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 (Ϯ)-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 concentrationdependent manner. Using a CYP2D6 promoter-luciferase construct, we found that NOR4 and another NO donor, Snitrosoglutathione (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 Ϫ80 to ϩ65 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.Cytochrome P450 (P450) enzymes are a major class of heme-containing proteins that participate in the oxidative metabolism of endogenous substrates such as steroid hormones and the biotransformation of xenobiotics. Many studies have shown that the decreases of P450 activity and content caused by lipopolysaccharides (LPS) and cytokines under conditions of inflammation and infection contribute to impaired drug metabolism in vivo and in vitro (Sonawane et al., 1982;Renton, 1983;Morgan, 1989). Lipopolysaccharides and cytokines evoke the excessive production of nitric oxide (NO) via the induction of inducible NO synthase (iNOS) in hepatocytes (Hoffman et al., 1992). Recently, it was reported that NO may be involved in impaired drug metabolism (Khatsenko, 1998).Nitric oxide has pleiotropic functions, such as the regulation of vascular tone, platelet aggregation, neurotransmission, and the cytotoxic action of activated macrophages (Moncada et al., 1991). Intracellular targets for NO are heme or nonheme iron and thiol-containing proteins. Therefore, the binding of NO to heme of such P450s as CYP1A1/2, CYP2B1, and CYP2E1 inactivated these enzymes (Wink et al., 1993;Gergel et al., 1997). In addition, LPS and inflammatory cytokines inhibit the expression of constitutive and inducible P450 mRNAs in vivo and in vitro, and the suppression of P450 mRNA is prevente...

Time-Dependent Interaction between Differentiated Embryo Chondrocyte-2 and CCAAT/Enhancer-Binding Protein α Underlies the Circadian Expression of CYP2D6 in Serum-Shocked HepG2 Cells

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D2-Dopaminergic Receptor-Linked Pathways: Critical Regulators of CYP3A, CYP2C, and CYP2D

“…It is well established that both of these enzymes exert negative regulatory control on several P450s (Hara and Adachi, 2002). Possible contributions of eNOS and iNOS to the down-regulation of CYP3A, CYP2C, and CYP2D cannot be excluded.…”

Roles of nitric oxide in inflammatory downregulation of human cytochromes P450

“…NO donors can down-regulate CYP mRNAs and proteins in colon carcinoma [25,26] and hepatocarcinoma [27] cell lines, and it was reported that in HT-29 cells, NOS2 inhibitors or antisense oligonucleotides could block the down-regulation of CYP3A4 protein by ceramide [28], a putative mediator of the effects of pro-inflammatory cytokines on CYP expression [29]. We are aware of one published study on the regulation of CYP expression by physiological or pharmacological NO in primary human hepatocytes, which is the optimal system to study regulation of the human drug-metabolizing CYPs.…”