Aryl Hydrocarbon Receptor Response Elements Research Articles

Dioxin Disrupts Thyroid Hormone and Glucocorticoid Induction of klf9, a Master Regulator of Frog Metamorphosis.

Frog metamorphosis, the development of an air-breathing froglet from an aquatic tadpole, is controlled by thyroid hormone (TH) and glucocorticoids (GC). Metamorphosis is susceptible to disruption by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AHR) agonist. Krüppel-like factor 9 (klf9), an immediate early gene in the endocrine-controlled cascade of expression changes governing metamorphosis, can be synergistically induced by both hormones. This process is mediated by an upstream enhancer cluster, the klf9 synergy module (KSM). klf9 is also an AHR target. We measured klf9 mRNA following exposures to triiodothyronine (T3), corticosterone (CORT), and TCDD in the Xenopus laevis cell line XLK-WG. klf9 was induced 6-fold by 50 nM T3, 4-fold by 100 nM CORT, and 3-fold by 175 nM TCDD. Cotreatments of CORT and TCDD or T3 and TCDD induced klf9 7- and 11-fold, respectively, whereas treatment with all 3 agents induced a 15-fold increase. Transactivation assays examined enhancers from the Xenopus tropicalis klf9 upstream region. KSM-containing segments mediated a strong T3 response and a larger T3/CORT response, whereas induction by TCDD was mediated by a region ∼1 kb farther upstream containing 5 AHR response elements (AHREs). This region also supported a CORT response in the absence of readily identifiable GC responsive elements, suggesting mediation by protein-protein interactions. A functional AHRE cluster is positionally conserved in the human genome, and klf9 was induced by TCDD and TH in HepG2 cells. These results indicate that AHR binding to upstream AHREs represents an early key event in TCDD's disruption of endocrine-regulated klf9 expression and metamorphosis.

Bioengineering of Genetically Encoded Gene Promoter Repressed by the Flavonoid Apigenin for Constructing Intracellular Sensor for Molecular Events.

In recent years, Synthetic Biology has emerged as a new discipline where functions that were traditionally performed by electronic devices are replaced by “cellular devices”; genetically encoded circuits constructed of DNA that are built from biological parts (aka bio-parts). The cellular devices can be used for sensing and responding to natural and artificial signals. However, a major challenge in the field is that the crosstalk between many cellular signaling pathways use the same signaling endogenous molecules that can result in undesired activation. To overcome this problem, we utilized a specific promoter that can activate genes with a natural, non-toxic ligand at a highly-induced transcription level with low background or undesirable off-target expression. Here we used the orphan aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that upon activation binds to specific AHR response elements (AHRE) of the Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) promoter. Flavonoids have been identified as AHR ligands. Data presented here show the successful creation of a synthetic gene “off” switch that can be monitored directly using an optical reporter gene. This is the first step towards bioengineering of a synthetic, nanoscale bio-part for constructing a sensor for molecular events.

Single Nucleotide Polymorphisms at a Distance from Aryl Hydrocarbon Receptor (AHR) Binding Sites Influence AHR Ligand-Dependent Gene Expression.

Greater than 90% of significant genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) are in noncoding regions of the genome, but only 25.6% are known expression quantitative trait loci (eQTLs). Therefore, the function of many significant GWAS SNPs remains unclear. We have identified a novel type of eQTL for which SNPs distant from ligand-activated transcription factor (TF) binding sites can alter target gene expression in a SNP genotype-by-ligand–dependent fashion that we refer to as pharmacogenomic eQTLs (PGx-eQTLs)—loci that may have important pharmacotherapeutic implications. In the present study, we integrated chromatin immunoprecipitation-seq with RNA-seq and SNP genotype data for a panel of lymphoblastoid cell lines to identify 10 novel cis PGx-eQTLs dependent on the ligand-activated TF aryl hydrocarbon receptor (AHR)—a critical environmental sensor for xenobiotic (drug) and immune response. Those 10 cis PGx-eQTLs were eQTLs only after AHR ligand treatment, even though the SNPs did not create/destroy an AHR response element—the DNA sequence motif recognized and bound by AHR. Additional functional studies in multiple cell lines demonstrated that some cis PGx-eQTLs are functional in multiple cell types, whereas others displayed SNP-by-ligand–dependent effects in just one cell type. Furthermore, four of those cis PGx-eQTLs had previously been associated with clinical phenotypes, indicating that those loci might have the potential to inform clinical decisions. Therefore, SNPs across the genome that are distant from TF binding sites for ligand-activated TFs might function as PGx-eQTLs and, as a result, might have important clinical implications for interindividual variation in drug response.SIGNIFICANCE STATEMENTMore than 90% of single-nucleotide polymorphisms (SNPs) that are associated with clinical phenotypes are located in noncoding regions of the genome. However, the mechanisms of action of many of those SNPs have not been elucidated, and drugs may unmask functional expression quantitative trail loci (eQTLs). In the current study, we used drugs that bind to the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and identified SNPs that were associated with interindividual variation in gene expression following drug exposure—termed pharmacogenomic (PGx)-eQTLs. Possibly of greater significance, those PGx-eQTL SNPs were outside of AHR binding sites, indicating that they do not interrupt AHR DNA recognition. PGx-eQTLs such as those described in this work may have crucial implications for interindividual variation in drug.

Increased MDR1 Transporter Expression in Human Brain Endothelial Cells Through Enhanced Histone Acetylation and Activation of Aryl Hydrocarbon Receptor Signaling.

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) is a critical efflux transporter that extrudes chemicals from the blood-brain barrier (BBB) and limits neuronal exposure to xenobiotics. Prior studies in malignant cells demonstrated that MDR1 expression can be altered by inhibition of histone deacetylases (HDAC), enzymes that modify histone structure and influence transcription factor binding to DNA. Here, we sought to identify the mechanisms responsible for the up-regulation of MDR1 by HDAC inhibitors in human BBB cells. Immortalized human brain capillary endothelial (hCMEC/D3) cells were treated with HDAC inhibitors and assessed for MDR1 expression and function. Of the HDAC inhibitors profiled, valproic acid (VPA), apicidin, and suberoylanilide hydroxamic acid (SAHA) increased MDR1 mRNA and protein levels by 30-200%, which corresponded with reduced intracellular accumulation of the MDR1 substrate rhodamine 123. Interestingly, induction of MDR1 mRNA by HDAC inhibitors mirrored increases in the expression of the aryl hydrocarbon receptor (AHR) and its target gene cytochrome P450 1A1. To explore the role of AHR in HDAC inhibitor-mediated regulation of MDR1, a pharmacological activator (β-naphthoflavone, βNF) and inhibitor (CH-223191, CH) of AHR were tested. The induction of MDR1 in cells treated with SAHA was amplified by βNF and attenuated by CH. Furthermore, SAHA increased the binding of acetylated histone H3K9/K14 and AHR proteins to regions of the MDR1 promoter that contain AHR response elements. In conclusion, HDAC inhibitors up-regulate the expression and activity of the MDR1 transporter in human brain endothelial cells by increasing histone acetylation and facilitating AHR binding at the MDR1 promoter.

The Role of the Aryl Hydrocarbon Receptor (AHR) in Immune and Inflammatory Diseases.

The aryl hydrocarbon receptor (AHR) is a nuclear receptor that modulates the response to environmental stimuli. It was recognized historically for its role in toxicology but, in recent decades, it has been increasingly recognized as an important modulator of disease—especially for its role in modulating immune and inflammatory responses. AHR has been implicated in many diseases that are driven by immune/inflammatory processes, including major depressive disorder, multiple sclerosis, rheumatoid arthritis, asthma, and allergic responses, among others. The mechanisms by which AHR has been suggested to impact immune/inflammatory diseases include targeted gene expression and altered immune differentiation. It has been suggested that single nucleotide polymorphisms (SNPs) that are near AHR-regulated genes may contribute to AHR-dependent disease mechanisms/pathways. Further, we have found that SNPs that are outside of nuclear receptor binding sites (i.e., outside of AHR response elements (AHREs)) may contribute to AHR-dependent gene regulation in a SNP- and ligand-dependent manner. This review will discuss the evidence and mechanisms of AHR contributions to immune/inflammatory diseases and will consider the possibility that SNPs that are outside of AHR binding sites might contribute to AHR ligand-dependent inter-individual variation in disease pathophysiology and response to pharmacotherapeutics.

SNPs Outside Response Elements Impact Aryl Hydrocarbon Receptor (AHR) Binding and Gene Regulation: Genome‐wide SNP‐dependent Transcriptional Regulation

BACKGROUNDMost functionally‐significant single nucleotide polymorphisms (SNPs) are outside of open reading frames. Mechanisms by which these SNPs contribute to gene regulation are poorly understood. Aryl hydrocarbon receptor (AHR) is a nuclear receptor that regulates drug metabolism, xenobiotic response and immune response. Our previous data indicate that SNPs outside response elements can impact transcription factor binding and gene regulation. We hypothesized that SNPs near but outside AHR response elements (AhREs) might impact AHR binding and gene regulation.METHODSWe have collected dense SNP‐genotypes across 300 lymphoblastoid cell lines (LCLs) – a powerful tool for generating and testing pharmacogenomic hypotheses. Eight LCLs were used to generate hypotheses about AHR SNP‐dependent transcription utilizing RNA‐ and ChIP‐seq, which were tested in additional LCLs using qRT‐PCR and ChIP after AHR agonist or antagonist treatment.RESULTS69 genes were highly significantly differentially expressed after agonist or antagonist treatment and 80% of those genes had an AhRE within 50kb. Five SNP‐gene pairs have been functionally validated in additional LCLs (Table 1).CONCLUSIONSNPs that are outside of AhREs can alter AHR binding and gene transcription. This is a general phenomenon that may have large‐scale influence on gene transcriptional regulation and pharmacogenomics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The aryl hydrocarbon receptor regulates the expression of TIPARP and its cis long non-coding RNA, TIPARP-AS1

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood.

Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice.

Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy-induced hepatic steatosis (HS). C57BL/6 mice were fed a high-methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment up-regulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by small interfering RNA (siRNA) knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A4 (LXA4 ), a metabolite of arachidonic acid, in HMD-fed mice. Furthermore, overexpression of 15-oxoprostaglandin 13-reductase 1, a LXA4 inactivation enzyme, inhibited Hcy-induced AHR activation, lipid uptake, and lipid accumulation. Moreover, LXA4 -induced up-regulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed HHcy-induced lipid accumulation by inhibiting the AHR-CD36 pathway in mice. HHcy activates the AHR-CD36 pathway by increasing hepatic LXA4 content, which results in hepatic steatosis. (Hepatology 2016;64:92-105).

NADPH Oxidase-Dependent Mechanism Explains How Arsenic and Other Oxidants Can Activate Aryl Hydrocarbon Receptor Signaling.

The mechanisms explaining arsenic toxicity are not well understood, but physiological consequences of stimulated aryl hydrocarbon receptor (AHR) signaling both directly and through cross-talk with other pathways have been indicated. The aim of this study was to establish how arsenic interacts with AHR-mediated transcription. The human hepatoma cell line (HepG2-XRE-Luc) carrying a luciferase reporter under the control of two AHR response elements (AHREs) and immortalized human keratinocytes (HaCaT) were exposed to sodium arsenite (NaAsO2; As(3+)), alone or in combination with the endogenous high affinity AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Luciferase activity, cytochrome P4501A1 (CYP1A1) activity, oxidative stress-related responses, metabolic clearance of FICZ, and NADPH oxidase (NOX) activity as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression were measured. Arsenic inhibited CYP1A1 enzyme activity and reduced the metabolic clearance of FICZ. Arsenic also led to activated CYP1A1 transcription but only in cells grown in medium containing trace amounts of the endogenous ligand FICZ, pointing to an indirect mechanism of activation. Initially, arsenic caused dose-dependent inhibition of FICZ-activated AHR signaling, disturbed intracellular GSH status, and increased expression of oxidative stress-related genes. Silencing of NOX4, addition of N-acetylcystein, or pretreatment with arsenic itself attenuated the initial dose-dependent inhibition of AHR signaling. Arsenic pretreatment led to elevated GSH levels and sensitized the cells to ligand-dependent AHR signaling, while silencing of Nrf2 significantly reduced arsenic-mediated activation of the AHR. In addition, influence of NOX on AHR activation was also observed in cells treated with the SH-reactive metals cadmium, mercury, and nickel. Together, the results suggest that SH-reactive agents via a new and possibly general NOX/H2O2-dependent mechanism can interfere with the endogenous regulation of the AHR.

Vehicular exhaust particles promote allergic airway inflammation through an aryl hydrocarbon receptor–notch signaling cascade

Traffic-related particulate matter (PM) has been linked to a heightened incidence of asthma and allergic diseases. However, the molecular mechanisms by which PM exposure promotes allergic diseases remain elusive. We sought to determine the expression, function, and regulation of pathways involved in promotion of allergic airway inflammation by PM. We used gene expression transcriptional profiling, in vitro culture assays, and in vivo murine models of allergic airway inflammation. We identified components of the Notch pathway, most notably Jagged 1 (Jag1), as targets of PM induction in human monocytes and murine dendritic cells. PM, especially ultrafine particles, upregulated TH cytokine levels, IgE production, and allergic airway inflammation in mice in a Jag1- and Notch-dependent manner, especially in the context of the proasthmatic IL-4 receptor allele Il4raR576. PM-induced Jag1 expression was mediated by the aryl hydrocarbon receptor (AhR), which bound to and activated AhR response elements in the Jag1 promoter. Pharmacologic antagonism of AhR or its lineage-specific deletion in CD11c(+) cells abrogated the augmentation of airway inflammation by PM. PM activates an AhR-Jag1-Notch cascade to promote allergic airway inflammation in concert with proasthmatic alleles.

The Ah Receptor Recruits IKKα to Its Target Binding Motifs to Phosphorylate Serine-10 in Histone H3 Required for Transcriptional Activation

Aryl hydrocarbon receptor (AHR) activation by xenobiotic ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is key to their toxicity. Following activation and nuclear translocation, AHR heterodimerizes with the AHR nuclear translocator (ARNT) and binds to AHR response elements (AhREs) in the enhancer of target genes, of which Cyp1a1 is the prototype. Previously, we showed that concomitant with AHR binding, histone H3 in the Cyp1a1 enhancer-promoter AhRE cluster became phosphorylated in serine-10 (H3S10), suggesting that the ligand-activated AHR recruited one or more kinases to the enhancer chromatin to phosphorylate this residue. To test this hypothesis, we used mouse hepatoma Hepa-1c1c7 cells and their c35 mutant derivative, lacking a functional AHR, to search for candidate kinases that would phosphorylate H3S10 in an AHR dependent manner. Using chromatin immunoprecipitation with antibodies to a comprehensive set of protein kinases, we identified three kinases, IκB kinase α (IKKα), mitogen and stress activated protein kinase 1 (MSK1), and mitogen and stress activated protein kinase 2 (MSK2), whose binding to the Cyp1a1 enhancer was significantly increased by TCDD in Hepa-1c1c7 cells and absent in control c35 cells. Complexes of AHR, ARNT, and IKKα could be coimmunoprecipitated from nuclei of TCDD treated Hepa-1c1c7 cells and shRNA-mediated IKKα knockdown inhibited both H3S10 phosphorylation in the Cyp1a1 enhancer and the induction of Cyp1a1, Aldh3a1, and Nqo1 in TCDD-treated cells. We conclude that AHR recruits IKKα to the promoter of its target genes and that AHR-mediated H3S10 phosphorylation is a key epigenetic requirement for induction of AHR targets. Given the role of H3S10ph in regulation of chromosome condensation, AHR-IKKα cross-talk may be a mediator of chromatin remodeling by environmental agents.

Reply to the comment of Ana I. Alvarez and Gracia Merino regarding “Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model” by Wassermann et al. 2013

June 2009) and GQ141082 (oABCG2: submitted on 3 July 2009). These generated ABCG2 full-length clones consist of the open reading frame (ORF) sequence as well as an 5′ untranslated region (5′-UTR) where regulatory elements are located. In this context, we identified besides other reg-ulatory elements such as SP-1 and AP-1 aryl hydrocarbon receptor response elements (AhRE) in the bovine as well as the caprine ABCG2 5′-UTR (Wassermann et2013 al. ). In further mechanistic studies, we showed that the func-tional expression of ABCG2 was induced due to binding of ligand-activated AhR to AhRE sequences in the ABCG2 5′-UTR (Halwachs et2012). Merino and coworkers al. (Merino et al. 2009) as well as Real and coworkers (Real et al. 2011) did not provide NCBI accession numbers for the hepatocellular ABCG2 cDNA sequences in their pub-lications. To our knowledge, the NCBI database entry (NM_001037478) from Real et al. was first published and accessible in June 2012. Since then, it was not possible to perform sequence analysis to determine cDNA sequence homology between the bovine hepatocellular and mam-mary ABCG2 clones. In addition, we did not only gener-ate a full-length bovine mammary ABCG2 cDNA sequence but also caprine and ovine mammary ABCG2 sequences. In this context, differences between the three species occurred regarding the ABCG2 cDNA as well as protein sequences (Wassermann etal. 2013 ). Former studies (Wassermann et al. 2013) as well as the present publication additionally revealed species-dependent differences regarding the sub-strate specificity of the generated MDCKII-ABCG2 cell lines. However, the aim of our research was to generate tis-sue-specific full-length ABCG2 cDNA clones to investigate the influence of xenobiotics on the tissue-specific expres-sion of ABCG2. In this context, the generated MDCKII cell lines stably expressing bABCG2, cABCG2 or oABCG2 represent a new in vitro model.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Comparative gene responses to collected ambient particles in vitro: endothelial responses

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 μg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

An Integrated Genomic Analysis of Aryl Hydrocarbon Receptor-Mediated Inhibition of B-Cell Differentiation

The aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters differentiation of B cells and suppresses antibody production. A combination of whole-genome, microarray-based chromatin immunoprecipitation (ChIP-on-chip), and time course gene expression microarray analysis was performed on the mouse B-cell line CH12.LX following exposure to lipopolysaccharide (LPS) or LPS and TCDD to identify the primary and downstream transcriptional elements of B-cell differentiation that are altered by the AHR. ChIP-on-chip analysis identified 1893 regions with a significant increase in AHR binding with TCDD treatment. Transcription factor binding site analysis on the ChIP-on-chip data showed enrichment in AHR response elements. Other transcription factors showed significant coenrichment with AHR response elements. When ChIP-on-chip regions were compared with gene expression changes at the early time points, 78 genes were identified as potential direct targets of the AHR. AHR binding and expression changes were confirmed for a subset of genes in primary mouse B cells. Network analysis examining connections between the 78 potential AHR target genes and three transcription factors known to regulate B-cell differentiation indicated multiple paths for potential regulation by the AHR. Enrichment analysis on the differentially expressed genes at each time point evaluated the downstream impact of AHR-regulated gene expression changes on B-cell-related processes. AHR-mediated impairment of B-cell differentiation occurred at multiple nodes of the B-cell differentiation network and potentially through multiple mechanisms including direct cis-acting effects on key regulators of B-cell differentiation, indirect regulation of B-cell differentiation-related pathways, and transcriptional coregulation of target genes by AHR and other transcription factors.

Dioxin Increases the Interaction Between Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha at Human Promoters

Recent studies have shown that activated aryl hydrocarbon receptor (AHR) induced the recruitment of estrogen receptor-alpha (ERalpha) to AHR-regulated genes and that AHR is recruited to ERalpha-regulated genes. However, these findings were limited to a small number of well-characterized AHR- or ERalpha-responsive genes with little knowledge of what was occurring at other genomic regions. In this study, we showed using chromatin immunoprecipitation followed by hybridization to promoter focused microarrays (ChIP-chip) that 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment significantly increased the overlap of genomic regions bound by both AHR and ERalpha. Conventional and sequential ChIPs confirmed the recruitment of AHR and ERalpha to many of the identified regions. Transcription factor binding site analysis revealed an overrepresentation of aryl hydrocarbon receptor response elements in regions bound by both AHR and ERalpha, suggesting that AHR was the important factor determining the recruitment of ERalpha to these regions. RNA interference-mediated knockdown of AHR confirmed its requirement for the recruitment of ERalpha to some, but not all, of the shared regions. Our findings demonstrate not only that dioxin induces the recruitment of ERalpha to AHR target genes but also that AHR is recruited to estrogen-responsive regions in a gene-specific manner, suggesting that AHR utilizes both of these mechanisms to modulate estrogen-dependent signaling.

The Aryl Hydrocarbon Receptor May Regulate Genes Involved in Cell Cycle and X Chromosome Inactivation in the Neonatal Mouse Ovary.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor known for its role in mediating the toxic effects of numerous environmental contaminants. Recent data also indicate that the AHR plays a role in normal ovarian physiology. Specifically, previous studies have shown that Ahr deletion results in a decrease in the number of antral follicles, slow follicular growth, decreased estradiol synthesis, and reduced fertility in adult female mice. However, the role of the AHR in early neonatal reproductive function is relatively unknown. Normally, just after birth, the mouse ovary contains a finite number of germ cells available for follicle formation. These germ cells are organized in clusters called oocyte nests that will break apart and form primordial follicles. During this period more than 70% of the germ cells die by apoptosis and the surviving germs cells are then surrounded by a single layer of flattened granulosa cells, forming primordial follicles. Very little is understood about what determines the fate of the germ cells and what signals the development of the neonatal ovarian cell types. Previous studies in our laboratory have shown that the AHR may play a role in regulating the timing of follicle formation. Thus, this study tested the hypothesis that on postnatal day (PND) 3, when oocyte nests are breaking apart to form primordial follicles, there are changes in expression of genes involved in regulating apoptosis and cell survival between aryl hydrocarbon receptor null (AhRKO) and wild type (WT) mouse ovaries. To test this hypothesis, ovaries were collected from AhRKO and WT mice on PND3 and subjected to Affymetrix GeneChip® Mouse Genome 430 2.0 arrays to compare the expression of more than 20,000 genes. A comparative analysis revealed differential expression of 147 genes: 123 genes were up-regulated, whereas 24 genes were down-regulated in the AhRKO ovaries compared to WT ovaries. Several of these are known to play essential roles in cell cycle regulation and apoptosis. For example, a gene necessary for maintaining spindle structure and proper movement of chromosomes during the cell cycle, kinesin family member 11 (Kif11), was significantly increased by 2 fold in the AhRKO ovaries compared to the WT ovaries. This result was confirmed by quantitative RT-PCR (AhRKO=3.32±0.55, WT=1.67±0.15, relative expression level, n=4, p≤0.05). Interestingly, a gene essential for initiation of X chromosome inactivation, inactive X specific transcripts (Xist), was decreased by 2 fold in the AhRKO ovary compared to the WT ovary. This finding was also confirmed by quantitative RT-PCR (AhRKO=0.68±0.04, WT=1.10±0.12 relative expression level, n=5, p≤0.05). We further screened both Kif11 and Xist as candidate target genes for the ligand activated AHR by searching for potential aryl hydrocarbon receptor response elements (AHREs) within the promoter regions of these genes. We found AHREs within the promoter regions of both Kif11 and Xist genes. Collectively, these data suggest that the AHR regulates genes involved in cell cycle and the X chromosome inactivation during early follicle formation. (platform)

Repression of Activated Aryl Hydrocarbon Receptor–Induced Transcriptional Activation by 5α-Dihydrotestosterone in Human Prostate Cancer LNCaP and Human Breast Cancer T47D Cells

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin– induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5α-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element–driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.

(−)-Epigallocatechin-3-gallate Is a Novel Hsp90 Inhibitor

(-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea, protects against certain types of cancers, although the mechanism has not yet been determined. It was previously demonstrated that EGCG blocks aryl hydrocarbon receptor (AhR)-mediated transcription induced by the potent carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Unlike other AhR antagonists that directly bind to the AhR, EGCG inhibits AhR-mediated transcription by binding to hsp90. We hypothesize that EGCG exerts anti-AhR and anticancer effects by acting as an hsp90 inhibitor. Using proteolytic footprinting, immunoprecipitation, and an ATP-agarose pull-down assay, EGCG was found to directly modulate the conformation of hsp90 and bind at or near to a C-terminal ATP binding site. Hsp90 chaperone function, as assessed by its ability to mediate refolding of denatured luciferase, was inhibited by EGCG treatment. Hsp90 dimerization, which occurs at the C-terminal end, was also inhibited by EGCG treatment. Coimmunoprecipitation studies showed that EGCG stabilizes an AhR complex that includes hsp90 and XAP2 (hepatitis B virus X-associated protein 2), and decreases the association of aryl hydrocarbon nuclear translocator (Arnt) with ligand-activated AhR. Thus, EGCG, through its ability to bind to hsp90, blocks AhR response element (AhRE) recognition. These studies indicate a novel mechanism whereby EGCG inhibits ligand-induced AhRE binding and AhR-mediated transcriptional activity. In EGCG-treated human ovarian carcinoma SKOV3 cells, decreased levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1 and phospho-AKT, were observed. EGCG also modified the association of hsp90 with several cochaperones. Overall, these data indicate that EGCG is a novel hsp90 inhibitor. Further studies are needed to determine if this has a role in the antitumor actions of EGCG.

Coordinate regulation of cellular proliferation and apoptosis in rodent liver

The balance between cellular proliferation and apoptosis is a co-ordinated process. Rodent hepatocytes proliferate rapidly in response to non-genotoxic liver growth agents such as peroxisome proliferators, a result of both increased DNA synthesis and suppression of apoptosis (Roberts et al. , 1995). This study aimed to identify groups of genes that were co-regulated in the response of rodent liver to mitogenic stimuli, and used three compounds known to induce liver growth via different processes: cyproterone acetate (60 mg/kg/day), dexamethasone (100 mg/kg/day) and Wy-14,643 (50 mg/kg/day). Each compound or vehicle was administered to groups of five male rats for up to four days, with animals being sacrificed at time-points between 6 and 96 hours post-intial dose. Total RNA from liver samples was processed and analysed by DNA microarray (Affymetrix). The expression profile for each gene was first compared between the three compounds across all time points, identifying 38 genes that could be clustered into groups showing similar expression profiles. From these 38 genes, two groups were selected for further investigation and validation by quantitative real-time PCR analysis using a range of samples dosed with the same compounds over the same time-points. One group, composed of malic enzyme 1 (Me1) and rat brain acyl CoA hydrolase (rBACH), was found to respond only to peroxisome-proliferators, and hence was not investigated further. The second group, comprising seven genes, showed inhibition of gene expression in response to the compounds administered. A similar response was observed during quantitative real-time PCR validation as in the microarray analysis, therefore this group was investigated further using a reporter gene assay. Promoter sequences immediately upstream of the transcriptional start site (1 kilobase) were successfully cloned into a plasmid upstream of a reporter gene (SEAP) for four of the genes, and were transfected into the FaO rat hepatoma cell line. Exposure of transfected cells to liver growth agents produced a similar profile of SEAP expression to that seen in the original in vivo analysis, suggesting regulation of the genes was at the transcriptional level. A deletion construct series was prepared for two of the genes, HCaRG and Nob1p, and the transcriptional response localised to within the first ~100 bp of the proximal promoter in each case. Exposure of the ~100 bp reporter gene constructs to β-naphthoflavone (100 μM), which activates both the aryl hydrocarbon receptor (AhR) and the NF-E2-related factor 2 (Nrf2), elicited a significant ≥2-fold decrease in reporter gene expression, similar to the decrease elicited by the liver growth agents. Dosing with the AhR ligands 3-methylcholanthrene and benzo(a)pyrene failed to mimic the response of the promoter constructs to β-naphthoflavone indicating that AhR was not solely responsible for the inhibition of HCaRG and Nob1p expression observed. However, site-directed mutagenesis of individual potential AhR and Nrf2 response elements within the HCaRG and Nob1p promoter constructs failed to ablate the response to β-naphthoflavone or dexamethasone. This indicated either interplay between the two transcription factors in the response, or that the two transcription factors were capable of compensation. This study has identified and confirmed a link between two genes exhibiting co-ordinate regulation in their expression in response to liver growth agent administration, with the potential of coregulation with five other genes. It has identified the mediators of this response as being the AhR, Nrf2 or more likely a combination of the two transcription factors, with the potential of supporting the increasing observation that Nrf2 and the AhR co-operate in the regulation of genes in response to xenobiotic stimuli.