Aryl Hydrocarbon Receptor Repressor Gene Research Articles

Non-cigarette tobacco products, aryl-hydrocarbon receptor repressor gene methylation and smoking-related health outcomes

IntroductionCigarette smoking leads to altered DNA methylation at the aryl-hydrocarbon receptor repressor (AHRR) gene. However, it remains unknown whether pipe or cigar smoking is associated with AHRR methylation. We evaluated...

Association of the AhR, ARNT, and AhRR gene polymorphisms and cord blood AhR levels with elevated cord blood IgE susceptibility in Taiwan mother-infant pairs: a nested case–control study

ABSTRACT The roles of aryl hydrocarbon receptor (AhR), AhR-nuclear translocator (ARNT), and AhR repressor (AhRR) genes in the elevation of cord blood IgE (CbIgE) remained unclear. Our aims were to determine the polymorphisms of AhR, ARNT, and AhRR genes, cord blood AhR (CBAhR) level, and susceptibility to elevation of CbIgE. 206 infant-mother pairs with CbIgE>=0.35 IU/ml and 421 randomly selected controls recruited from our previous study. Genotyping was determined using TaqMan assays. Statistical analysis showed AhR rs2066853 (GG vs. AA+AG: adjusted OR (AOR)=1.5, 95%CI=1.10–2.31 and AOR=1.60, 95%CI=1.06–2.43, respectively) and the combination of AhR rs2066853 and maternal total IgE (mtIgE)>=100 IU/ml were significantly correlated with CbIgE>=0.35 IU/ml or CbIgE>=0.5 IU/ml. CBAhR in a random subsample and CbIgE levels were significantly higher in infants with rs2066853GG genotype. We suggest that infant AhR rs2066853 and their interactions with mtIgE>=100 IU/ml significantly correlate with elevated CbIgE, but AhRR and ARNT polymorphisms do not.

Contrasting Association of Maternal Plasma Biomarkers of Smoking and 1-Carbon Micronutrients with Offspring DNA Methylation: Evidence of Aryl Hydrocarbon Receptor Repressor Gene–Smoking–Folate Interaction

BackgroundMaternal prenatal smoking is known to alter offspring DNA methylation (DNAm). However, there are no effective interventions to mitigate smoking-induced DNAm alteration. ObjectivesThis study investigated whether 1-carbon nutrients (folate, vitamins B6, and B12) can protect against prenatal smoking-induced offspring DNAm alterations in the aryl hydrocarbon receptor repressor (AHRR) (cg05575921), GFI1 (cg09935388), and CYP1A1 (cg05549655) genes. MethodsThis study included mother-newborn dyads from a racially diverse US birth cohort. The cord blood DNAm at the above 3 sites were derived from a previous study using the Illumina Infinium MethylationEPIC BeadChip. Maternal smoking was assessed by self-report and plasma biomarkers (hydroxycotinine and cotinine). Maternal plasma folate, and vitamins B6 and B12 concentrations were obtained shortly after delivery. Linear regressions, Bayesian kernel machine regression, and quantile g-computation were applied to test the study hypothesis by adjusting for covariables and multiple testing. ResultsThe study included 834 mother-newborn dyads (16.7% of newborns exposed to maternal smoking). DNAm at cg05575921 (AHRR) and at cg09935388 (GFI1) was inversely associated with maternal smoking biomarkers in a dose-response fashion (all P < 7.01 × 10−13). In contrast, cg05549655 (CYP1A1) was positively associated with maternal smoking biomarkers (P < 2.4 × 10−6). Folate concentrations only affected DNAm levels at cg05575921 (AHRR, P = 0.014). Regression analyses showed that compared with offspring with low hydroxycotinine exposure (<0.494) and adequate maternal folate concentrations (quartiles 2–4), an offspring with high hydroxycotinine exposure (≥0.494) and low folate concentrations (quartile 1) had a significant reduction in DNAm at cg05575921 (M-value, ß ± SE = −0.801 ± 0.117, P = 1.44 × 10−11), whereas adequate folate concentrations could cut smoking-induced hypomethylation by almost half. Exposure mixture models further supported the protective role of adequate folate concentrations against smoking-induced aryl hydrocarbon receptor repressor (AHRR) hypomethylation. ConclusionsThis study found that adequate maternal folate can attenuate maternal smoking-induced offspring AHRR cg05575921 hypomethylation, which has been previously linked to a range of pediatric and adult diseases.

Aryl Hydrocarbon Receptor Repressor Is Hypomethylated in Psoriasis and Promotes Psoriasis-like Inflammation in HaCaT Cells.

It is known that DNA hypomethylation of aryl hydrocarbon receptor repressor (AhRR), one of the epigenetic markers of environmental pollutants, causes skin diseases. However, the function and mechanisms are still unknown. We aimed to determine whether AhRR is hypomethylated in PBMC of psoriasis patients, as well as to examine the expression of psoriasis-related inflammatory cytokines and antimicrobial peptides after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in HaCaT cells overexpressing or silencing AhRR. AhRR was determined by qPCR, Western blot, immunohistochemistry, and immunocytochemistry in skin tissue and HaCaT cells. DNA methylation of AhRR was performed by Infinium Human Methylation450 BeadChip in PBMC of psoriasis patients and methylation-specific PCR (MSP) in HaCaT cells. NF-κB pp50 translocation and activity were performed by immunocytochemistry and luciferase reporter assay, respectively. We verified AhRR gene expression in the epidermis from psoriasis patients and healthy controls. AhRR hypomethylation in PBMC of psoriasis patients and pAhRR-HaCaT cells was confirmed. The expression level of AhRR was increased in both TCDD-treated HaCaT cells and pAhRR-HaCaT cells. NF-κB pp50 translocation and activity increased with TCDD. Our results showed that AhRR was hypomethylated and overexpressed in the lesional skin of patients with psoriasis, thereby increasing AhRR gene expression and regulating pro-inflammatory cytokines through the NF-κB signaling pathway in TCDD-treated HaCaT cells.

Meta-analysis of epigenome-wide association studies of carotid intima-media thickness

Common carotid intima-media thickness (cIMT) is an index of subclinical atherosclerosis that is associated with ischemic stroke and coronary artery disease (CAD). We undertook a cross-sectional epigenome-wide association study (EWAS) of measures of cIMT in 6400 individuals. Mendelian randomization analysis was applied to investigate the potential causal role of DNA methylation in the link between atherosclerotic cardiovascular risk factors and cIMT or clinical cardiovascular disease. The CpG site cg05575921 was associated with cIMT (beta = −0.0264, p value = 3.5 × 10–8) in the discovery panel and was replicated in replication panel (beta = −0.07, p value = 0.005). This CpG is located at chr5:81649347 in the intron 3 of the aryl hydrocarbon receptor repressor gene (AHRR). Our results indicate that DNA methylation at cg05575921 might be in the pathway between smoking, cIMT and stroke. Moreover, in a region-based analysis, 34 differentially methylated regions (DMRs) were identified of which a DMR upstream of ALOX12 showed the strongest association with cIMT (p value = 1.4 × 10–13). In conclusion, our study suggests that DNA methylation may play a role in the link between cardiovascular risk factors, cIMT and clinical cardiovascular disease.

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.

A comprehensive analysis of AHRR gene as a candidate for cleft lip with or without cleft palate

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. Methods: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. Results: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. Conclusion: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.

Transethnic associations among immune-mediated diseases and single-nucleotide polymorphisms of the aryl hydrocarbon response gene ARNT and the PTPN22 immune regulatory gene

BackgroundBecause immune responses are sensitive to environmental changes that drive selection of genetic variants, we hypothesized that polymorphisms of some xenobiotic response and immune response genes may be associated with specific types of immune-mediated diseases (IMD), while others may be associated with IMD as a larger category regardless of specific phenotype or ethnicity. ObjectiveTo examine transethnic gene-IMD associations for single nucleotide polymorphism (SNP) frequencies of prototypic xenobiotic response genes—aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), AHR repressor (AHRR) — and a prototypic immune response gene, protein tyrosine phosphatase, non-receptor type 22 (PTPN22), in subjects from the Environmental Polymorphisms Registry (EPR). MethodsSubjects (n = 3731) were genotyped for 14 SNPs associated with functional variants of the AHR, ARNT, AHRR, and PTPN22 genes, and their frequencies were compared among African Americans (n = 1562), Caucasians (n = 1838), and Hispanics (n = 331) with previously reported data. Of those genotyped, 2015 EPR subjects completed a Health and Exposure survey. SNPs were assessed via PLINK for associations with IMD, which included those with autoimmune diseases, allergic disorders, asthma, or idiopathic pulmonary fibrosis. Transethnic meta-analyses were performed using METAL and MANTRA approaches. ResultsARNT SNP rs11204735 was significantly associated with autoimmune disease by transethnic meta-analyses using METAL (odds ratio, OR [95% confidence interval] = 1.29 [1.08–1.55]) and MANTRA (ORs ranged from 1.29 to 1.30), whereas ARNT SNP rs1889740 showed a significant association with autoimmune disease by METAL (OR = 1.25 [1.06–1.47]). For Caucasian females, PTPN22 SNP rs2476601 was significantly associated with autoimmune disease by allelic association tests (OR = 1.99, [1.30–3.04]). In Caucasians and Caucasian males, PTPN22 SNP rs3811021 was significantly associated with IMD (OR = 1.39 [1.12–1.72] and 1.50 [1.12–2.02], respectively) and allergic disease (OR = 1.39 [1.12–1.71], and 1.62 [1.19–2.20], respectively). In the transethnic meta-analysis, PTPN22 SNP rs3811021 was significantly implicated in IMD by METAL (OR = 1.31 [1.10–1.56]), and both METAL and MANTRA suggested that rs3811021 was associated with IMD and allergic disease in males across all three ethnic groups (IMD METAL OR = 1.50 [1.15–1.95]; IMD MANTRA ORs ranged from 1.47 to 1.50; allergic disease METAL OR = 1.58 [1.20–2.08]; allergic disease MANTRA ORs ranged from 1.55 to 1.59). ConclusionsSome xenobiotic and immune response gene polymorphisms were shown here, for the first time, to have associations across a broad spectrum of IMD and ethnicities. Our findings also suggest a role for ARNT in the development of autoimmune diseases, implicating environmental factors metabolized by this pathway in pathogenesis. Further studies are needed to confirm these data, assess the implications of these findings, define gene-environment interactions, and explore the mechanisms leading to these increasingly prevalent disorders.

45P - Global and sex-specific epigenome-wide association studies for the identification of the main methylated loci related to smoking in a mediterranean population

BackgroundTobacco use has been reported to be the main cause of 90% of male and 80% of female lung cancers, as well as a relevant risk factor for other cancers such as oropharynx, larynx, esophagus, stomach, liver, pancreas, kidney, bladder, and colorectum. Smoking directly affects DNA methylation and although several genes have been reported to be differentially methylated in smokers, gender differences and population-specific differences remain to be further investigated. Our aim was to undertake global and sex-specific genome-wide epigenomic studies (EWAS) in a Mediterranean population to identify the main smoking DNA-methylated genes. MethodsWe analyzed 88 participants in the PREDIMED PLUS-Valencia study paired by age and sex (44 males and 44 females). We included current smokers, former and non-smokers according to the WHO classification. We performed genome-wide DNA methylation using Illumina 850K methylation EPIC arrays. Differential methylation (M-values) was statistically analyzed with Partek, including multivariate adjustement. ResultsIn the whole sample, the top-ranked differentially methylated CpG sites (blood) were: cg05575921 in the AHRR (aryl hydrocarbon receptor repressor) gene (P=2.73E-14), cg21566642 (P=1.51E-12); cg01940273 (P=2.10E-10); cg03636183 in the F2RL3 (coagulation factor II thrombin receptor-like 3) gene (P=6.90E-09); cg23576855 in the AHRR (9.05E-08); and cg17739917 in the RARA (retinoic acid receptor, alpha) gene (P=1.90E-07), replicating previous finding of the hypomethylation in the AHRR and F2RL3 sites in smokers. In the sex-specific analyses, we detected for both men and women similar hypomethylation in the cg05575921-AHRR site, but some heterogeneity for other loci including the BRCA1 gene. ConclusionsSmoking has consistent effect of on the methylation of the AHRR gene, but some gender differences for other genes have been detected. This may be related with further gender-smoking differences for some cancers. Clinical trial identification2011-005398-22. Legal entity responsible for the studyInstituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades. FundingInstituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades. DisclosureAll authors have declared no conflicts of interest.

Abstract 599: Smoking-associated AHRR demethylation in cord blood DNA: Impact of CD235a+ nucleated red blood cells

Abstract Prenatal tobacco smoke exposure is associated with numerous adverse health outcomes at birth and in later life. Numerous studies have demonstrated that Aryl Hydrocarbon Receptor Repressor gene (AHRR) hypomethylation in umbilical cord blood is significantly associated with prenatal tobacco smoke exposure and is suitable as a fetal exposure biomarker. The mechanism driving this demethylation and whether all cord blood cell types are impacted is unclear. Nucleated red blood cells (nucleated CD235a+ cells, nRBCs) are developmentally immature RBCs that display genome-wide demethylation and are observed at increased frequency in the cord blood of smoking mothers. We assessed if nRBC counts or methylation level affected smoking-associated DNA methylation in the AHRR gene as measured in whole cord blood. We analyzed methylation in DNA from whole cord blood, CD14+ monocytes, and nRBCs across four AHRR CpGs using pyrosequencing and also with Illumina 850K arrays (cg05575921) from smoking (n=34) and nonsmoking (n=19) mothers. nRBCs present in cord blood were determined by conventional CBCs and also estimated with a Houseman deconvolution model. AHRR methylation levels were significantly lower in nRBCs relative to whole blood and CD14+ monocytes. Methylation values at each AHRR CpG averaged 14.6% lower in nRBCs (range 0.4% to 24%, p=3.9E-13) relative to CD14+ monocytes with nonsmokers displaying significantly greater hypomethylation in nRBCs than smokers. Methylation levels at each CpG across the AHRR DMR were strongly associated with either self-reported smoking or cord blood cotinine levels in both CD14+ monocytes (r2=0.45, p=4.6E-08) and nRBCs (r2=0.26, p=1.2E-04). The most significant prenatal smoking effect on methylation occurred in CD14+ monocytes at the CpG located at chr5:373490 (-16.7%, p=6.7E-09), 112nt downstream of cg05575921. For subjects with 850K data, adjusting a regression analysis model with estimated cell-type composition, including nRBC counts or estimates, marginally increased the magnitude and significance of the prenatal smoke exposure effect on AHRR cg05575921 methylation. Prenatal tobacco smoke exposure strongly affects DNA methylation in the AHRR gene and this prenatal smoke exposure biomarker is largely unaffected by cord blood cell type composition. Citation Format: Matthew A. Bergens, Gary S. Pittman, Isabel J. Thompson, Michelle R. Campbell, Xuting Wang, Ma Wan, Cathrine Hoyo, Douglas A. Bell. Smoking-associated AHRR demethylation in cord blood DNA: Impact of CD235a+ nucleated red blood cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 599.

DNA Methylation and Smoking: Implications for Understanding Effects of Electronic Cigarettes

Use of electronic cigarettes (e-cigs) has increased sharply recently although understanding of toxicity is limited, particularly target organ effects. Altered DNA methylation is a reversible response to environmental exposures, including smoking, and may be useful as a biomarker of e-cig harm. Among studies examining DNA methylation in blood by smoking status, there is considerable variability in differentially methylated CpGs identified; certain CpGs are consistently found. These include AHRR (aryl hydrocarbon receptor repressor gene), particularly cg05575921, cg0363183 in the F2RL2 gene coding for the protease-activated receptor 4 (PAR-4), and several CpGs in the 2q37.1 genomic region. Differences are found even with short duration and light smoking; effects vary with pack-years and time since quitting among former smokers. For tissues other than blood, data are limited but also indicate altered methylation with smoking. DNA methylation changes are a consistent biomarker of smoke exposure. Most studies regarding smoke effects on methylation are of blood cells; further evidence regarding effects of smoke, secondhand smoke, and e-cigs on target tissues for smoking-related diseases are needed. Understanding biological effects of e-cigs is critically important to inform regulation; examination of e-cig effects on DNA methylation can significantly add to evidence-based regulation.

Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

Background:Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure.Objective:Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes.Method:We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating monocyte DNA collected from two independent human studies [ from Clinical Research Unit (CRU) and from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified T cells, T cells, granulocytes, B cells, and NK cells ( females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA.Results:RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers.Conclusions:Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type–specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395

Mechanistic Role of Cytochrome P450 1B1 in Hyperoxic Lung Injury

Supplemental oxygen is often used to assist those with respiratory dysfunction, including adults with acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD) in preterm infants. Experimental evidence has shown that increases in oxygen levels can create a hyperoxic environment that is capable of exacerbating existing pulmonary injury as well as independently inducing injury due to increases in reactive oxygen species (ROS) and oxidative stress. Our group has examined the role of cytochrome P450 (CYP) enzymes in hyperoxic toxicity because of their possible role in the generation of ROS. We have previously found that CYP1A1/1A2 as well as the aryl hydrocarbon receptor (AHR), protect against hyperoxic toxicity. We have also found that CYP1B1 may, in fact, contribute to hyperoxic toxicity in vitro and in Cyp1b1‐null mice. Further, we found that Cyp1b1‐null mice exposed to hyperoxia have decreased markers of oxidative stress and increased CYP1A gene expression compared to WT mice. In this study, we hypothesized that Cyp1b1‐null mice would be less susceptible to hyperoxic lung injury compared to WT mice and that CYP1A mechanistically contributes to the attenuated phenotype of Cyp1b1‐null mice. To test this hypothesis, we exposed male and female C57BL/6J (WT), Cyp1b1‐null, Cyp1a1/1b1‐null (DKO), and Cyp1a1/1a2/1b1‐null (TKO) mice to hyperoxia (&gt;95% O2) for 24–68 hours. Lung weight measurements were used to assess pulmonary injury and edema and qPCR was used to measure RNA. We found that all mice lacking CYP1B1, including DKO and TKO mice, had reduced pulmonary injury as indicated by reduced lung weights in male (P&lt;0.01) and female (P&lt;0.05) mice after 68 hours of hyperoxia. CYP1B1 gene expression was induced (P&lt;0.01) in WT mice during hyperoxia exposure, and CYP1A1 gene expression was increased in Cyp1b1‐null mice (P=0.01). NQO1 gene expression was significantly induced by hyperoxia (P&lt;0.05) in all groups with higher expression in TKO mice at room air (P&lt;0.001) and early phase of hyperoxia (P&lt;0.05). We found that pulmonary AHR repressor (AHRR) expression was increased in the DKO and TKO (P&lt;0.05) mice after hyperoxia exposure. Our results suggest that increased AHR‐dependent transcription, as evidenced by enhanced CYP1A1 and AHRR gene expression, may play a role in attenuating lung injury in mice lacking CYP1B1 and needs to be elucidated further. Future studies will thoroughly characterize the effect of CYP1B1 on oxidative stress in these animals and determine the viability of CYP1B1‐directed therapeutics to treat or prevent ARDS/BPD.Support or Funding InformationResearch reported in this publication was supported by the National Institutes of Health under award number R01ES019689, R01ES009132, R01HL12516, R01HL129794, and R01HL087174 to B.M. and T32GM088129 to A.V. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

Offspring DNA methylation of the aryl-hydrocarbon receptor repressor gene is associated with maternal BMI, gestational age, and birth weight

Prenatal smoke exposure, maternal obesity, aberrant fetal growth, and preterm birth are all risk factors for offspring metabolic syndrome. Cord blood aryl-hydrocarbon receptor repressor (AHRR) DNA methylation is responsive to maternal smoking during pregnancy. AHRR serves not only to inhibit aryl-hydrocarbon receptor (AHR) transcription, which is involved in mediating xenobiotic metabolism, but it is also involved in cell growth and differentiation. Other than maternal smoking, other predictors of offspring AHRR DNA methylation status remain unknown; we sought to identify them among newborns. We enrolled pregnant women in the PROGRESS birth cohort in Mexico City. Using pyrosequencing, we analyzed DNA methylation of 3 CpG sites within the AHRR gene promoter from the umbilical cord blood of 531 infants. We used generalized estimating equations to account for the correlation of DNA methylation between CpG sites. Multivariable models were used to adjust for maternal age, BMI, education, parity, smoke-exposure, infant sex, gestational age, and birth weight-for-gestational age. AHRR DNA methylation was positively associated with maternal BMI (P = 0.0009) and negatively associated with the length of gestation (P < 0.0001) and birth weight-for-gestational age (P < 0.0001). AHRR DNA methylation was 2.1% higher in offspring of obese vs. normal weight mothers and 3.1% higher in preterm vs. term infants, representing a third and a half standard deviation differences in methylation, respectively. In conclusion, offspring AHRR DNA methylation was associated with maternal obesity during pregnancy as well as infant gestational age and birth weight-for-gestational age. Further work to discover the health impacts of altered AHRR DNA methylation is warranted.

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

Association between polymorphisms in the aryl hydrocarbon receptor repressor gene and disseminated testicular germ cell cancer

In the Western world, testicular germ cell cancer (TGCC) is the most common malignancy of young men. The malignant transformation of germ cells is thought to be caused by developmental and hormonal disturbances, probably related to environmental and lifestyle factors because of rapidly increasing incidence of TGCC in some countries. Additionally, there is a strong genetic component that affects susceptibility. However, genetic polymorphisms that have been identified so far only partially explain the risk of TGCC. Many of the persistent environmental pollutants act through the aryl hydrocarbon receptor (AHR). AHR signaling pathway is known to interfere with reproductive hormone signaling, which is supposed to play a role in the pathogenesis and invasive progression of TGCC. The aim of the present study was to identify whether AHR-related polymorphisms were associated with risk as well as histological and clinical features of TGCC in 367 patients and 537 controls. Haplotype-tagging single-nucleotide polymorphisms (SNPs) were genotyped in genes encoding AHR and AHR repressor (AHRR). Binary logistic regression was used to calculate the risk of TGCC, non-seminoma versus seminoma, and metastasis versus localized disease. Four SNPs in AHRR demonstrated a significant allele association with risk to develop metastases (rs2466287: OR = 0.43, 95% CI 0.21–0.90; rs2672725: OR = 0.49, 95% CI: 0.25–0.94; rs6879758: OR = 0.27, 95% CI: 0.08–0.92; rs6896163: OR = 0.34, 95% CI: 0.12–0.98). This finding supports the hypothesis that compounds acting through AHR may play a role in the invasive progression of TGCC, either directly or through modification of reproductive hormone action.

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.

Epigenome-wide association study in the European Prospective Investigation into Cancer and Nutrition (EPIC-Turin) identifies novel genetic loci associated with smoking

A single cytosine-guanine dinucleotide (CpG) site within coagulation factor II (thrombin) receptor-like 3 (F2RL3) was recently found to be hypomethylated in peripheral blood genomic DNA from smokers compared with former and non-smokers. We performed two epigenome-wide association studies (EWAS) nested in a prospective healthy cohort using the Illumina 450K Methylation Beadchip. The two populations consisted of matched pairs of healthy individuals (n = 374), of which half went on to develop breast or colon cancer. The association was analysed between methylation and smoking status, as well as cancer risk. In addition to the same locus in F2RL3, we report several loci that are hypomethylated in smokers compared with former and non-smokers, including an intragenic region of the aryl hydrocarbon receptor repressor gene (AHRR; cg05575921, P = 2.31 × 10(-15); effect size = 14-17%), an intergenic CpG island on 2q37.1 (cg21566642, P = 3.73 × 10(-13); effect size = 12%) and a further intergenic region at 6p21.33 (cg06126421, P = 4.96 × 10(-11), effect size = 7-8%). Bisulphite pyrosequencing validated six loci in a further independent population of healthy individuals (n = 180). Methylation levels in AHRR were also significantly decreased (P < 0.001) and expression increased (P = 0.0047) in the lung tissue of current smokers compared with non-smokers. This was further validated in a mouse model of smoke exposure. We observed an association with breast cancer risk for the 2q37.1 locus (P = 0.003, adjusted for the smoking status), but not for the other loci associated with smoking. These data show that smoking has a direct effect on the epigenome in lung tissue, which is also detectable in peripheral blood DNA and may contribute to cancer risk.

Fusion of the AHRR and NCOA2 genes through a recurrent translocation t(5;8)(p15;q13) in soft tissue angiofibroma results in upregulation of aryl hydrocarbon receptor target genes

Soft tissue angiofibroma is a recently delineated tumor type of unknown cellular origin. Cytogenetic analysis of four cases showed that they shared a t(5;8)(p15;q13). In three of them it was the sole change, underlining its pathogenetic significance. FISH mapping suggested the involvement of the aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2) genes in 5p15 and 8q13, respectively. RT-PCR revealed in-frame AHRR/NCOA2 and NCOA2/AHHR transcripts in all four cases. Interphase FISH on paraffin-embedded tissue from 10 further cases without cytogenetic data showed that three were positive for fusion of AHRR and NCOA2. While AHRR has never been implicated in gene fusions before, NCOA2 is the 3'-partner in fusions with MYST3 and ETV6 in leukemias and with PAX3 and HEY1 in sarcomas. As in the previously described fusion proteins, NCOA2 contributes with its two activation domains to the AHRR/NCOA2 chimera, substituting for the repressor domain of AHRR. Because the amino terminal part of the transcription factor AHRR, responsible for the recognition of xenobiotic response elements in target genes and for heterodimerization, shows extensive homology with the aryl hydrocarbon receptor (AHR), the fusion is predicted to upregulate the AHR/ARNT signaling pathway. Indeed, global gene expression analysis showed upregulation of CYP1A1 as well as other typical target genes of this pathway, such as those encoding toll-like receptors. Apart from providing a diagnostic marker for soft tissue angiofibroma, the results also suggest that this tumor constitutes an interesting model for evaluating the cellular effects of AHR signaling.