Aryl Hydrocarbon Receptor Binding Research Articles

Aryl Hydrocarbon Receptor Activates NDRG1 Transcription under Hypoxia in Breast Cancer Cells.

Hypoxia has been intensively investigated over the past several decades based on the observations that hypoxic tumors are more resistant to therapy and have a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) is strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuating oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study was to identify the transcription factors that regulate NDRG1 and to investigate the functional roles of NDRG1 in hypoxia. We showed that binding sites of aryl hydrocarbon receptor (AHR) were predicted in the NDRG1 promoter. Nuclear AHR was up-regulated in the presence of cobalt and hypoxia. AHR translocated to nuclei and bound between base pairs −412 and −388 of the NDRG1 promoter in hypoxia. Moreover, hypoxia-mimetic induction of NDRG1 was attenuated by knockdown of AHR expression. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation of NDRG1. These results showed for the first time that AHR positively regulates NDRG1 transcription through an AHR binding site by way of hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regimen to prevent tumor malignancy under hypoxia.

Microrna 29b Mediates Immune Evasion of Natural Killer Cells in Acute Myeloid Leukemia

Natural killer (NK) cells are innate effector cells that can spontaneously recognize and kill cancer cells. While important in inducing long-term disease free survival (DFS) in the setting of killer immunoglobulin-like receptor (KIR) mismatch for acute myeloid leukemia (AML), harnessing NK cells to kill autologous or self AML blasts to extend DFS has had no success. In this study, we uncover one potential mechanism by which AML blasts can evade NK cell cytotoxicity. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that has now been shown to be expressed in immature human NK cells. Furthermore, activation of AHR in these immature cells suppresses human NK cell maturation and function. We and others have shown that human AML blasts secrete ligands that can activate AHR, leading us to hypothesize that AML may evade autologous NK cell cytotoxicity in part by inhibiting NK cell maturation. Expression of microRNA (miR)-29b has previously been shown to suppress the expression of transcription factors Tbx21 (TBET) and Eomesodermin (EOMES), both of which are critical for terminal NK cell differentiation and function. Here we show that AHR is able to directly regulate the expression of miR-29b and thus may serve as the link between AML and immune evasion of NK cells. We first identified putative AHR binding sites within the proximalpromoter of miR-29b, suggesting that AHR may directly regulate miR-29b expression. To test this, we transfected the AHR responsive HepG2 human cell line with a miR-29b promoter driven luciferase reporter and measured luciferase activity after treatment with the known AHR agonist, FICZ, compared to vehicle control. We discovered that cells treated with 6-formylindolol[3,2-b]carbazole (FICZ) had increased luciferase activity compared to cells treated with vehicle control (P<0.05). This effect was subsequently shown to be directly mediated by AHR, as mutation of the putative AHR binding site as well as siRNA targeting of AHR mRNA both significantly diminished the induced luciferase activity (P<0.01). We then established the importance of miR-29b in human NK cell development by transducing immature NK cells, characterized as Lin(-)CD117(+)CD94(-), with a miR-29b knockdown virus and testing for the ability of these cells to become mature [i.e., Lin(-)CD117(-)CD94(+)] NK cells, after two weeks in IL-15 and FICZ. Indeed, in early experiments, knockdown of miR-29b resulted in increased percentages of mature NK cells compared to cells transduced with control virus (16.8% compared to 3.6%, respectively), despite being cultured with an AHR agonist. Finally, utilizing a translational in vivo murine AML model developed by our laboratory that recapitulates human AML, we have found that AML blasts harvested from these leukemic mice release an AHR agonist (P < 0.01), similar to previous reports that have described AHR ligands being produced by human AML blasts. In addition,NK cells isolated from these mice have increased levels of miR-29b when leukemic, compared to NK cells from wild type control littermates, consistent with our hypothesis that AHR regulates miR-29b expression. Thus, we propose that AHR, when activated by AML-derived ligands, upregulates miR-29b in NK cells to ultimately suppress the primary regulators of NK cell maturation and function, resulting in immune evasion (See Figure). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 17671: TCF21 Regulates Coronary Artery Disease Causing Aryl-hydrocarbon Receptor Gene Expression and its Downstream Pathway Activation by Environmental Ligands

Background: Recently, the TCF21 gene was linked to the highly replicated coronary artery disease (CAD) associated polymorphism in the 6q23 locus. TCF21 is especially compelling as a causal gene since it is a transcription factor that is known to play a critical role in the development of coronary arteries. One downstream target gene identified through preliminary studies was AHR. AHR is a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (eg. cigarette smoke), and has recently been associated with CAD in mice and humans. We set out to identify the TCF21 downstream pathways that are related to CAD. Methods: We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-sequencing using primary human coronary artery smooth muscle cells (HCASMC) to survey the targets of TCF21 binding and the regulated downstream pathways. The peaks from ChIP-Seq were identified using MACSv2 according to ENCODE parameters. RNA-Seq was analyzed using edgeR to identify the differentially expressed genes. The effect of TCF21 on Aryl-hydrocarbon receptor (AHR) pathway activation was tested using knockdown and overexpression lentivirus transduction of HCASMC followed by 2,3,7,8-Tetrachlorodibenxo- p -dioxin (TCDD) treatment and qPCR. Results: RNA-Seq studies identified AHR as a significantly regulated mRNA downstream of TCF21 (p=1.4x10 -5 ). Also, the ChIP-Seq identified multiple TCF21 binding peaks in the AHR gene. We found a significant overlap of the AHR and TCF21 binding peaks (p&lt;1x10 -15 ) and genes differentially regulated by TCF21 and AHR (p=1x10 -5 ), among publicly available datasets. Furthermore, we found that the TCF21 level modulated the magnitude of the ligand-dependent activation of AHR pathway genes by TCDD. Conclusion: We identified a novel interaction between TCF21 and AHR, a gene involved in environmental xenobiotic response that was recently associated with CAD. The functional link between TCF21 and AHR provides an opportunity to explore gene by environment interactions in the pathogenesis of CAD.

Homocitrullination Is a Novel Histone H1 Epigenetic Mark Dependent on Aryl Hydrocarbon Receptor Recruitment of Carbamoyl Phosphate Synthase 1

The aryl hydrocarbon receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the nonconsensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in concomitant recruitment of carbamoyl phosphate synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin-modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome, resulting in a specific enzyme-independent post-translational modification of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.

The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region.

Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.

Abstract 1983: Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells

Abstract Hypoxia has been intensively investigated over past decades based on the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) was strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuation of oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study is to identify the novel transcription factors that regulate NDRG1 and to investigate the functional roles of NDRG1 in hypoxia. In silico analysis predicted binding sites of aryl hydrocarbon receptor (AHR) existing in the NDRG1 promoter. Western blotting showed that nuclear AHR was up-regulated in the presence of cobalt and hypoxia. Immunofluorescence staining and chromatin immunoprecipitation assays showed that AHR translocated to nuclei and bound to NDRG1 promoter in hypoxia. Luciferase reporter assays further showed that the binding site of AHR at -412 ∼ -388 bp played a crucial role in regulating NDRG1 under hypoxia-mimicking conditions. Moreover, hypoxia-mimetic induction of NDRG1 by was attenuated by knockdown of AHR expression using short interfering RNA. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation of NDRG1, whereas shRNA knockdown of NDRG1 reduced cell growth and motility. In summary, these results showed for the first time that AHR positively regulates NDRG1 transcription through a AHR binding site by hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regime to prevent tumor malignancy under hypoxia. Citation Format: En-Yu Li, Wei-Yung Huang, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2015-1983

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells.

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice.

Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues

Polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms are important components of crude oil. Both groups of PAHs have been reported to cause dioxin-like responses, mediated by aryl hydrocarbon receptor (AhR). Thus, characterization of binding affinity to the AhR of unsubstituted or alkylated PAHs is important to understand the toxicological consequences of oil contamination on ecosystems. We investigated the potencies of major PAHs of crude oil, e.g., chrysene, phenanthrene and dibenzothiophene, and their alkylated forms (n=17) to upregulate expression of AhR-mediated processes by use of the H4IIE-luc transactivation bioassay. In addition, molecular descriptors of different AhR activation potencies among PAHs were investigated by use of computational molecular docking models. Based on responses of the H4IIE-luc in vitro assay, it was shown that potencies of PAHs were determined by alkylation in addition to the number and conformation of rings. Potencies of AhR-mediated processes were generally greater when a chrysene group was substituted, especially in 1-methyl-chrysene. Significant negative correlations were observed between the in vitro dioxin-like potency measured in H4IIE-luc cells and the binding distance estimated from the in silico modeling. The difference in relative potency for AhR activation observed among PAHs and their alkylated forms could be explained by differences among binding distances in the ligand binding domain of the AhR caused by alkylation. The docking model developed in the present study may have utility in predicting risks of environmental contaminants of which toxicities are mediated by AhR binding.

The effect of 2, 3, 7, 8-Tetrachlorodibenzo -p-Dioxin in the human 3’IgH regulatory region and immunoglobulin expression. (IRC11P.440)

Abstract The transcription of Ig heavy chain (Igh) is regulated by numerous regulatory elements including 3’Igh regulatory region (3’IghRR).Several transcription factors are involved in modulating the 3’IghRR and aryl hydrocarbon receptor (AhR). TCDD is an environmental contaminant and a ligand for AhR known to interfere with Immunoglobulins (Ig) expression in animal models. Our lab have identified in previous study the mouse 3’Ig heavy chain regulatory region (3’IgH RR) as a sensitive transcriptional target of TCDD, which inhibits mouse 3’IgH RR activation and induces AhR binding to dioxin response element (DRE) within 3’IgH RR. In human the hs1, 2 enhancer is polymorphic which has been correlated with several autoimmune diseases. We have identified TCDD-induced activation of the hu-hs1, 2 enhancer and a decrease in enhancer activity with deletion of the polymorphic region. Pervious study in our lab suggested that the hu-hs1, 2 enhancer may be a negative regulator of 3’IgH RR activity and Ig expression. The objective of this study is to study the cooperative effect of the Human 3’IgHRR Enhancers in intronic promoters and the possibility of TCDD interfering with this activity in the presence and absence of the stimulation and to study the polymorphic alleles effect in the human 3’IgHRR basal activity. We used human CL-01 B cell for this study and multiple luciferase reporter constructs driven by human Igγ intronic promoter and regulated by the human 3’IgHRR enhancers .

Aryl hydrocarbon receptor mediates both proinflammatory and anti-inflammatory effects in lipopolysaccharide-activated microglia.

The aryl hydrocarbon receptor (AhR) regulates peripheral immunity; but its role in microglia-mediated neuroinflammation in the brain remains unknown. Here, we demonstrate that AhR mediates both anti-inflammatory and proinflammatory effects in lipopolysaccharide (LPS)-activated microglia. Activation of AhR by its ligands, formylindolo[3,2-b]carbazole (FICZ) or 3-methylcholanthrene (3MC), attenuated LPS-induced microglial immune responses. AhR also showed proinflammatory effects, as evidenced by the findings that genetic silence of AhR ameliorated the LPS-induced microglial immune responses and LPS-activated microglia-mediated neurotoxicity. Similarly, LPS-induced expressions of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were reduced in the cerebral cortex of AhR-deficient mice. Intriguingly, LPS upregulated and activated AhR in the absence of AhR ligands via the MEK1/2 signaling pathway, which effects were associated with a transient inhibition of cytochrome P450 1A1 (CYP1A1). Although AhR ligands synergistically enhance LPS-induced AhR activation, leading to suppression of LPS-induced microglial immune responses, they cannot do so on their own in microglia. Chromatin immunoprecipitation results further revealed that LPS-FICZ co-treatment, but not LPS alone, not only resulted in co-recruitment of both AhR and NFκB onto the κB site of TNFα gene promoter but also reduced LPS-induced AhR binding to the DRE site of iNOS gene promoter. Together, we provide evidence showing that microglial AhR, which can be activated by LPS, exerts bi-directional effects on the regulation of LPS-induced neuroinflammation, depending on the availability of external AhR ligands. These findings confer further insights into the potential link between environmental factors and the inflammatory brain disorders.

Aryl hydrocarbon receptor-dependent stanniocalcin 2 induction by cinnabarinic acid provides cytoprotection against endoplasmic reticulum and oxidative stress.

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor historically known for its role in xenobiotic metabolism. Although AhR activity has previously been shown to play a cytoprotective role against intrinsic apoptotic stimuli, the underlying mechanism by which AhR confers cytoprotection against apoptosis is largely unknown. Here, we demonstrate that activation of AhR by the tryptophan catabolite cinnabarinic acid (CA) directly upregulates expression of stanniocalcin 2 (Stc2) to elicit cytoprotection against apoptosis induced by endoplasmic reticulum stress and oxidative stress. Chromatin immunoprecipitation studies demonstrated that CA treatment induces direct AhR binding to a region of the Stc2 promoter containing multiple xenobiotic response elements. Using isolated primary hepatocytes from AhR wild-type (AhR floxed) and liver-specific AhR conditional knockout mice, we showed that pretreatment with CA conferred cytoprotection against hydrogen peroxide (H(2)O(2))-, thapsigargin-, and ethanol-induced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H(2)O(2), thapsigargin, and ethanol injury were absolutely dependent on Stc2. Immunochemistry revealed the presence of Stc2 in the endoplasmic reticulum and on the cell surface, consistent with Stc2 secretion and autocrine and/or paracrine signaling. Finally, in vivo data using a mouse model of acute alcohol hepatotoxicity demonstrated that CA provided cytoprotection against ethanol-induced apoptosis, hepatic microvesicular steatosis, and liver injury. Collectively, our data uncovered a novel mechanism for AhR-mediated cytoprotection in the liver that is dependent on CA-induced Stc2 activity.

The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology.

The aryl hydrocarbon receptor (AhR) is an evolutionarily old transcription factor belonging to the Per-ARNT-Sim-basic helix-loop-helix protein family. AhR translocates into the nucleus upon binding of various small molecules into the pocket of its single-ligand binding domain. AhR binding to both xenobiotic and endogenous ligands results in highly cell-specific transcriptome changes and in changes in cellular functions. We discuss here the role of AhR for immune cells of the barrier organs: skin, gut, and lung. Both adaptive and innate immune cells require AhR signaling at critical checkpoints. We also discuss the current two prevailing views-namely, 1) AhR as a promiscuous sensor for small chemicals and 2) a role for AhR as a balancing factor for cell differentiation and function, which is controlled by levels of endogenous high-affinity ligands. AhR signaling is considered a promising drug and preventive target, particularly for cancer, inflammatory, and autoimmune diseases. Therefore, understanding its biology is of great importance.

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells.

Highly reactive quinone species produced by photooxidation and/or metabolic activation of mono- or bi-aromatic hydrocarbons modulate cellular homeostasis and electrophilic signal transduction pathways through the covalent modification of proteins. Polycyclic aromatic hydrocarbons, but not mono- or bi-aromatic hydrocarbons, are well recognized as ligands for the aryl hydrocarbon receptor (AhR). However, quinone species produced from mono- and bi-aromatic hydrocarbons could potentially cause AhR activation. To clarify the AhR response to mono- and bi-aromatic hydrocarbon quinones, we studied Cyp1a1 (cytochrome P450 1A1) induction and AhR activation by these quinones. We detected Cyp1a1 induction during treatment with quinones in Hepa1c1c7 cells, but not their parent compounds. Nine of the twelve quinones with covalent binding capability for proteins induced Cyp1a1. Cyp1a1 induction mediated by 1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone (1,4-BQ) and tert-butyl-1,4-BQ was suppressed by a specific AhR inhibitor and was not observed in c35 cells, which do not have a functional AhR. These quinones stimulated AhR nuclear translocation and interaction with the AhR nuclear translocator. Interestingly, 1,2-NQ covalently modified AhR, which was detected by an immunoprecipitation assay using a specific antibody against 1,2-NQ, resulting in enhancement of xenobiotic responsive element (XRE)-derived luciferase activity and binding of AhR to the Cyp1a1 promoter region. While mono- and bi-aromatic hydrocarbons are generally believed to be poor ligands for AhR and hence unable to induce Cyp1a1, our study suggests that the quinones of these molecules are able to modify AhR and activate the AhR/XRE pathway, thereby inducing Cyp1a1. Since we previously reported that 1,2-NQ and tert-butyl-1,4-BQ also activate NF-E2-related factor 2, it seems likely that some of quinones are bi-functional inducers for phase-I and phase-II reaction of xenobiotics.

Chemometrics-Assisted Effect-Directed Analysis of Crude and Refined Oil Using Comprehensive Two-Dimensional Gas Chromatography–Time-of-Flight Mass Spectrometry

An effect-directed analysis (EDA) of fresh and artificially weathered (evaporated, photooxidized) samples of North Sea crude oil and residual heavy fuel oil is presented. Aliphatic, aromatic, and polar oil fractions were tested for the presence of aryl hydrocarbon receptor (AhR) agonist and androgen receptor (AR) antagonist, demonstrating for the first time the AR antagonist effects in the aromatic and, to a lesser extent, polar fractions. An extension of the typical EDA strategy to include an N-way partial least-squares (N-PLS) model capable of relating the comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS) data set to the bioassay data obtained from normal-phase LC fractions is proposed. The predicted AhR binding effects in the fresh and artificially weathered aromatic oil fractions facilitated the identification of alkyl-substituted three- and four-ring aromatic systems in the active fractions through the weighting of their contributions to the observed effects. A N-PLS chemometric model is demonstrated as a potentially useful strategy for future EDA studies that can streamline the compound identification process and provide additional reduction of samples' complexity. The AhR binding effects of the suspected compounds predicted by N-PLS and identified by GC × GC-TOFMS were confirmed using quantitative structure-activity relationship (QSAR) estimates.

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.

Ah Receptor–Mediated Suppression of Liver Regeneration through NC-XRE–Driven p21Cip1Expression

Previous studies in hepatocyte-derived cell lines and the whole liver established that the aryl hydrocarbon receptor (AhR) can disrupt G1-phase cell cycle progression following exposure to persistent AhR agonists, such as TCDD (dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin). Growth arrest was attributed to inhibition of G1-phase cyclin-dependent kinase 2 (CDK2) activity. The present study examined the effect of TCDD exposure on liver regeneration following 70% partial hepatectomy in mice lacking the Cip/Kip inhibitors p21(Cip1) or p27(Kip1) responsible for regulating CDK2 activity. Assessment of the regenerative process in wild-type, p21(Cip1) knockout, and p27(Kip1) knockout mice confirmed that TCDD-induced inhibition of liver regeneration is entirely dependent on p21(Cip1) expression. Compared with wild-type mice, the absence of p21(Cip1) expression completely abrogated the TCDD inhibition, and accelerated hepatocyte progression through G1 phase during the regenerative process. Analysis of the transcriptional response determined that increased p21(Cip1) expression during liver regeneration involved an AhR-dependent mechanism. Chromatin immunoprecipitation studies revealed that p21(Cip1) induction required AhR binding to the newly characterized nonconsensus xenobiotic response element, in conjunction with the tumor suppressor protein Kruppel-like factor 6 functioning as an AhR binding partner. The evidence also suggests that AhR functionality following partial hepatectomy is dependent on a p21(Cip1)-regulated signaling process, intimately linking AhR biology to the G1-phase cell cycle program.

Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan

Water and sediment samples from river systems located in Southern Taiwan were investigated for the presence of aryl hydrocarbon receptor (AhR) agonists and genotoxicants by a combination of recombinant cell assays and gas chromatography–mass spectrometry analysis. AhR agonist activity and genotoxic response were frequently detected in samples collected during different seasons. In particular, dry-season water and sediment samples from Erren River showed strong AhR agonist activity (201–1423ngL−1 and 1374–5631ngg−1 β-naphthoflavone equivalents) and high genotoxic potential. Although no significant correlation was found between AhR agonist activity and genotoxicity, potential genotoxicants in sample extracts were suggested to be causative agents for yeast growth inhibition in the AhR-responsive reporter gene assay. After high performance liquid chromatography fractionation, AhR agonist candidates were detected in several fractions of Erren River water and sediment extracts, while possible genotoxicants were only found in water extracts. In addition, polycyclic aromatic hydrocarbons, the typical contaminants showing high AhR binding affinity, were only minor contributors to the AhR agonist activity detected in Erren River sediment extracts. Our findings displayed the usefulness of bioassays in evaluating the extent of environmental contamination, which may be helpful in reducing the chances of false-negative results obtained from chemical analysis of conventional contaminants. Further research will be undertaken to identify major candidates for xenobiotic AhR agonists and genotoxicants to better protect the aquatic environments in Taiwan.

Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells

Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P<.001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P<.001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P<.001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P<.001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.

Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines

The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity.

Aryl Hydrocarbon Receptor Deficient Donor T Cells Display Decreased Migration and Cause Reduced Or Enhanced Graft-Versus-Host Diseases In Different Transplant Models

The transcription factor aryl hydrocarbon receptor (AhR) is a cytosolic sensor of numerous small synthetic compounds (xenobiotics) and natural chemicals. Ligand binding of AhR causes a conformational change of the receptor, allowing its translocation to nucleus to regulate an array of genes. Different ligands could induce different sets of genes. AhR was first discovered as the mediator of dioxin toxicity. Its role in immunity was recently extensively investigated and expanded exponentially. Here we investigated the role of AhR in donor T cell-induced graft-versus-host diseases (GvHD) after bone marrow transplant in two MHC-mismatched models. B6D2 or Balb/c recipients were irradiated with one dose (950 rads for B6D2 and 800 rads for Balb/c) one day before transplant. C57BL/6 WT or AhR deficient (AhR KO) T cells were transplanted with T cell-depleted bone marrow to B6D2 or Balb/c, at a dose of three million T cells or five hundred thousand T cells, respectively. At different time points, recipient lymphoid organs or target organs were harvested and used for flow analysis or ELISA. A group of recipient mice were observed for an extended time to establish GvHD scores and survival curves. In B6 to B6D2 model, recipient mice transplanted with AhR KO T cells displayed lower GvHD scores and survived longer than mice transplanted with WT T cells (Figure 1C). We found that AhR KO donor T cells failed to accumulate in lymph nodes and spleen as rapidly as WT T cells as evident on day 1 and day 3 post-transplant (Figure 1A). Further investigation showed that the proliferation and cell death were similar between WT and AhR donor T cells, suggesting decreased migration of AhR KO T cells to lymphoid organs. It has been shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced AhR activation leads to the up-regulation of CCR4 and CCR5 in T cells, supporting our data that AhR affects cell migration. We also observed that lower percentage of AhR KO T cells produced IFN-g and IL-17 in lymph nodes and spleen early after transplant, but the differences disappeared at later time points (day 7). Colonic homogenates of AhR KO transplants contained less IFN-g, TNF-a, and IL-1a than WT transplants, which indicated that AhR KO T cells caused less inflammation in the gut (Figure 1B). Gut-associated injury due to inflammation is the major cause of death in this model. However, in B6 to Balb/c model, recipient mice transplanted with AhR KO T cells displayed the opposite survival curve. They had diminished survival compared to mice receiving WT T cells with most mice succumbing to GvHD around day 10 to day 15 before the onset of diarrhea. AhR KO T cells in this model still accumulated less in lymph nodes at early time points, which suggested that AhR played a role in cell migration in both models. It was noteworthy that after day 6, the number of AhR KO T cells in the spleen was actually similar to or higher than WT T cells in both models, suggesting cell proliferation was not affected by AhR deficiency. In conclusion, AhR KO T cells demonstrate decreased migration to lymphoid organs after bone marrow transplant compared to WT T cells. This decrease in migration may be the reason why mice transplanted with AhR KO T cells exhibit less inflammation in the gut and subsequently survive better from GvHD in the B6 to B6D2 model. However, mice transplanted with AhR KO T cells have diminished survival in B6 to Balb/c model despite of lower T cell numbers in lymph nodes at early time points. Thus, certain function of AhR in donor T cells is model dependent while other function is conserved among different models. These data suggest that investigators should be cautious regarding the supplementation of AhR ligands to diminish GvHD in clinical studies. Disclosures: No relevant conflicts of interest to declare.