Hydrocarbon Nuclear Translocator Research Articles

Abstract P2127: A Novel Arnt Dependent Hif2a Singling In Protecting Against Cardiac Microvascular Endothelial Barrier Dysfunction Following Myocardial Infarction

Rationale: Cardiac microvascular leakage and inflammation are triggered during myocardial infarction (MI) and contribute to heart failure. Hypoxia-inducible factor 2α ( Hif2α ) is highly expressed in endothelial cells (ECs) and rapidly activated by myocardial ischemia, but its impact in microvascular endothelial barrier function during MI is unclear. Objective: To test our hypothesis that the expression of Hif2α in ECs regulates cardiac microvascular permeability in infarcted hearts, which is through its binding partner aryl hydrocarbon nuclear translocator. (ARNT). Methods and Results: Experiments were conducted with mice carrying an inducible EC-specific Hif2α -knockout ( ecHif2α -/- ) mutation, with mouse cardiac microvascular endothelial cells (CMVECs) isolated from the hearts of ecHif2α -/- mice after the mutation was induced, and with human CMVECs and umbilical-vein endothelial cells transfected with ecHif2α siRNA. After MI induction, echocardiographic assessments of cardiac function were significantly lower, while measures of cardiac microvascular leakage (Evans blue assay), plasma IL6 levels, and cardiac neutrophil accumulation and fibrosis (histology) were significantly greater, in ecHif2α -/- mice than in control mice, and RNA-sequencing analysis of heart tissues from both groups indicated that the expression of genes involved in vascular permeability and collagen synthesis was enriched in ecHif2α -/- hearts. In cultured ECs, ec Hif2α deficiency was associated with declines in endothelial barrier function (electrical cell impedance assay) and the reduced abundance of tight-junction proteins, as well as an increase in the expression of inflammatory markers, all of which were largely reversed by the overexpression of ARNT. We also found that ARNT, but not Hif2α, binds directly to the IL6 promoter and suppresses IL6 expression. Conclusions: Endothelial HIF-2a protects from hypoxia-induced cardiac microvascular barrier dysfunction, promotes inflammation damage and represents a potential therapeutic target for cardioprotection, and prevention of fibrosis following acute ischemic injury.

OR33-07 ARNT2: A Potential Novel Candidate Gene for Monogenic Obesity in Humans

Introduction: Aryl hydrocarbon nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH)-PAS (Per/Arnt/Sim) transcription factor shown to be critical to the development of paraventricular nucleus of the hypothalamus (PVN), key region for energy homeostasis and feeding response. In vivo and in vitro studies have shown that ARNT2 is an obligate heterodimer for SIM1, known cause of monogenic obesity. Null mutations in Arnt2 in animals are not viable, but hypomorphic mutation results in hyperphagic obesity and its associated consequences (1). Due to the critical role of ARNT2 in the development of PVN, we hypothesize that hypomorphic mutations may result in early onset obesity in humans. Methods: The Genetics of Early Childhood Obesity (GECO) study recruits children with severe obesity (BMI > 120% of 95th percentile) of early onset (< 6 years). Whole exome sequencing (WES) was performed in a subset of proband-parent trios. The functional validation of the mutation(s) in ARNT2 is ongoing with co-transfection of tagged Arnt2 and Sim1 in HEK293 cells, with the induction of a luciferase reporter gene under the control of 6 repeats of bHLH-PAS core binding element by the Arnt2-Sim1 complex. Results: Two adolescents from unrelated families were found to have genetic variants in ARNT2. Subject 1 has a novel de novo heterozygous coding variant in ARNT2, c.388 C>G (p.P130A, CADD 25), predicted to be deleterious by 8/12 in silico algorithms. She is a 14-year old Caucasian girl with severe early onset obesity, BMI 28.1 kg/m2 (BMIz +4.72) at 2.5 years of age that has increased to 53.54 kg/m2 (BMIz + 3.25) at 14-years, and height > 95th %tile. She is non-dysmorphic, has developmental delay, absence seizures, behavior abnormalities & glucose intolerance/dyslipidemia secondary to obesity. Using genematcher, we identified another proband with the phenotype of obesity: an African American girl (BMIz +1.9) with biallelic inherited heterozygous variants in ARNT2, c.1228T>A (p.W410R, CADD 29) and c.916G>A (p.G306S, CADD 22). An only child conceived by IVF, she is non-dysmorphic and on treatment for bilateral focal epilepsy. All 3 variants are rare, with mean allele frequency < 0.005 in population-based databases such as gNOMAD. Both the patients have early onset obesity and a significant neurological phenotype. ARNT2 is a highly constrained gene of 717 amino acids with a significant depletion of missense variants in the N-terminus (1-244 aa) and overall fewer loss of function variants in ~282,644 alleles sequenced in gNOMAD. Conclusions: We propose that hypomorphic mutations in ARNT2 could be a potential novel cause of monogenic obesity in humans. Future studies will investigate the molecular mechanisms causing weight dysregulation in patient specific disease relevant hypothalamic neurons.Reference: (1) Turer et al., Dis Model Mech. 2018; 11(12)

Cholesterol Starvation and Hypoxia Activate the FVII Gene via the SREBP1-GILZ Pathway in Ovarian Cancer Cells to Produce Procoagulant Microvesicles.

Interaction between the transcription factors, hypoxia-inducible factor (HIF1α and HIF2α) and Sp1, mediates hypoxia-driven expression of FVII gene encoding coagulation factor VII (fVII) in ovarian clear cell carcinoma (CCC) cells. This mechanism is synergistically enhanced in response to serum starvation, a condition possibly associated with tumor hypoxia. This transcriptional response potentially results in venous thromboembolism, a common complication in cancer patients by producing procoagulant extracellular vesicles (EVs). However, which deficient serum factors are responsible for this characteristic transcriptional mechanism is unknown. Here, we report that cholesterol deficiency mediates synergistic FVII expression under serum starvation and hypoxia (SSH) via novel sterol regulatory element binding protein-1 (SREBP1)-driven mechanisms. Unlike conventional mechanisms, SREBP1 indirectly enhances FVII transcription through the induction of a new target, glucocorticoid-induced leucine zipper (GILZ) protein. GILZ expression induced in response to hypoxia by a HIF1α-dependent mechanism activates SREBP1 under SSH, suggesting reciprocal regulation between SREBP1 and GILZ. Furthermore, GILZ binds to the FVII locus. Xenograft tumor samples analyzed by chromatin immunoprecipitation confirmed that HIF1α-aryl hydrocarbon nuclear translocator and GILZ bind to the TSC22D3 (GILZ) and FVII gene loci, respectively, thereby potentially modulating chromatin function to augment FVII transcription. Thus, deficiency of both O2 and cholesterol, followed by interplay between HIFs, Sp1, and SREBP1-GILZ pathways synergistically induce fVII synthesis, resulting in the shedding of procoagulant EVs.

Apoptin-derived peptide reverses cisplatin resistance in gastric cancer through the PI3K-AKT signaling pathway.

The prognosis of gastric cancer (GC) remains poor due to clinical drug resistance, and novel drugs are urgently needed. Apoptin‐derived peptide (AdP) is an antitumor polypeptide constructed in our laboratory that has been used to combat cisplatin (CDDP) resistance in GC cells. MTT and colony‐formation assays and Hoechst 33342 staining were used to measure the cytotoxicity of CDDP and AdP in GC cells. Cell apoptosis was measured using an Annexin‐V‐FITC/PI dual staining assay. Western blot analysis was conducted to detect the expression of proteins in the PI3K/AKT signaling pathway and resistance‐related markers. AdP exerted a specific cytotoxic effect on GC cells and CDDP‐resistant GC cells in a concentration‐ and time‐dependent manner. AdP also suppressed cell invasion and migration. Additionally, AdP inhibited the expression of p85, AKT, p‐p85, p‐AKT, multidrug resistance 1 (MDR1), and aryl hydrocarbon nuclear translocator (ARNT) in the PI3K/AKT/ARNT signaling pathway, which promoted apoptosis and necrosis in GC cells. AdP promoted apoptosis in CDDP‐resistant GC cells by suppressing the PI3K/AKT/ARNT signaling pathway and might be considered a candidate agent for the clinical treatment of cisplatin‐resistant GC.

A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism’s ability to respond on a phenotypic level to various stimuli within an inconstant environment.

Abstract 15414: Ablation of Aryl Hydrocarbon Nuclear Translocator (ARNT) in the Heart Leads to Diabetic Cardiomyopathy Phenotype Through PPARα Activity

Background: Metabolic changes decrease cardiac efficiency and energetics in diabetes. Impaired hypoxia inducible factor (HIF) signaling may contribute to cardiac metabolic abnormalities. ARNT, also known as HIF1β, is a transcription factor that regulates several cellular processes by dimerizing with certain proteins, including HIF-1α or -2α, and its function in the heart is unknown. We hypothesized that cardiac deletion of ARNT leads to metabolic derangement and cardiac dysfunction. Methods and Results: Cardiac-specific ARNT knockout (csARNT-KO) mice were generated by our lab as previously described. ARNT deletion in the heart resulted in dilated cardiomyopathy and significant lipid accumulation, which was determined by electron microscopy, oil-red staining and triglyceride level quantification. We found that loss of ARNT in the heart increased PPARα activity, as confirmed by 2-fold increase in fatty acid oxidation (FAO) in ex-vivo csARNT+/- working hearts, increased PPARα protein content and increased PPARα target gene expression.To study the mechanism by which ARNT regulates PPARα, luciferase reporter assays revealed that ARNT knockdown resulted in increased PPARα promoter activity and deletion of the second hypoxia response element (HRE) upstream of the initiation site removed the inhibitory effect of ARNT on the PPARα promoter, suggesting that this site is likely involved in the regulation of the gene. ARNT binding to this sequence was confirmed by chromatin immunoprecipitation analysis. Moreover, a reduction in HIF-2α, but not HIF-1α or AHR, also significantly increased the levels of PPARα. These results suggest that the effects of ARNT deletion on PPARα are, at least partially, through its association with HIF-2α and through the binding of this complex to the second HRE upstream of the PPARα initiation site. Finally, mice with double deletion of ARNT and PPARα demonstrated rescue of the cardiac dysfunction phenotype, improved survival, and complete reversal of FA accumulation. Conclusion: Reduction in ARNT levels leads to cardiomyopathy by increasing lipid accumulation through a PPARα pathway. Thus, ARNT is a critical regulator of fatty acid metabolism in the heart, and a potential target for the treatment of diabetic cardiomyopathy.

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy.

Patients with type 2 diabetes often present with cardiovascular complications; however, it is not clear how diabetes promotes cardiac dysfunction. In murine models, deletion of the gene encoding aryl hydrocarbon nuclear translocator (ARNT, also known as HIF1β) in the liver or pancreas leads to a diabetic phenotype; however, the role of ARNT in cardiac metabolism is unknown. Here, we determined that cardiac-specific deletion of Arnt in adult mice results in rapid development of cardiomyopathy (CM) that is characterized by accumulation of lipid droplets. Compared with hearts from ARNT-expressing mice, ex vivo analysis of ARNT-deficient hearts revealed a 2-fold increase in fatty acid (FA) oxidation as well as a substantial increase in the expression of PPARα and its target genes. Furthermore, deletion of both Arnt and Ppara preserved cardiac function, improved survival, and completely reversed the FA accumulation phenotype, indicating that PPARα mediates the detrimental effects of Arnt deletion in the heart. Finally, we determined that ARNT directly regulates Ppara expression by binding to its promoter and forming a complex with HIF2α. Together, these findings suggest that ARNT is a critical regulator of myocardial FA metabolism and that its deletion leads to CM and an increase in triglyceride accumulation through PPARα.

Dose- and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBT-exposed intertidal copepod Tigriopus japonicus

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon nuclear translocator (ARNT) genes from the copepod Tigriopus japonicus (Tj) were cloned to examine their potential functions in the invertebrate putative AhR-CYP signaling pathway. The amino acid sequences encoded by the Tj-AhR and Tj-ARNT genes showed high similarity to homologs of Daphnia and Drosophila, ranging from 68% and 70% similarity for the AhR genes to 56% for the ARNT genes. To determine whether Tj-AhR and Tj-ARNT are modulated by environmental pollutants, transcriptional expression of Tj-AhR and Tj-ARNT was analyzed in response to exposure to five concentrations of polychlorinated biphenyl (PCB 126) (control, 10, 50, 100, 500μgL−1), benzo[a]pyrene (B[a]P) (control, 5, 10, 50, 100μgL−1), and tributyltin (TBT) (control, 1, 5, 10, 20μgL−1) 24h after exposure. A time-course experiment (0, 3, 6, 12, 24h) was performed to analyze mRNA expression patterns after exposure to PCB, B[a]P, and TBT. T. japonicus exhibited dose-dependent and time-dependent upregulation of Tj-AhR and Tj-ARNT in response to pollutant exposure, and the degree of expression was dependent on the pollutant, suggesting that pollutants such as PCB, B[a]P, and TBT modulate expression of Tj-AhR and Tj-ARNT genes in the putative AhR-CYP signaling pathway.

Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2.

The bHLH (basic helix-loop-helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational 'hot-spot' in SIM1 that is critical for protein function.

Regulation of hypoxia inducible factors by Sprouty2 (842.9)

Sprouty2 (Spry2) is an inhibitor of receptor tyrosine kinase signaling. Spry2 levels are decreased in cancers of the breast, lung, prostate and liver, thereby permitting excessive receptor tyrosine kinase signaling in these pathologies. Furthermore, a decrease in Spry2 levels has been correlated with poor patient prognosis. In development and tumor growth, cells experience a hypoxic environment to which they adapt to by up regulating the transcription factors, Hypoxia Inducible Factors (HIFs). The HIFs are composed of a beta subunit (HIF1β or aryl hydrocarbon nuclear translocator (ARNT)) and an oxygen sensitive alpha subunit (HIF1α or HIF2α). Using a hepatocellular carcinoma cell line (HuH7), we have shown that Spry2 decreases the protein levels of HIF1α, HIF2α, and ARNT without altering the mRNA levels. Additionally, Spry2 reduces the ability of the HIFs to induce transcription as measured by luciferase assays and mRNA levels of their target genes. We also show that Spry2 interacts with HIF1α and ARNT and that Spry2 decreases the stability of ARNT and HIF1α. To begin to deduce the mechanism, we have shown that Spry2 enhances the ubiquitination of HIF1α. Collectively, our data reveal a novel regulation of the HIFs by Spry2.Grant Funding Source: Supported in part by NIH grant GM 073181

Establishment of a stable aryl hydrocarbon receptor-responsive HepG2 cell line

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. It heterodimerizes with aryl hydrocarbon nuclear translocator, binds to the xenobiotic-responsive element (XRE), and enhances the transcription of genes encoding xenobiotic metabolizing enzymes. AHR also plays important roles in the inhibition of intestinal carcinogenesis and the modulation of gut immunity. It is very important to screen for AHR-activating compounds because those are expected to produce the AHR-mediated physiological functions. Until now, AHR-mediated transcriptional activity represented by the transcriptional activity of CYP1A1 in luciferase assay has been applied as a screening procedure for AHR-activating compounds. However, the AHR-mediated transcriptional activity did not necessarily correspond with the CYP1A1 transcriptional activity. To evaluate AHR-mediated transcriptional activity more specifically, and to screen for AHR-activating compounds, we establish a stable AHR-responsive HepG2 cell line by co-transfection of an AHR expression vector and an AHR-responsive vector (pGL3-XRE) containing a luciferase gene and three tandemly arranged XRE elements into a human hepatoma derived cell line, HepG2. The induction of luciferase activity in the stable AHR-responsive HepG2 cell line by typical AHR activators occurred in time- and concentration-dependent manners. By assessing the AHR target genes CYP1A1, UGT1A1, and ABCG2, an AHR activator-mediated induction was observed at mRNA level. Furthermore, the AHR activator-mediated induction of luciferase activity was positively correlated with the mRNA levels of CYP1A1, UGT1A1, and ABCG2. These findings verified the usefulness of the established stable AHR-responsive HepG2 cell line for the screening of AHR-activating compounds.

Role of the aryl hydrocarbon receptor nuclear translocator in establishing lymphocyte and innate lymphoid cells of the intestinal tract and the skin

BackgroundThe aryl hydrocarbon receptor (AHR) is a transcription factor that controls development of innate and adaptive lymphoid cells in the intestinal mucosa and skin. Pharmacological ligands for AHR include dioxins, whereas physiological ligands include tryptophan metabolites from nutritional components or tryptophan catabolites produced by microbiota. AHR is thus an important link between environmental stimuli and immunity at barrier organs. One binding partner for AHR is the aryl hydrocarbon nuclear translocator (ARNT). A key question is whether the immune functions of AHR are solely mediated by AHR–ARNT complexes. We aimed to assess the importance of ARNT for lymphocyte populations of the intestine and the skin. MethodsMice expressing a Vav-Cre transgene were backcrossed to mice with floxed Arnt alleles to produce Vav-Cre Arntfl/fl mice that have deleted ARNT in all haemopoietic cells. The effect of ARNT loss for lymphocytes in the skin and in the small intestine was analysed by flow cytometry and confocal microscopy. FindingsVav-Cre Arntfl/fl mice had a three-fold reduction of TCRαβCD8αα+ and four-fold reduction of TCRγδ+ cells in the intraepithelial layer of the small intestine compared with wild type controls. They also lacked the TCRαβ+CD4+CD8+ cells in this layer. They had a loss of Roryt+ innate lymphoid cells in the lamina propria. Epidermal TCRγδ+ cells were also absent in the Vav-Cre Arntfl/fl mice. We also noted reductions in conventional natural killer cells in the lamina propria of Vav-Cre Arntfl/fl mice compared with wild type controls, which has not been reported in AHR null mice. InterpretationARNT is crucial for establishing intraepithelial lymphocytes, lamina propria innate lymphoid cells, and epidermal TCRγδ+ cells. These data suggest that AHR–ARNT complexes control the development and survival of lymphocytes at barrier surfaces. ARNT also seems to have a role in conventional natural killer cells in the lamina propria independently of the AHR. FundingWellcome Trust.

Molecular Cloning and Expression Analysis of Hypoxia Inducible Factor 1α in Tongue Sole, &lt;I&gt;Cynoglossus Semilaevis&lt;/I&gt; (Actinopterygii: Pleuronectiformes: Cynoglossidae), Subjected to Acute Hypoxia

Hypoxia-inducible factor-1 (HIF-1) is the master regulator of oxygen homeostasis by regulating more than 100 genes expression (Manolescu et al. 2009). HIF-1 is a heterodimer which is composed of two subunits, HIF-1α and HIF-1β. The two subunits belong to the helix-loop-helix (Bhlh)-Per-Arnt-Sim (PAS) family, which contain conserved structural domains, such as a bHLH domain and two PAS domains (Kewley et al. 2004). The HIF-1β subunit, also known as hydrocarbon nuclear translocator (ARNT), is constitutively expressed, and is not affected by oxygen tension (Wang et al. 1995). HIF-1α is constitutively transcribed under normoxic conditions, but it is rapidly degraded via an ubiquitin-dependent pathway. However, during hypoxia this pathway is inhibited and HIF-1α is translocated to the nucleus and dimerizes with HIF-1β, causing HIF-1 to bind with hypoxia response elements (HREs) of target genes to activate their transcription (Maynard and Ohh 2004). Currently, HIF-1 has been cloned and characterized in mammals (Gassmann et al. 2002), birds, amphibians, and some fish species (Cao et al. 2008, Kodama et al. 2012). ACTA ICHTHYOLOGICA ET PISCATORIA (2013) 43 (3): 201–209 DOI: 10.3750/AIP2013.43.3.04

Genome-Wide RNAi High-Throughput Screen Identifies Proteins Necessary for the AHR-Dependent Induction of CYP1A1 by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.

Genetic polymorphisms in the aryl hydrocarbon receptor-signaling pathway and sleep disturbances in middle-aged women

ObjectiveWe aimed to determine if selected genetic polymorphisms in the aryl hydrocarbon receptor (AHR)-signaling pathway and circadian locomotor output cycles kaput (CLOCK) are associated with insomnia and early awakening in middle-aged women. MethodsWomen aged 45 to 54years (n=639) were recruited into a middle-aged health study and agreed to complete questionnaires and donate blood samples. Questionnaires were used to assess sleep outcomes. Blood samples were processed for genotyping for the selected polymorphisms: AHR (rs2066853), AHR repressor (AHRR) (rs2292596), aryl hydrocarbon nuclear translocator (ARNT) (rs2228099), and CLOCK (rs1801260). Data were analyzed using multivariable logistic regression. ResultsWomen heterozygous for the AHRR alleles (GC) had decreased odds of insomnia compared to women homozygous for the AHRR_C allele (adjusted odds ratio [aOR], 0.69; 95% confidence interval [CI], 0.49–0.96). Women with at least one of the AHRR_G or CLOCK_C alleles had significantly decreased odds of insomnia compared to women homozygous for the AHRR_C and CLOCK_T alleles (aOR, 0.64; 95% CI, 0.43–0.96). Additionally, women homozygous for the AHRR_G and CLOCK_C alleles had significantly decreased odds of insomnia compared to women homozygous for the AHRR_C and CLOCK_T alleles (aOR, 0.56; 95% CI, 0.35–0.89). None of the selected single nucleotide polymorphisms (SNPs) or combinations of SNPs were significantly associated with early awakening. ConclusionsSelected genetic polymorphisms in the AHR-signaling pathway (i.e., AHRR) and CLOCK may play a role in decreasing the risk for experiencing insomnia during the menopausal transition.

MicroRNA-221 overexpression accelerates hepatocyte proliferation during liver regeneration

The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR-221, which is frequently up-regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR-221 in hepatocytes. Here, we hypothesize that modulation of miR-221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two-thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno-associated virus-mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR-221, which contributes to the pro-proliferative activity of miR-221. miR-221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR-221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration.

First Phase Insulin Secretion and Type 2 Diabetes

Type 2 diabetes (T2D) is a metabolic disorder characterised by the inability of β-cells to secrete enough insulin to maintain glucose homeostasis. Pancreatic β-cells secrete insulin in a biphasic manner, first and second phase insulin secretion, and loss of first phase insulin secretion is an independent predictor of T2D onset. Restoration of first phase insulin secretion has been shown to improve blood glucose in T2D by suppressing hepatic glucose production and priming insulin sensitive tissue to more readily take up glucose and has thus prompted numerous studies into its regulation. First phase insulin secretion is initiated primarily by the classical triggering pathway, a complex system comprised of multiple stimulatory signals. Recent studies have identified a number of novel regulatory factors that are crucial for first phase insulin secretion and glucose homeostasis. These include, among others, hypoxia inducible factor 1α, von Hippel-Lindau, factor inhibiting HIF, nicotinamide phospho-ribosyl-transferase, and the sirtuin family. This review will outline how first phase insulin secretion is initiated and detail some of the recent findings in its regulation. Keywords: Aryl hydrocarbon nuclear translocator, glucose homeostasis, hypoxia inducible factor-1α, insulin secretion, regulatory genes, type 2 diabetes

Genetic susceptibility to dioxin-like chemicals’ induction of cytochrome P4501A2 in the human adult linked to specific AhRR polymorphism

BackgroundDioxin-like chemicals are known to exert their effect by binding to aryl hydrocarbon receptor (AhR), forming complexes with aryl hydrocarbon nuclear translocator (ARNT), and binding to dioxin responsive elements (DREs) in promoter region to regulate the transcription of specific genes. In a previous study of the Yucheng cohort of humans who were exposed to high toxic levels of dioxin-like chemicals (PCDFs and PCBs), we reported marked induction of cytochrome P450 1A2 (CYP1A2) activity and this induction was an excellent biomarker of the exposure and adverse human health effects seen in the Yucheng cohort. ObjectivesThe goal of this study was to determine the relationship between inducibility of CYP1A2 and genetic polymorphisms of AhR, ARNT, and AhRR in human. MethodsThe Yucheng victims who completed blood sample collecting in 1994–1995 for serum concentrations of PCB, PCDF, and PCDD congeners, and also completed the caffeine breath tests for CYP1A2 activity were identified. From the collected blood samples, six single nucleotide polymorphisms were selected for genotyping, including AhR (rs2066853), AhRR (rs2292596), ARNT (rs7517566), ARNT (rs3820541), ARNT (rs3768016), and ARNT (rs2228099). ResultsAhRR (rs2292596) polymorphism was significantly related to CYP1A2 inducibility (p=0.01). A linear trend test was observed between people with AhRR (rs2292596) GG, GC, and CC genotype (p=0.0014). ConclusionOverall, AhRR (rs2292596) genotypes predict the inducibility of CYP1A2 in people highly exposed to toxic dioxin-like chemicals. Future studies and analysis will determine to what degree these polymorphisms can predict a human’s susceptibility to dioxin-related adverse human health effects.

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

The heterodimer HIF-1α (hypoxia inducible factor)/HIF-β (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics "hotspot identification" strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.

Abstract 5312: Identification of novel HIF-1-dependent target genes in a murine model of breast cancer

Abstract Introduction: Hypoxia is a hallmark of most solid tumors. Under hypoxic stress tumor cells adapt by regulating survival, metabolism and angiogenesis. The heterodimeric Hypoxia-Inducible Factor (HIF)-1 transcription factor is a master regulator of this response, and is comprised of HIF-1α, the oxygen-regulated subunit, and aryl hydrocarbon nuclear receptor translocator (ARNT), which is constitutively expressed. HIF-1α protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases. Over-expression of HIF-1α is independently correlated with poor prognosis and decreased survival in breast cancer patients. In agreement with these observations, conditional deletion of Hif1a represses tumor initiation and lung metastasis in the MMTV-polyoma virus middle T (MMTV-PyMT) model of breast cancer. Methods: Primary mammary tumor epithelial cells (MTECs) were established from late stage carcinomas originating in PyMT+; Hif1a floxed mice maintained on an inbred background (FVB/Nj). MTECs were exposed ex vivo to Adenovirus-β-gal or -Cre to create wild type (WT) and knockout (KO) cells, respectively; cells were then re-introduced into the mammary fat pad of FVB/Nj recipients. HIF-1α deletion reduced primary tumor growth by ∼60% and lung macrometastases by ∼90%. To identify HIF-dependent target genes, microarray profiling was performed. Several genes were differentially expressed between both WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) and end-stage WT and KO tumors. Of particular interest, creatine kinase brain isoform (Ckb) mRNA levels were reduced &amp;gt;100-fold in KO cells and &amp;gt;2-fold in KO end-stage tumors; down-regulation at the protein level was confirmed by western blotting. Two independent shRNA constructs (shc59 and shc61) were generated to stably knockdown Ckb in WT cells to assay for effects on cell proliferation and metastasis. Results: In 2-D culture, no significant difference in cell growth was observed in either knockdown line versus the WT cells. In contrast, when one million cells were introduced into the circulation by tail vein injection, 100% of mice injected with WT cells developed macrometastases, whereas no macromets were derived from shc61 cells and only 20% of mice injected with shc59 cells developed macromets. Four putative hypoxic response elements (HREs) have been identified in the Ckb promoter; therefore, ChIP experiments are in progress to determine if Ckb is a direct HIF-1 target. Additional experiments will test for a role of Ckb in regulating primary tumor growth, cell migration or invasion, and, if cyclocreatine, a CKB inhibitor, is effective in reducing primary tumor growth and/or metastasis. Conclusions: Further characterization of the function of Ckb, and other genes downstream of HIF-1α that drive metastasis, may identify pathways amenable to therapeutic intervention to treat patients with metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5312. doi:1538-7445.AM2012-5312