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-a 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.

Pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolines: Novel compounds that reverse ABCG2-mediated resistance in cancer cells

“…HepG2 cells were transiently transfected with pcDNA3-hPXR and CYP3A4-luc plasmids using FuGENE 6 (Promega, Madison, WI) [17,18]. After 24 h in the growth media, the cells were seeded at a density of approximately 10,000 cells/well in 96-well assay plates (PerkinElmer, Waltham, MA) in phenol red-free DMEM containing 5% charcoal/dextran-treated fetal bovine serum.…”

“…HEK293/pcDNA3.1, NCI-H460 and the multidrug-resistant cell lines HEK293/pcDNA3.1, ABCG2/T10, ABCG2/R2, H460-MX20, and S1-M1-80 were obtained from Dr. Gary Kruh's lab at University of Chicago, Illinois. HepG2 human liver carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) [17,18]. All the cell lines were grown as adherent monolayers in flasks with DMEM culture medium (Hyclone) containing 10% bovine serum and 1% penicillin/ streptomycin at 37°C in a humidified atmosphere of 5% CO2.…”

“…After 24 h in the growth media, the cells were seeded at a density of approximately 10,000 cells/well in 96-well assay plates (PerkinElmer, Waltham, MA) in phenol red-free DMEM containing 5% charcoal/dextran-treated fetal bovine serum. The cells were then treated with the vehicle control DMSO, RIF, or IND compounds for an additional 24 h before performing luciferase assays by measuring luminescence using the using the Neolite Reporter Gene Assay System (PerkinElmer) and a FLUOstar Optima microplate reader (BMG Labtech, Cary, NC) [17,18]. CYP3A4 promoter activity was expressed as luminescence units.…”

“…All other reagents and chemicals were purchased from VWR (Atlanta, GA). The pcDNA3-hPXR and pGL3-CYP3A4-luc plasmids were as previously described [17,18].…”

Stearidonic acid, a plant-based dietary fatty acid, enhances the chemosensitivity of canine lymphoid tumor cells

“…The cells were then either untreated or treated with PBS, DMSO, ETOH, VCR, DOXO, SDA ± VCR, or SDA ± DOX for 24 h. Next, the CellTiter-Glo luminescent cell viability assays (Promega) were performed to determine the number of viable cells by quantifying the ATP present, which indicates the presence of metabolically active cells [23,24].…”