Abstract
In our study, we tested the hypothesis whether valproic acid (VPA) in therapeutic concentrations has potential to affect expression of CYP3A4 and MDR1 via constitutive androstane receptor (CAR) and pregnane X receptor (PXR) pathways. Interaction of VPA with CAR and PXR nuclear receptors was studied using luciferase reporter assays, real-time reverse transcriptase polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA), and analysis of CYP3A4 catalytic activity. Using transient transfection reporter assays in HepG2 cells, VPA was recognized to activate CYP3A4 promoter via CAR and PXR pathways. By contrast, a significant effect of VPA on MDR1 promoter activation was observed only in CAR-cotransfected HepG2 cells. These data well correlated with up-regulation of CYP3A4 and MDR1 mRNAs analyzed by real-time RT-PCR in cells transfected with expression vectors encoding CAR or PXR and treated with VPA. In addition, VPA significantly up-regulated CYP3A4 mRNA in primary hepatocytes and augmented the effect of rifampicin. EMSA experiments showed VPA-mediated augmentation of CAR/retinoid X receptor α heterodimer binding to direct repeat 3 (DR3) and DR4 responsive elements of CYP3A4 and MDR1 genes, respectively. Finally, analysis of specific CYP3A4 catalytic activity revealed its significant increase in VPA-treated LS174T cells transfected with PXR. In conclusion, we provide novel insight into the mechanism by which VPA affects gene expression of CYP3A4 and MDR1 genes. Our results demonstrate that VPA has potential to up-regulate CYP3A4 and MDR1 through direct activation of CAR and/or PXR pathways. Furthermore, we suggest that VPA synergistically augments the effect of rifampicin in transactivation of CYP3A4 in primary human hepatocytes.
Footnotes
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This work was supported in part by grants from The Grant Agency of the Charles University in Prague (94/2005/C), the Czech Science Foundation (170/53/75301), and the Ministry of Education of the Czech Republic (MSM 6198959216).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.014456.
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ABBREVIATIONS: VPA, valproic acid; HDAC, histone deacetylase; CITCO, (6-(4-chlorophenyl) imidazo [2,1-b][1,3] thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime); NR, nuclear receptor; RT-PCR, reverse transcriptase polymerase chain reaction; PXR, pregnane X receptor; CAR, constitutive androstane receptor; RXRα, retinoid X receptor α (9-cis retinoic acid receptor-α); FCS, fetal calf serum; DMSO, dimethyl sulfoxide; DR, direct repeat; ER, everted repeat; HPRT, hypoxanthine-guanine phosphoribosyl transferase; B2M, β2-microglobulin; PBREM, phenobarbital responsive enhancer module; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance.
- Received December 20, 2006.
- Accepted March 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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