Aryl Hydrocarbon Receptor Nuclear Translocator 2 Research Articles

Gene Profiling of Cannabis-sativa-mediated Apoptosis in Human Melanoma Cells.

Malignant melanoma is an aggressive skin cancer, accounting for the majority of skin cancer deaths. Prognosis is often poor and finding effective treatment remains a challenge. Tetrahydrocannabinol (THC) and cannabidiol (CBD) are main bioactive components of Cannabis sativa plant extracts that have been shown to exert anti-tumor effects. In this study, we aimed to perform gene expression analysis of human melanoma A375 cells following stimulation with C. sativa extracts. Gene expression profiles of A375 human melanoma and Vero (control) cell lines were evaluated by RNA sequencing and quantitative real-time PCR. Flow cytometry showed that the THC+CBD cannabis fractions induced apoptosis on A375 cells. Induction of apoptosis was accompanied by a notable up-regulation of DNA damage inducible transcript 3 (DDIT), nerve growth factor receptor (NGFR), colony-stimulating factor 2 (CSF2), growth arrest and DNA damage inducible beta (GADD45B), and thymic stromal lymphopoietin (TSLP) genes and down-regulation of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), cyclin E2 (CCNE2), integrin subunit alpha 9 (ITGA9), proliferating cell nuclear antigen (PCNA) and E2F transcription factor 1 (E2F1) genes. Treatment of A375 cells with the THC+CBD fraction inhibited the phosphorylation of ERK1/2 signaling pathway, which regulates melanoma cell proliferation. We showed that the THC+CBD combination disrupted melanoma cell migration. Use of C. sativa-derived extracts containing equal amounts of THC and CBD is proposed as a potential treatment of melanoma.

Potential Molecular Mechanism of Upregulated Aryl Hydrocarbon Receptor Nuclear Translocator 2 in Nasopharyngeal Carcinoma.

Background Currently, the benefits of nasopharyngeal carcinoma (NPC) therapy are limited, and it is necessary to further explore possible therapeutic targets. Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) has been extensively studied in other cancer species, but little has been explored in NPC. The aim of this study was to verify the expression level of ARNT2 and its underlying mechanism in NPC. Methods Datasets containing ARNT2 mRNA expression levels were retrieved and collected from various databases to explore the expression status of ARNT2 in NPC. ARNT2-related coexpressed genes, differential expressed genes, and target genes were obtained for functional enrichment analysis. The potential target gene of ARNT2 and their regulatory relationship were studied through ChIP-seq data. CIBERSORTx was used to assess the immune infiltration of NPC, and the association with ARNT2 expression was calculated through correlation analysis. Results ARNT2 was upregulated and possessed an excellent discriminatory capability in NPC samples. ARNT2 positively correlated target genes were clustered in pathways in cancer, while negatively correlated target genes were enriched in immune-related pathway. The ChIP-seq information of ARNT2 and histone showed that prostaglandin-endoperoxide synthase 2 (PTGS2) was a potential target gene of ARNT2. CIBERSORTx revealed the immunity status in NPC, and ARNT2 expression was correlated with infiltration of five immune cells. Conclusions ARNT2 is overexpressed in NPC and may regulate PTGS2 to participate in the cancer process. ARNT2 serves as a key oncogenic target in NPC patients.

Exosomal transfer of miR-106a-5p contributes to cisplatin resistance and tumorigenesis in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), a subclass of cancers of the neck and head, is a predominant cause of cancer‐associated death worldwide. Hence, there is a critical need for research into NPC‐related treatment strategies. Cisplatin is a promising therapy option for NPCs and other cancers that is frequently utilized. Some patients acquire resistance to cisplatin therapy, which complicates the successful use of cisplatin treatment in NPCs. Although exosomal transfer of oncogenic miRNAs has been shown to improve recipient cell proliferation, metastasis and chemoresistance, the molecular mechanism behind this effect on NPC has yet to be fully understood. Exosomal microRNAs (miRNAs) from cisplatin‐resistant cells were identified as significant mediators of chemoresistance in NPC cells in this investigation. Initially, we found that exosomal miR‐106a‐5p levels in the serum of chemoresistant and last‐cycle patients were greater than in that of non‐resistant and first‐cycle patients. Also, exosomal miR‐106a‐5p enhanced the proliferative ability of NPC cells. Mechanistically, exosomal miR‐106a‐5p targets ARNT2, which further activates AKT phosphorylation, and thus promotes NPC cell proliferation, decreases apoptosis and in turn regulates tumorigenesis. We found similar results using in vivo NPC models, where exosomal miR‐106a‐5p through regulation of ARNT2 (aryl hydrocarbon receptor nuclear translocator 2) promoted tumorigenesis. Taken together, these findings indicate that exosomal miR‐106a‐5p could be a promising diagnostic biomarker and drug target for patients with NPC.

Elevation of DNA Methylation in the Promoter Regions of the Brain-Derived Neurotrophic Factor Gene is Associated with Heroin Addiction.

To study the potential role of brain-derived neurotrophic factor (BDNF) methylation in heroin addiction, we first detected the methylation level of seven CpG islands that included 106 CpG sites in the promoter regions of BDNF from 120 people addicted to heroin and 113 controls. Methylation quantitative trait locus (mQTL) analysis was then employed to determine the association between the single-nucleotide polymorphism rs6265, a well-known locus shown to be correlated with heroin addiction, and the methylation levels of these CpG sites. Finally, we used the JASPAR database to predict whether transcription factors could bind to these CpG sites. We found that the methylation levels of CpG islands 6 and 7 and the methylation levels of BDNF_45 and BDNF_80 were significantly higher in the heroin addiction group than in the control group. We also found that rs6265 was an mQTL and was associated with the methylation level of BDNF_58. Using the JASPAR database, we found that ALX homeobox 3 (ALX3), achaete-scute family bHLH transcription factor 1 (ASCL1) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) could bind to CpG island 6, and ALX3 could bind to CpG island 7. In summary, we showed that increased DNA methylation in the promoter regions of the BDNF gene was associated with heroin addiction in Han Chinese.

Generation of the iPSC line CUIMCi003-A derived from a patient with severe early onset obesity

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH/PAS) transcription factor involved in the development of paraventricular nucleus of the hypothalamus (PVH) through the heterodimerization with Single-minded 1 (SIM1) (Michaud et al., 2000). Using a Sendai virus-based approach, the four reprogramming factors OCT3/4, SOX2, KLF4 and C-MYC were delivered into Peripheral Blood Mononuclear Cell (PBMCs) from a 14-year-old girl with early onset obesity carrying a de novo variant (p.P130A) in ARNT2. The resulting iPSC line CUIMCi003-A had a normal karyotype, showed pluripotency and three germ layer differentiation capacity in vitro and was heterozygous for the de novo ARNT2 variant.

Oligodendrocyte ARNT2 expression is altered in models of MS.

ObjectiveWe examined expression of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a basic-loop-helix transcription factor implicated in neuronal development and axonal health, in oligodendrocyte (OL) cultures and over the course of chronic experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis (MS).MethodsWe assessed OL ARNT2 expression in EAE compared with sham-immunized controls and also in OL primary cultures and over the course of dibutyryl cyclic adenosine monophosphate (dbcAMP)-mediated maturation of the immortalized Oli-neu cell line. We also tested the functional role of ARNT2 in influencing OL characteristics using small interfering RNA (siRNA).ResultsARNT2 is localized to Olig2+ cells in healthy spinal cord gray and white matter. Despite a significant expansion of Olig2+ cells in the white matter at peak disease, ARNT2 is reduced by almost half in OLs, along with a reduction in the percentage of ARNT2+/Olig2+ cells. Mature OLs in mixed cortical cultures or OLs matured from embryonic progenitors express negligible ARNT2. Similarly, Oli-neu cells express high levels of ARNT2, which are reduced following dbcAMP maturation. siRNA-mediated knockdown of ARNT2 affected OL viability, which led to an enrichment of myelin-producing OLs.ConclusionThe analysis of ARNT2 expression in OLs demonstrates that OL ARNT2 expression is altered in EAE and during OL maturation. Findings point to ARNT2 as an important mediator of OL viability and differentiation and warrant further characterization as a target for intervention in demyelinating disorders such as MS.

Expression of the neuroprotective protein aryl hydrocarbon receptor nuclear translocator 2 correlates with neuronal stress and disability in models of multiple sclerosis

BackgroundAxonal degeneration and neuronal loss have been described as the major causes of irreversible clinical disability in multiple sclerosis (MS). The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) protein has been associated with neuroprotection in models of ischemia and neuronal responses to stressors.MethodsTo characterize its potential to influence inflammatory neurodegeneration, we examined ARNT2 expression in the experimental autoimmune encephalomyelitis (EAE) model of MS and characterized mediators that influence ARNT2 expression as well as plausible partners and targets.ResultsArnt2 message and protein levels dropped significantly in EAE spinal cords as disease developed and were lowest at peak disability. ARNT2 expression is prominent in neuronal cell bodies within the gray matter with some staining in glial fibrillary acidic protein (GFAP)+ astrocytes in healthy animals. At peak disease, ARNT2 expression is reduced by 20–50% in gray matter neurons compared to healthy controls. ARNT2 intensity in neurons throughout the EAE spinal cord correlated inversely with the degree of immune cell infiltration (r = − 0.5085, p < 0.01) and axonal damage identified with SMI32 staining (r = − 0.376, p = 0.032). To understand the relationship between ARNT2 expression and neuronal health, we exposed enriched cortical cultures of neurons to hydrogen peroxide (H2O2) to mimic oxidative stress. H2O2 at lower doses rapidly increased ARNT2 protein levels which returned to baseline within 3–4 h. Exposure to higher doses of H2O2) dropped ARNT2 levels below baseline, preceding cytotoxicity measured by morphological changes and lactate dehydrogenase release from cells. Decreases in ARNT2 secondary to staurosporine and H2O2 preceded increases in cleaved caspase 3 and associated apoptosis. We also examined expression of neuronal pas 4 (Npas4), whose heterodimerization with ARNT2 drives expression of the neurotrophic factor brain-derived neurotrophic factor (Bdnf). Like ARNT2, Npas4 levels also decline at the onset of EAE and are linked to decreases in Bdnf. In vitro, H2O2 exposure drives Npas4 expression that is tied to increases in Bdnf.ConclusionOur data support ARNT2 as a neuronal transcription factor whose sustained expression is linked to neuronal and axonal health, protection that may primarily be driven through its partnering with Npas4 to influence BDNF expression.

Emotion recognition associated with polymorphism in oxytocinergic pathway gene ARNT2.

The ability to correctly understand the emotional expression of another person is essential for social relationships and appears to be a partly inherited trait. The neuropeptides oxytocin and vasopressin have been shown to influence this ability as well as face processing in humans. Here, recognition of the emotional content of faces and voices, separately and combined, was investigated in 492 subjects, genotyped for 25 single nucleotide polymorphisms (SNPs) in eight genes encoding proteins important for oxytocin and vasopressin neurotransmission. The SNP rs4778599 in the gene encoding aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a transcription factor that participates in the development of hypothalamic oxytocin and vasopressin neurons, showed an association that survived correction for multiple testing with emotion recognition of audio–visual stimuli in women (n = 309). This study demonstrates evidence for an association that further expands previous findings of oxytocin and vasopressin involvement in emotion recognition.

Hepatoprotective and antioxidant effects of dietary Glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2, ARNT2, CYP1A mRNA in Jian Carp (Cyprinus carpio var. Jian)

To evaluate the protective effects of Glycyrrhiza polysaccharide (GPS) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatotoxicity in Jian carp, the fish were fed diets containing GPS at doses of 0.1, 0.5 and 1.0g/kg for 60days before an intraperitoneal injection of 0.6μg/kg TCDD at a volume of 0.05mL/10g body weight. At 72hr post-injection, blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices. The results showed that increases in alanine aminotransferase (GPT), aspartate aminotransferase (GOT), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP) in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0g/kg GPS. Following the 1.0g/kg GPS pre-treatment, total protein (TP), albumin (Alb), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in liver tissue increased significantly, malondialdehyde (MDA) formation (P<0.05 or P<0.01) was significantly inhibited, and the expression of cytochrome P4501A (CYP1A), aryl hydrocarbon receptor 2 (AHR2) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) mRNA (P<0.05) was significantly enhanced. Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining, and the results showed that histological changes were obviously reduced by 0.5 and 1.0g/kg GPS. GPS significantly reduced liver tissue damage caused by TCDD. Overall, these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD, and supported the use of GPS (1.0g/kg) as a hepatoprotective and antioxidant agent in fish.

A Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induced Liver Injury in Jian Carp (Cyprinus carpio var. Jian) Using Precision-Cut Liver Slices.

The aim of this study was to establish a model for the study of liver injury induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Jian carp using precision-cut liver slices (PCLS). PCLS were treated with TCDD at concentrations of 0, 0.05, 0.1, 0.3, and 0.6μg/L for 6h, followed by collection of the culture supernatant and PCLS for analysis. Several biochemical indices were analyzed, including glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Expression of mRNA was also estimated for cytochrome P4501A (CYP1A), aryl hydrocarbon receptor2 (AhR2), and aryl hydrocarbon receptor nuclear translocator2 (ARNT2). Results showed that some significant effects (p<0.05) in MDA, GSH-Px and PCLS viability were observed at a TCDD concentration as low as 0.05µg/L, and the observed effects increased with exposure concentration. Following exposure to TCDD for 6h at a concentration of 0.3μg/L, significant increases (p<0.01) in the content of GPT, GOT, MDA, and LDH were observed, while SOD activity, GSH-Px activity, and PCLS viability were decreased (p<0.01 or p<0.05). Exposure to 0.3μg/L TCDD also resulted in increased expression of mRNA for CYP1A, AhR2, and ARNT2. Overall, these results provide evidence of TCDD-induced liver injury and oxidative stress in Jian carp. These results also support the use of PCLS as an in vitro model for the evaluation of hepatotoxicity in Jian carp.

Downregulation of ARNT2 promotes tumor growth and predicts poor prognosis in human hepatocellular carcinoma.

Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is a transcriptional regulator and member of the basic helix-loop-helix/Per-ARNT-SIM (bHLH/PAS) superfamily. Recently, evidence of that ARNT is involved in carcinogenesis and cancer progression has emerged. The aim of current study was to investigate the role of ARNT2, a homolog of ARNT, in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC). Tissue microarray and immunohistochemical staining were used to examine the expression of ARNT2 in 195 HCC tissues. Factors associated with ARNT2 levels were assessed by univariate and multivariate Cox regression analyses. Cell proliferation, migration, and invasion assays were performed by using ARNT2 silencing and overexpressing HCCLM6 cell line. Orthotopic xenograft HCC model was used to elucidate the effects of ARNT2 on HCC progression in vivo. High intratumoral of ARNT2 level was well correlated with longer overall survival (OS) and lower tumor to recurrence (TTR) of HCC patients after resection. Multivariate analysis revealed that intratumoral ARNT2 overexpression was an independent prognostic factor for both OS and TTR. Knockdown of ARNT2 in HCCLM6 cells was significantly enhanced while overexpression of ARNT2 significantly inhibited the ability of cell proliferation, invasion, and migration. In animal studies, downregulation of ARNT2 in HCCLM6 cells promoted, whereas upregulation of ARNT2 in HCCLM6 cells reduced HCCLM6 growth in vivo. Our data demonstrate that ARNT2 plays an inhibitory role in HCC progression and suggest that ARNT2 may be a potential prognostic predictor and therapeutic target for HCC.

ARNT2 is downregulated and serves as a potential tumor suppressor gene in non-small cell lung cancer.

The present study aims to investigate the expression pattern of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) protein and its clinical significance in human non-small cell lung cancer (NSCLC). We investigated the expression levels of ARNT2 in 104 NSCLC surgical specimens by immunohistochemistry and then analyzed its clinical significance. Additionally, the role of ARNT2 on the biological properties of the NSCLC line HCC827 was experimentally tested in vitro and in vivo to confirm the clinical observations. We found that the expression level of ARNT2 was significantly higher in normal lung tissues compared with NSCLC tissues (P < 0.01). Overall survival (OS) of patients with a high intratumoral ARNT2 level was significantly longer than survival of those with a low ARNT2 level (P = 0.004). In addition, intratumoral ARNT2 expression was an independent prognostic factors for OS (hazard ratio [HR] = 0.529; P = 0.001). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the ARNT2 overexpression inhibited cell viability, while ARNT2 knockdown promoted cell growth in NSCLC cell lines HCC827 and A549. Annexin V/PI assay showed that ARNT2 overexpression increased cell apoptosis, while ARNT2 knockdown decreased cell apoptosis in HCC827 and A549 cells. Moreover, in vivo study showed that attenuated ARNT2 expression in HCC827 cells greatly promoted tumor growth, while overexpressed ARNT2 remarkably inhibited tumor growth in a HCC827 xenograft model. Taken together, our data demonstrate that ARNT2 might serve as a tumor suppressor in NSCLC progression.

Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome.

BackgroundAutism Spectrum Conditions (ASC) are neurodevelopmental conditions characterized by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is one subgroup of ASC and differs from classic autism in that in AS there is no language or general cognitive delay. Genetic, epigenetic and environmental factors are implicated in ASC and genes involved in neural connectivity and neurodevelopment are good candidates for studying the susceptibility to ASC. The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) gene encodes a transcription factor involved in neurodevelopmental processes, neuronal connectivity and cellular responses to hypoxia. A mutation in this gene has been identified in individuals with ASC and single nucleotide polymorphisms (SNPs) have been nominally associated with AS and autistic traits in previous studies.MethodsIn this study, we tested 34 SNPs in ARNT2 for association with AS in 118 cases and 412 controls of Caucasian origin. P values were adjusted for multiple comparisons, and linkage disequilibrium (LD) among the SNPs analysed was calculated in our sample. Finally, SNP annotation allowed functional and structural analyses of the genetic variants in ARNT2. We tested the replicability of our result using the genome-wide association studies (GWAS) database of the Psychiatric Genomics Consortium (PGC).ResultsWe report statistically significant association of rs17225178 with AS. This SNP modifies transcription factor binding sites and regions that regulate the chromatin state in neural cell lines. It is also included in a LD block in our sample, alongside other genetic variants that alter chromatin regulatory regions in neural cells.ConclusionsThese findings demonstrate that rs17225178 in the ARNT2 gene is associated with AS and support previous studies that pointed out an involvement of this gene in the predisposition to ASC.

Associations between oxytocin-related genes and autistic-like traits

Oxytocin has repeatedly been shown to influence human behavior in social contexts; also, a relationship between oxytocin and the pathophysiology of autism spectrum disorder (ASD) has been suggested. In the present study, we investigated single-nucleotide polymorphisms (SNPs) in the oxytocin gene (OXT) and the genes for single-minded 1 (SIM1), aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) and cluster of differentiation 38 (CD38) in a population of 1771 children from the Child and Adolescent Twin Study in Sweden (CATSS). Statistical analyses were performed to investigate any association between SNPs and autistic-like traits (ALTs), measured through ASD scores in the Autism-Tics, ADHD and other Co-morbidities inventory. Firstly, we found a statistically significant association between the SIM1 SNP rs3734354 (Pro352Thr) and scores for language impairment (p = .0004), but due to low statistical power this should be interpreted cautiously. Furthermore, nominal associations were found between ASD scores and SNPs in OXT, ARNT2 and CD38. In summary, the present study lends support to the hypothesis that oxytocin and oxytocin neuron development may have an influence on the development of ALTs and suggests a new candidate gene in the search for the pathophysiology of ASD.

Hippocampal expression of aryl hydrocarbon receptor nuclear translocator 2 and neuronal PAS domain protein 4 in a rat model of depression

The transcription factors aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) and neuronal PAS domain protein 4 (NPAS4) may influence emotion and cognitive function by regulating brain-derived neurotrophic factor expression in the hippocampus. We estimated hippocampal ARNT2 and NPAS4 expression in chronic unexpected mild stress (CUMS) rat model. The possible association was investigated between expression of these transcription factors and depressive behaviors. Behavioral tests were conducted before, during, and after 28 days of group housing or isolation plus CUMS. The sucrose solution consumption test was used to assess changes in interest and pleasure-seeking, and the open field test (OFT) was conducted to measure spontaneous activity and exploratory behavior. Expression levels of ARNT2 and NPAS4 were estimated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Compared to controls, rats subjected to isolation plus CUMS exhibited significantly reduced weight gain (t = 9.317, P = 0.000), sucrose consumption (t = 3.756, P = 0.003), horizontal ambulation (t = 2.362, P = 0.041), and number of rearings (vertical motion) (t = 2.268, P = 0.040). Relative hippocampal NPAS4 expression was significantly lower in depression model rats compared to controls (t = 2.995, P = 0.010) but there was no significant difference in hippocampal ARNT2 expression between groups (t = 0.091, P = 0.929). The relationship between the CUMS model of depression and NPAS4 expression requires further exploration.

Altered subcellular localization of heat shock protein 90 is associated with impaired expression of the aryl hydrocarbon receptor pathway in dogs.

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.

Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients

Background/PurposeWe hypothesized that polymorphic differences among individuals might cause variations in the effect that environmental endocrine disruptors (EEDs) have on male genital malformations (MGMs). In this study, individual variation in the genetic response to low-dose bisphenol A (BPA) was investigated in human foreskin fibroblast cells (hFFCs) derived from child cryptorchidism (CO) and hypospadias (HS) patients.Methodology/Principal FindingshFFCs were collected from control children without MGMs (n = 5) and child CO and HS patients (n = 8 and 21, respectively). BPA exposure (10 nM) was found to inhibit matrix metalloproteinase-11 (MMP11) expression in the HS group (0.74-fold, P = 0.0034) but not in the control group (0.93-fold, P = 0.84) and CO group (0.94-fold, P = 0.70). Significantly lower levels of MMP11 expression were observed in the HS group compared with the control group (0.80-fold, P = 0.0088) and CO group (0.79-fold, P = 0.039) in response to 10 nM BPA. The effect of single-nucleotide polymorphism rs5000770 (G>A), located within the aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) locus, on individual sensitivity to low-dose BPA was investigated in the HS group. A significant difference in neurotensin receptor 1 (NTSR1) expression in response to 10 nM BPA was observed between AA and AG/GG groups (n = 6 and 15, respectively. P = 0.031). However, no significant difference in ARNT2 expression was observed (P = 0.18).Conclusions/SignificanceThis study advances our understanding of the specificity of low-dose BPA effects on human reproductive health. Our results suggest that genetic variability among individuals affects susceptibility to the effects of EEDs exposure as a potential cause of HS.

Replication and Extension of Association Between Common Genetic Variants in SIM1 and Human Adiposity

Haplo-insufficiency of the bHLH (basic helix-loop-helix) transcription factor single-minded 1 (SIM1) causes severe obesity in mice and humans. We hypothesized that common genetic variations in/near SIM1 could exert more subtle effects on its function and associate with human adiposity. First, SIM1 coding regions were sequenced in severely obese subjects, and two common nonsynonymous single-nucleotide polymorphisms (nsSNPs) in complete linkage disequilibrium (LD) were identified: Pro352Thr (rs3734354) and Ala371Val (rs3734355). We next carried out a SNP association study of five adiposity traits (BMI, % body fat, abdominal visceral and subcutaneous fat, and leptin concentrations) in 1,699 whites and 1,173 blacks. TagSNPs covering SIM1 and nearby conserved regions, and the only common nsSNP in SIM1's binding partner aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) (Gly679Ser/rs4072568), were investigated. The effects of rs3734355/4 on SIM1 activity were tested using an in vitro reporter assay. We replicated previous observations that homozygosity for the 371Val allele was associated with higher BMI in white males (P = 0.003). Together with previous findings in white males (combined n = 3,479), BMI was increased by 1.10kg/m(2) in 371Val homozygotes (95% confidence interval (CI): 0.25-1.95kg/m(2), P = 0.01). In vitro, the 352Thr-371Val haplotype impaired SIM1 transcriptional activity by 22% (P < 0.0001). TagSNP analysis of SIM1 revealed two SNPs in the 3' region (rs9390322 and rs7746743) and another in intron 5 (rs3734353) to be significantly associated with various adiposity measures in ethnicity- and sex-specific manners after multiple testing correction. In white males, rs4072568 in ARNT2 was also associated with BMI (P = 9 × 10(-4)) and % body fat (P = 0.001). Our findings implicate heritable defects of the SIM1-ARNT2 axis in the predisposition to human obesity.

SiRNA-mediated knockdown of aryl hydrocarbon receptor nuclear translocator 2 affects hypoxia-inducible factor-1 regulatory signaling and metabolism in human breast cancer cells

Recent human studies found that the mRNA expression level of aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) was positively associated with the prognosis of breast cancer. In this study, we used small interfering RNA techniques to knockdown ARNT2 expression in MCF7 human breast cancer cells, and found that an almost 40% downregulation of ARNT2 mRNA expression increased the expression of sensitive to apoptosis gene (3.36-fold), and decreased the expression of von Hippel-Lindau (0.27-fold) and matrix metalloproteinase-1 (0.35-fold). The metabolite analysis revealed the contents of glucose, glycine, betaine, phosphocholine, pyruvate and lactate involved in the hypoxia-inducible factor (HIF)-1-dependent glycolytic pathway were significantly lower in cells treated with siARNT2. Our results suggested that ARNT2 might play an important role in the modulation of HIF-1-regulated signaling and metabolism.

Xenoestrogens down-regulate aryl-hydrocarbon receptor nuclear translocator 2 mRNA expression in human breast cancer cells via an estrogen receptor alpha-dependent mechanism

Environmental chemicals with estrogenic activity, known as xenoestrogens, may cause impaired reproductive development and endocrine-related cancers in humans by disrupting endocrine functions. Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is believed to play important roles in a variety of physiological processes, including estrogen signaling pathways, that may be involved in the pathogenesis and therapeutic responses of endocrine-related cancers. However, much of the underlying mechanism remains unknown. In this study, we investigated whether ARNT2 expression is regulated by a range of representative xenoestrogens in human cancer cell lines. Bisphenol A (BPA), benzyl butyl phthalate (BBP), and 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p′-DDT) were found to be estrogenic toward BG1Luc4E2 cells by an E-CALUX bioassay. ARNT2 expression was downregulated by BPA, BBP, and o,p′-DDT in a dose-dependent manner in estrogen receptor 1 (ESR1)-positive MCF-7 and BG1Luc4E2 cells, but not in estrogen receptor-negative LNCaP cells. The reduction in ARNT2 expression in cells treated with the xenoestrogens was fully recovered by the addition of a specific ESR1 antagonist, MPP. In conclusion, we have shown for the first time that ARNT2 expression is modulated by xenoestrogens by an ESR1-dependent mechanism in MCF-7 breast cancer cells.