Onica Tania, Nichols Kathleen, Larin Meghan, Ng Lorraine, Maslen Ann, Dvorak Zdenek, Pascussi Jean-Marc, Vilarem Marie-Josée, Maurel Patrick, Kirby Gordon M
Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada.
Mol Pharmacol. 2008 Feb;73(2):451-60. doi: 10.1124/mol.107.039354. Epub 2007 Oct 31.
Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) alpha response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4alpha- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4alpha increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4alpha mRNA levels by 4-fold; however, the amount of HNF4alpha nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4alpha to the HNF4-RE and that an interaction of GR and HNF4alpha occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4alpha to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4alpha with an HNF4-RE rather than a glucocorticoid response element.
人类细胞色素P450 2A6（CYP2A6）可代谢多种临床相关化合物，包括尼古丁和烟草特异性致癌物；然而，该基因的转录调控机制尚不清楚。我们研究了糖皮质激素受体（GR）在CYP2A6转录调控中的作用。地塞米松（DEX）可增加原代培养的人肝细胞中CYP2A6的mRNA和蛋白质水平。GR受体拮抗剂米非司酮（RU486；17β-羟基-11β-[4-二甲基氨基苯基]-17α-[1-丙炔基]雌甾-4,9-二烯-3-酮）可减弱这种作用，提示DEX对CYP2A6的诱导作用是由GR介导的。在基因报告试验中，DEX可导致荧光素酶活性呈剂量依赖性增加，RU486也可抑制这种增加，对CYP2A6启动子进行逐步截短后发现，DEX反应性定位于包含肝细胞核因子4（HNF4）α反应元件（HNF4-RE）的-95至+12区域。HNF4-RE的突变消除了HNF4α和DEX介导的CYP2A6反式激活。此外，HNF4α的过表达使CYP2A6转录活性增加了3倍。DEX可使HNF4α mRNA水平增加4倍；然而，HNF4α核蛋白的量未发生改变。电泳迁移率变动分析、染色质免疫沉淀（ChIP）和链霉抗生物素蛋白DNA结合试验显示，DEX可增加HNF4α与HNF4-RE的结合，并且GR和HNF4α在该位点发生相互作用。此外，ChIP试验表明，DEX可增加人肝细胞中CYP2A6近端启动子染色质的组蛋白H4乙酰化，这可能有助于HNF4α与HNF4-RE的结合增加。这些发现表明，DEX增加CYP2A6表达是由GR通过一种非常规转录机制介导的，该机制涉及HNF4α与HNF4-RE而非糖皮质激素反应元件的相互作用。
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