Aryl Hydrocarbon Receptor Repressor Expression Research Articles

Aryl hydrocarbon receptor gene expression in ankylosing spondylitis and its correlation with interleukin-17, RAR-related orphan receptor gamma t expression, and disease activity indices.

Considering the role of T helper (Th)17 cells in the pathogenesis of ankylosing spondylitis (AS), the aim of this study was to determine the correlation between aryl hydrocarbon receptor (AHR) gene expression and the expression of Th17-related genes including interleukin (IL)-17 and RAR-related orphan receptor gamma t (RORγt) transcription factor. Thirty patients with AS (26 males, 4 females; mean age: 36.1±8.1 years) and 30 age- and sex-matched healthy individuals (26 males, 4 females; mean age: 36.2±14.6 years) were recruited for the case-control study between June 2021 and January 2022. Ribonucleic acid (RNA) was extracted from peripheral blood cells and expression levels of AHR, IL-17, RORγt, and AHR repressor (AHRR) genes were evaluated using real-time polymerase chain reaction technique. The serum level of IL-17 was evaluated with enzyme-linked immunosorbent assay. The results showed a nonsignificant elevation of AHR, IL-17, and RORγt gene expression in the patient group compared to the control. There was a direct correlation between AHR gene expression and IL-17 and RORγt genes and a negative correlation between AHR and AHRR expression. Moreover, AHR gene expression showed a weak correlation with disease activity indices, including Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, Bath Ankylosing Spondylitis Metrology Index, Bath Ankylosing Spondylitis Global Score, and Ankylosing Spondylitis Quality of Life. Moreover, the serum level of IL-17 was higher in AS patients compared to the healthy group (p=0.02). Upregulated expression of the AHR gene in ankylosing spondylitis and its correlation with IL-17 and ROR-γ t gene expression suggests that it could be a potential diagnostic and therapeutic target for AS.

From Differential DNA Methylation in COPD to Mitochondria: Regulation of AHRR Expression Affects Airway Epithelial Response to Cigarette Smoke.

Cigarette smoking causes hypomethylation of the gene Aryl Hydrocarbon Receptor Repressor (AHRR), which regulates detoxification and oxidative stress-responses. We investigated whether AHRR DNA methylation is related to chronic obstructive pulmonary disease (COPD) and studied its function in airway epithelial cells (AECs). The association with COPD was assessed in blood from never and current smokers with/without COPD, and in AECs from ex-smoking non-COPD controls and GOLD stage II-IV COPD patients cultured with/without cigarette smoke extract (CSE). The effect of CRISPR/Cas9-induced AHRR knockout on proliferation, CSE-induced mitochondrial membrane potential and apoptosis/necrosis in human bronchial epithelial 16HBE cells was studied. In blood, DNA methylation of AHRR at cg05575921 and cg21161138 was lower in smoking COPD subjects than smoking controls. In vitro, AHRR DNA methylation at these CpG-sites was lower in COPD-derived than control-derived AECs only upon CSE exposure. Upon AHRR knockout, we found a lower proliferation rate at baseline, stronger CSE-induced decrease in mitochondrial membrane potential, and higher CSE-induced late apoptosis/necroptosis. Together, our results show lower DNA methylation of AHRR upon smoking in COPD patients compared to non-COPD controls. Our data suggest that higher airway epithelial AHRR expression may lead to impaired cigarette smoke-induced mitochondrial dysfunction and apoptosis/necroptosis, potentially promoting unprogrammed/immunogenic cell death.

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Functional Analysis of the Enhancer at cg05575921, a DNA Methylation Site that is Tobacco Smoke‐Responsive and Highly Associated with Lung Cancer Risk

This project aims to investigate the molecular role of epigenetic changes from tobacco smoke exposure in the development of lung cancer, the largest cause of cancer-related deaths. The epigenome consists of the heritable chemical groups on the genome that regulate gene expression. One well-studied epigenetic mark is DNA methylation, which occurs at CpG dinucleotides and is usually associated with gene silencing. Environmental and lifestyle factors such as smoking can induce heritable alterations to the epigenome and can thereby influence disease risk. However, the mechanisms by which tobacco smoke exposure leads to increased lung cancer risk are not fully understood. Past studies have linked hypomethylation of cg05575921, a CpG in the aryl hydrocarbon receptor repressor (AHRR) gene, a regulator of the xenobiotic response pathway, as a strong indicator of lung cancer risk. Tobacco smoke exposure results in hypomethylation of cg05575921 in blood and lung tissue. Our previous research showed that cg05575921 flanks a tobacco smoke-inducible enhancer linked to the induction of AHRR expression. Our guiding hypothesis is that hypomethylation of cg05575921 is a byproduct of enhancer activation by tobacco smoke, and that it is the enhancer activity that has functional consequences, not the loss of methylation at cg05575921. To evaluate the functional role of this enhancer, we used CRISPR/Cas9 gene editing technology to generate monoclonal A549 cell lines with targeted DNA deletions of either the enhancer region, the CpG site adjacent to it, or a negative control region lacking regulatory marks. Resulting clones will be treated with cigarette smoke condensate (CSC) and analyzed for AHRR expression through whole-transcriptome mRNA sequencing (RNA-seq). Initial findings show a high rate of successful deletions in transfected cells, bolstering the technique's potential for studying enhancers’ roles in cancerous transformation. Thus far, clones in the enhancer deletion group have been screened via PCR for the desired deletion, further characterized via Sanger sequencing across deletion sites and heterozygous SNPs, exposed to CSC for 48 hours, and RNA has been subjected to RNA-seq. Analysis of RNA-seq data is ongoing. CpG deletion clones have been screened and sequenced and are ready to undergo CSC exposure. Negative control deletion clones have been generated and are growing in preparation for screening. Our analyses promise to shed light on the mechanism underlying the link between cg05575921 hypomethylation and lung cancer risk.

Functional analysis of an enhancer flanked by four tobacco smoke‐responsive DNA methylation sites in the aryl hydrocarbon receptor repressor gene Samuel Jump 1,2 , Chunli Yan 1,2 , Ite Offringa 123 * 1 USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA, 2 Department of Surgery, 3 Department of Biochemistry and Molecular

Lung cancer is the leading cause for cancer-related deaths worldwide. Research suggests that lung cancers arising in smokers show many different molecular alterations compared to those in non-smokers. While mutations contribute to lung cancer development, environmental exposures also lead to epigenetic changes –changes in the decorations and protein occupancy of the DNA—which have been shown to increase lung cancer risk. We are thus focused on studying how epigenetic changes caused by tobacco smoke exposure affect the development of lung adenocarcinoma. Specifically, the objective is to better understand the significance of tobacco smoke-induced DNA hypomethylation near regulatory elements of the aryl hydrocarbon receptor repressor (AHRR) genein increasing lung cancer risk. Differential DNA methylation can be an epigenetic marker of regulatory elements and is usually inversely associated with activity of these elements. It can be associated with changes in expression levels of their respective tobacco smoke responsive genes like AHRR. Previous epigenome-wide association studies have identified hypomethylation of cg05575921 upon exposure to cigarette smoke (a CpG which flanks an enhancer located in the third intron of AHRR) as predictive of lung cancer risk (1,2). Due to the importance of detoxification, it is expected that multiple enhancers may drive AHRR leading to identification of other tobacco smoke-induced hypomethylated CpGs in AHRR. From the literature I identified four smoking-induced hypomethylated CpGs on the flanks of another enhancer (as marked by histone enhancer marks H3K4me1 and H3K27ac) potentially establishing a link between cigarette smoke, this enhancer, hypomethylation, and altered levels of AHRR expression (3,4,5,6). By using a modified version of the CRISPR/Cas9 system, we are deleting this enhancer from A549 lung cancer cells to test the connection between differential DNA methylation flanking this enhancer and changes in the activity AHRR or other genes within 1 megabase. Our analyses promise to shed light on the mechanism underlying the link between hypomethylation in AHRR and lung cancer risk.

Dietary AhR Ligands Regulate AhRR Expression in Intestinal Immune Cells and Intestinal Microbiota Composition

A diet rich in vegetables and fruit is generally considered healthy because of a high content of phytochemicals, vitamins, and fiber. The phytochemical indole-3-carbinol (I3C), a derivative of glucobrassicin, is sold as a dietary supplement promising diverse health benefits. I3C metabolites act as ligands of the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals. Here, we investigated how dietary AhR ligand supplementation influences AhR target gene expression and intestinal microbiota composition. For this, we used AhR repressor (AhRR)-reporter mice as a tool to study AhR activation in the intestine following dietary I3C-supplementation in comparison with AhR ligand-deprived diets, including a high fat diet. AhRR expression in intestinal immune cells was mainly driven by dietary AhR ligands and was independent of microbial metabolites. A lack of dietary AhR ligands caused enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis and correlated with the expansion of Enterobacteriaceae, whereas Clostridiales, Muribaculaceae, and Rikenellaceae were strongly reduced. I3C supplementation largely reverted this effect. Comparison of I3C-induced changes in microbiota composition using wild-type (WT), AhRR-deficient, and AhR-deficient mice revealed both AhR-dependent and -independent alterations in the microbiome. Overall, our study demonstrates that dietary AhR ligand supplementation has a profound influence on Ahrr expression in intestinal immune cells as well as microbiota composition.

芳香烃受体在特应性皮炎患者外周血及皮损组织中的表达及意义

Objective To detect levels of aryl hydrocarbon receptor (AhR) and its downstream molecules in peripheral blood mononuclear cells (PBMCs) and sera from patients with atopic dermatitis (AD) , and to analyze the correlation of their expression with serum cytokines and the severity of AD. Methods Real-time quantitative PCR (RT-PCR) was performed to analyze mRNA expression of AhR, cytochrome P4501A (CYP1A1) , AhR repressor (AHRR) , AhR nuclear translocator (ARNT) in PBMCs from 29 AD patients and 17 healthy controls, enzyme -linked immunosorbent assay (ELISA) to detect serum levels of interleukin (IL) -1β, IL-6, tumor necrosis factor (TNF) -α, IL-4, IL-22 and AhR in the AD patients, and immunohistochemical study to determine AhR expression in skin lesions of the AD patients and normal skin tissues of 21 patients with pigmented nevus. Measurement data were compared by using unpaired Student′s t test, enumeration data were compared by using chi-square test, and correlations between indices were analyzed by using Pearson correlation analysis. Results The serum level of AhR was significantly higher in the AD group (41.26 ± 4.52 pmol/L) than in the healthy control group (33.73 ± 2.49 pmol/L, t = 6.507, P<0.001) . Compared with the healthy control group, the AD group showed significantly increased mRNA expression of AhR (1.572 ± 0.392 vs. 1.000 ± 0.173, t = 6.819, P = 0.007) , AHRR (2.402 ± 1.716 vs. 1.000 ± 0.788, t = 3.722, P = 0.039) , CYP1A1 (2.258 ± 1.598 vs. 1.000 ± 0.796, t = 3.400, P = 0.002) and ARNT (1.383 ± 0.842 vs. 1.000 ± 0.586, t = 1.653, P = 0.105) in PBMCs. The AhR expression in skin lesions in the AD group was significantly higher than that in normal skin tissues in the control group (0.191 ± 0.041 vs. 0.087 ± 0.017, t = 10.036, P<0.001) . In the AD group, the mRNA expression of AhR in PBMCs was positively correlated with eczema area and severity index score (r = 0.448, P = 0.019) and the serum IL-6 level (r = 0.377, P = 0.046) , and the AHRR mRNA expression was positively correlated with the serum IL-1β level (r = 0.467, P = 0.021) . Conclusion AhR and its downstream molecules were highly expressed in the AD patients compared with healthy controls, and the AhR expression was positively correlated with the serum IL-6 level and AD severity in AD patients, suggesting that the AhR signaling pathway may play a certain role in pathogenesis of AD and AhR may serve as an efficient index for evaluating AD severity. Key words: Dermatitis, atopic; Hydrocarbons, aromatic; Cytochrome P-450 enzyme system; Interleukin-6; Aryl hydrocarbon receptor

Expression analysis of aryl hydrocarbon receptor repressor (AHRR) gene in gallbladder cancer.

Background:The aryl hydrocarbon receptor repressor (AHRR), a member of the growing superfamily, is a basic helix-loop-helix/PerAHR nuclear translocator (ARNT)-Sim (bHLH-PAS) protein. AHRR has been proposed to function as a putative new tumor suppressor gene based on studies in multiple types of human cancers. This current study aims to investigate AHHR expression and its prognostic significance in gallbladder cancer.Methods:The study includes 48 gallbladder cancer and 34 chronic cholecystitis cases as controls. The expression level of AHRR was analyzed by using semi-quantitative PCR and immunohistochemical staining. The results were correlated with different clinical parameters.Results:We demonstrate that the expression of AHRR is significantly down-regulated in gallbladder cancer tissue samples as compared to that in chronic cholecystitis tissue samples by reverse transcriptase PCR (RT-PCR) (P = 0.017) and immunohistochemistry analysis (P = 0.002). Interestingly, our RT-PCR data revealed that AHRR mRNA expression is frequently down-regulated (45.8%; 22/48) in cases as compared to 14.7% (5/34) in controls. Similarly, immunohistochemical analysis data show significant down-regulation of AHRR expression in 77.1% (37/48) of gallbladder cancer cases than 44.1% (15/34) in controls (P < 0.017). Reduced mRNA and protein expression is significantly associated with advanced T-stage (P = 0.001), histological differentiation (P = 0.001), and tumors with nodal metastasis (P = 0.001). Decreased expression of AHRR is significantly associated with poor prognosis in gallbladder cancer patients.Conclusion:In conclusion, the present study suggests that low AHRR expression may be critical in gallbladder cancer development. Our data suggests that AHRR may act as a tumor suppressor gene and its expression profile may be useful as a diagnostic marker in gallbladder cancer.

Suppressive effects of aryl-hydrocarbon receptor repressor on adipocyte differentiation in 3T3-L1 cells

The aryl-hydrocarbon receptor repressor (AhRR) negatively regulates aryl-hydrocarbon receptor (AhR) signaling via its inhibitory transactivation. AhR is well known to suppress adipocyte differentiation, but the function of AhRR during adipogenesis is unclear. The purpose of this study was to investigate the role of AhRR in adipocyte differentiation using 3T3-L1 cells. During the early phase of differentiation, AhRR expression was transiently induced, but throughout the entire differentiation process, low levels of AhR expression were maintained. AhRR knockdown significantly increased not only glycerol-3-phosphate dehydrogenase (GPDH) activity but also lipid accumulation inside the cells. AhRR overexpression clearly reduced GPDH activity and lipid accumulation, indicating that AhRR upregulation during the early stage of adipogenesis suppresses adipocyte differentiation. Since AhRR knockdown increases the expression and activity of peroxisome proliferator-activated receptor γ (PPARγ), AhRR negatively regulates PPARγ during adipogenesis. In summary, similar to AhR, AhRR acts as an inhibitor of adipocyte differentiation. In addition to controlling the negative feedback loop of AhR, AhRR might be involved in other functions, especially in adipocyte differentiation processes.

Involvement of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Repressor in Helicobacter Pylori-related Gastric Pathogenesis.

Background: Persistent Helicobacter pylori (H. pylori) infection leads to various gastric diseases. Multiple studies have demonstrated that aryl hydrocarbon receptor (AHR) plays roles in the antibacterial response and aryl hydrocarbon receptor repressor (AHRR) is downregulated in stomach cancer. However, the role of AHR or AHRR in H. pylori-related gastric diseases remains unclear.Aims: To investigate whether AHR or AHRR is involved in H. pylori-related gastric diseases.Methods: Patients with gastritis or gastric adenocarcinoma were enrolled randomly, and gastric tissue specimens were diagnosed pathologically. AHR, AHRR, and H. pylori infection status in tissues were detected by immunohistochemistry. Human gastric cells were cocultured with H. pylori. siRNAs were used to silence AHR or AHRR, and a C57bl/6 mouse model colonized by H. pylori was established. Protein expression was determined by western blotting analysis, and TNF, IL-8 and IL-1β in cell supernatants were measured by ELISA.Results: AHR and AHRR were expressed in gastritis tissues and gastric cancer tissues without H. pylori infection, and principally located in the cytoplasm and nucleus. AHR expression was significantly correlated with AHRR expression in gastric tissues without H. pylori infection (P=0.008). However, their expressions were negatively correlated with H. pylori infection status. H. pylori coculture inhibited AHR and AHRR expression in stomach mucosa in vitro and in vivo. Gastric cells produced more TNF, IL-8 and IL-1β when AHR or AHRR was silenced.Conclusions: This preliminary study indicates that AHR and AHRR may be involved in H. pylori-related gastric pathogenesis, and helps toward understanding of inflammation-initiated carcinogenesis of gastric cancer.

Influence of AHRR Pro189Ala polymorphism on kidney functions.

The function of aryl hydrocarbon receptor repressor (AHRR) in the kidney is unclear. The present study investigated associations between AHRR Pro189Ala polymorphism and estimated glomerular filtration rates (eGFR), serum creatinine, and hemoglobin levels in 2775 Japanese adults without diabetes. In addition, we examined whether AHRR expression levels in the kidney of control and chronic kidney disease (CKD) rats were changed. Multiple linear regression analyses showed that carriers of the Ala allele had increased eGFR and lower concentrations of serum creatinine and hemoglobin (p<0.05). Immunohistochemical analysis showed that the expression of AHRR was upregulated in the kidneys of rats with CKD. These findings suggest that AHRR plays distinct roles in kidney functions and hemoglobin values. The effects of the AHRR polymorphism might be intensified in the kidneys of patients with CKD.

AhR mediates an anti-inflammatory feedback mechanism in human Langerhans cells involving FcεRI and IDO.

Aryl hydrocarbon receptor (AhR), an important regulator of immune responses, is activated by UVB irradiation in the skin. Langerhans cells (LC) in the epidermis of patients with atopic dermatitis (AD) carry the high-affinity receptor for IgE, FcεRI, and are crucially involved in the pathogenesis of AD by inducing inflammatory responses and regulating tolerogenic processes. We investigated AhR and AhR repressor (AhRR) expression and functional consequences of AhR activation in human ex vivo skin cells and in in vitro-generated LC. Epidermal cells from healthy skin were analyzed for their expression of AhR and AhRR. LC generated from CD34+ hematopoietic stem cells (CD34LC) were treated with the UV photoproduct and AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Cell surface receptors, transcription factors, and the tolerogenic tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) were analyzed using flow cytometry and quantitative PCR. Epidermal LC and CD34LC express AhR and AhRR. AhR was also found in keratinocytes, which lack AhRR. AhR activation of LC by FICZ caused downregulation of FcεRI in CD34LC without affecting their maturation. AhR-mediated regulation of FcεRI did not involve any known transcription factors related to this receptor. Furthermore, we could show upregulation of IDO mediated by AhR engagement. Our study shows that AhR activation by FICZ reduces FcεRI and upregulates IDO expression in LC. This AhR-mediated anti-inflammatory feedback mechanism may dampen the allergen-induced inflammation in AD.

313 - Role of MiRNA-125b in the Modulation of Endothelial Anti-Oxidative Response to Cigarette Smoking

Cigarette smoking is an important cardiovascular risk factor. Recent studies revealed that vascular cells can communicate via extracellular vesicles (EV). EV have been shown to transfer miRNAs to target vascular cells. The aryl hydrocarbon receptor (AhR) is a receptor of cigarette smoke components, including dioxin. Bound to its ligand, it translocates to the nucleus and acts as a transcription factor for many genes including anti-oxidative enzymes like NAD(P)H quinone dehydrogenase 1 (NQO1). The aryl hydrocarbon receptor repressor (AHRR) is a competitive inhibitor of the activity of AhR. We aim to analyze the miRNA profile in EV released from vascular endothelial cells upon exposure to aqueous cigarette smoke extract (CSEaq). In this study, the role of miRNAs in fine tuning of the activity of the anti-oxidative machinery of vascular endothelial cells was investigated. Primary human umbilical vein endothelial cells (HUVEC) were exposed for 24 hours to aqueous CSEaq under laminar flow conditions. Using ultracentrifugation, EV were harvested from cell culture supernatants and lysed for miRNA isolation. miRNA expression was quantified by stem-loop reverse transcription and qPCR. AHR, AHRR, and NQO1 gene expression was significantly increased with high laminar flow (1.77±0.47, 28.90±13.07, and 12.93±7.79, respectively) or without it (1.97±0.54, 21.00±16.55, 3.66±2.19, respectively) (P

Regulation of CD40 signaling in colon cancer cells and its implications in clinical tissues.

CD40 is a member of the tumor necrosis factor receptor family. We reveal here a correlation between CD40 expression and colon cancer differentiation. Upon CD40 ligand (CD40L) binding, CD40/CD40L signaling inhibited colon cancer proliferation, induced apoptosis, stalled cells at G0/G1, and influenced cell adhesion and metastasis. Clustering analysis identified the elevation of aryl hydrocarbon receptor repressor (AHRR) expression along with activation of CD40/CD40L signaling. Examination of clinical specimens revealed that both AHR and AHRR levels correlated with colon cancer histological grade. In addition, high expression of AHRR was associated with high expression of CD40 in tumor cells, with CD40L expression being particularly high in the tumor interstitium. Real-time PCR and western blotting analysis showed that AHRR expression in colon cancer cells was up-regulated by CD40L binding. The likely mediating signaling pathways for the effects of CD40 are described herein.

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

Wood smoke enhances cigarette smoke-induced inflammation by inducing the aryl hydrocarbon receptor repressor in airway epithelial cells.

Our previous studies showed that cigarette smokers who are exposed to wood smoke (WS) are at an increased risk for chronic bronchitis and reduced lung function. The present study was undertaken to determine the mechanisms for WS-induced adverse effects. We studied the effect of WS exposure using four cohorts of mice. C57Bl/6 mice were exposed for 4 or 12 weeks to filtered air, to 10 mg/m(3) WS for 2 h/d, to 250 mg/m(3) cigarette smoke (CS) for 6 h/d, or to CS followed by WS (CW). Inflammation was absent in the filtered air and WS groups, but enhanced by twofold in the bronchoalveolar lavage of the CW compared with CS group as measured by neutrophil numbers and levels of the neutrophil chemoattractant, keratinocyte-derived chemokine. The levels of the anti-inflammatory lipoxin, lipoxin A4, were reduced by threefold along with cyclo-oxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in airway epithelial cells and PGE2 levels in the bronchoalveolar lavage of CW compared with CS mice. We replicated, in primary human airway epithelial cells, the changes observed in mice. Immunoprecipitations showed that WS blocked the interaction of aryl hydrocarbon receptor (AHR) with AHR nuclear transporter to reduce expression of COX-2 and mPGES-1 by increasing expression of AHR repressor (AHRR). Collectively, these studies show that exposure to low concentrations of WS enhanced CS-induced inflammation by inducing AHRR expression to suppress AHR, COX-2, and mPGES-1 expression, and levels of PGE2 and lipoxin A4. Therefore, AHRR is a potential therapeutic target for WS-associated exacerbations of CS-induced inflammation.

Abstract 1791: Tristetraprolin downregulates AHRR expression through mRNA destabilization.

Abstract Background. The aryl hydrocarbon receptor repressor (AHRR) inhibits the transcription activity of the aryl hydrocarbon receptor (AHR) by competing for dimerization with the AHR nuclear translocator (ARNT) and subsequently binding to XRE. Interestingly, the AHRR has two AU-rich elements (AREs) in its 3’UTR which relates to the post-transcriptional regulation by ARE-binding proteins including Tristetraprolin (TTP), BRF1, HuR and so on. In previous study, we determined that TTP plays a critical role in the decaying of VEGF, LATS2, cIAP2, COX2 and Pim-1 through binding these AREs in 3’ UTR. Methodology/principal Findings. We investigated the post-transcriptional regulation of the AHRR expression by TTP. Our results show that the expression level of AHRR is inversely correlated with TTP expression in human breast cancer cell lines using the real-time PCR and western blot. The overexpression of TTP decreased the expression level of AHRR and inhibited the proliferation of MDA-MB435 human breast cancer cells. On the contrary, the treatment with a small interfering RNA against TTP increased the expression of AHRR mRNA and protein. AHRR mRNA contains two AREs within the 3’UTR and TTP destabilized a luciferase mRNA containing AHRR ARE. Moreover, RNA electrophoretic mobility shift assay revealed that TTP binds directly to the ARE of AHRR mRNA. Conclusions/Significance. These studies demonstrated that the TTP expression is inversely correlated with AHRR expression in several human breast cancer cell lines using the real-time PCR and western blot. We suggest that TTP acts as a negative regulator of the AHRR gene expression and it may have an effect on tumor development through inducing AHR activation and the expression of tumor suppressors containing XRE in human breast cancer cells. Citation Format: Hyun Hee Lee, Won Tae Kim, Dong Hee Kim, Sun-Hee Leem. Tristetraprolin downregulates AHRR expression through mRNA destabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2013-1791

Tristetraprolin suppresses AHRR expression through mRNA destabilization

The aryl hydrocarbon receptor repressor (AHRR) inhibits the transcription of the aryl hydrocarbon receptor (AHR) by binding to XRE. We report that AHRR expression is inhibited by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP decreased the mRNA stability and protein expression of AHRR, and TTP-overexpressing cells made smaller colonies than the control. Contrarily, inhibition of TTP by siRNA increased AHRR expression. Analyses of point mutants of the AREs demonstrated that AREs were responsible for the TTP-mediated destabilization of AHRR mRNA. RNA EMSA revealed that TTP directly binds to the AHRR 3′UTR. Taken together, we demonstrate that TTP acts as a negative regulator of AHRR and may affect tumor development through induction of tumor suppressor genes as observed in MDA-MB435.

Aryl Hydrocarbon Receptor Repressor (AhRR) Function Revisited: Repression of CYP1 Activity in Human Skin Fibroblasts Is Not Related to AhRR Expression

The skin reacts to environmental noxae by inducing cytochrome P450 (CYP)-catalyzed reactions via activation of the aryl hydrocarbon receptor (AhR). A drawback of this response is the generation of oxidative stress, which is especially dangerous for postreplicative cells such as dermal fibroblasts, in which damage may accumulate over time. Accordingly, in dermal fibroblasts, CYP1 expression is repressed and it has been proposed that this is due to the AhR repressor (AhRR), which is supposedly overexpressed in fibroblasts as compared with other skin cells. Here, we revisited this "AhRR hypothesis", which has been mainly based on ectopic overexpression studies and correlation analyses of high AhRR gene expression with CYP1A1 repression in certain cell types. In primary human skin fibroblasts (NHDFs) of 25 individuals, we found that (i) the AhRR was expressed only at moderate RNA copy numbers and that, against the common view, (ii) in some fibroblast strains, CYP1A1 mRNA expression could be induced by AhR activators. However, even the highest induction did not translate into measurable CYP1 enzyme activity, and neither basal expression nor mRNA inducibility correlated with AhRR expression. In addition, enhancement of CYP1A1 mRNA expression by trichostatin A, which inhibits AhRR-recruited histone deacetylases at the CYP1A1 promoter, failed to induce measurable CYP1 activity. Finally, AhRR-deficient ((-/-)) mouse embryonic fibroblasts were not induced to biologically relevant CYP1 enzyme activity despite impressive mRNA induction. These data clearly indicate that repressed CYP1 activity in NHDFs is not causally related to AhRR expression, which may serve a different, yet unknown, biological function.

Effects of dioxin isomers on induction of AhRs and CYP1A1 in early developmental stage embryos of medaka ( Oryzias latipes)

The aryl hydrocarbon receptor ( AhR) binds to polyaromatic compounds, including dioxins, and enhances the expression of several target genes, including drug-metabolizing cytochrome P450s ( CYP1As). Four AhR genes ( AhR1b-1, AhR1b-2, AhR2a, and AhR2b) were identified in the medaka genome. The molecular machinery involved in the dioxin response has been clarified chiefly in mammals, although fish models, such as zebrafish ( Danio rerio), medaka ( Oryzias latipes), and Fundulus, are excellent candidates for examining the mechanisms of developmental dioxin toxicity. Using these fish models, several experimental studies investigating the induced expression of CYP1A1 and AhRs, including functional evaluations by 2378T4CDD exposure, have been performed. However, few studies have examined the exposure to other dioxin isomers and it is not certain whether similar induced expressions patterns and toxicity-mediating functions of CYP1A1, AhRs, and AhR repressor ( AhRR) compare with 2378T4CDD exposure. In this study, we investigated the toxicity of 13 dioxin isomers, including 2378T4CDD, and the induced expression of AhRs, AhRR, and CYP1A1 ( CYP1A1_ORYLA) in the early life stages of medaka embryos. After exposure to dioxin isomers for 24–48 h, the expression of AhR2a and CYP1A1_ORYLA correlated to the dioxin toxicity, and AhRR mRNA was widely expressed indicating it modulates AhR activity during the early stages of medaka embryos.