Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden.
J Pharmacol Exp Ther. 2011 Jul;338(1):302-9. doi: 10.1124/jpet.110.175075. Epub 2011 Apr 14.
Cytochrome P4502C9 (CYP2C9) is an important drug-metabolizing enzyme responsible for the metabolism of approximately 16% of all clinically relevant drugs. It was shown previously that the activity of CYP2C9 in vivo is inhibited by oral contraceptives. The mechanisms of this effect have not been elucidated. We hypothesize that this may occur because of the sex steroid-dependent activation of estrogen receptor α (ERα) with further transactivation of the CYP2C9 gene. Here, we show that the CYP2C9 promoter indeed contains a functionally relevant estrogen responsive element (ERE) half-site at position -149/-145. Its ERα binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ERα into Huh7 hepatoma cells and treated with various ERα ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17β-estradiol (EE), and 17α-ethinylestradiol (ETE). The luciferase activity driven by the wild-type CYP2C9 promoter construct was up-regulated by 4-OHT and R and significantly or marginally suppressed by ETE and EE, respectively. An identical effect was observed in primary hepatocytes treated with these compounds. Mutations introduced into the ERE half-site abolished the observed effects in the Huh7 cells. Electrophoretic mobility-shift assay revealed sequence-specific binding of a nuclear protein to the oligonucleotide containing the ERE half-site, which was identified as ERα by antibody supershift analysis. In addition, the association of ERα with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ERα and its ligands play an important role in the regulation of CYP2C9 expression.
细胞色素 P4502C9（CYP2C9）是一种重要的药物代谢酶，负责代谢大约 16%的所有具有临床相关性的药物。先前已经表明，口服避孕药会抑制 CYP2C9 在体内的活性。但这种作用的机制尚未阐明。我们假设，这可能是因为雌激素受体 α（ERα）的性激素依赖性激活，进而使 CYP2C9 基因的转录激活。在这里，我们表明 CYP2C9 启动子确实在位置-149/-145 处包含一个具有功能相关性的雌激素反应元件（ERE）半位点。通过荧光素酶基因报告基因检测法测试了其 ERα 结合活性。带有该位点的启动子构建体与 ERα 共转染到 Huh7 肝癌细胞中，并用各种 ERα 配体（包括 4-羟基他莫昔芬（4-OHT）、雷洛昔芬（R）、17β-雌二醇（EE）和 17α-乙炔雌二醇（ETE））处理。野生型 CYP2C9 启动子构建体驱动的荧光素酶活性被 4-OHT 和 R 上调，而 ETE 和 EE 分别显著或轻微抑制。用这些化合物处理原代肝细胞也观察到了相同的效果。引入 ERE 半位点的突变消除了 Huh7 细胞中观察到的效应。电泳迁移率变动分析显示，一种核蛋白与含有 ERE 半位点的寡核苷酸特异性结合，通过抗体超迁移分析鉴定为 ERα。此外，染色质免疫沉淀数据强烈支持 ERα 与 CYP2C9 启动子的结合。总之，这些结果表明 ERα 及其配体在 CYP2C9 表达的调节中发挥重要作用。
