title: Contribution of hepatocyte nuclear factor-4 to down-regulation of CYP2D6 gene expression by nitric oxide.
abstract: Nitric oxide (NO) released under inflammatory and infectious conditions has been implicated in the down-regulation of many cytochrome P450 genes, but its mechanism of action remains unknown. We showed that the expression of the CYP2D6 gene is down-regulated at the transcriptional level by NO in HepG2 cells. The NO donor (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4) decreased the expression of CYP2D6 mRNA in a concentration-dependent manner. Using a CYP2D6 promoter-luciferase construct, we found that NOR4 and another NO donor, S-nitrosoglutathione (GSNO), reduced the luciferase activity in a concentration-dependent manner. A guanylate-cyclase inhibitor failed to prevent suppression of CYP2D6 promoter activity by GSNO, indicating that the activity of the CYP2D6 promoter is suppressed via an NO-guanylate cyclase-independent pathway. Deletion analysis of the CYP2D6 promoter revealed that the -80 to +65 region, which contains the nuclear receptor hepatocyte nuclear factor-4 (HNF4) binding site, was responsible for the suppression of CYP2D6 promoter activity by NO. Therefore, we examined NO responsiveness of the HNF4 binding site by electrophoretic mobility-shift assays and site-direct mutagenesis. The DNA-binding activity of HNF4 was directly inhibited by NO donors, GSNO, and S-nitroso-N-acetyl-penicillamine in a concentration-dependent manner. Mutation of the HNF4 binding site in the CYP2D6 promoter partially restored the suppression of the promoter activity by NO donors. These results demonstrated that NO down-regulates CYP2D6 gene expression, at least in part, by directly inhibiting HNF4 binding to the CYP2D6 promoter.
authors.affiliation: Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan. harah@gifu-pu.ac.jp
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Hara
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Adachi
mesh_terms: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D006152:Cyclic GMP / Q000494:pharmacology; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D004347:Drug Interactions; D015870:Gene Expression / Q000187:drug effects* / Q000502:physiology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D020030:Nitric Oxide Donors; D010750:Phosphoproteins; D012333:RNA, Messenger; D014157:Transcription Factors; D009569:Nitric Oxide; D004247:DNA; D019389:Cytochrome P-450 CYP2D6; D006162:Guanylate Cyclase; D006152:Cyclic GMP