Hepatocyte nuclear factor (HNF)-4 alpha/gamma, HNF-1 alpha, and vHNF-1 regulate the cell-specific expression of the human dihydrodiol dehydrogenase (DD)4/AKR1C4 gene.
Recently, we found a region A (a hepatocyte nuclear factor (HNF)-4-binding site from nucleotides -701 to -684) and a region B (an HNF-1-binding site from nucleotides -682 to -666) as cis-acting elements necessary for the transcriptional activation of the human dihydrodiol dehydrogenase (DD)4 gene in human hepatoblastoma HepG2 cells, which express DD4 mRNA. Thus, to investigate the mechanism(s) responsible for the cell-type-specific expression of DD4 mRNA, we constructed a reporter plasmid, pDD4 Foot A+B: -95/+28, in which regions A and B were linked to the human DD4 minimal promoter (-95 to +28) fused to the luciferase gene. The luciferase activity was detectable in HepG2 cells but not in human renal adenocarcinoma ACHN cells transfected with the pDD4 Foot A+B: -95/+28, which do not express DD4 mRNA. A supershift assay using antibodies to HNF-4 alpha, -4 gamma, -1 alpha, or valiant HNF (vHNF)-1 revealed that HNF-4 alpha, -4 gamma, and -1 alpha recognized regions A and B in HepG2 cells and that only vHNF-1 bound to regions A and B in ACHN cells. Semiquantitative reverse-transcribed polymerase chain reaction showed that a large amount of vHNF-1-C, which lacked transcriptional activation domain, was expressed in ACHN cells. Transfection of ACHN cells with an expression plasmid of vHNF-1-C did not activate the pDD4 Foot A+B: -95/+28 reporter gene. Taken together, we conclude that the cell-type-specific expression of DD4 mRNA is regulated by vHNF-1-C.
Ozeki
Takeshi
T
Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Hokkaido, Japan. ozeki-tky@umin.ac.jp
Takahashi
Yoshiki
Y
Nakayama
Kazuo
K
Kamataki
Tetsuya
T
D000230:Adenocarcinoma / Q000201:enzymology; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D018197:Hepatoblastoma / Q000201:enzymology; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D007680:Kidney Neoplasms / Q000201:enzymology; D008113:Liver Neoplasms / Q000201:enzymology; D009687:Nuclear Proteins; D009928:Organ Specificity; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; C496597:HNF4G protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase