The small/short heterodimer partner (SHP, NR0B2) is a nuclear receptor corepressor lacking a DNA binding domain. SHP is induced by bile acid-activated farnesoid X receptor (FXR) resulting in CYP7A1 gene suppression. In contrast, Pregnane X receptor (PXR) activation by its ligands was recently suggested to inhibit SHP gene transactivation to maximize the induction of PXR target genes. However, there are also conflicting reports in literature whether PXR or rodent Pxr activation down-regulates SHP/Shp expression. Moreover, the PXR-mediated regulation of the SHP gene has been studied only at the SHP mRNA and transactivation (gene reporter assay) levels. In this study, we studied the effect of rifampicin, a prototype PXR ligand, on SHP mRNA, and protein expression in three primary human hepatocyte cultures. We found that SHP mRNA is not systematically down-regulated in hepatocyte in culture after 24 h treatment with rifampicin. Consistently, we did not observe down-regulation of SHP protein in primary human hepatocytes after 24 and 48 h of incubation with rifampicin. We can conclude that although we observed slight down-regulation of SHP mRNA and protein in several hepatocyte preparations, the phenomenon is unlikely critical for PXR-mediated induction of its target genes.

To cite this article: Rulcova A, Prokopova I, Krausova L, Bitman M, Vrzal R, Dvorak Z, Blahos J, Pavek P. Stereoselective interactions of warfarin enantiomers with pregnane X nuclear receptor in gene regulation of major drug-metabolizing cytochrome P450 enzymes. J Thromb Haemost 2010; 8: 2708-17.See also Miller GP. Warfarin therapy: how the less interesting half just got interesting. This issue, pp 2705-7.Summary. Background: Warfarin, an antagonist of vitamin K, is an oral coumarin anticoagulant widely used to control and prevent thromboembolic disorders. Warfarin is clinically available as a racemic mixture of R-and S-warfarin. The Senantiomer has three to five times greater anticoagulation potency than its optical congener. Recently, vitamin K 2 function has been proposed via the pregnane X receptor (PXR) in osteocytes. PXR acts as a xenobiotic sensor that controls expression of many genes involved in drug/xenobiotic metabolic clearance. Objective: The aim was to examine whether enantiomers of warfarin stereoselectively interact with PXR to upregulate main drug/xenobiotic-metabolizing enzymes of the cytochrome P450 superfamily. Methods: Interactions of warfarin enantiomers with PXR were tested by gene reporter assays and time-resolved fluorescence resonance energy transfer technology (TR-FRET) ligand binding assay. Up-regulation of PXR-target gene mRNAs by warfarin enantiomers was studied using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in primary human hepatocytes. Results: We found that R-warfarin interacts with the PXR nuclear receptor. Consistently, R-warfarin significantly induced CYP3A4 and CYP2C9 mRNAs in cultures of primary human hepatocytes or in LS174T intestinal cells. On the other hand, S-warfarin is a less potent inducer of PXR-target genes in human hepatocytes and activates PXR only at supraphysiological concentrations. In addition, we showed that racemic 10-and 4¢-hydroxywarfarins are also highly potent PXR ligands and inducers of CYP3A4 and CYP2C9 mRNA in human hepatocytes. Conclusion: We showed that R-warfarin can significantly up-regulate major drug-metabolizing enzymes CYP3A4 and CYP2C9 in the liver and thus may cause drug-drug interactions (DDI) with coadministered drugs. The results warrant reconsideration of racemic warfarin usage in clinics.

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Hepatocyte tumor cell lines lack the expression or induction properties of major cytochrome P450 (CYP) enzymes compared to primary human hepatocytes. The Ras/Raf/MEK/ERK signaling cascade contributes to hepatocarcinogenesis, dedifferentiation and loss of hepatocyte drug metabolism in hepatocyte tumors. In the present study, we examined whether MEK1/2 inhibitors can restore the expression of CYP genes in hepatocarcinoma HepG2 cells. We found that U0126, a prototype dual MEK1/2 inhibitor, is a potent inducer of CYP3A4, CYP3A5 and CYP3A7 mRNA expression (>100-fold) in HepG2 cells and CYP3A4 mRNA expression in primary human hepatocytes. This U0126-mediated induction is sensitive to the transcriptional inhibitor actinomycin D and was not detected for CYP2B6 or MDR1 mRNA expression. In gene reporter assays, U0126 activates a CYP3A4 promoter luciferase reporter construct containing PXR response elements (PXREs), but not a construct containing mutated PXREs. Based on a ligand binding assay and the examination of a PXR mutant expressing an obstructed ligand binding pocket, we found that U0126 is a ligand of PXR. We also found that U0126 up-regulates the mRNA expression of the nuclear receptors HNF4α, CAR, VDR and PXR but abolishes small heterodimer partner (SHP) corepressor expression in HepG2 cells. The MEK1/2 inhibitors PD0325901 and PD184352, as well as dominant-negative MEK1 expression, also down-regulate SHP mRNA expression. In contrast, dominant-negative MEK1 expression does not significantly induce CYP3A4 gene in HepG2 cells. In conclusion, we found that U0126 is an atypical PXR ligand that via direct (binding and activation of PXR) and indirect (SHP dowregulation) mechanisms selectively restores CYP3A genes in HepG2 cells.

Aim. Valproic acid (VPA) is a widely-used anticonvulsant and mood-stabilizing agent. VPA is also known to inhibit histone deacetylases (HDACs) affecting the expression of numerous genes. Methods. In the present study, we examined the effect of VPA on the extracellular signal-related kinase (ERK, p42/p44) pathway (Ras-Raf-MEK-ERK) belonging to the mitogen-activated protein kinases (MAPKs) pathways in primary human hepatocytes. In the liver, the pathway is associated with progression of hepatocellular carcinoma. Results. We found that VPA in a therapeutically relevant concentration (500 μM) activates the ERK pathway, as indicated by increased ERK Thr202/Tyr204 phosphorylation. Interestingly, a prototype HDAC inhibitor, trichostatin A, also activated ERK phosphorylation in primary human hepatocytes. These data suggest that HDAC inhibition might be the primary stimulus for ERK pathway activation in primary human hepatocytes. Notably, U0126, a MEK1 inhibitor, was ineffective in inhibiting ERK pathway activation, likely due to its metabolic deactivation in metabolically competent primary human hepatocytes. Conclusion. We conclude that VPA activates the ERK pathway in primary human hepatocytes.