Mg2+/Mn2+-dependent phosphatase 1A is involved in regulating pregnane X receptor-mediated cytochrome p450 3A4 gene expression

Abstract

Variations in the expression of human pregnane X receptor (hPXR)-mediated cytochrome p450 3A4 (CYP3A4) in liver can alter therapeutic response to a variety of drugs and may lead to potential adverse drug interactions. We sought to determine whether Mg(2+)/Mn(2+)-dependent phosphatase 1A (PPM1A) regulates hPXR-mediated CYP3A4 expression. PPM1A was found to be coimmunoprecipitated with hPXR. Genetic or pharmacologic activation of PPM1A led to a significant increase in hPXR transactivation of CYP3A4 promoter activity. In contrast, knockdown of endogenous PPM1A not only attenuated hPXR transactivation, but also increased proliferation of HepG2 human liver carcinoma cells, suggesting that PPM1A expression levels regulate hPXR, and that PPM1A expression is regulated in a proliferation-dependent manner. Indeed, PPM1A expression and hPXR transactivation were found to be significantly reduced in subconfluent HepG2 cells compared with confluent HepG2 cells, suggesting that both PPM1A expression and hPXR-mediated CYP3A4 expression may be downregulated in proliferating livers. Elevated PPM1A levels led to attenuation of hPXR inhibition by tumor necrosis factor-α and cyclin-dependent kinase-2, which are known to be upregulated and essential during liver regeneration. In mouse regenerating livers, similar to subconfluent HepG2 cells, expression of both PPM1A and the mouse PXR target gene cyp3a11 was found to be downregulated. Our results show that PPM1A can positively regulate PXR activity by counteracting PXR inhibitory signaling pathways that play a major role in liver regeneration. These results implicate a novel role for PPM1A in regulating hPXR-mediated CYP3A4 expression in hepatocytes and may explain a mechanism for CYP3A repression in regenerating livers.

Fig. 1.

PPM1A coimmunoprecipitates with hPXR, and activation of PPM1A enhances transactivation function of hPXR. (A) COS-7 cells were cotransfected with FLAG-hPXR and PPM1A or FLAG-pcDNA. Twenty-four hours post-transfection, the cells were treated with either DMSO or 10 µM rifampicin for 24 hours and lysed with Triton lysis buffer. The interaction of FLAG-hPXR with PPM1A was examined by immunoprecipitation with the anti-FLAG antibody, followed by Western blot analysis with anti-PPM1A antibody. The same lysates were also Western blotted with the anti-PPM1A antibody. Activation of PPM1A enhances transactivation function of hPXR. HepG2 (B, D, and E) and COS-7 (C) cells were cotransfected with hPXR, CYP3A4-luc, PPM1A, and CMV-Renilla (transfection control), and treated with DMSO, 1 or 10 μM rifampicin (RIF), 10 μM C6-ceramide ± 10 μM RIF, or 10 μM dihydro-C6-ceramide ± 10 μM RIF. Firefly and Renilla luciferase activities were measured 24 hours after the treatments, and the relative luminescence units were determined by normalizing with the Renilla luciferase control. Data represent mean ± S.D. from eight independent experiments. Statistical significance (*P < 0.05) was determined using unpaired Student’s t test. In (B) and (C), PPM1A-cotransfected samples were compared with hPXR-transfected samples in each treatment group.

Fig. 2.

Downregulation of PPM1A levels impairs hPXR activity. (A) HepG2 cells stably expressing hPXR and CYP3A4-luc were transduced with lentiviral vectors carrying control nonsilencing shRNA or PPM1A shRNA. Whole-cell lysates were collected and subjected to Western blot analysis using anti-PPM1A and anti-actin antibodies (as a loading control). Data shown are from a representative experiment. (B) Knockdown of PPM1A impairs hPXR transactivation of CYP3A4 promoter activity. The transduced and nontransduced HepG2 cells were treated for 24 hours with DMSO or 10 µM rifampicin (RIF), and Firefly luciferase activity was measured and normalized with the total number of live cells. The relative luciferase activity is shown as the mean ± S.D. from six independent observations. Statistical significance (*P < 0.05) was determined using unpaired Student’s t test by comparing PPM1A shRNA samples with no shRNA samples in each treatment group.

Fig. 3.

PPM1A protein levels (B) and hPXR activity (C) are downregulated in subconfluent proliferating HepG2 cells. (A) Both confluent and subconfluent HepG2 cells were analyzed for cell cycle distribution using flow cytometry, as described in Materials and Methods. (B) Whole-cell lysates were collected from confluent and subconfluent HepG2 cells and subjected to Western blot analysis using anti-PPM1A and anti-actin antibodies. Data shown are from a representative experiment. (C) hPXR transactivation of CYP3A4 promoter activity was determined in confluent and subconfluent HepG2 cells. The cells were transiently cotransfected with pGL3-CYP3A4-luc, CMV-Renilla (transfection control), and pcDNA3 or pcDNA3-hPXR plasmids. After 24-hour transfection, the cells were treated with DMSO or 10 µM rifampicin for another 24 hours. Firefly and Renilla luciferase activities were measured 24 hours after the treatments using Dual-Glo luciferase assay system. CYP3A4 promoter activity was determined by normalizing the Firefly luciferase activity with the Renilla luciferase control. The results are presented as fold increase over DMSO, and the values represent the mean ± S.D. of four experiments. *P < 0.05, determined by unpaired Student’s t test.

Fig. 4.

PPM1A activation attenuates hPXR inhibition by TNFα (A) and CDK2 (B), and Cyp3a11 (C) levels correlate positively with PPM1A (D) levels in mouse regenerating livers. HepG2 cells were cotransfected with FLAG-hPXR, CYP3A4-luc, PPM1A, CDK2 (and cyclin E; data not shown), and CMV-Renilla. The cells were treated with DMSO, 10 μM rifampicin (RIF), or 20 ng/ml TNFα ± 10 µM RIF. The luciferase activities were measured 20 hours after the treatments. The relative luminescence units were shown as the means ± S.D. of five to six experiments. *P < 0.05, determined by unpaired Student’s t test. Cyp3a11 levels correlate positively with PPM1A levels in mouse regenerating livers. mRNA levels of Cyp3a11 (C) and mPXR (E) and protein levels of PPM1A (D) were shown in mouse regenerating livers at different time points (0 hours to 14 days) after PHX. mRNA levels were determined by using quantitative reverse-transcription polymerase chain reaction and by normalizing to 18S mRNA level. Protein levels were determined using Western blot analysis and by normalizing to actin protein level. Data represent mean ± S.D. (n = 3). *P < 0.05; compared with control (0 hours) by unpaired Student’s t test. Proposed mechanism for PPM1A regulation of PXR-mediated CYP3A expression in liver. (F) The current model of PXR activation via PPM1A directly or indirectly by desensitizing PXR inhibitory signaling pathways that are essential for liver regeneration. (G) One of the proposed mechanisms of CYP3A repression during liver regeneration.