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Regulation mechanism of expression of drug-metabolizing enzymes,UGT1A1 and CYP3A4,at cell-cycle check-point

细胞周期检查点药物代谢酶UGT1A1和CYP3A4表达的调控机制

基本信息

批准号：
22590068
负责人：
SUGATANI Junko
金额：
$ 2.91万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

We have found that CDK inhibitor roscovtine markedly stimulated the expression of UGT1A1 mRNA and protein, whereas it slightly stimulated the expression of CYP1A1, CYP2B6, and CYP3A4 mRNAs and proteins in HepG2 cells. PXR-mediated transacriptional activity of the reporter gene in the absence of PXR ligand rifampicin by roscovitine was more prominently enhanced, compared with the basal-, CAR-, and AhR/ARNT-mediated transacriptional activities. Phosphomimetic mutations at positions T57, T290, S350, and T408 attenuated the induction of UGT1A1 and CYP3A4 mRNAs by roscovitine, while the residues T57, T290, S305, and T408 were involved in the suppression for rifampicin stimulation. Transfection with anti-CDK2 siRNA but not anti-CDK1 and CDK5siRNAs led to stimulated expression of UGT1A1. Phosphomimetic mutant at S350 of PXR was detected in the nucleus, and co-transfection with co-activator SRC-2but not SRC-1 recovered the PXR activity. Immunoprecipitation analysis revealed the binding of PXR with HDAC1 in the nucleus. T290D mutant YFP-PXR fusion proteins retained in the cytoplasm and were not translocated to the nucleus of the cells stimulated with roscovitine. These results indicate that phosphorylation at positions T290 retained PXR protein in the cytoplasm, and that roscovitine stimulated expression of UGT1A1 and CYP3A4 through inhibiting CDK2, which phosphorylated PXR at S350 to suppress the transactivation in the nucleus, that is, the binding of PXR with RXR and the deacetylation of PXR.

我们发现CDK抑制剂罗斯古汀显著刺激UGT1A1 mRNA和蛋白的表达，而轻微刺激HepG2细胞中CYP1A1、CYP2B6和CYP3A4 mRNA和蛋白的表达。与基础、CAR-和AhR/ arnt介导的转录活性相比，罗斯科维汀在缺乏PXR配体利福平的情况下，PXR介导的报告基因转录活性更显著增强。T57、T290、S350和T408位点的拟磷突变减弱了罗斯科维汀对UGT1A1和CYP3A4 mrna的诱导作用，而T57、T290、S305和T408位点则参与抑制利福平的刺激。转染抗cdk2 siRNA而不转染抗cdk1和cdk5sirna可刺激UGT1A1的表达。在细胞核中检测到PXR的S350处的拟磷突变体，与共激活剂src -2共转染而不与SRC-1共转染可恢复PXR的活性。免疫沉淀分析显示PXR与细胞核内HDAC1结合。T290D突变体YFP-PXR融合蛋白保留在细胞质中，不易位到罗斯科维汀刺激的细胞核中。这些结果表明，T290位点的磷酸化使PXR蛋白保留在细胞质中，罗斯科维汀通过抑制CDK2刺激UGT1A1和CYP3A4的表达，CDK2使PXR在S350位点磷酸化，从而抑制核内的反活化，即PXR与RXR的结合和PXR的去乙酰化。