Cyclin-dependent Kinase 2 Negatively Regulates Human Pregnane X Receptor-mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells
Cyclin-dependent Kinase 2 Negatively Regulates Human Pregnane X Receptor-mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells
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DOI：
10.1074/jbc.m806132200
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发表时间：
2008-11-07
影响因子：
4.8
通讯作者：
Chen, Taosheng
中科院分区：
文献类型：
--
作者：
Lin, Wenwei;Wu, Jing;Chen, Taosheng
人类孕烷X受体(HPXR)调节药物代谢关键酶的表达。细胞色素P450 3A4(细胞色素P450 3A4)是其中的一种酶，在处于静止或处于细胞周期中的肝细胞的药物代谢中起着关键作用。已经证实，在肝脏发育或再生过程中，P450的表达显著减少，如CYP3A4。大量研究表明，细胞信号通路参与调节包括hPXR在内的核受体的功能。在此，我们报道了细胞周期蛋白依赖性蛋白激酶(CDKs)的抑制作用可导致hPXR介导的细胞色素P3A4基因在人肝癌细胞中的表达被激活。与这一发现一致的是，CDK2的激活减弱了CYP3A4基因表达的激活。体外激酶分析表明，CDK2直接磷酸化hPXR。CDK磷酸化位点Ser(350)的拟磷酸化突变显著削弱了hPXR的功能，而磷酸化缺陷突变则使人对CDK2产生抗性。利用稳定表达的hPXR和细胞色素P3A4-荧光素酶报告基因，在细胞周期的不同时相中，我们发现hPXR介导的细胞色素P3A4在S期的表达显著降低。我们的结果首次表明，CDK2负性调节hPXR的活性，并提示CDK2在细胞周期中调节hPXR活性和CYP3A4表达方面发挥重要作用，例如在胚胎或再生成人肝脏中。
The human pregnane X receptor (hPXR) regulates the expression of critical drug metabolism enzymes. One of such enzymes, cytochrome P450 3A4 (CYP3A4), plays critical roles in drug metabolism in hepatocytes that are either quiescent or passing through the cell cycle. It has been well established that the expression of P450, such as CYP3A4, is markedly reduced during liver development or regeneration. Numerous studies have implicated cellular signaling pathways in modulating the functions of nuclear receptors, including hPXR. Here we report that inhibition of cyclin-dependent kinases (Cdks) by kenpaullone and roscovitine (two small molecule inhibitors of Cdks that we identified in a screen for compounds that activate hPXR) leads to activation of hPXR-mediated CYP3A4 gene expression in HepG2 human liver carcinoma cells. Consistent with this finding, activation of Cdk2 attenuates the activation of CYP3A4 gene expression. In vitro kinase assays revealed that Cdk2 directly phosphorylates hPXR. A phosphomimetic mutation of a putative Cdk phosphorylation site, Ser(350), significantly impairs the function of hPXR, whereas a phosphorylation-deficient mutation confers resistance to Cdk2. Using HepG2 that has been stably transfected with hPXR and the CYP3A4-luciferase reporter, enriched in different phases of the cell cycle, we found that hPXR-mediated CYP3A4 expression is greatly reduced in the S phase. Our results indicate for the first time that Cdk2 negatively regulates the activity of hPXR, and suggest an important role for Cdk2 in regulating hPXR activity and CYP3A4 expression in hepatocytes passing through the cell cycle, such as those in fetal or regenerating adult liver.