The ligands of estrogen receptor alpha regulate Cytochrome P4502C9 (CYP2C9) expression

Mwinyi, Jessica; Cavaco, Isa; Yurdakok, Begum; Mkrtchian, Souren; Ingelman-Sundberg, Magnus

http://hdl.handle.net/10400.1/11471

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Authors

Ingelman-Sundberg, Magnus

Abstract(s)

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 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.

Keywords

Transcription factor Gata-4 Pregnane X receptor Nuclear factor-4-Alpha Drug-metabolism Gene-expression Human liver Er-Alpha Polymorphisms Induction Promoter