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.
- MeSH
- fosforylace účinky léků MeSH
- hepatocyty účinky léků metabolismus MeSH
- inhibitory histondeacetylas farmakologie MeSH
- kultivované buňky MeSH
- kyselina valproová farmakologie MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buňky Hep G2 MeSH
- butadieny farmakologie MeSH
- cytochrom P-450 CYP3A genetika MeSH
- hepatocyty účinky léků enzymologie MeSH
- inhibitory enzymů farmakologie MeSH
- izoenzymy MeSH
- lidé MeSH
- ligandy MeSH
- luciferasy renil genetika MeSH
- MAP kinasa-kinasa 1 antagonisté a inhibitory MeSH
- MAP kinasa-kinasa 2 antagonisté a inhibitory MeSH
- MAP kinasový signální systém účinky léků MeSH
- nitrily farmakologie MeSH
- plazmidy MeSH
- primární buněčná kultura MeSH
- promotorové oblasti (genetika) MeSH
- steroidní receptory metabolismus MeSH
- transfekce MeSH
- upregulace MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
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.
- Publikační typ
- časopisecké články MeSH
Valproic acid (VPA) is a wide spread anticonvulsant and mood-stabilizing agent, the use of which is associated with hepatotoxicity, bone marrow suppression and osteomalacia. In the current paper we propose a possible mechanism of VPA-induced osteomalacia involving accelerated catabolism of 1α,25(OH)(2)-vitamin D3 (VD3) due to increased expression of CYP24. We demonstrate that VPA strongly potentiates CYP24 mRNA expression by VD3 in human hepatocytes (HH) and in human embryonic kidney cells (HEK293). By the method of gene reporter assay we found that VPA increases basal and VD3-inducible activity of CYP24 promoter (pCYP24-luc) in human liver adenocarcinoma (HepG2) and in HEK293 cells in dose-dependent manner. In order to delineate the role of inhibitory effects of VPA on histone deacetylase 1 (HDAC1), we compared the effects of VPA with trichostatin A (TSA) on basal and inducible levels of CYP24 mRNA and pCYP24-luc transactivation. Transactivation of CYP24 promoter by VD3 was enhanced in the presence of both TSA and VPA. In contrast, VD3-inducible expression of CYP24 mRNA was enhanced by VPA but not by TSA, implying that HDAC1 inhibition is not the major reason for VPA effects on CYP24. We examined the effects of VPA on mitogen-activated protein kinases as the important transcriptional regulators of VDR. VPA activated extracellular signal-regulated kinase (ERK) but not c-Jun-N-terminal kinase (JNK) and p38 MAPKs. In conclusion, VPA enhances transcriptional activity of VDR and increases expression of CYP24 mRNA in the presence of VD3 in physiological concentrations. The mechanism involves activation of ERK and partly the inhibition of HDAC1.
- MeSH
- antikonvulziva toxicita MeSH
- cholekalciferol farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy genetika metabolismus MeSH
- HEK293 buňky MeSH
- hepatocyty účinky léků MeSH
- histondeacetylasa 1 biosyntéza genetika MeSH
- kyselina valproová toxicita MeSH
- lidé MeSH
- luciferasy genetika MeSH
- messenger RNA biosyntéza genetika MeSH
- osteomalacie chemicky indukované patologie MeSH
- plazmidy genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- receptory kalcitriolu účinky léků MeSH
- steroidhydroxylasy biosyntéza genetika MeSH
- steroidní receptory účinky léků MeSH
- transfekce MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: 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 S-enantiomer has three to five times greater anticoagulation potency than its optical congener. Recently, vitamin K2 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 up-regulate 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 co-administered drugs. The results warrant reconsideration of racemic warfarin usage in clinics.
- MeSH
- aktivace transkripce MeSH
- antikoagulancia chemie farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese enzymů účinky léků MeSH
- reportérové geny MeSH
- rezonanční přenos fluorescenční energie MeSH
- stereoizomerie MeSH
- steroidní receptory účinky léků MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- techniky dvojhybridového systému MeSH
- upregulace účinky léků MeSH
- warfarin chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- cytochrom P-450 CYP3A genetika účinky léků MeSH
- farmakogenetika metody trendy MeSH
- financování organizované MeSH
- jednonukleotidový polymorfismus genetika účinky léků MeSH
- lidé MeSH
- reportérové geny genetika účinky léků MeSH
- steroidní receptory genetika účinky léků MeSH
- transkripční faktory účinky léků MeSH
- virové receptory genetika účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- abstrakty MeSH
- MeSH
- buněčné linie účinky léků MeSH
- cytochrom P-450 CYP1A1 metabolismus účinky léků MeSH
- dexamethason farmakologie MeSH
- dioxiny farmakologie metabolismus MeSH
- financování organizované MeSH
- glukokortikoidy farmakologie MeSH
- interakce mezi receptory a ligandy účinky léků MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody využití MeSH
- receptory aromatických uhlovodíků účinky léků MeSH
- represorové proteiny účinky léků MeSH
- trofoblasty účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- abstrakty MeSH
