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

• Published in: Biochemical journal Vol. 355; no. 2; pp. 537 - 544
• Main Authors: OZEKI, Takeshi, TAKAHASHI, Yoshiki, KUME, Toshiyuki, NAKAYAMA, Kazuo, YOKOI, Tsuyoshi, NUNOYA, Ken-Ichi, HARA, Akira, KAMATAKI, Tetsuya
• Format: Journal Article
• Language: English
• Published: 15.04.2001
• Subjects: a1-antitrypsin; DD4 gene; dihydrodiol dehydrogenase; HNF-1^a protein; HNF-4^g protein
• ISSN: 0264-6021
• DOI: 10.1042/0264-6021:3550537

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5'-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two 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 HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha sub(1)-antitrypsin gene. A supershift assay using antibodies to human HNF-4 alpha , HNF-4 gamma and HNF-1 alpha showed that HNF-4 alpha and HNF-4 gamma bound to the HNF-4 motif, and that HNF-1 alpha interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif 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 , HNF-4 gamma and HNF-1 alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.