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

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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.