CYP1A1 Promoter Research Articles

P-Anilinoaniline Enhancement of Dioxin-Induced CYP1A1 Transcription and Aryl Hydrocarbon Receptor Occupancy of CYP1A1 Promoter: Role of the Cell Cycle

The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1-200 μM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 μM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 μM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 μM pAA was cytostatic and induced a G(1) arrest that lasted ∼7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G(1) and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.

Effects of atorvastatin metabolites on induction of drug‐metabolizing enzymes and membrane transporters through human pregnane X receptor

Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.

DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.

Identification and Characterization of a Novel Retinoic Acid Response Element in Zebrafish cyp26a1 Promoter

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previously, we demonstrated that zebrafish cyp26a1 promoter possesses two conserved RA response elements (RAREs; proximal R1 and distal R2) in response to RA. Here, we report that it contains a novel RARE (R3) lying between R1 and R2. Mutagenesis analysis reveals that R3 works together with R1 and R2 to ensure the maximum RA inducibility of cyp26a1 promoter. Performing electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we show that RA receptor alpha can bind the novel RARE. Creating and analyzing transgenic zebrafish of Tg(cyp26a1-R3mut:eYFP)nju3/+ that harbor enhanced yellow fluorescent protein reporter gene (eYFP) driven by cyp26a1 promoter with mutated R3, we demonstrate that the reporter is mainly expressed in tissues of endogenous RA independent but not regions of RA dependent. Like Tg(cyp26a1:eYFP)nju1/+, which harbor eYFP driven by wild-type cyp26a1 promoter, the reporter in Tg(cyp26a1-R3mut:eYFP)nju3/+ responds to excessive RA dose dependently. However, it is expressed in a significantly lower level than the reporter in Tg(cyp26a1:eYFP)nju1/+ in response to exogenous RA. Taken together, our results demonstrate that zebrafish cyp26a1 promoter contains a novel RARE that plays crucial roles in regulating cyp26a1 expression during early development of zebrafish.

Wnt Signaling in Ovarian Development Inhibits Sf1 Activation of Sox9 via the Tesco Enhancer

Genome analysis of patients with disorders of sex development, and gain- and loss-of-function studies in mice indicate that gonadal development is regulated by opposing signals. In females, the Wnt/β-catenin canonical pathway blocks testicular differentiation by repressing the expression of the Sertoli cell-specific gene Sox9 by an unknown mechanism. Using cell and embryonic gonad culture models, we show that activation of the Wnt/β-catenin pathway inhibits the expression of Sox9 and Amh, whereas mRNA and protein levels of Sry and steroidogenic factor 1 (Sf1), two key transcriptional regulators of Sox9, are not altered. Ectopic activation of Wnt/β-catenin signaling in male gonads led to a loss of Sf1 binding to the Tesco enhancer and absent Sox9 expression that we also observed in wild-type ovaries. Moreover, ectopic Wnt/β-catenin signaling induced the expression of the female somatic cell markers, Bmp2 and Rspo1, as a likely consequence of Sox9 loss. Wnt/β-catenin signaling in XY gonads did not, however, affect gene expression of the steroidogenic Leydig cell Sf1 target gene, Cyp11a1, or Sf1 binding to the Cyp11a1 promoter. Our data support a model in ovary development whereby activation of β-catenin prevents Sf1 binding to the Sox9 enhancer, thereby inhibiting Sox9 expression and Sertoli cell differentiation.

CCAAT/ enhancer‐binding protein β activation by capsaicin contributes to the regulation of CYP1A1 expression, mediated by the aryl hydrocarbon receptor

Capsaicin, a constituent of peppers, has been linked to the suppression of tumorigenesis and carcinogenesis. The influence of capsaicin on cytochrome P450 (CYP) 1A1, which is involved in metabolism of carcinogens, and the underlying mechanisms remain unclear. Here, we examined the effect of capsaicin on CYP1A1 expression in mouse hepatoma cells. Murine hepatoma Hepa-1c1c7 cells were incubated with capsaicin and/or 3-methylcholanthrene (3-MC). Effects of capsaicin on CYP1A1 levels were determined by analysing mRNA expression, transcription activity and protein expression. Regulation of CYP1A1 was investigated by determining transcriptional factor expression, activation and binding activity with cotreatment with target signal antagonists. Capsaicin alone slightly induced CYP1A1 activity, mRNA expression, protein level and promoter activity. Treatment with transient receptor potential vanilloid type-1 receptor (TRPV1) or aryl hydrocarbon receptor (AhR) antagonist decreased induction of CYP1A1 expression by capsaicin. Additionally, capsaicin significantly inhibited 3-MC-induced CYP1A1 mRNA and protein level and xenobiotic response element-luciferase activity. Capsaicin also inhibited 3-MC-induced AhR transactivation and nuclear localization of AhRs. Moreover, capsaicin increased Ca(2+) /calmodulin (CaM)-dependent protein kinase (CaMK) and CCAAT/ enhancer-binding protein β (C/EBPβ) activation, downstream of TRPV1 receptors. Capsaicin-induced C/EBPβ activation inhibited induction of CYP1A1 mRNA and protein by 3-MC. Capsaicin alone weakly induced CYP1A1 expression, and 3-MC-induced CYP1A1 levels were suppressed by capsaicin. Activation of C/EBPβ and inhibition of 3-MC-induced AhR transactivation by capsaicin contributed to the suppression of CYP1A1 expression. Capsaicin has a potential chemopreventive effect through inhibiting induction of CYP1A1 by poly aryl hydrocarbons.

Detection of water toxicity using cytochrome P450 transgenic zebrafish as live biosensor: For polychlorinated biphenyls toxicity

Cytochrome P450 (CYPs) is significant in degradation of endogenous substrates and detoxification of carcinogens, therefore it is a biomarker for assessment of polycyclic aromatic hydrocarbons (PAHs) level in aquatic environment. In the present study, a transgenic line of zebrafish had been generated using a CYP-green fluorescence protein (CYP-GFP) construct, driven by CYP1A1 promoter. Polychlorinated biphenyls (PCBs) were used as toxicant, in concentrations of 0.02μg/ml, 0.04μg/ml, 0.08μg/ml, 0.4μg/ml, and 0.8μg/ml. The transgenic control fish showed low intensity of fluorescence in the liver. After exposed to PCBs, zebrafish had morphological changes such as expansion of yolk, contortion of tails and inflation of pericardial area. Green fluorescence signals were found to express according to concentrations and time. The green fluorescence signal was most intense after treatment with 0.08μg/ml PCBs. However, the maximum area of green fluorescent signal was found at 0.04μg/ml PCBs. GFP started to express at 3h exposure to PCBs, increasing its intensity until 6h exposure, and then level off. Since the GFP expression is fast responding and is sensitive to low PAHs concentrations, transgenic fish is a good tool for live imaging and monitoring of aquatic contamination.

Intrinsic Sexually Dimorphic Expression of the Principal Human CYP3A4 Correlated with Suboptimal Activation of GH/Glucocorticoid-Dependent Transcriptional Pathways in Men

Cytochrome P450 (CYP)3A4 is the principal and most abundant human isoform of CYP responsible for the metabolism of more than 50% of all consumed drugs and innumerable endogenous compounds. Expression of CYP3A4 is sexually dimorphic and regulated by the combined actions of GH and glucocorticoids. In the case of the rat, nearly all of the CYPs are "intrinsically" or "inherently" sexually dimorphic, meaning that the expressed sex differences are permanent and irreversible. Using primary hepatocyte cultures derived from men and women exposed to physiologic-like levels of continuous GH (the feminine circulating profile) alone, dexamethasone alone, and the combined regimen, we observed a dramatic inherent CYP3A4 sexual dimorphism (women more than men) with all treatments. The molecular basis for this intrinsic sexually dimorphic expression of CYP3A4 appears to be due, at least in part, to a greater level of hormone-dependent activation and nuclear translocation of both hepatocyte nuclear factor-4α (HNF-4α) and pregnane X receptor in female hepatocytes. Furthermore, these transcription factors exhibited significantly higher DNA binding levels to their specific motifs on the CYP3A4 promoter in female hepatocytes, inferring a possible explanation for the elevated expression of CYP3A4 in women. Accordingly, experiments using HepG2 cells treated with small inhibitory RNA-induced knockdown of HNF-4α and/or transfected with luciferase reporter constructs containing a CYP3A4 promoter lacking HNF-4α-binding motifs demonstrated that GH, to a greater extent dexamethasone, and to the greatest extent the combine hormone regimen, stimulated HNF-4α and pregnane X receptor promoter transactivation, signifying enhanced transcription of CYP3A4 and, thus, identifying a molecular mechanism contributing to the intrinsic sexual dimorphic expression of human CYP3A4.

CYP1A1 is overexpressed upon incubation of breast cancer cells with a polyphenolic cocoa extract

To evaluate the effect of cocoa flavonoids in breast cancer cells at the molecular level, a functional genomic analysis was performed using a polyphenolic cocoa extract (PCE) in MCF-7 and SKBR3 cell lines. The expression profile of 84 genes included in the Stress & Toxicity PathwayFinder™ PCR Array was analyzed after PCE incubation for 24 h. mRNA and protein levels were analyzed by RT-PCR and western blot, respectively. Gel shift assays were used to evaluate DNA-protein complexes. Protein complexes were identified by co-immunoprecipitation. Cell viability was evaluated by MTT assays. Upon PCE incubation, 7 genes were overexpressed and 1 underexpressed in MCF-7 cells, whereas 9 genes were overexpressed in SKBR3 cells. Among the differentially expressed genes in both cell lines, cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) was chosen for further study. CYP1A1 mRNA and protein levels and enzymatic activity increased upon PCE incubation. CYP1A1 transcriptional activation by PCE was mediated through AhR binding to XRE elements within the CYP1A1 promoter in MCF-7 cells. A protein complex including AhR and ERα was detected. The combination of PCE with tamoxifen caused a synergistic cytotoxicity in both cell lines and was due to an increase in apoptosis in MCF-7 cells. The interaction between ERα and AhR upon incubation with PCE leads to CYP1A1 induction in breast cancer cells. The synergy between PCE and non-cytotoxic tamoxifen concentrations opens the possibility for a combination therapy based on polyphenols from cocoa that increased tamoxifen efficacy.

Astaxanthin rich crude extract of Haematococcus pluvialis induces cytochrome P450 1A1 mRNA by activating aryl hydrocarbon receptor in rat hepatoma H4IIE cells

The xanthophyll, astaxanthin (Ax), strongly induces cytochrome P450 (CYP) 1A-dependent activity in rats and mice, via increased transcription of Cyp1a1 mRNA through an undetermined mechanism. In this study, we have investigated how aryl hydrocarbon receptor (AhR)-related events are affected by the addition of Ax-rich crude extract from Haematococcus pluvialis in H4IIE rat hepatoma cells. The expression of Cyp1a1 mRNA was significantly increased by exposure to H. pluvialis extract (100 μmol/l, 17.6-fold). The highest level of Cyp1a1 mRNA was observed after a 6-h exposure to H. pluvialis extract (25-fold), but 12- and 24-h exposures resulted in lower levels of induction. Green fluorescence protein-tagged AhR was translocated into the nucleus in COS-7 cells after a 10 min exposure to Ax. Moreover, H. pluvialis extract containing Ax activated the human CYP1A1 enhancer region in a luciferase assay, and a gel shift assay showed that nuclear proteins stimulated by H. pluvialis extract can interact specifically with xenobiotic responsive elements (XREs) in the CYP1A1 promoter. Together these results indicate the possibility that Ax, contained in H. pluvialis extract, caused the induction of Cyp1a1 mRNA through the activation and translocation of an XRE-binding form of AhR.

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.

4S polycyclic aromatic hydrocarbon receptor (glycine N-methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor-1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH-R/ARNT hetero-oligomers?

Rat CYP1A1 promoter-luciferase, transiently transfected wild-type and 4S PAH receptor (glycine N-methyl transferase, GNMT)-transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild-type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT-transformed CHO cells and wild-type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno-precipitated with anti-GNMT antibodies, separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti-aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero-oligomer (dimer?) with ARNT/HIF-1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor-1 function with respect to the HIF-1β subunit (ARNT).

Aflatoxins upregulate CYP3A4 mRNA expression in a process that involves the PXR transcription factor

Pregnane X receptor (PXR) is a member of the nuclear hormone receptor (NHR) superfamily, which regulates xenobiotic and endobiotic metabolism in the liver. This transcription factor is activated by structurally diverse ligands, including drugs and environmental pollutants. PXR regulates the expression of numerous genes that function in biotransformation and the disposition of xenobiotics upon binding to an AG(G/T)TCA DNA motif in target promoter regions. We performed a screen of mycotoxins that pose a known environmental threat to human and animal health for the ability to activate PXR function in a human hepatocyte cell line, HepG2. We found that aflatoxins B1, M1, and G1 activated PXR. This activation was associated with upregulation of CYP3A4 expression and increased occupancy of PXR protein on the CYP3A4 promoter. Using a microarray approach, we also found that aflatoxin B1 upregulated the expression of multiple genes involved in xenobiotic metabolism, including genes known to be regulated in a PXR-dependent fashion. We also observed an effect of aflatoxin B1 on the expression in other functional groups of genes, including the downregulation of genes involved in cholesterologenesis. The results of this study indicate that aflatoxin B1 is able to activate PXR, a known regulator of liver xenobiotic metabolism, in human hepatocytes, and it can upregulate the expression of PXR-dependent genes responsible for aflatoxin B1 biotransformation, including CYP3A4.

Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals

CYP11A1 encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression of CYP11A1 in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controlling CYP11A1 expression is steroidogenic factor-1 (SF-1), but the transcriptional regulation of CYP11A1 in the brain is unclear. We recently used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional-specific expression of Cre reporter in the SCC-Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF-1 binding site did not affect brain CYP11A1 promoter activity. The upstream SF-1 binding site, however, is required for CYP11A1 promoter function in the embryonic adrenals. The 3.8-kb promoter, like the 4.4-kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7-kb promoter was only observed in the caudal part of midbrain. This suggests that the 5'-flanking region between 3.8 and 2.7 kb contains a crucial element for activation of CYP11A1 promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that control CYP11A1 expression in the brain and embryonic adrenals.

Is the Hospital Course in Hypertriglyceridemia-Induced Pancreatitis (HIP) Different From That of Gallstone Pancreatitis (GSP)?

Cryptochinones A–D are tetrahydroflavanones isolated from the leaves of Cryptocarya chinensis, an evergreen tree whose extracts are believed to have a variety of health benefits. The origin of their possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and for hyperglycemia. We studied whether cryptochinones A–D, which are structurally similar to known FXR ligands, may act at this target. Indeed, in mammalian one-hybrid and transient transfection reporter assays, cryptochinones A–D transactivated FXR to modulate promoter action including GAL4, SHP, CYP7A1, and PLTP promoters in dose-dependent manner, while they exhibited similar agonistic activity as chenodeoxycholic acid (CDCA), an endogenous FXR agonist. Through molecular modeling docking studies we evaluated their ability to bind to the FXR ligand binding pocket. Our results indicate that cryptochinones A–D can behave as FXR agonists.

Coordinate Regulation of Cytochrome P450 1A1 Expression in Mouse Liver by the Aryl Hydrocarbon Receptor and the β-Catenin Pathway

The expression of cytochrome P450 (CYP) 1a1 and other drug-metabolizing enzymes is controlled by the aryl hydrocarbon receptor (AhR), which is activated by dioxin-type inducers leading to transcriptional induction of target genes. Here, we show that a second level of transcriptional control exists in hepatocytes, which is tightly linked to the Wnt/β-catenin/T-cell factor (TCF) signaling pathway. In transgenic mice, hepatic expression of CYP1A (and other CYP isoforms) is stimulated by the expression of mutationally activated β-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of β-catenin. These effects were further analyzed in vitro, and the stimulatory role of β-catenin was ascribed to a TCF-binding site within the CYP1A1 promoter. Moreover, β-catenin signaling acted cooperatively with AhR agonists via AhR-binding sites on the DNA during the induction of Cyp1a1 in vivo and in vitro. Activation of β-catenin enhanced the transactivation potential of ligand-activated AhR at its DNA-binding sites without altering the total amount of DNA-bound AhR. Coimmunoprecipitation demonstrated a physical interaction between AhR and β-catenin. Furthermore, the present results suggest that transcriptional induction of the AhR by β-catenin does not play a major role in β-catenin-dependent regulation of Cyp1a1 expression and that inhibition of β-catenin signaling by ligand-activated AhR, as recently observed in the intestine does not occur in mouse liver. In conclusion, signaling through β-catenin activates basal CYP1A1 expression and augments CYP1A1 induction by AhR ligands through enhancement of the transactivation potential of the AhR.

Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

Supplemental oxygen administration is frequently administered to pre-term and term infants having pulmonary insufficiency. However, hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Cytochrome P450 (CYP)A enzymes have been implicated in hyperoxic lung injury. In this study, we tested the hypothesis that hyperoxia induces CYP1A1 and 1A2 enzymes by transcriptional activation of the corresponding promoters in vivo, and transgenic mice expressing the human CYP1A1 or the mouse 1A2 promoter would be more susceptible to hyperoxic lung injury than wild type (WT) mice. Adult WT (CD-1) (12 week-old) mice, transgenic mice carrying a 10 kb human CYP1A1 promoter and the luciferase (luc) reporter gene (CYP1A1-luc), or mice expressing the mouse CYP1A2 promoter (CYP1A2-luc) were maintained in room air or exposed to hyperoxia for 24–72 h. Hyperoxia exposure of CYP1A1-luc mice for 24 and 48 h resulted in 2.5- and 1.25-fold increases, respectively, in signal intensities, compared to room air controls. By 72 h, the induction had declined to control levels. CYP1A2-luc mice also showed enhanced luc expression after 24–48 h, albeit to a lesser extent than those expressing the CYP1A1 promoter. Also, these mice showed decreased levels of endogenous CYP1A1 and 1A2 expression after prolonged hyperoxia, and were also more susceptible to lung injury than similarly exposed WT mice, with CYP1A2-luc mice showing the greatest injury. Our results support the hypothesis that hyperoxia induces CYP1A enzymes by transcriptional activation of its corresponding promoters, and that decreased endogenous expression of these enzymes contribute to the increased susceptibilities to hyperoxic lung injury in the transgenic animals. In summary, this is the first report providing direct evidence of hyperoxia-mediated induction of CYP1A1 and CYP1A2 expression in vivo by mechanisms entailing transcriptional activation of the corresponding promoters, a phenomenon that has implications for hyperoxic lung injury, as well as other pathologies caused by oxidative stress.

CYP7A1 genotypes and haplotypes associated with hypertension in an obese Han Chinese population

This study investigated the association between single-nucleotide polymorphisms (SNPs; rs3808607 and rs1125226) within the CYP7A1 promoter and hypertension susceptibility in a Han Chinese population. From 2003 through 2006, a population-based case-control study was performed in a cohort of 1187 randomly selected Han Chinese subjects. A sib-pair study for a transmission disequilibrium test analysis was carried out in 76 hypertensive (HT) families (n=312) from northeastern Liaoning province. SNPs were detected using real-time PCR. No significant differences were found in the genotype or allele frequencies of either SNP (P>0.05), with no excessive allele sharing. For rs3808607, the frequency of the AA genotype in obese hypertensive patients was 31.91%, significantly higher than in normotensive (NT) subjects (12.73%; odds ratio (OR)=3.21, 95% confidence interval (CI)=1.35-7.66). For rs3808607, the AA genotype frequency was significantly higher in obese male HT subjects (27.87%) than in matched NTs (7.41%; OR=4.83, 95% CI=1.03-22.65). After adjustment for environmental risk factors in obese participants, the AA genotype was associated with hypertension (OR=3.395, 95% CI=1.412-8.162). Among subjects with body mass index 28 kg m(-2), the HT and NT groups had significantly different frequencies of Hap I (C/C) and Hap IV (A/A). The frequencies of rs3808607 alleles in the CYP7A1 gene differed significantly between obese HT and NT men. Haplotypes I and IV were associated with hypertension in obese participants.

Aryl Hydrocarbon Receptor Ligands of Widely Different Toxic Equivalency Factors Induce Similar Histone Marks in Target Gene Chromatin

Posttranslational histone modifications are a critical regulatory mechanism of gene transcription. Previous studies from our laboratory have shown that contingent on binding to its cognate promoter motifs in the Cyp1a1 gene, activation of the aryl hydrocarbon receptor (AHR) by benzo[a]pyrene (BaP) treatment induces histone modifications in the Cyp1a1 promoter that are required for activation of gene transcription. Here, we have studied different AHR ligands, including polychlorinated biphenyls (PCBs) of different toxic equivalency factors (TEF), to determine whether changes in histone modifications are linked to different levels of Cyp1a1 expression or dependent on AHR-ligand affinity. We find that all ligands lead to the same pattern of histone modifications in a relationship that parallels the strength of their AHR-ligand affinity. Thus, whereas PCB126 (TEF 0.1), 3-methylcholanthrene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) initiate a pattern of histone marks similar to those induced by BaP, PCB77 (TEF 0.0001) causes a lower level of change in the same marks and requires a longer activation time than PCB126, BaP, or TCDD. In contrast, the non-dioxin-like PCB153 recruits AHR to the Cyp1a1 enhancer causing a displacement of enhancer-associated histone H3 but does not cause the other observed histone mark changes nor does it induce transcription. These results indicate that AHR recruitment to the promoter is not sufficient to induce the histone modifications needed to activate gene expression and show that there is a good correlation between the regulatory chromatin changes associated with ligand-induced AHR target gene transcription and the resultant toxicity of the ligand.

NCOA6 differentially regulates the expression of the CYP2C9 and CYP3A4 genes

CYP2Cs and CYP3A4 sub families of enzymes of the Cytochrome P450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR–HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors.