Aryl Hydrocarbon Receptor Nuclear Translocator Expression Research Articles

Targeting ARNT attenuates chemoresistance through destabilizing p38α-MAPK signaling in glioblastoma

Glioblastoma (GBM) is the most aggressive and lethal brain tumor in adults. This study aimed to investigate the functional significance of aryl hydrocarbon receptor nuclear translocator (ARNT) in the pathogenesis of GBM. Analysis of public datasets revealed ARNT is upregulated in GBM tissues compared to lower grade gliomas or normal brain tissues. Higher ARNT expression correlated with the mesenchymal subtype and poorer survival in GBM patients. Silencing ARNT using lentiviral shRNAs attenuated the proliferative, invasive, and stem-like capabilities of GBM cell lines, while ARNT overexpression enhanced these malignant phenotypes. Single-cell RNA sequencing uncovered that ARNT is highly expressed in a stem-like subpopulation and is involved in regulating glycolysis, hypoxia response, and stress pathways. Mechanistic studies found ARNT activates p38 mitogen-activated protein kinase (MAPK) signaling to promote chemoresistance in GBM cells. Disrupting the ARNT/p38α protein interaction via the ARNT PAS-A domain restored temozolomide sensitivity. Overall, this study demonstrates ARNT functions as an oncogenic driver in GBM pathogenesis and represents a promising therapeutic target.

#6635 ARNT AND ALK3 MEDIATE RENO PROTECTION AFTER ISCHEMIC PRECONDITIONING

Abstract Background and Aims Ischemia and following hypoxia are a common cause of acute kidney injury (AKI). Strategies for prevention and treatment of AKI are limited. In several animal models, ischemic preconditioning (IPC) has been established as an effective intervention to prevent ischemic organ injury. An IPC protocol of ischemia and reperfusion mediates organ protection for a few hours. This first phase of organ protection is followed by a second, protective phase occurring after 24–48 h. The second phase, named second window of protection (SWOP), persists for 3–4 d. Mechanisms and transcriptional mediators during SWOP are mostly unknown. The aim of the underlying study was to investigate effectors and target genes during SWOP and to identify a potential pharmacological therapeutic target. Method 6–8-week-old C57BL/6 mice were undergoing an IPC intervention; five minutes of renal sinistra artery ischemia was followed by five minutes of reperfusion. This cycle was performed five times. Mice were sacrificed 2, 4, 8, 12, 25, 48, 72 h after IPC. Two additional groups of mice were pharmacologically preconditioned for 48 h with low dose FK506 and small molecule GPI1046. Analysis of murine kidneys were performed by SDS-Page and Western blot. Results The first phase after IPC (2–4 h) was characterized by an increasing expression of hypoxia inducible factor-1α (HIF1α); 48 h after intervention a significant increase of expression of aryl hydrocarbon receptor nuclear translocator (ARNT) and consecutive activin receptor-like kinase 3 (ALK3) was observed. Comparable induction of ARNT and ALK3 expression was obtained by pharmacological preconditioning with FK506 or GPI1046. Conclusion Affected by hypoxia, HIF1α activates transcriptional upregulation of ARNT 48 h after IPC. The increasing expression of ARNT, follows an increased ARNT homodimerization. ARNT homodimer binds to the palindromic E-box 5′-CACGTG-3′ of the proximal ALK3 promoter. By this it induces the expression of the renoprotective ALK3 during SWOP. The organ-protective effects of constitutively expressed ARNT during SWOP can be mimicked pharmacologically by administration of FK506 or GPI1046 and may be used as a future therapeutic target.

Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 μmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 μmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Effects of intranasal instillation of nanoparticulate matter in the olfactory bulb

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague–Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P < 0.001) and 1.60-fold (P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

Daytime Restricted Feeding Modifies the Temporal Expression of CYP1A1 and Attenuated Damage Induced by Benzo[a]pyrene in Rat Liver When Administered before CYP1A1 Acrophase.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that heterodimerizes with the AhR nuclear translocator (ARNT) to modulate CYP1A1 expression, a gene involved in the biotransformation of benzo[a]pyrene (BaP). The AhR pathway shows daily variations under the control of the circadian timing system. Daytime restricted feeding (DRF) entrains the expression of genes involved in the processing of nutrients and xenobiotics to food availability. Therefore, we evaluate if temporal AhR, ARNT, and CYP1A1 hepatic expression in rats are due to light/dark cycles or fasting/feeding cycles promoted by DRF. Our results show that AhR oscillates throughout the 24 h period in DRF and ad libitum feeding rats (ALF), showing maximum expression at the same time points. DRF modified the peak of ARNT expression at ZT5; meanwhile, ALF animals showed a peak of maximum expression at ZT17. An increased expression of CYP1A1 was linked to the meal time in both groups of animals. Although a high CYP1A1 expression has been previously associated with BaP genotoxicity, our results show that, compared with the ALF group, DRF attenuated the BaP-CYP1A1 induction potency, the liver DNA-BaP adducts, the liver concentration of unmetabolized BaP, and the blood aspartate aminotransferase and alanine aminotransferase activities when BaP is administered prior to the acrophase of CYP1A1 expression. These results demonstrate that DRF modifies the ARNT and CYP1A1 expression and protects from BaP toxicity.

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

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

ARNT deficiency represses pyruvate dehydrogenase kinase 1 to trigger ROS production and melanoma metastasis

The metabolic changes in melanoma cells that are required for tumor metastasis have not been fully elucidated. In this study, we show that the increase in glucose uptake and mitochondrial oxidative phosphorylation confers metastatic ability as a result of aryl hydrocarbon receptor nuclear translocator (ARNT) deficiency. In clinical tissue specimens, increased ARNT, pyruvate dehydrogenase kinase 1 (PDK1), and NAD(P)H quinine oxidoreductase-1 (NQO1) was observed in benign nevi, whereas lower expression was observed in melanoma. The depletion of ARNT dramatically repressed PDK1 and NQO1 expression, which resulted in an increase of ROS levels. The elimination of ROS using N-acetylcysteine (NAC) and inhibition of oxidative phosphorylation using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and rotenone inhibited the ARNT and PDK1 deficiency-induced cell migration and invasion. In addition, ARNT deficiency in tumor cells manipulated the glycolytic pathway through enhancement of the glucose uptake rate, which reduced glucose dependence. Intriguingly, CCCP and NAC dramatically inhibited ARNT and PDK1 deficiency-induced tumor cell extravasation in mouse models. Our work demonstrates that downregulation of ARNT and PDK1 expression serves as a prognosticator, which confers metastatic potential as the metastasizing cells depend on metabolic changes.

Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

Aryl hydrocarbon receptor nuclear translocator promotes the proliferation and invasion of clear cell renal cell carcinoma cells potentially by affecting the glycolytic pathway.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that has been reported to play a vital role in regulating glycolysis, angiogenesis and apoptosis. Recently, ARNT has been reported to a play role in pancreatic-islet function in type 2 diabetes. However, the role of ARNT in kidney cancer has not yet been investigated. In the present study, ARNT expression was detected in tissues from patients with renal cell carcinoma (RCC) and in RCC cell lines. Oncomine, The Cancer Genome Atlas and cBioPortal were used to investigate the roles of ARNT in RCC. Cell migration and invasion assays were used to explore the molecular mechanisms involved. It was found that ARNT protein expression was elevated both in tissues from patients with clear cell RCC (ccRCC) and in different RCC cell lines. ARNT disruption using siRNA knockdown inhibited the migratory abilities and cell proliferation, potentially by altering the glycolysis pathway in vitro, as evidenced by decreased M2 type acetone kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 and hexokinase 2 expression. Taken together, the findings in the present study revealed a novel function of ARNT in ccRCC and indicated that ARNT promotes the proliferation and invasion of ccRCC, possibly through changes to the glycolytic pathway.

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

Aryl hydrocarbon receptor nuclear translocators (ARNT1, ARNT2, and ARNT3) of white sturgeon (Acipenser transmontanus): Sequences, tissue-specific expressions, and response to β-naphthoflavone

Sturgeons (Acipenseridae) are ancient fishes that have tissue-specific profiles of transcriptional responses to dioxin-like compounds (DLCs) that are unique from those generally measured in teleost fishes. Because DLCs exert their critical toxicities through activation of the aryl hydrocarbon receptor (AHR), this transcription factor has been the subject of intensive study. However, less attention has focused on the aryl hydrocarbon receptor nuclear translocator (ARNT), which is the dimerization partner of the AHR and required for AHR-mediated transcription. The present study sequenced ARNT1, ARNT2, and ARNT3 in a representative species of sturgeon, the white sturgeon (Acipenser transmontanus), and quantified tissue-specific basal transcript abundance for each ARNT and the response following exposure to the model agonist of the AHR, β-naphthoflavone. In common with other proteins in sturgeons, the amino acid sequences of ARNTs are more similar to those of tetrapods than are ARNTs of other fishes. Transcripts of ARNT1, ARNT2, and ARNT3 were detected in all tissues investigated. Expression of ARNTs are tightly regulated in vertebrates, but β-naphthoflavone caused down-regulation in liver and up-regulation in gill, while an upward trend was measured in intestine. ARNTs are dimeric partners for multiple proteins, including the hypoxia inducible factor 1α (HIF1α), which mediates response to hypoxia. A downward trend in abundance of HIF1α transcript was measured in liver of white sturgeon exposed to β-naphthoflavone. Altered expression of ARNTs and HIF1α caused by activation of the AHR might affect the ability of certain tissues in sturgeons to respond to hypoxia when co-exposed to DLCs or other agonists.

Overexpression of the aryl hydrocarbon receptor nuclear translocator partially rescues fetoplacental angiogenesis in severe fetal growth restriction.

Pregnancies complicated by severe fetal growth restriction with abnormal umbilical artery Doppler velocimetry (FGRadv) are at substantial risk for adverse perinatal and long-term outcomes. Impaired angiogenesis of the placental vasculature in these pregnancies results in a sparse, poorly branched vascular tree, which structurally contributes to the abnormally elevated fetoplacental vascular resistance that is clinically manifested by absent or reversed umbilical artery Doppler indices. Previous studies have shown that aryl hydrocarbon receptor nuclear translocator (ARNT) is a key mediator of proper placental angiogenesis, and within placental endothelial cells (ECs) from human FGRadv pregnancies, low expression of ARNT leads to decreased vascular endothelial growth factor A (VEGFA) expression and deficient tube formation. Thus, the aim of the present study was to determine the effect of VEGFA administration or ARNT overexpression on angiogenic potential of FGRadv ECs. ECs were isolated and cultured from FGRadv or gestational age-matched control placentas and subjected to either vehicle vs VEGFA treatment or transduction with adenoviral-CMV (ad-CMV) vs adenoviral-ARNT (ad-ARNT) constructs. They were then assessed via wound scratch and tube formation assays. We found that VEGFA administration nominally improved FGRadv EC migration (P<0.01) and tube formation (P<0.05). ARNT overexpression led to significantly enhanced ARNT expression in FGRadv ECs (P<0.01), to a level similar to control ECs. Despite this, FGRadv EC migration (P<0.05) and tube formation (P<0.05) were still only partially rescued. This suggests that although ARNT does play a role in fetoplacental EC migration, other factors in addition to ARNT are likely also important in placental angiogenesis.

Abstract 17332: Endothelial-Specific Deletion of Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Leads to Microvascular Barrier Dysfunction and Cardiomyopathy

Introduction: Cardiac microvascular hyperpermeability is a key contributor to heart disease in patients with diabetes. Although the link between diabetes and microvascular barrier dysfunction is largely unknown, expression of the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) is significantly downregulated in the cardiac microvascular endothelial cells (CMECs) of both diabetic mouse hearts (n=5, p&lt;0.01) and the explanted hearts of patients with diabetes mellitus (n=3, p&lt;0.05). We hypothesize that cardiac microvascular permeability is limited by the expression of ARNT in the endothelium and, consequently that endothelial-cell ARNT (ecARNT) expression is essential for normal cardiac function. Methods and Results: We have recently generated tamoxifen-inducible, endothelial-cell-specific, VE-cadherin-Cre ETR2 ARNT-knockout mice (ecARNT -/- mice). ecARNT deletion is achieved by tamoxifen oral administration for two weeks. Littermates controls are either mice without tamoxifen chow or ARNT flox/flox treated with tamoxifen diet. Induction of the ecARNT -/- mutation led to vascular leakage (as determined via in-vivo endothelial permeability assay) (n=5, p&lt;0.05), which occurred predominantly in the heart, and to increases in matrix metalloproteinase (MMP) expression (microarray analysis), including a 3.8-fold increase in the expression of MMP3 (n=3, p&lt;0.01). Furthermore, MMP3 inhibition attenuated the increase in cell permeability observed in CMECs from ecARNT -/- mouse hearts (as measured via electrical cell-substrate impedance sensing), as well as cardiac vascular leakage in ecARNT -/- mice, while long-term studies indicated that ejection fractions were significantly lower (ecARNT -/- : 22.3±2.6%, Control: 33.2±1.8%;), and Left ventricular end-diastolic diameters were significantly greater (ecARNT -/- : 4.8±0.8 mm, Control: 3.6±0.6 mm; n=8,p&lt;0.01) six months after ecARNT deletion (echocardiography). Conclusion: ARNT-mediated MMP3 downregulation is required for maintaining cardiac microvascular barrier integrity and preserving cardiac function; thus, modulation of the ARNT/MMP3 axis could be a novel approach for the treatment of cardiovascular diseases such as diabetic cardiomyopathy.

ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

BackgroundThe transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) participates in the hypoxia-inducible factor (HIF) pathway which senses a decline in cellular oxygen tension. In hypoxia, HIF-1α and ARNT form the transcriptional active complex HIF-1 followed by the expression of target genes. ARNT is considered as constitutively expressed and unaffected by hypoxia. However, certain tumour cell lines derived from different entities are capable to elevate ARNT expression under hypoxic conditions which implies a survival benefit. It was demonstrated that high ARNT protein levels mediate radioresistance in tumour cells. Furthermore, a HIF-1α-driven feed-forward loop leading to augmented HIF signalling was discovered in Hep3B cells. Herein HIF-1α elevates the mRNA and protein expression of its binding partner ARNT in hypoxia. However, the detailed mechanism remained unclear. The objective of this study was to test whether HIF-1α might directly regulate ARNT expression by recruitment to the ARNT promoter.MethodsChromatin immunoprecipitation (ChIP), CRISPR/Cas9 genome editing, Western blotting, quantitative RT-PCR and reporter gene assays were applied. The unpaired t test was used for statistical analysis.ResultsChIP assays revealed the binding of both HIF-1α and ARNT to the ARNT promoter in hypoxia. The relevance of this particular region for hypoxic ARNT induction was confirmed by CRISPR/Cas9 genome editing. ARNT normoxic basal expression and hypoxic inducibility was reduced in genome-edited Hep3B cells. This phenotype was accompanied with impaired HIF signalling and was rescued by ARNT overexpression.ConclusionsThe results indicate ARNT to be a putative HIF-1 target gene and a limiting factor in this model.

Adiponectin Upregulates Filaggrin Expression via SIRT1-Mediated Signaling in Human Normal Keratinocytes.

BackgroundFilaggrin (FLG) is the major component of the epidermal granular layer and binds to and condenses the keratin cytoskeleton. FLG thus contributes to cell compaction and serves as a natural moisturizing factor by promoting unfolding and degradation into hygroscopic amino acids. Loss or downregulation of FLG has been shown to result in a weak stratum corneum, which causes water loss and increases the possibility of skin barrier-related seizure. Adiponectin (Acrp30) contributes to the functional recovery of somatic cells, including human normal epidermal keratinocytes (NHEKs).ObjectiveTo investigate the effect of Acrp30 in FLG expression and identifying its signal transduction mechanism.MethodsNormal human keratinocytes were treated with Acrp30 and the levels of FLG were examined. Silent mating type information regulation 2 homolog (SIRT)-targeting siRNA and aryl hydrocarbon receptor nuclear translocator (ARNT)-targeting siRNA were used to identify the role of various signal transduction pathway components.ResultsAcrp30 upregulated SIRT1 and ARNT expression in NHEKs, resulting in increased FLG expression. Treatment with both SIRT1-targeting siRNA and ARNT-targeting siRNA blocked Acrp30 stimulation and silenced FLG expression.ConclusionAdiponectin upregulates FLG expression through a SIRT1-mediated pathway. Our results suggest that Acrp30 is a promising agent for skin barrier permeability improvement.

Possible role for HIF-1?/ARNT in the regulation of vascular function in diabetes and cardiovascular disease

Aryl hydrocarbon receptor nuclear translocator (ARNT), also known as hypoxia-inducible factor-1 beta (HIF-1β), is a transcription factor that functions as a master regulator of glucose homeostasis. We have previously demonstrated that ARNT expression is highest in the heart, where it is required for maintaining normal cardiac metabolism and cardiac function. Diabetes is associated with a loss of ARNT, although the specific role of ARNT in the diabetic heart is still unknown. Diabetes is also characterized by endothelial dysfunction. Thus, investigating the role of endothelial ARNT in diabetic cardiovascular disease could lead to new therapies for treatment or prevention.

Role of Glucocorticoid Receptor and Pregnane X Receptor in Dexamethasone Induction of Rat Hepatic Aryl Hydrocarbon Receptor Nuclear Translocator and NADPH-Cytochrome P450 Oxidoreductase.

The aryl hydrocarbon receptor (AHR) nuclear translocator (ARNT), as the AHR's heterodimerization partner, and NADPH-cytochrome P450 oxidoreductase (POR), as the key electron donor for all microsomal P450s, are independent and indispensable components in the adaptive and toxic responses to polycyclic aromatic hydrocarbons. Expression of both ARNT and POR in rat liver is induced by dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). To better understand the role of GR and PXR in the in vivo DEX induction of rat hepatic ARNT and POR at the mRNA and protein levels, we studied the following: 1) the effects of DEX doses that activate GR (≥0.1 mg/kg) or PXR (≥10 mg/kg); 2) responses produced by GR- and PXR-selective agonists; 3) the impact of GR antagonism on DEX's inducing effects; and 4) whether biologic responses to DEX are altered in PXR-knockout rats. Our findings are consistent with a role for GR as a key mediator of the induction of rat hepatic ARNT expression by glucocorticoids; a role for PXR in the modulation of ARNT protein levels could not be excluded. Although GR activation may contribute to POR mRNA induction, regulation of POR expression and function by DEX is primarily PXR-mediated. This work suggests that the hepatic expression and function of ARNT and POR may be modulated by exposure to exogenous PXR activators and/or conditions that alter glucocorticoid levels such as stress, steroidal therapies, and diseases of excess or deficiency.

A HIF-1α-driven feed-forward loop augments HIF signalling in Hep3B cells by upregulation of ARNT.

Oxygen-deprived (hypoxic) areas are commonly found within neoplasms caused by excessive cell proliferation. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) is part of the hypoxia-inducible factor (HIF) pathway, which mediates adaptive responses to ensure cellular survival under hypoxic conditions. HIF signalling leads to metabolic alterations, invasion/metastasis and the induction of angiogenesis in addition to radio-chemoresistance of tumour cells. Activation of the HIF pathway is based on the abundance of HIF-α subunits, which are regulated in an oxygen-dependent manner and form transcriptional active complexes with ARNT or ARNT2 (also referred as HIF-1β and HIF-2β, respectively). ARNT is considered to be unaffected by hypoxia but certain cell lines, including Hep3B cells, are capable to elevate this transcription factor in response to oxygen deprivation, which implies an advantage. Therefore, the aim of this study was to elucidate the mechanism of hypoxia-dependent ARNT upregulation and to determine implications on HIF signalling. Gene silencing and overexpression techniques were used to alter the expression pattern of HIF transcription factors under normoxic and hypoxic conditions. qRT-PCR and western blotting were performed to measure gene and protein expression, respectively. HIF activity was determined by reporter gene assays. The results revealed a HIF-1α-dependent mechanism leading to ARNT upregulation in hypoxia. Forced expression of ARNT increased reporter activity under normoxic and hypoxic conditions. In conclusion, these findings indicate a novel feed-forward loop and suggest that ARNT might be a limiting factor. Augmented HIF signalling in terms of elevated target gene expression might be advantageous for tumour cells.

Loss of HIF-1α in macrophages attenuates AhR/ARNT-mediated tumorigenesis in a PAH-driven tumor model.

Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1αLysM−/− macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1αLysM−/− macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.

The expression level of the transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) determines cellular survival after radiation treatment.

BackgroundTumour hypoxia promotes radioresistance and is associated with poor prognosis. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as Hypoxia-inducible factor (HIF)-1β, is part of the HIF pathway which mediates cellular adaptations to oxygen deprivation and facilitates tumour progression.The subunits HIF-1α and ARNT are key players within this pathway. HIF-1α is regulated in an oxygen-dependent manner whereas ARNT is considered to be constitutively expressed. However, there is mounting evidence that certain tumour cells are capable to elevate ARNT in hypoxia which suggests a survival benefit.Therefore the objective of this study was to elucidate effects of an altered ARNT expression level on the cellular response to radiation.MethodsDifferent human cell lines (Hep3B, MCF-7, 786-Owt, 786-Ovhl, RCC4wt and RCC4vhl) originating from various tumour entities (Hepatocellular carcinoma, breast cancer and renal cell carcinoma respectively) were X-irradiated using a conventional linear accelerator. Knockdown of ARNT expression was achieved by transient siRNA transfection. Complementary experiments were performed by forced ARNT overexpression using appropriate plasmids. Presence/absence of ARNT protein was confirmed by Western blot analysis. Clonogenic survival assays were performed in order to determine cellular survival post irradiation. Statistical comparison of two groups was achieved by the unpaired t-test.ResultsThe results of this study indicate that ARNT depletion renders tumour cells susceptible to radiation whereas overexpression of this transcription factor confers radioresistance.ConclusionsThese findings provide evidence to consider ARNT as a drug target and as a predictive marker in clinical applications concerning the response to radiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0539-9) contains supplementary material, which is available to authorized users.