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- Regulation of CYP2C19 Expression by Estrogen Receptor alpha: Implications for Estrogen-Dependent Inhibition of Drug MetabolismPublication . Mwinyi, Jessica; Cavaco, Isa; Pedersen, Rasmus Steen; Persson, Anna; Burkhardt, Sabrina; Mkrtchian, Souren; Ingelman-Sundberg, MagnusCytochrome P4502C19 (CYP2C19) is an important drug-metabolizing enzyme involved in the biotransformation of, for example, proton pump inhibitors and antidepressants. Several in vivo studies have shown that the CYP2C19 activity is inhibited by oral contraceptives, which can cause important drug interactions. The underlying molecular mechanism has been suggested to be competitive inhibition. However, the results presented here indicate that estradiol derivatives down-regulate CYP2C19 expression via estrogen receptor (ER)alpha, which interacts with the newly identified ER-binding half site [estrogen response element (ERE)] at the position -151/-147 in the CYP2C19 promoter. In gene reporter experiments in Huh-7 hepatoma cells, the activity of the luciferase construct carrying a 1.6-kb long CYP2C19 promoter fragment cotransfected with ER alpha was down-regulated upon treatment with 17 beta-estradiol (EE) or 17 alpha-ethinylestradiol (ETE) at half-maximum concentrations of 10(-7) and 10(-8) M, respectively. Mutations introduced into the ERE half site -151/-147 significantly inhibited these ligand-dependent effects. Electrophoretic mobility shift assays and quantitative chromatin immunoprecipitation experiments revealed that estrogen receptor alpha binds to this element. A significant suppression of CYP2C19 transcription by female sex steroids was confirmed by reverse transcription polymerase chain reaction after hormonal treatment of human hepatocytes. Inhibition experiments using a stable human embryonic kidney 293 CYP2C19 cell line revealed competitive inhibition at much higher concentrations of EE and ETE compared with those required for transcriptional inhibition. These results indicate that both EE and ETE inhibit CYP2C19 expression via an ER alpha-dependent regulatory pathway, thus providing a new insight into the molecular mechanism behind the inhibitory effect of oral contraceptives on CYP2C19 activity.
- The ligands of estrogen receptor alpha regulate Cytochrome P4502C9 (CYP2C9) expressionPublication . Mwinyi, Jessica; Cavaco, Isa; Yurdakok, Begum; Mkrtchian, Souren; Ingelman-Sundberg, MagnusCytochrome 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 alpha (ER alpha) 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 ER alpha binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ER alpha into Huh7 hepatoma cells and treated with various ER alpha ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17 beta-estradiol (EE), and 17 alpha-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 ER alpha by antibody supershift analysis. In addition, the association of ER alpha with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ER alpha and its ligands play an important role in the regulation of CYP2C9 expression.