Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

Abstract

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 (PP2C␤l), a unique phosphatase that was originally cloned from human liver. In this study, we sought to determine whether PP2C␤l is involved in regulating hPXR's transactivation activity and whether PP2C␤l affects CDK2 regulation of this activity in HepG2 liver carcinoma cells. In transactivation assays, transiently coexpressed PP2C␤l 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 PP2C␤l promoted cell proliferation, inhibited the interaction of hPXR with steroid receptor coactivator-1, and attenuated the hPXR transcriptional activity. The levels of PP2C␤l did not affect hPXR expression. Our results show for the first time that PP2C␤l 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 PP2C␤l 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.

Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

“…For example, co-treatment of patients with St. John’s wort and other drugs caused failure of the therapeutic drugs, mainly due to activation of PXR by St. John’s wort, which causes CYP3A4 induction resulting in enhanced drug metabolism [49, 50]. Given that xenobiotics activate hPXR by post-translational modifications such as phosphorylation [34, 51], further investigation is needed to determine whether and how MET could indirectly activate hPXR in addition to the direct-binding mechanism we report here.…”

Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

“…The pcDNA3, pcDNA3-hPXR, FLAG-pcDNA3, FLAG-pcDNA3-hPXR, pEF-rPXR WT, pEF-rPXR F305L, pGL3-CYP3A4-luc and pGL3-CMV-Renilla luciferase plasmids were previously described (Kittayaruksakul et al, 2013; Lin et al, 2008; Mu et al, 2005; Pondugula et al, 2009a, 2010). pcDNA3-mPXR plasmid was provided by Dr. Steven Kliewer (University of Texas Southwestern Medical Center).…”

Mg2+/Mn2+-Dependent Phosphatase 1A Is Involved in Regulating Pregnane X Receptor–Mediated Cytochrome p450 3A4 Gene Expression

“…Dimethylsulfoxide (DMSO), rifampicin, C 6 -ceramide, dihydro-C 6 -ceramide, and tumor necrosis factor a (TNFa) were purchased from Sigma-Aldrich (St. Louis, MO). The pcDNA3, FLAGpcDNA3, pcDNA3-hPXR, FLAG-pcDNA3-hPXR, pcDNA3-PPM1A, CDK2, cyclin E, pGL3-CYP3A4-luc, and pGL3-CMV-Renilla plasmids were previously described (Lin et al, 2008;Pondugula et al, 2010Pondugula et al, , 2014Shohat et al, 2012).…”

“…Reverse transcription was performed with the QuantiTect Reverse Transcription Kit (Qiagen), and quantitative polymerase chain reaction was performed by using the QuantiTect SYBR Green Kit (Qiagen), iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA), and gene-specific primers of 18S rRNA (housekeeping gene), mouse PXR, and Cyp3a11 (Supplemental Table 1). Relative mRNA expression was calculated as described previously (Pondugula et al, 2010(Pondugula et al, , 2014.…”