title: Hepatocyte nuclear factor 4α (HNF4α) in coordination with retinoic acid receptors increases all-trans-retinoic acid-dependent CYP26A1 gene expression in HepG2 human hepatocytes.
abstract: CYP26A1 expression is very highly induced by retinoic acid (RA) in the liver, compared to most other tissues, suggesting that a liver-enriched factor may be required for its physiological transcriptional response. HNF4α is a highly conserved liver-specific/enriched member of nuclear receptor superfamily. In this study, we hypothesized that HNF4α and RARs may cooperate in an RA-dependent manner to induce a high level of CYP26A1 expression in liver cells. Partial inhibition of endogenous HNF4α by siRNA reduced the level of RA-induced CYP26A1 mRNA in HepG2 cells. Cotransfection of HNF4α, with or without RARs, demonstrated RA-dependent activation of a human CYP26A1 promoter-luciferase construct. Analysis of a 2.5-kbp putative CYP26A1 promoter sequence identified five potential HNF4α DNA response elements: H1 located in a proximal region overlapping with an RAR element-1 (RARE1 or R1); H2 and H3 in the distal region, close to RARE2 (R2) and RARE3 (R3); and H4 and H5 in intermediary regions. In EMSA and ChIP analyses HNF4α and RARs binding in the proximal and distal CYP26A1 promoter regions was significantly higher in RA-treated cells. Mutational analysis of the individual HNF4α DNA-response elements identified H1 as the major site for HNF4α binding because mutation of H1 inhibited the promoter activity by ~90%, followed by H2 mutation with less than 40% inhibition. Our results indicate that HNF4α coordinates with RARs in an RA-dependent manner to strongly induce CYP26A1 gene expression in the liver, which may explain the high level of response to RA observed in vivo.
authors.affiliation: Department of Nutritional Sciences, Pennsylvania State University, University Park, PA, 16802, USA.
authors.forename: Reza
authors.initials: R
authors.lastname: Zolfaghari
authors.forename: A Catharine
authors.initials: AC
authors.lastname: Ross
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D000072516:Retinoic Acid 4-Hydroxylase; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D000072516:Retinoic Acid 4-Hydroxylase
keywords: HEPATOCYTE; LIVER-SPECIFIC GENE EXPRESSION; RETINOIC ACID RESPONSE ELEMENT; TRANSCRIPTION FACTOR