Downregulation Of Aryl Hydrocarbon Receptor Research Articles

A188 ENHANCED MICROBIAL TRYPTOPHAN METABOLISM ACTIVATES THE ARYL HYDROCARBON RECEPTOR AND AMELIORATES INTESTINAL INFLAMMATION

Abstract Background Intestinal microbiota, diet, and the immune system have been proposed to contribute to the development of inflammatory bowel diseases (IBD). The aryl hydrocarbon receptor (AhR) is a critical regulator of intestinal immunity and mucosal barrier homeostasis that is activated by agonists such as host and microbial tryptophan metabolites. IBD patients have altered microbiota and reduced AhR agonists in intestinal content resulting in downregulation of AhR. Our recent findings indicate that mice harbouring microbiota with impaired tryptophan metabolism and AhR agonist production develop more severe inflammation during colitis. Aims To study the influence of diet-microbe interventions on AhR activation and intestinal inflammation in mice with impaired microbial tryptophan metabolism. Methods Germ-free C57BL/6 mice from McMaster’s Axenic Gnotobiotic Unit were colonized with cecal content of mice harbouring reduced microbiota with impaired tryptophan metabolism with and without tryptophan metabolizing Clostridium sporogenes. Following three weeks of microbial stabilization, mice were provided a high tryptophan (HT) diet or a low tryptophan (LT) diet as a control. Experimental colitis was induced using dextran sulfate sodium in drinking water (DSS; 2.5% w/v). Briefly, DSS was provided ad libitum for five days, followed by two days of recovery before tissue collection. Activation of AhR was measured in stool and C. sporogenes cultures using an in vitro AhR luciferase reporter assay. AhR agonists were quantified by capillary-electrophoresis-mass spectrometry. Colonic expression of AhR pathway genes was evaluated by RT-qPCR. Susceptibility to colitis was assessed by histologic analysis of distal colon sections, fecal lipocalin-2, weight loss, and clinical scores. Inflammatory gene transcripts were quantified in colon tissue by Nanostring. Results C. sporogenes efficiently produces AhR agonists in vitro and in vivo. In mice with impaired tryptophan metabolism, the HT diet increased AhR agonists in intestinal content and colonic AhR pathway gene expression, which was further enhanced in mice colonized with C. sporogenes. Compared to mice fed LT and HT diets, mice colonized with C. sporogenes and fed HT were most protected from weight loss, tissue damage, and immune cell infiltration. Conclusions These findings highlight the potential of tryptophan-rich diets and targeted microbe interventions as a novel approach to enhance AhR activation and mitigate inflammation during colitis. Consequently, exploring methods to improve tryptophan metabolism through diet and manipulation of the microbiota could be a promising therapeutic avenue for IBD patients with impaired AhR activation. Funding Agencies CCC, CIHR

A51 INTESTINAL MICROBIOTA DETERMINES ARYL HYDROCARBON RECEPTOR ACTIVATION AND SUSCEPTIBILITY TO COLITIS

Abstract Background Intestinal microbiota, diet, and the immune system have been proposed to contribute to the development of inflammatory bowel diseases (IBD). The aryl hydrocarbon receptor (AhR) is a critical regulator of intestinal immunity and mucosal barrier homeostasis that is activated by agonists such as host and microbial tryptophan metabolites. IBD patients have altered microbiota and reduced AhR agonists in intestinal content resulting in the downregulation of AhR. These findings necessitate further study to understand how diet-microbiota interactions contribute to intestinal inflammation. Purpose To study the influence of intestinal microbiota on tryptophan metabolism, AhR activation, and colitis severity. Method 8- to 10-week-old germ-free C57BL/6 mice were colonized with cecal content of mice harbouring specific pathogen-free (SPF) or a limited and well-defined altered Schaedler flora (ASF) microbiota. Germ-free mice were used as controls. Three weeks following colonization, mucosal injury was induced in a subset of mice with dextran sulfate sodium (DSS) in drinking water for five days followed by two days of water recovery. Activation of AhR was measured in stool using an in vitro AhR luciferase reporter assay. Stool AhR agonists were determined using high-performance liquid chromatography coupled to high-resolution mass spectrometry. Colonic expression of AhR pathway genes Cyp1a1, Il22, Ahrr, Ahr, and Il17 was evaluated by RT-qPCR. Susceptibility to colitis was assessed by analysing stool consistency and stool blood, colonic microscopic damage, immune infiltration by immunohistochemistry, and pro-inflammatory gene expression (NanoString). Fecal microbiota was analysed by 16S rRNA gene sequencing (Illumina). Result(s) AhR agonists, AhR activation in vitro, and AhR pathway gene expression were elevated in mice colonized with SPF microbiota in comparison to mice colonized with ASF microbiota and germ-free mice. In ASF-colonized mice, DSS induced more severe inflammation than in SPF-colonized mice, as demonstrated by worsened mucosal injury, greater weight loss, and softer stools. SPF-colonized mice developed less mucosal immune cell infiltration and pro-inflammatory gene signaling. Conclusion(s) Our findings suggest that intestinal microbiota composition determines the metabolic capacity to degrade tryptophan into agonists that homeostatically activate AhR. When mucosal injury is induced, mice with elevated microbiota-derived AhR agonists and AhR activation develop less severe mucosal injury and signs of colitis. This study presents a useful tool for evaluating dietary and microbial therapies in the context of a microbiota with impaired tryptophan metabolism. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

The absence of AhR in CD4+ T cells in patients with acute graft-versus-host disease may be related to insufficient CTCF expression

BackgroundAcute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Accumulating evidence suggests that imbalanced Treg/Th17 ratio accelerates the progression of aGVHD. The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated through cognate ligand binding. Current evidence supports that AhR plays a critical regulatory role in the differentiation of Treg and Th17 cells. However, the relationship between AhR and aGVHD remains unclear.ResultsOur results showed that AhR expression was downregulated significantly in CD4+ T cells from patients with aGVHD compared with the non-aGVHD group. We also discovered that after activating AhR deficient CD4+ T cells, the expression levels of the activation markers-CD40L, CD134 and CD137 and cell proliferation activity were significantly higher than those of AhR-expressing CD4+ T cells. Restoring the expression of AhR in aGVHD CD4+ T cells resulted in significantly increased percentage of Tregs and associated gene transcripts, including Foxp3, IL-10 and CD39. In contrast, Th17 cell amounts and the transcription of related genes, including RORγt, IL-17A and IL-17F, were significantly reduced. We confirmed that CTCF recruited EP300 and TET2 to bind to the AhR promoter region and promoted AhR expression by mediating histone H3K9/K14 hyperacetylation and DNA demethylation in this region. The low expression of CTCF caused histone hypoacetylation and DNA hypermethylation of the AhR promoter, resulting in insufficient expression in aGVHD CD4+ T cells.ConclusionsCTCF is an important inducer of AhR transcription. Insufficient expression of CTCF leads to excessive AhR downregulation, resulting in substantial CD4+ T cell activation and Th17/Treg ratio increase, thereby mediating the occurrence of aGVHD.

Upregulation of Aryl Hydrocarbon Receptor in Granulosa Cells by Endoplasmic Reticulum Stress Contributes to the PCOS Pathophysiology

Studies have demonstrated that endocrine disrupting chemicals (EDC) are involved in the pathophysiology of PCOS, and aryl hydrocarbon receptor (AHR) mediates the cellular effect of EDC by inducing xenobiotic metabolizing enzymes including cytochrome P450 1B1 (CYP1B1). However, recent studies suggest the novel role of AHR in various diseases, including obesity and cancer progression, independent from the EDC metabolism. We previously demonstrated that endoplasmic reticulum (ER) stress, a newly recognized local factor, contributes to PCOS pathology by affecting diverse functions of granulosa cells. We hypothesized that ER stress induces the expression of AHR and activates its downstream signaling in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. At first, we determined the upregulation of AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) in the granulosa cells of PCOS patients and model mice by immunohistochemical staining and qPCR. We examined CYP1B1 as a representative AHR target gene. Treatment of cultured human granulosa-lutein cells (GLCs) with tunicamycin (ER stress inducer) upregulated the expression of AHR, ARNT and CYP1B1. Knockdown of AHR decreased the tunicamycin-induced expression and activity of CYP1B1, suggesting the intermediary role of AHR in upregulation of AHR activity by ER stress. To confirm the role of AHR in vivo, we administered the AHR antagonist CH223191 to PCOS model mice. The administration of the antagonist restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, this study indicates that AHR and downstream signaling are activated by ER stress in GLCs of PCOS. Moreover, downregulation of local AHR expression and activation restores a normal reproductive phenotype in a PCOS mouse model. Our findings demonstrate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Suppressed Hepatic Production of Indoxyl Sulfate Attenuates Cisplatin-Induced Acute Kidney Injury in Sulfotransferase 1a1-Deficient Mice.

Endogenous factors involved in the progression of cisplatin nephropathy remain undetermined. Here, we demonstrate the toxico-pathological roles of indoxyl sulfate (IS), a sulfate-conjugated uremic toxin, and sulfotransferase 1A1 (SULT1A1), an enzyme involved in its synthesis, in cisplatin-induced acute kidney injury using Sult1a1-deficient (Sult1a1-/- KO) mice. With cisplatin administration, severe kidney dysfunction, tissue damage, and apoptosis were attenuated in Sult1a1-/- (KO) mice. Aryl hydrocarbon receptor (AhR) expression was increased by treatment with cisplatin in mouse kidney tissue. Moreover, the downregulation of antioxidant stress enzymes in wild-type (WT) mice was not observed in Sult1a1-/- (KO) mice. To investigate the effect of IS on the reactive oxygen species (ROS) levels, HK-2 cells were treated with cisplatin and IS. The ROS levels were significantly increased compared to cisplatin or IS treatment alone. IS-induced increases in ROS were reversed by downregulation of AhR, xanthine oxidase (XO), and NADPH oxidase 4 (NOX4). These findings suggest that SULT1A1 plays toxico-pathological roles in the progression of cisplatin-induced acute kidney injury, while the IS/AhR/ROS axis brings about oxidative stress.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Down-regulation of aryl hydrocarbon receptor intensifies carcinogen-induced retinal lesion via SOCS3-STAT3 signaling.

The aryl hydrocarbon receptor (AHR) is a ligand-activated receptor that regulates the metabolism of several xenobiotics and participates in ocular inflammation. Although severe inflammation is a major risk of retinal damage, the underlying mechanism is not well established. In this study, to elucidate how AHR mediates inflammation homeostasis, we hypothesized that AHR expression may diminish during long-term exposure to benzo [a] pyrene (B [a]P), a carcinogen in cigarette smoke. The blockage of AHR function considerably impaired suppressor of cytokine signaling 3 (SOCS3) negative feedback regulation and upregulated B [a]P-induced pro-inflammation. Signal transducer and activator of transcription 3 (STAT3) was activated by B [a] P due to AHR dysfunction in human adult retinal pigment epithelial cells (ARPE-19). The STAT3-inducible element revealed higher activity in AHR knockout cells with B [a] P treatment, but not in wild type ARPE-19 cells. Moreover, AHR dysfunction led to STAT3 hypo-ubiquitination and changed the STAT3-SOCS3 interaction. Increased STAT3-SOCS3 complex during AHR dysfunction by B [a] P was suppressed by nifuroxazide in ARPE-19 cells. Furthermore, the in vivo results showed that STAT3 inhibition during AHR impairment by long-term B [a] P exposure preserved the retina thickness and reversed the visual function in male C57Bl/6 mice. Overall, long-term B [a] P exposure may attenuate AHR function, dysregulating the homeostasis of the SOCS3-STAT3 axis with intensive STAT3 activation. This finding is significant given that the disintegration of the AHR-SOCS3 axis is a sensitive factor involved in AMD-like lesion development in the retina, revealing that the low AHR level may be associated with cigarette smoking or xenobiotics exposure, causing retina inflammation and damage.

Quinazolinone derivative BNUA-3 ameliorated [NDEA+2-AAF]-induced liver carcinogenesis in SD rats by modulating AhR-CYP1B1-Nrf2-Keap1 pathway.

Cytochrome P450 1B1, considered as one of the novel chemotherapeutic targets involved in cancer prevention and therapy is also associated with the conversion of procarcinogens into their active metabolites. The aryl hydrocarbon receptor (AhR) is responsible for mediating different biological responses to a wide variety of environmental pollutants and also causes transcriptional activation of cytochrome P450 enzymes including CYP1B1 and thus plays a pivotal role for initiating cancer and its progression. On the other hand, active carcinogenic metabolites and reactive oxygen species-mediated stress alter different molecular signalling pathways and gene expressions. Quinazoline derivatives are recognized for their diversified biological activities including anticancer properties. The current study was designed for evaluation of chemotherapeutic efficacy of a synthetic quinazolinone derivative BNUA-3 against hepatocellular cancer in Sprague-Dawley (SD) rats. A detailed in vivo analysis was performed by administrating BNUA-3 (15, 30mg/kg b.w. for 28days, i.p.) in N-Nitrosodiethylamine+2-Acetylaminofluorene induced partially hepatectomized liver cancer in SD rats. This was followed by morphological evaluations, biochemical estimations and analysis of different mRNA and protein expressions. The results demonstrated the potency of BNUA-3 in efficient restoration of the altered morphology of liver, its protective effect against lipid peroxidation, enzymic and non-enzymic antioxidants levels in liver tissue which was disrupted after cancer induction. The study also demonstrated downregulation of AhR, CYP1B1 and Keap1 expressions with subsequent augmentation of protective Nrf2, HO-1, NQO1 and GSTA1 expressions thus, revealing the chemotherapeutic potency of BNUA-3 in inhibiting liver carcinogenesis through AhR/CYP1B1/Nrf2/Keap1 pathway.

Elevated Notch1 enhances interleukin-22 production by CD4+ T cells via aryl hydrocarbon receptor in patients with lung adenocarcinoma.

Notch signaling induced interleukin (IL)-22 secretion by CD4+ T cells via retinoid-related orphan nuclear receptor γt (RORγt) or aryl hydrocarbon receptor (AhR). Previous studies have demonstrated that Notch-AhR-IL-22 axis took part in the pathogenesis of chronic viral infection, however, its role in cancer has not been fully elucidated. Thus, the aim of current study was to investigate the involvement of Notch-AhR-IL-22 axis in the pathogenesis of lung adenocarcinoma. A total of 37 late-stage lung adenocarcinoma patients and 17 healthy individuals were enrolled. CD4+ T cells were purified from peripheral bloods and bronchoalveolar lavage fluids (BALF), and were stimulated with γ-secretase inhibitor (GSI). mRNA corresponding to Notch receptors and transcriptional factors were measured by real-time PCR. IL-22 concentration was investigated by ELISA. The bioactivity (including cellular proliferation, cell cycle, apoptosis, and invasion) of lung adenocarcinoma cell line A549 was also assessed in response to recombinant IL-22 stimulation in vitro. Notch1 mRNA expression was significantly elevated in CD4+ T cells purified from peripheral bloods and tumor site BALF in lung adenocarcinoma patients. IL-22 expression and RORγt/AhR mRNA in BALF was also remarkably increased in tumor site. Inhibition of Notch signaling by GSI did not affect cellular proliferation, but reduced IL-22 production in CD4+ T cells from BALF, along with down-regulation of AhR, but not RORγt. Moreover, IL-22 stimulation promoted A549 cells invasion. The current data indicated that elevated Notch1 induced higher IL-22 secretion by CD4+ T cells in lung adenocarcinoma patients, and Notch-AhR-IL-22 axis took part in the pathogenesis of lung adenocarcinoma.

Aryl hydrocarbon receptor/IL-22/Stat3 signaling pathway is involved in the modulation of intestinal mucosa antimicrobial molecules by commensal microbiota in mice.

Compelling evidence demonstrates the crucial role of the commensal microbiota in host physiology and the detrimental effects of its perturbations following antibiotic treatment. However, the effects of commensal microbiota on intestinal mucosa antimicrobial molecules have not been elucidated systematically. Here, we investigate the impacts of antibiotic-induced depletion and subsequent restoration of the intestinal microbiota on the murine antimicrobial molecules in intestinal mucosa. Our results demonstrate that depletion of commensal microbiota leads to intestinal mucosa atrophy and reduction of antimicrobial molecules, including lysozyme, regenerating islet-derived protein 3 gamma (RegIIIγ), and cryptdin 5 mRNA, whereas subsequent reconstitution of intestinal microbiota by fecal microbiota transplantation (FMT) rescues mucosa morphology and antimicrobials. Importantly, our study shows that down-regulation of aryl hydrocarbon receptor (AhR), interleukin-22 (IL-22), and phosphorylated Stat3 (p-Stat3) is associated with decreased antimicrobials, which might mediate the antibiotic-associated intestinal mucosa injury. Last, exogenous activation of the AhR/IL-22/Stat3 signaling pathway with the AhR agonist 6-formylindolo(3,2-b)carbazole (Ficz) rescued antimicrobial molecule levels markedly after antibiotic treatment to levels similar to those following reconstitution of intestinal microbiota by FMT. Together, our results demonstrate that the AhR/IL-22/Stat3 signaling pathway is involved in the modulation of intestinal mucosa antimicrobial molecules by commensal microbiota and suggest this pathway as a promising target in the treatment of antibiotic-associated gut barrier damage.

Exacerbated Immune Complex-Mediated Vascular Injury in Mice with Heterozygous Deficiency of Aryl Hydrocarbon Receptor through Upregulation of Fcγ Receptor III Expression on Macrophages.

Aryl hydrocarbon receptor (AhR), which was discovered as a receptor for environmental concomitants, plays an important role widely in the immune system. In this study, we assessed AhR involvement in immune-complex-mediated vascular injury by examining the reverse-passive Arthus reaction in AhR heterozygous knockout (AhR+/-) mice. In the cutaneous Arthus reaction, dermal edema was severer in AhR+/- mice than in wild-type mice. The number of infiltrating neutrophils and mRNA expression levels of CXC chemokine ligand 1 and IL-6 were also increased in AhR+/- mice. Similarly, in the peritoneal Arthus reaction, infiltration of neutrophils was increased in AhR+/- mice. Peritoneal macrophages from AhR+/- mice expressed higher levels of Fcγ receptor III and produced higher levels of CXC chemokine ligand 1 and IL-6 after immune complex treatment. In addition, AhR occupied the promoter regions of Fcγ receptor III gene in peritoneal macrophages in a ligand-dependent manner. Depletion of macrophages reduced the cutaneous Arthus reaction in AhR+/- mice, and adoptive transfer of AhR+/- mice macrophages into wild-type mice exacerbated the peritoneal Arthus reaction. Furthermore, AhR expression was decreased and Fcγ receptor III expression was increased in CD14+ monocytes in peripheral blood from patients with immune-complex-mediated vasculitis compared with those from healthy controls. These results suggest that downregulation of AhR is associated with the exacerbation of immune-complex-mediated vascular injury.

A multidimensional blood stimulation assay reveals immune alterations underlying systemic juvenile idiopathic arthritis.

The etiology of sporadic human chronic inflammatory diseases remains mostly unknown. To fill this gap, we developed a strategy that simultaneously integrates blood leukocyte responses to innate stimuli at the transcriptional, cellular, and secreted protein levels. When applied to systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory disease of unknown etiology, this approach identified gene sets associated with specific cytokine environments and activated leukocyte subsets. During disease remission and off treatment, sJIA patients displayed dysregulated responses to TLR4, TLR8, and TLR7 stimulation. Isolated sJIA monocytes underexpressed the IL-1 inhibitor aryl hydrocarbon receptor (AHR) at baseline and accumulated higher levels of intracellular IL-1β after stimulation. Supporting the demonstration that AHR down-regulation skews monocytes toward macrophage differentiation, sJIA monocytes differentiated in vitro toward macrophages, away from the dendritic cell phenotype. This might contribute to the increased incidence of macrophage activation syndrome in these patients. Integrated analysis of high-dimensional data can thus unravel immune alterations predisposing to complex inflammatory diseases.

Abstract 2427: Alterations in gene expression after exposure of MDA-MB-231 cells to isobutylparaben

Abstract Parabens are a group of chemical compounds extensively used as preservatives in consumer products such as cosmetics, pharmaceuticals and processed food. Parabens have been shown to have endocrine disrupting properties, to accumulate in breast tissue and to increase the proliferation of hormone receptor positive breast cancer cell lines through competitive binding to the estrogen receptor. Studies have shown that isobutylparaben has the highest binding affinity for the aryl hydrocarbon receptor (AhR), reported to bind environmental toxins. This study examined the alterations in gene expression correlated to exposure of the hormone receptor negative breast cancer cell line MDA-MB-231 to isobutylparaben over a 72 hour time frame. Exposure of the cells to concentrations of isobutylparabens commonly found in consumer products alters the genetic expression profile of the cells. In our study, detectable expression levels of AhR mRNA decreased, while cytochrome P450 (CYP1A1) and receptor activator of NFκB ligand (RANKL) mRNA levels increased, as analyzed by qPCR. The downregulation of AhR and altered expression levels of invasion related genes is suggestive of an increased metastatic phenotype with exposure to isobutylparaben. Citation Format: Christine Strelchuk, Holly Jones Taggart. Alterations in gene expression after exposure of MDA-MB-231 cells to isobutylparaben [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2017-2427

RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expression by Creating MicroRNA Recognition Sequence

Adenosine to inosine (A-to-I) RNA editing is the most frequent type of post-transcriptional nucleotide conversion in humans, and it is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. In this study we investigated the effect of RNA editing on human aryl hydrocarbon receptor (AhR) expression because the AhR transcript potentially forms double-stranded structures, which are targets of ADAR enzymes. In human hepatocellular carcinoma-derived Huh-7 cells, the ADAR1 knockdown reduced the RNA editing levels in the 3'-untranslated region (3'-UTR) of the AhR transcript and increased the AhR protein levels. The ADAR1 knockdown enhanced the ligand-mediated induction of CYP1A1, a gene downstream of AhR. We investigated the possibility that A-to-I RNA editing creates miRNA targeting sites in the AhR mRNA and found that the miR-378-dependent down-regulation of AhR was abolished by ADAR1 knockdown. These results indicated that the ADAR1-mediated down-regulation of AhR could be attributed to the creation of a miR-378 recognition site in the AhR 3'-UTR. The interindividual differences in the RNA editing levels within the AhR 3'-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA-editing-dependent down-regulation of AhR by miR-378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered for the first time that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver.

Ligand-independent activation of aryl hydrocarbon receptor signaling in PCB3-quinone treated HaCaT human keratinocytes

Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a critical role in metabolism, cell proliferation, development, carcinogenesis, and xenobiotic response. In general, dioxin-like polychlorinated biphenyls (PCBs) exhibit a ligand-dependent activation of AhR-signaling. Results from this study show that a quinone-derivative (1-(4-Chlorophenyl)-benzo-2,5-quinone; 4-ClBQ) of a non-dioxin like PCB (PCB3) also activates AhR-signaling. Treatments of HaCaT human keratinocytes with 4-ClBQ and dioxin-like PCB126 significantly increased AhR-target gene expression, CYP1A1 mRNA and protein levels. 4-ClBQ-induced increase CYP1A1 expression was associated with an increase in the nuclear translocation of AhR protein as well as an increase in the luciferase-reporter activity of a human CYP1A1 xenobiotic response element (XRE). 6,2′,4′-Trimethoxyflavone (TMF), a well-characterized AhR-ligand antagonist significantly suppressed PCB126-induced increase in CYP1A1 expression, while the same treatment did not suppress 4-ClBQ-induced increase in CYP1A1 expression. However, siRNA-mediated down-regulation of AhR significantly inhibited 4-ClBQ-induced increase in CYP1A1 expression, suggesting that AhR mediates 4-ClBQ-induced increase in CYP1A1 expression. Interestingly, treatment with the antioxidant N-acetyl-l-cysteine significantly suppressed 4-ClBQ-induced increase in CYP1A1 expression. Furthermore, CYP1A1 expression also increased in cells treated with hydrogen peroxide. These results demonstrate that a ligand-independent and oxidative stress dependent pathway activates AhR-signaling in 4-ClBQ treated HaCaT cells. Because AhR signaling is believed to mediate xenobiotics response, our results may provide a mechanistic rationale for the use of antioxidants as effective countermeasure to environmental pollutant-induced adverse health effects.

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.

AHR Regulates WT1 Genetic Programming during Murine Nephrogenesis

Mounting evidence suggests that the blueprint of chronic renal disease is established during early development by environmental cues that dictate alterations in differentiation programming. Here we show that aryl hydrocarbon receptor (AHR), a lig-and-activated basic helix-loop-helix-PAS homology domain transcription factor, disrupts murine renal differentiation by interfering with Wilms tumor suppressor gene (WT1) signaling in the developing kidney. Embryonic kidneys of C57BL/6J Ahr⁻/⁻ mice at gestation d (GD) 14 showed reduced condensation in the nephrogenic zone and decreased numbers of differentiated structures compared with wild-type mice. These deficits correlated with increased expression of the (+) 17aa Wt1 splice variant, decreased mRNA levels of Igf-1 rec., Wnt-4 and E-cadherin, and reduced levels of 52 kDa WT1 protein. AHR knockdown in wild-type embryonic kidney cells mimicked these alterations with notable increases in (+) 17aa Wt1 mRNA, reduced levels of 52 kDa WT1 protein, and increased (+) 17aa 40-kDa protein. AHR downregulation also reduced Igf-1 rec., Wnt-4, secreted frizzled receptor binding protein-1 (sfrbp-1) and E-cadherin mRNAs. In the case of Igf-1 rec. and Wnt-4, genetic disruption was fully reversed upon restoration of cellular Wt1 protein levels, confirming that functional interactions between AHR and Wt1 represent a likely molecular target for renal developmental interference.

A Role of the Aryl Hydrocarbon Receptor in Attenuation of Colitis

The aryl hydrocarbon receptor (AhR), which is a member of the basic helix-loop-helix/Per-Arnt-Sim homology superfamily, plays an important role in multiple biological functions, and AhR knockout (AhR KO) animals suffer from a variety of organ disorders including a decline in the efficacy of their immune system. In addition, AhR activation is known to aid the maintenance of homeostasis in vivo. In this study, we investigated whether AhR is functionally associated with intestinal immunity. In in vivo experiments, it was found that dextran sodium sulfate (DSS)-evoked colitis was more severe in AhR KO mice than in C57BL/6J wild type mice. It was also revealed that the administration of DSS increased the expression levels of AhR and CYP1A1 mRNA in the colon epithelium. In addition, oral administration of β-naphthoflavone (βNF), a non-toxic agonist of AhR, suppressed the pathogenesis of DSS-induced colitis. βNF also attenuated DSS-induced colitis. In cell culture experiments, downregulation of AhR in human colon carcinoma SW480 cells enhanced the inflammatory responses evoked by lipopolysaccharide (LPS), and furthermore, AhR activation attenuated LPS-induced inflammatory responses, suggesting that AhR expressing intestinal epithelial cells are involved in the prevention of colitis. Our findings about the potential role of AhR activators in epithelial immune regulation aid our understanding of mucosal homeostasis and inflammatory bowl disease (IBD) and suggest that AhR activation has therapeutic value for the treatment of IBD.

Activation of the aryl hydrocarbon receptor induces hepatic steatosis via the upregulation of fatty acid transport

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix/Per-ARNT-Sim domain transcription factor, which is activated by various xenobiotic ligands. AHR is known to be abundant in liver tissue and to be associated with hepatic steatosis. However, it has not yet been elucidated how the activation of AHR promotes hepatic steatosis. The aim of this study is to clarify the role of AHR in hepatic steatosis. The intraperitoneal injection of 3-methylcholanthrene (3MC), a potent AHR ligand, into C57BL/6J mice significantly increased the levels of triglycerides and six long-chain monounsaturated fatty acids in the livers of mice, resulting in hepatic microvesicular steatosis. 3MC significantly enhanced the expression level of fatty acid translocase (FAT), a factor regulating the uptake of long-chain fatty acids into hepatocytes, in the liver. In an in vitro experiment using human hepatoma HepG2 cells, 3MC increased the expression level of FAT, and the downregulation of AHR by AHR siRNA led to the suppression of 3MC-induced FAT expression. In addition, the mRNA level of peroxisome proliferator-activated receptor (PPAR) α, an upstream factor of FAT, was increased in the livers of 3MC-treated mice. Taking together, AHR activation induces hepatic microvesicular steatosis by increasing the expression level of FAT.

In Vitro and In Vivo Studies Demonstrate Anticancer Property of Root Extract of Polygala senega

Polygala senega is extensively used in traditional systems of medicine against various lung diseases including cancer. In the present study we tested the anticancer potentials of ethanolic extract of roots of P. senega (generally used as a homeopathic drug) in a mammalian model, where mice, in vivo, were treated chronically with benzo[a] pyrene and in vitro where lung adenocarcinoma cell line (A549) were used. We deployed various parameters like cell viability assay, chromatin condensation studies with Hoechst 333258 staining, and maintained suitable controls. To understand the possible signal transduction pathways, expression of various signal proteins such as Aryl Hydrocarbon receptor (AhR), cytochrome P450 (CYP1A1), Bcl-2, proliferating cell nuclear antigen (PCNA), Bax and Caspase-3 was studied. Additionally, reverse transcriptase polymerase chain reaction analysis of AhR, p53, PCNA and β-actin (housekeeping) genes was made. Immunohistochemical localization of PCNA proteins was also conducted in vivo. Feeding of root extract of P. senega to mice (at the rate of 50 mg/kg and 100 mg/kg bw) chronically treated with the carcinogen (50 mg/kg bw dissolved in olive oil) showed positive modulation in expression of signal proteins. Upregulation of apoptotic signals such as p53, Caspase-3 and Bax, and downregulation of AhR, cytochrome P450 (CYP1A1), Bcl-2 and PCNA were observed. Addition of root extract of Polygala Senega (at doses of 50 μg and 100 μg) into culture medium containing A549 cells induced recovery of decreased cell viability and increased chromatin fragmentation (apoptosis). Therefore, results of both in vivo and in vitro studies scientifically validate its potential use as an anticancer agent, particularly against lung cancer, and provide important information potentially helpful in drug designing.