The human pregnane X receptor (hPXR) regulates the expression of cytochrome P450 3A4 (CYP3A4), which plays vital roles in hepatic drug metabolism and has considerably reduced expression levels in proliferating hepatocytes. We have recently shown that cyclin-dependent kinase 2 (CDK2) negatively regulates hPXR-mediated CYP3A4 gene expression. CDK2 can be dephosphorylated and inactivated by protein phosphatase type 2C beta isoform long (PP2Cβl), a unique phosphatase that was originally cloned from human liver. In this study, we sought to determine whether PP2Cβl is involved in regulating hPXR's transactivation activity, and whether PP2Cβl affects CDK2 regulation of this activity in HepG2 liver carcinoma cells. In transactivation assays, transiently coexpressed PP2Cβl significantly enhanced the hPXR-mediated CYP3A4 promoter activity and decreased the inhibitory effect of CDK2 on hPXR transactivation activity. In addition, shRNA-mediated downregulation of endogenous PP2Cβl promoted cell proliferation, inhibited the interaction of hPXR with steroid receptor coactivator-1 (SRC-1), and attenuated the hPXR transcriptional activity. The levels of PP2Cβl did not affect hPXR expression. Our results show for the first time that PP2Cβl is essential for hPXR activity and can positively regulate this activity by counteracting the inhibitory effect of CDK2. Our results implicate a novel and important role for PP2Cβl in regulating hPXR activity and CYP3A4 expression by inhibiting or desensitizing signaling pathways that negatively regulate the function of PXR in liver cells and are consistent with the notion that both the activity of hPXR and the expression of CYP3A4 are regulated in a cell cycle¡Vdependent and cell proliferation-dependent manner.
- Received January 8, 2010.
- Accepted June 9, 2010.
- The American Society for Pharmacology and Experimental Therapeutics