Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor [kappa]B pathway

HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti- inflammatory cytokine TGF beta (transforming growth factor beta ) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNF alpha (tumour necrosis factor alpha ) antagonizes TGF beta for the regulation of APOC3 gene expression in hepatocytes. TNF alpha was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNF alpha -induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNF alpha involved NF-[kappa]B (nuclear factor [kappa]B). Latent membrane protein 1 of the Epstein-Barr virus, which is an established potent activator of NF-[kappa]B as well as wild-type forms of various NF-[kappa]B signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNF alpha had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast- transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNF alpha and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor- gamma co-activator 1), CBP [CREB (cAMP- response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNF alpha , or other factors that trigger an NF-[kappa]B response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.