Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo-keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4 alpha/gamma and HNF-1 alpha

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a m ajor isoform of hepatic DD that oxidizes transdihydrodiols of polycyclic ar omatic hydrocarbons to reactive and redox-active o-quinones and that reduce s several ketone-containing drugs. To investigate the mechanism of transcri ptional regulation of the human DD4 gene, the 5'-flanking region of the gen e was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that t wo positive regulatory regions were located in regions from - 701 to - 684 and from - 682 to - 666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HN F-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif g ave a shifted band in a gel-shift assay with nuclear extracts from HepG2 ce lls. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha (1)-antitrypsin gene. A supershift assay using an tibodies to human HNF-4 alpha, HNF-4 gamma and HNF-1 alpha showed that HNF- 4a and HNF-4 gamma bound to the HNF-4 motif, and that HNF-1 alpha interacte d with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 m otif lowered the luciferase activity to 10 or 8%, respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4 alpha, H NF-4 gamma and HNF-1 alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.