CCAAT/Enhancer Binding Protein Isoforms in Hepatocytes

Hepatocytes were cultured in the presence of recombinant tumor necrosis factor (TNF) α or mutated TNF α peptides that specifically activate either p55 or p75 TNF receptors to determine if TNF α can activate cytokine-inducible CCAAT/enhancer binding protein (C/EBP) isoforms by post-transcriptional mechanisms that are initiated by TNF receptors. Within 5-10 min after treatment with any of these agents, nuclear concentrations of C/EBP β and C/EBP δ double and remain 2-4-fold greater than control cultures for 30 min (p < 0.01). Consistent with these results, gel mobility shift assays demonstrate 3-fold increased nuclear C/EBP β- and C/EBP δ-DNA binding activity in TNF α-treated cells, and immunocytochemistry confirms rapid redistribution of these C/EBP isoforms into the nucleus. In contrast, mRNA and whole cell protein concentrations of C/EBP β and δ are not altered by TNF α exposure, and nuclear concentrations of another C/EBP isoform, C/EBP α, are decreased by 80%. This novel evidence that TNF α initiates post-transcriptional activation of cytokine-inducible C/EBP isoforms identifies a mechanism that enables hepatocytes to respond immediately to inflammatory stress. Hepatocytes were cultured in the presence of recombinant tumor necrosis factor (TNF) α or mutated TNF α peptides that specifically activate either p55 or p75 TNF receptors to determine if TNF α can activate cytokine-inducible CCAAT/enhancer binding protein (C/EBP) isoforms by post-transcriptional mechanisms that are initiated by TNF receptors. Within 5-10 min after treatment with any of these agents, nuclear concentrations of C/EBP β and C/EBP δ double and remain 2-4-fold greater than control cultures for 30 min (p < 0.01). Consistent with these results, gel mobility shift assays demonstrate 3-fold increased nuclear C/EBP β- and C/EBP δ-DNA binding activity in TNF α-treated cells, and immunocytochemistry confirms rapid redistribution of these C/EBP isoforms into the nucleus. In contrast, mRNA and whole cell protein concentrations of C/EBP β and δ are not altered by TNF α exposure, and nuclear concentrations of another C/EBP isoform, C/EBP α, are decreased by 80%. This novel evidence that TNF α initiates post-transcriptional activation of cytokine-inducible C/EBP isoforms identifies a mechanism that enables hepatocytes to respond immediately to inflammatory stress.