Aryl Hydrocarbon Receptor In Regulation Research Articles

Aryl hydrocarbon receptor deficiency protects mice from diet-induced adiposity and metabolic disorders through increased energy expenditure.

BACKGROUND/OBJECTIVESEpidemics of obesity and diabetes are escalating. High-calorie/high-fat food is a major cause for these global health issues, but molecular mechanisms underlying high-fat, diet-induced obesity are still not well understood. The aryl hydrocarbon receptor (AhR), a transcription factor that acts as a xenobiotic sensor, mediates environmental toxicant-induced obesity, insulin resistance and development of diabetes. AhR also influences lipid metabolism and diet-induced obesity. The effects of AhR deficiency on diet-induced obesity, hepatic steatosis and insulin resistance were examined.METHODS: Male wild type (WT), AhR null (AhR−/−) and AhR heterozygote (AhR+/−) mice were fed a normal chow diet (NCD, 10% kcal from fat) or a high-fat diet (HFD, 60% kcal from fat) for up to 14 weeks. Adiposity, adipose and liver morphology, insulin signaling, metabolic parameters and gene profiles were assessed.RESULTSAhR deficiency protected against HFD-induced obesity, hepatic steatosis, insulin resistance and inflammation. Moreover, AhR deficiency preserved insulin signaling in major metabolic tissues. These protective effects result from a higher energy expenditure in AhR-deficient mice compared to WT. Levels of transcript for both the thermogenic gene, uncoupling protein 1 (Ucp1), in brown adipose tissue and mitochondrial β-oxidation genes in muscle were significantly higher in AhR−/− and AhR+/− mice compared to WT.CONCLUSIONSThis work documents a physiologically relevant function for AhR in regulation of body weight, hepatic fat deposition, insulin sensitivity and energy expenditure under HFD exposure, suggesting that AhR signaling may be developed as a potential therapeutic target for treatment of obesity and metabolic disorders.

Regulation of aryl hydrocarbon receptor interacting protein (AIP) protein expression by MiR-34a in sporadic somatotropinomas.

IntroductionPatients with germline AIP mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA).MethodsTo study the mechanism of low AIP protein expression 31 sporadic somatotropinomas with low (n = 13) or high (n = 18) AIP protein expression were analyzed for expression of AIP messenger RNA (mRNA) and 11 microRNAs (miRNAs) predicted to bind the 3’UTR of AIP. Luciferase reporter assays of wild-type and deletion constructs of AIP-3’UTR were used to study the effect of the selected miRNAs in GH3 cells. Endogenous AIP protein and mRNA levels were measured after miRNA over- and underexpression in HEK293 and GH3 cells.ResultsNo significant difference was observed in AIP mRNA expression between tumors with low or high AIP protein expression suggesting post-transcriptional regulation. miR-34a was highly expressed in low AIP protein samples compared high AIP protein adenomas and miR-34a levels were inversely correlated with response to SSA therapy. miR-34a inhibited the luciferase-AIP-3’UTR construct, suggesting that miR-34a binds to AIP-3’UTR. Deletion mutants of the 3 different predicted binding sites in AIP-3’UTR identified the c.*6–30 site to be involved in miR-34a’s activity. miR-34a overexpression in HEK293 and GH3 cells resulted in inhibition of endogenous AIP protein expression.ConclusionLow AIP protein expression is associated with high miR-34a expression. miR-34a can down-regulate AIP-protein but not RNA expression in vitro. miR-34a is a negative regulator of AIP-protein expression and could be responsible for the low AIP expression observed in somatotropinomas with an invasive phenotype and resistance to SSA.

Stability of the aryl hydrocarbon receptor and its regulated genes in the low activity variant of Hepa-1 cell line

We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two types of cell. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability.

Cross-species transcriptomic analysis elucidates constitutive aryl hydrocarbon receptor activity.

BackgroundResearch on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances.ResultsWe found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements.ConclusionsThese findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1053) contains supplementary material, which is available to authorized users.

Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways.

The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR−/− and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR−/− mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1+) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C–C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that are consistent with a possible role of AhR in wet AMD. The data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus; GEO Submission No. GSE56983, NCBI Tracking System No. 17021116.

AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

BackgroundThe aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).ResultsCells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.ConclusionAhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Expression and functional regulation of the nuclear receptors AHR, PXR, and CAR, and the transcription factor Nrf2 in rat parotid gland.

Nuclear receptors and transcription factors regulate the functions of many genes involved in cellular physiology and pathology (e.g. tumorigenesis and autoimmune diseases). The present study was performed to define the expression and the regulation of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), and nuclear factor E2-related factor 2 (Nrf2) in the rat parotid gland. Constitutive expression, as well as expression after stimulation with specific inducers for AhR [2,3,7,8-tetrachloro-dibenzylo-p-dioxin (TCDD)], Nrf2(oltipraz), PXR (dexamethasone), and CAR (phenobarbital), was evaluated using the quantitative PCR. Cellular localization of the nuclear receptors and the transcription factor was visualized by immunohistochemical staining. The study revealed constitutive expression of AhR as well as Nrf2, and their induction by TCDD andoltipraz, respectively. Immunohistochemical analysis revealed constitutive, predominantly cytoplasmic, expression of the AhR receptor, especially in interlobular striated duct cells, with nuclear shift upon exposure to TCDD. Inducible expression of Nfr2 was found mainly in the cytoplasm of intralobular striated duct cells. Constitutive expression of PXR and CAR was not found. Bearing in mind the involvement of AhR and Nrf2 in the regulation of many genes, it seems that these factors may play also a role in salivary gland physiology and pathology.

Co-expression network analysis reveals immune response pathways important for pathogenesis of Chlamydial pelvic inflammatory disease (PID). (HUM8P.329)

Abstract Processes driving development of immune pathology after Chlamydia trachomatis infection are poorly understood. To provide insight into the pathways that may be underappreciated by individual gene analysis, weighted gene co-expression network analysis was performed to identify network modules using blood transcriptional profiles of women with chlamydial PID (N=15) and asymptomatically infected women (N=84). Modules, defined as centrally connected genes, that were significantly associated with chlamydial PID were further accessed via Ingenuity Pathway analysis. Type I and type II interferon (IFN) signaling pathways were significantly up regulated in women with chlamydial PID. Up regulation of both of these pathways has been detected in the transcriptomes of patients with active tuberculosis, suggesting a parallel role in PID. Up regulation of aryl hydrocarbon receptor, agranulocyte/granulocyte adhesion and diapedesis pathways was also noted, while EIF2/EIF4, mTOR and ILK signaling, granzyme A/B signaling, cross talk between dendritic cells and natural killer cells, signaling in T helper cells and T cell receptor pathways were down-regulated. Pathways likely to contribute to immunopathogenesis, including migration of leukocytes and inflammation were activated in chlamydial PID, while those involved in cell growth/proliferation and clearance of infected cells were decreased. Bioinformatics provides a new avenue to improve understanding of chlamydial disease mechanisms.

IL-1 and TNF-α regulation of aryl hydrocarbon receptor (AhR) expression in HSY human salivary cells

ObjectivesRecent findings demonstrate that nuclear receptor – aryl hydrocarbon receptor (AhR) may play an important role in the pathogenesis of Sjögren's syndrome (SS) via involvement in Epstein–Barr virus reactivation. In that study a reporter system was used. Therefore, it was decided to define AhR expression in human salivary cell line (HSY) and its functional regulators. DesignThe expression and functional regulation of AhR was studied in HSY cells. The cells were incubated with dioxin (TCDD) – AhR model inducer, IL-1 and TNF-α. qRT-PCR was applied to assess the expression of AHR, AHRR (AhR repressor), ARNT (AhR nuclear translocator) as well as AhR dependent genes: CYP1A1 and CYP1B1. Enzymatic activity of CYP1A1 and CYP1B1 was evaluated using luciferin-labelled CYPs substrate. ResultsIn general, dioxin did not significantly influence the expression of AHR and ARNT, but reduced AHRR level. AhR dependent gene expression, i.e. CYP1A1 and CYP1B1 increased gradually with TCDD incubation time. TNF-α significantly induced AHR along with CYP1A1 and CYP1B1 expression. IL-1β did not affect AHR expression, and had minimal effects on CYP1 mRNA levels. Exposure of HSY cells to TCDD resulted in time-dependent induction of CYP1A1 and CYP1B1 enzymatic activity. ConclusionsThis study documents functional expression of AhR in HSY as well as induction of AhR and its dependent genes by TNF-α.

AhR is negatively regulated by miR-203 in response to TCDD or BaP treatment

The aryl hydrocarbon receptor (AhR) is an important nuclear receptor and mediates the biological consequences in response to environmental stimuli. However, the mechanism of AhR regulation in this process is still unclear. In this study, we attempt to identify which microRNAs (miRNAs) specifically modify the expression of AhR in A549 and HepG2 cells. miR-203 was predicted to target AhR by bioinformatic analysis. We performed a luciferase reporter assay and found miR-203 specifically binding to the mRNA 3′-UTR region of AhR, leading to the suppression of the expression of AhR at both the mRNA and protein levels. When exposed to TCDD or BaP, the expression of miR-203 could be induced and resulted in a reduction of AhR expression. The ectopic expression of miR-203 in A549 and HepG2 cells attenuated the activation of AhR induced by TCDD and subsequently suppressed its downstream regulated genes including cytochrome P450 1A1, 1A2 (CYP1A1, CYP1A2), and NADPH dehydrogenase 1 (NQO1). Moreover, overexpression of miR-203 affects the inducible enzyme activity of CYP1A1 by TCDD induction and the cytotoxicity induced by BaP. Taken together, we identify a novel miRNA that negatively regulates the expression of AhR, demonstrating that epigenetic modifications play a critical role in the metabolic activation of environmental chemicals.

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.

8: Epigenetic regulation of Th-17 differentiation

Differentiation of Th17 cells requires the combination of IL-6 and TGF-β and is further maintained by IL-23. This T cell subtype is important for both host defense against extracellular pathogens and the pathogenesis of autoimmune diseases. The main cytokines produced by Th17 cells are IL-17, which induces strong proinflammatory effects, and IL-22, an enhancer of epithelial proliferation and healing responses. Previous research has demonstrated that the expression of the transcription factor c-Maf is important for inhibition of IL-22 production in Th17 cells. The transcription factor AHR (aryl hydrocarbon receptor) has also been shown crucial for proper Th17 differentiation. The adapter protein PTIP (Pax transactivation domain-interacting protein) has recently been shown to have a role in the maturation of thymocytes. PTIP stably associates with complexes containing the chromatin methyltransferases MLL3 and MLL4, which modify chromatin in order to activate gene transcription. Here we demonstrate that PTIP contributes to Th17 differentiation and function via regulation of both c-Maf and AHR. Using CD4+ T cells from Ptipf/fCD4 Cre+ (PTIP−/−) mice and Cre- (WT) controls in a T cell transfer model of chronic colitis, we determined that RAG2−/− mice receiving WT cells had aggressive colitis with high expression of IL-17 in the intestine. However, in mice receiving PTIP−/− cells there was a reduction in IL-17 and an increase in IL-22 expression. The change in IL-17 and IL-22 expression in PTIP−/− mice was confirmed in vitro. Further analysis by chromatin immunoprecipitation (ChIP) assay showed that MLL3 binding and its dependent chromatin modifications at the loci for IL-17, c-Maf and AHR were enhanced in WT Th17 cells when compared to Th0 conditions and reduced in PTIP−/− Th17 cells. These data highlight, for the first time, that PTIP is essential for proper differentiation of Th17 cells.

The role of microRNA miR-34a in the regulation of aryl hydrocarbon receptor interacting protein

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The aryl hydrocarbon receptor in inflammatory bowel disease

The aryl hydrocarbon receptor (AhR), a transcription factor activated by a large number of environmental agents, modulates the activity of immune and nonimmune cells in the gut, and may represent an important link between the environment and the immune perturbations which underlie the pathogenesis of inflammatory bowel disease. This review will summarize the current knowledge of the role of AhR in regulation of intestinal immune homeostasis and inflammation. Activation of AhR by dietary ligands is necessary for the maintenance or expansion of innate immune cells in the gut, such as intraepithelial lymphocytes (IELs) and interleukin (IL)-22-producing lymphoid cells (ILC22). AhR-deficient mice lack IELs, have reduced number of ILC22 cells, and are more susceptible to bacterial infections and experimental colitis. In animal models, AhR activators inhibit proinflammatory cytokine synthesis and attenuate colitis by a pathway that involves IL-22. Analysis of AhR in the human gut reveals that intestinal T cells and natural killer cells isolated from Crohn's disease patients express low levels of AhR and respond to AhR ligands by downregulating inflammatory cytokines and upregulating IL-22. These novel findings may help explain how environmental factors may regulate mucosal immune responses.

The aryl hydrocarbon receptor (AhR) ligand VAF347 selectively acts on monocytes and naïve CD4+ Th cells to promote the development of IL-22-secreting Th cells

The low molecular weight compound VAF347, and its pro-drug version VAG539, interact with the transcription factor aryl hydrocarbon receptor (AhR) on monocytes to mediate its anti-inflammatory activity in vitro and in vivo. AhR is a crucial factor for IL-22 production, which regulates skin and gut homeostasis. Here we investigated whether VAF347 might control the differentiation of naïve T cells into IL-22-secreting cells and/or regulate IL-22 production by memory T cells. Human monocytes exposed to VAF347 differentiated into dendritic cells capable of instructing a naïve CD4(+) T cell differentiation program that promoted IL-22 secretion and concomitantly inhibited IL-17 production. Whilst AhR ligation by VAF347 on naïve CD4(+) T cells favored the development of single IL-22-secreting cells (Th22), it suppressed the generation of T cells secreting either IL-22 and IFN-γ or IL-17 and IFN-γ. In contrast, memory T cells were refractory to AhR regulation since VAF347, AhR antagonist or AhR gene silencing did not modulate the production of any of these cytokines. Interfering with AhR functions using VAF347 may provide an efficient way to intervene with autoimmune disease since it would enhance the host protective function mediated by IL-22 while preventing the development of Th cells secreting pro-inflammatory cytokines.

All-trans retinoic acid inhibits the recruitment of ARNT to DNA, resulting in the decrease of CYP1A1 mRNA expression in HepG2 cells

Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP) 1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RARα. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1–100nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions.

Aryl Hydrocarbon Receptor–Mediated Induction of Stearoyl-CoA Desaturase 1 Alters Hepatic Fatty Acid Composition in TCDD-Elicited Steatosis

2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) induces hepatic dyslipidemia mediated by the aryl hydrocarbon receptor (AhR). Stearoyl-CoA desaturase 1 (Scd1) performs the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis, desaturating 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. To further examine the role of Scd1 in TCDD-induced hepatotoxicity, comparative studies were performed in Scd1(+/+) and Scd1(-/-) mice treated with 30 μg/kg TCDD. TCDD induced Scd1 activity, protein, and messenger RNA (mRNA) levels approximately twofold. In Scd1(+/+) mice, hepatic effects were marked by increased vacuolization and inflammation and a 3.5-fold increase in serum alanine aminotransferase (ALT) levels. Hepatic triglycerides (TRGs) were induced 3.9-fold and lipid profiling by gas chromatography-mass spectroscopy measured a 1.9-fold increase in fatty acid (FA) levels, consistent with the induction of lipid transport genes. Induction of Scd1 altered FA composition by decreasing saturated fatty acid (SFA) molar ratios 8% and increasing MUFA molar ratios 9%. Furthermore, ChIP-chip analysis revealed AhR enrichment (up to 5.7-fold), and computational analysis identified 16 putative functional dioxin response elements (DREs) within Scd1 genomic loci. Band shift assays confirmed AhR binding with select DREs. In Scd1(-/-) mice, TCDD induced minimal hepatic vacuolization and inflammation, while serum ALT levels remained unchanged. Although Scd1 deficiency attenuated TCDD-induced TRG accumulation, overall FA levels remained unchanged compared with Scd1(+/+) mice. In Scd1(-/-) mice, TCDD induced SFA ratios 8%, reduced MUFA ratios 13%, and induced polyunsaturated fatty acid ratios 5% relative to treated Scd1(+/+) mice. Collectively, these results suggest that AhR regulation of Scd1 not only alters lipid composition but also contributes to the hepatotoxicity of TCDD.

Indolylfuran, a potent aryl hydrocarbon receptor agonist from sauerkraut, interacts with the oestrogen pathway

Numerous ligands of the aryl hydrocarbon receptor (AhR) have been found in plants, especially edible plants, such as cruciferous vegetables, which exert beneficent health effects. A potent activator of the AhR was found in sauerkraut juice. The isolated active ingredient was identified as the novel AhR ligand, (1-(2-furanyl)2-(3-indolyl)ethanone, common name indolylfuran. The isolated and the synthesised compound exerted similar potencies; their EC 50-values in an AhR transactivation assay were 160 and 123 nM, respectively. Our in vivo studies confirm and enlighten basic interactions between the AhR and oestrogen receptors (ERs). Further anti-oestrogenic effects of sauerkraut extract were shown. Indolylfuran regulates ER α and β expression, most likely via the AhR pathway, since indolylfuran had no effect on uterus weight and did not agonise ERα. Sauerkraut and indolylfuran may have potential for the prevention or treatment of diseases through modulation of AhR regulation and, indirectly, the ER pathway.

Hes1 Expression Impacts Megakaryocytic Polyploidization and May Contribute to the Platelet Defects Found In Aryl Hydrocarbon Receptor (AhR)-Null Mice.

Abstract 2612 Microarray analyses (n = 3) from our lab uncovered that Aryl hydrocarbon receptor (AhR) mRNA increased 4–6 fold in Megakaryocytes (Mks) compared to isogenic granulocytic cultures and identified the Aryl hydrocarbon receptor as a novel transcriptional activator of megakaryocytic differentiation and maturation (Lindsey et al. Blood, 2009). (Q)-RT-PCR and Western Blot analyses confirmed these findings in differentiating Mks derived from primary human CD34+ cells and differentiating CHRF cells (human megakaryoblastic leukemia cell line). Unpublished data from our lab found that AhR-null mice (Schmidt et al. Proc Natl Acad Sci, 1996) exhibited a 9% decrease in platelet counts compared to WT mice. There were also 10.4% fewer reticulated young, RNA-containing platelets in AhR-null mice compared to WT mice, indicating a defect in either platelet generation or Mk maturation. We examined platelet functionality and found that while the average bleeding time for WT mice was approximately 1.5 minutes, AhR-null mice bled on average 8 minutes, or roughly 5.3 times longer. We also calculated the volume of blood loss and found that AhR-null mice lost 3 times as much blood during these assays as WT mice. To investigate if these platelet defects were due to abnormal Mk maturation, we examined the level of steady-state polyploidization of Mks residing within the murine bone marrow niche and found that compared to WT mice, AhR-null mice had ca. 25% fewer high ploidy (≥ 32n) Mks, but more Mks in the lower ploidy classes of 8n and 16n. Together, these data suggest that AhR-null mice exhibit platelet defects of both number and function, and steady-state Mks from AhR-null mice are less polyploid than WT mice. Previous EMSA experiments (n = 4) had found that AhR transcriptional activity, measured by the binding of AhR to a consensus DNA binding site, increased during Mk polyploidization and that decreased Mk ploidy correlated to a 2.7 fold decrease in expression of Hes1, an AhR target gene (n = 3; p= 0.018). Therefore, we hypothesized that AhR impacts Mk ploidy through transcriptional regulation of Hes1. In our current work, EMSA experiments (n = 3) showed a 3.5 fold increase of AhR binding to oligonucleotides corresponding to a putative AhR binding site within the Hes1 promoter, indicating that AhR could transcriptionaly regulate Hes1 during megakaryopoiesis. As an important component and regulator of the Notch signaling cascade, Hes1 regulates the cell cycle and cellular quiescence (Sang et al. Science, 2008), but its role during normal Mk differentiation remains unexplored. Interestingly, mRNA expression of Dlk1, a Hes1 transcriptional target, is increased in CD34+ cells isolated from MDS patients compared to CD34+ cells from normal individuals (Sakajiri et al. Leukemia, 2005), suggesting that Hes1 target genes may be deregulated in megakaryoblastic leukemia. To determine if Hes1 could be involved in Mk differentiation, we performed (Q)-RT-PCR experiments on differentiating Mks, granulocytes, and erythrocytes ex vivo expanded from primary human CD34+ cells. We found that by day 12 Hes1 expression increases 2.8 fold in Mks, but decreases more than 2.9 fold in isogenic granulocytes (n = 2 for each; p = 0.044). Hes1 expression appears to remain unchanged during erythroid development (n = 2). To investigate the functional impact of Hes1 during megakaryopoiesis, we used RNAi-mediated knockdown of Hes1 to generate two independent KD-Hes1 cell lines. These cell lines express 68 and 59 percent less Hes1 mRNA (n = 3; p = 0.008 for Hes1-A and p = 0.015 for Hes1-B), 31 and 47 percent less Hes1 protein (n = 3), without impacting AhR expression (n = 3; p = 0.46). Similar to knocking down AhR, Mk-ploidy distributions in KD-Hes1 cells were shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥ 32n) seen in control cells. Although there was little impact in undifferentiated CHRF cells, ploidy levels on day 7 (maximal ploidy in control cells) were between 25.3 and 26.7 percent less than control cells depending on the cell line (n = 3; p &lt; 0.05 for both cell lines). These experiments genetically link Hes1 and AhR and suggest that AhR regulation of Hes1 during megakaryopoiesis mediates megakaryocytic polyploidization. As such, we provide a novel molecular model of endomitotic entry and Mk polyploidization, while also providing a mechanistic explanation for the platelet defects found in AhR null mice. Disclosures: No relevant conflicts of interest to declare.

Molecular and Functional Characterization of Aryl Hydrocarbon Receptor Repressor from the Chicken (Gallus gallus): Interspecies Similarities and Differences

The aryl hydrocarbon receptor (AHR) repressor (AHRR) has been recognized as a negative feedback modulator of AHR-mediated responses in fish and mammals. However, the repressive mechanism by the AHRR has not been investigated in other animals. To understand the molecular mechanism of dioxin toxicity and the evolutionary history of the AHR signaling pathway in avian species, the present study addresses chicken AHRR (ckAHRR). The complementary DNA sequence of ckAHRR encodes an 84-kDa protein sharing 29-52% identities with other AHRRs. High levels of ckAHRR messenger RNA were recorded in the kidney and intestine of nontreated chicks. In hepatoma LMH cells, the 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) 50% effective concentration value for ckAHRR induction (0.0016nM) was the same as that for chicken cytochrome P450 1A5 (ckCYP1A5), implying a shared transcriptional regulation of ckAHRR and ckCYP1A5 by chicken AHR (ckAHR). In ckAHRR transient transfection assays, ckAHRR repressed both ckAHR1- and ckAHR2-mediated transcriptional activities. Deletion and mutation assays revealed that basic helix-loop-helix/Per-ARNT-Sim A domains of ckAHRR, particularly 217-402 amino acid residues, are indispensable for the repression, but the AHR nuclear translocator sequestration by ckAHRR and SUMOylation of ckAHRR are not involved in its repressive mechanism. Additionally, subcellular localization assay of ckAHR1-enhanced green fluorescent protein fusion protein showed that ckAHRR did not affect nuclear translocation of the ckAHR1. Furthermore, ckAHRR inhibited the TCDD- and 17β estradiol-enhanced ckCYP1A5 transcription through AHR-estrogen receptor α (ERα) cross talk. Taken together, the function of AHRR is conserved in chicken in terms of the negative regulation of AHR and ERα activities, but its functional mechanism is likely distinct from those of the mammalian and fish homologues.