Lucie Stejskalova

Publications (8)28.33 Total impact

• Article: Rifampicin Does not Significantly Affect the Expression of Small Heterodimer Partner in Primary Human Hepatocytes.

  Petr Pavek, Lucie Stejskalova, Lucie Krausova, Michal Bitman, Radim Vrzal, Zdenek Dvorak
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  ABSTRACT: 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.
  Frontiers in pharmacology. 01/2012; 3:1.
• Article: Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene.

  Lucie Krausova, Lucie Stejskalova, Hongwei Wang, Radim Vrzal, Zdenek Dvorak, Sridhar Mani, Petr Pavek
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  ABSTRACT: Metformin is widely used in the treatment of type-2 diabetes. The pleotropic effects of metformin on glucose and lipid metabolism have been proposed to be mediated by the activation of AMP-activated protein kinase (AMPK) and the subsequent up-regulation of small heterodimer partner (SHP). SHP suppresses the functions of several nuclear receptors involved in the regulation of hepatic metabolism, including pregnane X receptor (PXR), which is referred to as a "master regulator" of drug/xenobiotic metabolism. In this study, we hypothesize that metformin suppresses the expression of CYP3A4, a main detoxification enzyme and a target gene of PXR, due to SHP up-regulation. We employed various gene reporter assays in cell lines and qRT-PCR in human hepatocytes and in Pxr(-/-) mice. We show that metformin dramatically suppresses PXR-mediated expression of CYP3A4 in hepatocytes. Consistently, metformin significantly suppressed the up-regulation of Cyp3a11 mRNA in the liver and intestine of wild-type mice, but not in Pxr(-/-) mice. A mechanistic investigation of the phenomenon showed that metformin does not significantly up-regulate SHP in human hepatocytes. We further demonstrate that AMPK activation is not involved in this process. We show that metformin disrupts PXR's interaction with steroid receptor coactivator-1 (SRC1) in a two-hybrid assay independently of the PXR ligand binding pocket. Metformin also inhibited vitamin D receptor-, glucocorticoid receptor- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4 mRNA in human hepatocytes. We show, therefore, a suppressive effect of metformin on PXR and other ligand-activated nuclear receptors in transactivation of the main detoxification enzyme CYP3A4 in human hepatocytes.
  Biochemical pharmacology 09/2011; 82(11):1771-80. · 4.25 Impact Factor
• Article: Aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator expression in human and rat placentas and transcription activity in human trophoblast cultures.

  Lucie Stejskalova, Lenka Vecerova, Laura Mesa Peréz, Radim Vrzal, Zdenek Dvorak, Petr Nachtigal, Petr Pavek
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  ABSTRACT: Aryl hydrocarbon receptor (AHR) and its heterodimer aryl hydrocarbon nuclear translocator (ARNT) form a ligand-activated transcription complex that regulates expression of the AHR battery of target genes that includes the most important placental biotransformation enzyme cytochrome CYP1A1. Expression, placental localization, and ontogeny of AHR/Ahr and ARNT/Arnt have not been systematically studied in either human or rat placentas. Moreover, induction of such AHR target genes as CYP1A1, CYP1A2, CYP1B1, UGT1A1, and breast cancer resistance protein (BCRP), as well as of AHR, ARNT, and aryl hydrocarbon receptor repressor (AHRR) genes, after exposure to AHR ligands have not been studied in human placental trophoblast cultures. In this article, we show that only CYP1A1 messenger RNA (mRNA), but not CYP1A2, CYP1B1, UGT1A1, BCRP, AHR, ARNT, and AHRR mRNAs, is significantly induced in human term placental trophoblast cultures after exposure to prototype AHR ligands/activators 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, omeprazole, and β-naphthoflavone. We localized AHR/Ahr and ARNT/Arnt in rat placental trophoblasts throughout gestation and in first trimester and term human placental trophoblast, which comprise the crucial component of the maternal-fetal barrier. We demonstrate that rat Ahr and Cyp1a1 reached highest expression during gestation days 15 and 18, which might indicate different response to Ahr ligands in placental Cyp1a1 induction during rat gestation. We also propose the JEG3 choriocarcinoma cell line as a cellular model for human trophoblast induction studies through AHR. In conclusion, we describe expression and ontogeny of AHR/Ahr and ARNT/Arnt and systematically characterize induction of major AHR target genes in human placental trophoblast forming the placental maternal-fetal morphological and metabolic barrier.
  Toxicological Sciences 06/2011; 123(1):26-36. · 4.65 Impact Factor
• Article: Endogenous and exogenous ligands of aryl hydrocarbon receptor: current state of art.

  Lucie Stejskalova, Zdenek Dvorak, Petr Pavek
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  ABSTRACT: Aryl hydrocarbon receptor (AhR) is an important transcriptional regulator of drug metabolizing enzymes that dominantly controls the expression of cytochrome P450 CYP1 family genes and some phase II enzymes. AhR also has many endogenous functions including cell cycle control, immune response, and cell differentiation. In addition, AhR is well-known to be involved in chemically-induced carcinogenesis. AhR is activated by a variety of endogenous and exogenous ligands. While exogenous activation of AhR has deleterious effects on human organism, sustained activation of AhR by endogenous ligands is indispensable for proper cell functions. Therefore, the effects of exogenous and endogenous ligands on AhR resemble the Dr. Jekyll and Mr. Hyde story. The aim of the current paper is to summarize and update the knowledge on exogenous and endogenous AhR ligands.
  Current Drug Metabolism 03/2011; 12(2):198-212. · 5.11 Impact Factor
• Article: The function of cytochrome P450 1A1 enzyme (CYP1A1) and aryl hydrocarbon receptor (AhR) in the placenta.

  Lucie Stejskalova, Petr Pavek
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  ABSTRACT: CYP1A1, an enzyme of the cytochrome P450 superfamily, is the most important xenobiotic-metabolizing enzyme of the placenta for which relevant inducible activity has been demonstrated throughout pregnancy. CYP1A1 metabolizes several drugs and compounds widely used in pharmacotherapy or present in diets. At the same time, this enzyme plays a key role in the bioactivation of procarcinogens and proteratogens, such as arylamines and polycyclic aromatic hydrocarbons (PAHs), which bind to placental and foetal DNA as DNA-adducts. The expression of CYP1A1 is transcriptionally up-regulated through the ligand-activated aryl hydrocarbon receptor (AhR). AhR plays an important role as mediator of an adaptive response to xenobiotics, as well as in normal physiology and embryonic development. Several exogenous AhR ligands, such as PAHs, polychlorinated biphenyls and halogenated dioxins, can be found in the constituents of numerous commercial products, including insulators and flame retardants, or as products of combustion processes, including chimney soot, charbroiled foods and cigarette smoke, or as the product of waste incineration. Exposure to these compounds subsequently affects cellular growth and differentiation, homeostasis, level of growth factors, reproduction function and hormonal regulation. Importantly, elevated CYP1A1 activity through activated AhR in placentas of women smokers has been associated with pregnancy complications, such as premature birth, intrauterine growth retardation (IUGR), structural abnormalities, foetal death or placenta abruption, risk of low birth weight, low birth length and low head circumference. We summarize the recent findings related to toxicological consequences of AhR activation and CYP1A1 induction in the human placenta during pregnancy.
  Current pharmaceutical biotechnology 02/2011; 12(5):715-30. · 3.40 Impact Factor
• Source

  Available from: Jana Blazkova

  Article: Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene.

  Petr Pavek, Katerina Pospechova, Lucie Svecova, Zdenka Syrova, Lucie Stejskalova, Jana Blazkova, Zdenek Dvorak, Jaroslav Blahos
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  ABSTRACT: CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene. In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRalpha heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4. Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRalpha heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells. We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.
  Biochemical pharmacology 09/2009; 79(2):277-87. · 4.25 Impact Factor
• Article: Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines.

  Katerina Pospechova, Veronika Rozehnal, Lucie Stejskalova, Radim Vrzal, Nada Pospisilova, Gabriela Jamborova, Karen May, Werner Siegmund, Zdenek Dvorak, Petr Nachtigal, Vladimir Semecky, Petr Pavek
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  ABSTRACT: Vitamin D receptor (VDR) regulates the expression of many genes involved in mineral metabolism, cellular proliferation, differentiation and drug biotransformation. We studied the expression and activity of VDR and its heterodimerization partner retinoid X receptor-alpha (RXRalpha) in choriocarcinoma trophoblast cell lines BeWo and JEG-3, in comparison with human isolated placental cytotrophoblasts and human full term placenta. We found that VDR and RXRalpha are localised in the human term placenta trophoblast and expressed in isolated cytotrophoblasts. However, we found low expression and no transcriptional activity of VDR in used choriocarcinoma cell lines. The inhibitor of DNA methylation, 5-deoxy-3'-azacytidine, and histone deacetylase inhibitor sodium butyrate partially restored the expression of VDR, suggesting an epigenetic suppression of the gene in choriocarcinoma cells. Differentiation of BeWo cells resulted in up-regulation of VDR mRNA. Finally, we observed a non-genomic effect of 1,25(OH)(2)D(3) in the activation of the extracellular signal-regulated kinase (ERK) signalling pathway in JEG-3 cells. In conclusion, our results suggest an epigenetic repression of VDR gene expression and activity in choriocarcinoma cell lines, and a non-genomic effect of 1,25(OH)(2)D(3) in JEG-3 cells.
  Molecular and Cellular Endocrinology 01/2009; 299(2):178-87. · 4.19 Impact Factor
• Article: Dexamethasone controls aryl hydrocarbon receptor (AhR)-mediated CYP1A1 and CYP1A2 expression and activity in primary cultures of human hepatocytes.

  Radim Vrzal, Lucie Stejskalova, Katalin Monostory, Patrick Maurel, Petr Bachleda, Petr Pavek, Zdenek Dvorak
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  ABSTRACT: CYP1A1 and CYP1A2 genes encode members of the cytochrome P450 superfamily of enzymes primarily involved in xenobiotic and drug metabolism. In this paper we examined the effects of synthetic glucocorticoid dexamethasone (DEX) on aryl hydrocarbon receptor (AhR)-mediated regulation of CYP1A1 and CYP1A2 genes and their enzymatic activity in primary cultures of human hepatocytes obtained from 17 donors and prepared in 3 countries. Dexamethasone significantly reduced both basal and inducible CYP1A1/2 ethoxyresorufin-O-deethylase (EROD) activities by more than 75 and 50%, respectively. Glucocorticoid receptor (GR) antagonist RU486 abolished this effect suggesting the involvement of GR in the process. In contrast, dexamethasone significantly augmented transcriptional activation of CYP1A2 mRNA but not CYP1A1 gene by prototype AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). Dexamethasone had no effect on basal and TCDD-inducible levels of CYP1As proteins; however, it reduced the levels of AhR and GRalpha mRNAs and AhR protein levels. In addition, using RT(2) Profiler PCR Array, we found the effect of dexamethasone on the expression of several co-activators of AhR and GR nuclear receptors in the primary human hepatocytes. We conclude that dexamethasone controls CYP1A1 and CYP1A2 expression and activity in human hepatocytes via multiple mechanisms, which remain to be elucidated.
  Chemico-biological interactions 12/2008; 179(2-3):288-96. · 2.46 Impact Factor