Vol. 61, Issue 1, 194-200, January 2002

Contribution of Hepatocyte Nuclear Factor-4 to Down-Regulation of CYP2D6 Gene Expression by Nitric Oxide

Hirokazu Hara and Tetsuo Adachi

Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, Gifu, Japan

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.