Protein phosphatase 2Cbetal regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells

Abstract

The human pregnane X receptor (hPXR) regulates the expression of CYP3A4, which plays a vital role 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 (PP2Cbetal), a unique phosphatase that was originally cloned from human liver. In this study, we sought to determine whether PP2Cbetal is involved in regulating hPXR's transactivation activity and whether PP2Cbetal affects CDK2 regulation of this activity in HepG2 liver carcinoma cells. In transactivation assays, transiently coexpressed PP2Cbetal significantly enhanced the hPXR-mediated CYP3A4 promoter activity and decreased the inhibitory effect of CDK2 on hPXR transactivation activity. In addition, shRNA-mediated down-regulation of endogenous PP2Cbetal promoted cell proliferation, inhibited the interaction of hPXR with steroid receptor coactivator-1, and attenuated the hPXR transcriptional activity. The levels of PP2Cbetal did not affect hPXR expression. Our results show for the first time that PP2Cbetal 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 PP2Cbetal in regulating hPXR activity and CYP3A4 expression by inhibiting or desensitizing signaling pathways that negatively regulate the function of pregnane X receptor 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-dependent and cell proliferation-dependent manner.

Fig. 1.

PP2Cβl positively regulates hPXR function. A, PP2Cβl enhances hPXR-mediated CYP3A4 promoter activity and counteracts the inhibitory effect of CDK2 on hPXR. HepG2 cells were cotransfected with CYP3A4-luc− and hPXR−, as well as cyclin E−, CDK2−, or PP2Cβl− plasmids, as indicated, and CMV-Renilla luciferase plasmid (as a transfection control). Transactivation assays were performed as described under Materials and Methods. The values represent the means of six independent experiments, and the bars denote S.D. The p value was ascertained using the Student's t test, and differences were considered significant for p < 0.05 (*) or 0.001 (***) and nonsignificant for p > 0.05. B, cotransfection of PP2Cβl does not alter hPXR protein levels. Whole-cell lysates of the transiently transfected cells in A were subjected to Western blot analysis using rabbit polyclonal anti-hPXR serum. The pcDNA3 vector transfection was used as a negative control, and actin expression was analyzed as a loading control. Data shown are from a representative experiment.

Fig. 2.

Knockdown of PP2Cβl attenuates hPXR-mediated CYP3A4 promoter activity. A, PP2Cβl shRNA knocks down the protein expression of endogenous PP2Cβl. HepG2 cells stably expressing hPXR and CYP3A4-luc (lane 1) were transduced with control nonsilencing shRNA (lane 2) or PP2Cβl shRNA (lane 3). Whole-cell lysates were collected and subjected to Western blot analysis using anti-PP2Cβl and anti-actin antibodies (as a loading control). Data shown are from a representative experiment. B, knockdown of PP2Cβl attenuates hPXR-mediated CYP3A4 promoter activity. HepG2 cells stably expressing hPXR and CYP3A4-luc were transduced with either control nonsilencing shRNA or PP2Cβl shRNA, and transactivation assays were performed as described under Materials and Methods. The RLA is shown as the mean value and S.D. from six independent observations. The Student's t test was used to determine statistical significance of unpaired samples by comparing the RLA obtained from the samples transduced with PP2Cβl shRNA to that of samples that were either untransduced or transduced with control shRNA. Differences were considered significant for p < 0.01 (**) or 0.001 (***) and nonsignificant (NS) for p > 0.05. C, overexpressing PP2Cβl rescues hPXR-mediated CYP3A4 promoter activity in PP2Cβl knockdown cells (HepG2/PP2Cβl shRNA). HepG2/PP2Cβl shRNA cells were transiently transfected with CMV-Renilla and either pcDNA3 or pcDNA3-PP2Cβl, and then the transactivation assays were performed as described under Materials and Methods section. The activity of firefly luciferase was normalized with that of the Renilla to determine relative luminescence units (RLU). The data represent the means and S.D.s of six independent experiments. The p value was determined using the Student's t test by comparing the samples transfected with pcDNA3 to PP2Cβl. Differences were considered significant for p < 0.05 (*) or 0.01 (**). D, PP2Cβl affects agonist-induced hPXR interaction with SRC-1. Mammalian two-hybrid assays were performed in HepG2/Control shRNA and HepG2/PP2Cβl shRNA cells as described under Materials and Methods. Induction of pG5-luc was used to measure the rifampicin-induced hPXR/SRC-1 interaction.

Fig. 3.

Knockdown of PP2Cβl affects cell cycle distribution and cell proliferation. A, flow cytometry cell cycle analysis of HepG2/Control shRNA and HepG2/ PP2Cβl shRNA cells. The Student's t test was used to determine statistical significance of unpaired samples by comparing the percentage of cells transduced with control shRNA or PP2Cβl shRNA in different phases of the cell cycle. Differences were considered significant for p < 0.05 (*) or 0.001 (***) and nonsignificant (NS) for p > 0.05. B, RT-CES was used to monitor the cell proliferation of HepG2/Control shRNA and HepG2/PP2Cβl shRNA cells. The cell index represents the means of eight independent samples, and the bars indicate the S.D. The Student's t test was used to determine statistical significance of unpaired samples by comparing the cell index of HepG2/Control shRNA cells to that of HepG2/PP2Cβl shRNA cells. Differences were considered significant for p < 0.05 (*) or 0.001 (***). p values are shown for two time points (i.e., 33 and 44 h after monitoring was started) corresponding to 45 and 56 h after cell seeding.