RETINOIC ACID MEDIATES DOWN-REGULATION OF THE ALPHA-FETOPROTEIN GENE THROUGH DECREASED EXPRESSION OF HEPATOCYTE NUCLEAR FACTORS

alpha-Fetoprotein (AFP), a protein highly induced during fetal liver d evelopment, is down-regulated by retinoids in the human hepatoma cell line Hep3B, in contrast to up-regulation observed in other cell types. Previously, we have documented that such up-regulation involves direc t effects through cis-retinoid X receptor-binding sites in the AFP enh ancer. In this report, we show a distinctive effect of all-trans-retin oic acid (RA) in Hep3B cells. RA caused a marked decrease in AFP trans cripts. Deletion analysis of the upstream regulatory region of the AFP gene revealed that cis-acting sites required for down-regulation resi ded near the promoter. Gel mobility shift assays for factors binding t o key elements in the AFP promoter region demonstrated that hepatocyte nuclear factor (HNF) 1 binding was diminished in nuclear extracts fro m RA-treated cells. In addition, HNF4, which is not known to bind to t he AFP promoter but does regulate HNF1, was also diminished. The level s of HNF1 and HNF4 mRNA were also decreased following RA treatment. AF P promoter-chloramphenicol acetyltransferase transient transfection as says demonstrated that the level of HNF1 had a direct impact on basal transcription as well as RA-mediated down-regulation of the AFP gene, and that co-transfection of HNF1 and HNF4, but not transfection of eit her factor alone, reversed the RA-mediated inhibition. Taken together these data point to an interaction among the RA, HNF1, and HNF4 signal s, which is reflected in decreased expression of AFP.