Dexamethasone accelerates degradation of aryl hydrocarbon receptor (AHR) and suppresses CYP1A1 induction in placental JEG-3 cell line.

The JEG-3 choriocarcinoma cell line has been proposed as a model cell line of human placental trophoblast for induction studies via aryl hydrocarbon receptor (AHR). We examined whether glucocorticoid dexamethasone influences AHR-mediated induction of CYP1A1 enzyme in the JEG-3 cell line. We found that dexamethasone dose- and time-dependently suppresses CYP1A1 transactivation in gene reporter assays, CYP1A1 mRNA induction, and upregulation of 7-ethoxyresorufin-O-deethylase (EROD) activity by 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in JEG-3 cells. Co-transfection of JEG-3 cells with glucocorticoid receptor (GR) expression construct and treatment with dexamethasone abolished the effect of MC on CYP1A1 promoter construct in transient transfection gene reporter assays. RU486, a GR antagonist, suppressed the effect of dexamethasone on MC-induced transactivation of AHR responsive reporter constructs. We also found that dexamethasone stimulates both ligand-dependent and ligand-independent degradation of AHR but not of aryl hydrocarbon receptor nuclear translocator (ARNT) protein in JEG-3 cells. In experiments with proteasome inhibitors MG132 and bortezomib, we found that the degradation is not sensitive to proteasome inhibition in JEG-3. We can conclude that dexamethasone suppresses AHR-mediated CYP1A1 induction in JEG-3 cells through the unique mechanism of AHR-GR crosstalk, which involves accelerated degradation of AHR.

Aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator expression in human and rat placentas and transcription activity in human trophoblast cultures.

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 ß-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.

Endogenous and exogenous ligands of aryl hydrocarbon receptor: current state of art.

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.

The function of cytochrome P450 1A1 enzyme (CYP1A1) and aryl hydrocarbon receptor (AhR) in the placenta.

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.

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

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.

Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines.

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.