Arpiainen Satu, Raffalli-Mathieu Françoise, Lang Matti A, Pelkonen Olavi, Hakkola Jukka
Department of Pharmacology and Toxicology, P.O. Box 5000, University of Oulu, FIN-90014 Oulun yliopisto, Finland.
Mol Pharmacol. 2005 Apr;67(4):1325-33. doi: 10.1124/mol.104.008078. Epub 2005 Jan 18.
We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.
我们研究了芳烃受体（AHR）在Cyp2a5基因调控中的作用。利用在AHR功能上存在遗传决定差异的C57BL/6和DBA/2小鼠品系，研究典型AHR配体2,3,7,8-四氯二苯并对二恶英（TCDD）和3-甲基胆蒽对CYP2A5的诱导作用。这些小鼠品系中CYP2A5 mRNA的上调在反应上存在差异，这是AHR调控基因的典型特征，无论是在培养的原代肝细胞中TCDD诱导的，还是在体内3-甲基胆蒽诱导的。在原代肝细胞中，TCDD使CYP2A5蛋白水平升高了3倍，并对CYP2A5催化的香豆素7-羟化活性产生了类似的诱导作用。在报告基因分析中，Cyp2a5启动子区域-3033至+10在原代肝细胞和具有完整AHR/AHR核转运体（ARNT）复合物的Hepa-1肝癌细胞中，经TCDD处理后介导荧光素酶活性2至5倍的诱导。在AHR/ARNT复合物存在缺陷的Hepa-1变异细胞系中，ARNT的缺失消除了诱导作用。在Cyp2a5启动子的-2514至-2492位置鉴定出一个假定的AHR反应元件（XRE），并发现其与AHR/ARNT异二聚体相互作用。结合XRE位点突变的转染实验表明，该位点部分介导了TCDD对Cyp2a5的诱导作用。另一种AHR依赖性机制也调节Cyp2a5基因的近端启动子。总之，我们的研究表明，AHR配体通过AHR/ARNT依赖性机制转录上调Cyp2a5，并将Cyp2a5确立为一个新的AHR调控基因。
