ARNT Expression Research Articles

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPβ1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPβ1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

In vivo localisation and vascular effects of nano-vesicles derived from normal first trimester human placentae

In vivo localisation and vascular effects of nano-vesicles derived from normal first trimester human placentae

Abstract 392: Decreased ARNT Mediates Cardiovascular Endothelial Dysfunction in Diabetic Cardiomyopathy

Background: Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy. Methods and Results: Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT -/- ) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT -/- mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway. Conclusion: Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.

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.

907P - Association Between Angiogenesis-Related Genes and the Response to Multimodal Therapy in High Grade Serous Advanced Ovarian Carcinoma (Aoc)

ABSTRACT Aim: Cytoreductive surgery plus chemotherapy is the standard of care in high grade serous AOC. To date, the outcome prediction after multimodal therapy remains one of the most important challenges for patients (pts) stratification according to effective treatments. Angiogenesis has maintained its prominent role in the ovarian cancer research field for years, being one of the most studied processes for the identification of alternative therapies in AOC. Previous studies identified several genes involved in angiogenesis as prognostic markers in this tumour. Methods: We included 39 pts with stages III or IV high grade serous ovarian cancer. These pts underwent surgical cytoreduction and received a carboplatin plus paclitaxel chemotherapy regimen. RNAs were collected from formalin-fixed paraffin-embedded samples. Expression levels of 82 angiogenesis-related genes were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Results: 74% of pts (29 out of 39) achieved a complete response after surgical and chemotherapy treatments. The only clinical factor associated to treatment complete response was the remaining tumour after debulking surgery (HR 18.9, 95% CI (2.0 - 2458.5)). Regarding the genetic variables analyzed, we found that the expression of ANGPT1, ARNT, CD34, EGF and MMP3 genes were related to response in the univariate analysis. We tried also to generate a genetic model for response prediction, being the most interesting one composed of the combined expression of 7 genes (AGT, CD34, EGF, EPOR, IL8, MMP3 and MMP7). After leave one out cross validation (LOOCV), we obtained an AUC of 0.67 and the accuracy assigned was 0.74. The only factor that remains statistically significant in the multivariate analysis (including clinical and genetic factors) was the residual tumour after surgery. Conclusions: Remaining tumour after debulking surgery remains the most important factor to date to successfully achieve a complete response after the multimodal treatment in high-grade serous AOC pts. The specific roles of genes identified in this study will need to be further studied. Disclosure: All authors have declared no conflicts of interest.

Role of AHR, AHRR and ARNT in response to dioxin-like PCBs in Spaurus aurata.

The aryl hydrocarbon receptor (AHR) mediates a variety of biological responses to ubiquitous dioxin and PCB dioxin-like. AHR together with ARNT, AHRR, represent a novel basic helix-loop-helix/PAS family of transcriptional regulators. Their interplay may affect the xenobiotic response. The aim of this study was to investigate, by histological, immunohistochemical investigations and western-blot analysis, the expression of AHR, ARNT and AHRR in liver of seabrem (Spaurus aurata) after exposure at different time to dioxin-like PCB126 in order to deep the knowledge about their specific role. The findings showed a significant increase of AHR and ARNT expression in juvenile fishes after 12 h than control group. The induction of AHR and ARNT is also significant at 24 and 72 hours compared to the control group. Furthemore, induction of AHRR expression has proved to increase both 12 h but this induction does not seem significant to 24 and 72 hours. The most important data of this work is that the induction of AHRR, when the action of the toxic persistence substances, as dioxin and PCB-126, it is not enough to reduce AHR signaling and thus its hyperactivation leads to toxic effects in seabrem (Spaurus aurata). All this confirms the importance of AHR ligands as new class of drugs that can be directed against severe disease such as cancer.

Antiangiogenic effect of combined treatment with curcumin and genistein on human prostate cancer cell line

High cost and toxicity, low success rate and patient incompliance associated with existing anticancer drugs necessitated for finding new anticancer drugs which can overcome the aforementioned drawbacks. Toward this direction effort has been made to understand the anticancer activity of curcumin (cur) and genistein (gen) combination in human prostate cancer cell line (PC3) cells with respect to their antiangiogenic effect. Cur and gen has shown dose and time dependent decrease in cell viability, increase in apoptosis and cell cycle arrest at G0 phase. These effects were more noticeable when cur (20 µM) and gen (100 μM) were used in combination. To understand antiangiogenic effect of this combination, expression of ARNT and HIF-1α was studied. Significant decline in expression of ARNT and HIF-1α protein level was seen in comparison to control group and their respective monotherapy treated groups. Cur and gen are shown to be effective in abrogating the VEGF production by evading ARNT and HIF-1α complex formation as proved by immunoprecipitation assay. Thus this combination seems to be promising toward cancer treatment.

Resveratrol inhibition of human keratinocyte proliferation via SIRT1/ARNT/ERK dependent downregulation of aquaporin 3

BackgroundAquaporin 3 (AQP3) is the predominant aquaporin in the skin and is overexpressed in hyperplastic epidermal disorders. Upregulation of AQP3 contributes to keratinocyte proliferation and epidermal hyperplasia. Resveratrol, a natural polyphenol, has an anti-proliferative effect on normal human epidermal keratinocytes (NHEKs), but its exact mechanism remains largely unknown. ObjectiveTo investigate the ability and mechanism of resveratrol to affect the proliferation and the AQP3 expression in NHEKs. MethodsNHEKs treated with resveratrol were analyzed. BrdU incorporation assay, real-time PCR, Western blotting and RNA interference using small interfering RNA were employed. ResultsAt non-toxic concentrations (less than 40μM), resveratrol inhibited the proliferation of NHEKs. Resveratrol inhibited the ERK phosphorylation and the AQP3 expression with reciprocal upregulation of ARNT expression in a concentration-dependent manner. The inhibitory effects of resveratrol on the ERK phosphorylation and the AQP3 expression were canceled by transfection of siRNA for ARNT, but not by that for AhR. Furthermore, the induction effect of resveratrol on ARNT expression was canceled after SIRT1 was knocked down in NHEKs. ConclusionResveratrol inhibited NHEK proliferation by downregulating the expression of AQP3 in an SIRT1/ARNT/ERK dependent fashion. This novel mechanism may facilitate drug innovation for hyperplastic skin disorders.

The aryl hydrocarbon receptor nuclear translocator (ARNT) modulates the antioxidant response in AML cells

We observed AML cell lines vary in their sensitivity to induction of apoptosis by troglitazone (TG), which induces apoptosis through the generation of intracellular reactive oxygen species (ROS). TG-resistant cell lines had increased abundance of ARNT transcripts and protein. Expression of ARNT in TG-sensitive cells made these cells resistant to both TG and daunorubicin. ARNT-expressing cells had increased expression of SOD2 and Nrf2 transcripts and elevated intracellular GSH concentration. Our results indicate that ARNT expression in AML cells augments antioxidant response and confers resistance to ROS inducers. This suggests ARNT may modulate ROS signaling and drug response in AML.

Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells

Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4).By conducting gene expression studies using the islets of Wfs1−/− Ay/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.

The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT/HIF-1β) is Influenced by Hypoxia and Hypoxia-Mimetics

Background: The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT, HIF-1β) is a member of the basic-Helix-Loop-Helix PER/ARNT/SIM (bHLH/PAS) protein family and a vital transcriptional regulator regarding development and physiological adaptation processes. ARNT is discussed to be linked with cancer, and other diseases. ARNT is known to be translocated into the cell nucleus, where accumulation of the protein takes place. ARNT is a heterodimerisation partner of the xenobiotic ligand activated Aryl Hydrocarbon Receptor (AhR), the Single Minded proteins (SIM), the cardiovascular helix-loop-helix factor 1 and the Hypoxia Inducible Factor proteins (HIF-α). ARNT is obligatory for HIF-1, HIF-2 and HIF-3 binding to DNA. Whereas degradation of the HIF-α subunits is suppressed by hypoxia, ARNT is generally regarded as constitutively expressed in excess within the cell, and stabilisation is commonly thought to be oxygen-independent. However, we provide evidence that the regulation of ARNT is far more complex. The aim of our study was to reevaluate the regulation of ARNT expression. Methods: We examined cell lines of different origin like MCF-7 and T47D (human breast cancer), HeLa (human cervix carcinoma), Hep3B and HepG2 (human hepatoma), Kelly (human neuroblastoma), REPC (human kidney) and Cos7 (primary primate kidney) cells. We used immunoblot analysis, densitometry, RT-PCR and transient transfection. Results and Conclusion: Our results show that ARNT protein levels are influenced by hypoxia and hypoxia mimetics such as cobalt(II)-chloride (CoCl₂) and dimethyloxalylglycine (DMOG) in a cell line specific manner. We demonstrate that this effect might be triggered by HIF-1α which plays an important role in the process of stabilizing ARNT in hypoxia.

Cigarette smoke condensate induces aryl hydrocarbon receptor-dependent changes in gene expression in spermatocytes

Cigarette smoke contains numerous compounds that cause oxidative stress and alter gene expression in many tissues, and cigarette smoking is correlated with male infertility. To identify mechanisms by which this occurs, we evaluated expression of antioxidant genes in mouse spermatocytes in response to cigarette smoke condensate (CSC). CSC exposure led to oxidative stress and dose-dependent up-regulation of Hsp90aa1, Ahr, Arnt, Sod1, Sod2, and Cyp1a1 expression in a mouse spermatocyte cell line. An antagonist of the aryl hydrocarbon receptor (AHR) abrogated several CSC-mediated changes in mRNA and protein levels. Consistent with these results, spermatocytes isolated by laser-capture microdissection from CSC-treated mice showed increased expression of several antioxidant genes. In vivo exposure to CSC was genotoxic to spermatocytes, resulting in apoptosis and disruptions to the seminiferous tubules. Our in vivo and in vitro data indicate that CSC-mediated damage to murine spermatocytes is AHR-dependent and is mediated by oxidative stress.

Regulation of uterine AHR battery gene expression by 17β-Estradiol is predominantly mediated by estrogen receptor α

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular response to numerous xenobiotics including dioxin. Surprisingly AHR knockout mice provide evidence for the involvement of the AHR signalling cascade in estrogen regulated physiological functions of the female reproductive system. Several studies already aimed to investigate the impact of the AHR mediated xenobiotic response pathway on estrogen receptor (ER) signalling, whereas on contrary availability of data describing the effect of 17β-Estradiol (E2) on the AHR signalling cascade is rather limited. In this study we observed an inhibitory effect of E2 treatment on uterine Ahr, Arnt, Arnt2, Ahrr, Cyp1a1, Ugt1 and Nfe2l2 gene expression in ovariectomized Wistar rats, whereas Cyp1b1, Nqo1 and Gsta2 displayed an increased transcription. The usage of the ER selective agonists, 16α-LE(2) (ERα selective) and 8β-VE(2) (ERβ selective), enabled us to distinguish between ER subtype specific responses. On mRNA level the observed changes in gene expression were mainly mediated by ERα except for the expression of Nqo1. In most cases the activation of ERβ caused effects opposite to the ones observed following activation of ERα. Despite the significant changes in AHR mRNA levels immunohistochemical staining uterine tissue section did not reveal changes of the AHR protein level. Taken together our results validate, support and extend the hypothesis of uterine crosstalk between AHR and ER signalling pathways. Furthermore they give an insight into how the AHR and its related genes may participate in E2 dependent uterine physiological processes and provide another potential mechanism of action for xenoestrogens.

Abstract 4228: Aryl hydrocarbon receptor nuclear translocator is associated with cisplatin resistance in cancer cells

Abstract The (ARNT) belongs to the basic-helix-loop-helix (bHLH) transcription factors containing Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α, ARNT can also form homodimers. Our previous study had shown that ARNT is a new factor involved in EGF regulated COX-2 gene expression under normoxia involving tumorigenesis in cervical cancer tissues. Resistence to cisplatin is often observed in cervical cancer therapy. Here we showed that ARNT expression is critical for cisplatin resistance. Specifically, we showed that in HeLa cervical cancer lines, cisplatin decreased ARNT expression level. Over-expression of ARNT rendered HeLa cells resistant to cisplatin. In addition, inhibition of ARNT by small interfering RNA silencing increased cisplatin induced cell death by activation of caspase 3 and sensitized drug resistant cells to cisplatin. Using ARNT deficient and ARNT fully expressed cell lines showed that induction of apoptosis by cisplatin correlates with ARNT. Importantly down regulation of ARNT decreased drug efflux pump protein MDR1 expression, suggesting that MDR1 gene expression depends on ARNT activity. In conclusion, ARNT mediates the efflux of cisplatin and has a functional role in cervical cancer cell sensitivity to cisplatin. In a xenograft analysis of SCID mice, cisplatin also efficiently inhibited ARNT-deficient tumor formation. These results suggest that targeting ARNT could overcome cisplatin resistance in human cervical 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 4228. doi:1538-7445.AM2012-4228

ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways

Previously we showed that spatial and developmental modulation of ARNT (HIF1β) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) - the most highly expressed EGFR ligand in human keratinocytes - whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation of the AREG-EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results suggest that ARNT controls AREG expression and the downstream EGFR-ERK pathway in keratinocytes, at least in part, by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.

AHR and ARNT expression in the human and rat placentas and their transcription activity in human trophoblast cultures in transactivation AHR battery genes

AHR and ARNT expression in the human and rat placentas and their transcription activity in human trophoblast cultures in transactivation AHR battery genes

Hypoxia-mediated control of HIF/ARNT machinery in epidermal keratinocytes

Transcriptional activity of hypoxia-induced factor 1 (HIF1) – a heterodimer of HIF1α and ARNT (HIF1β) – is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O 2) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.

ARNT/HIF-1β: An AML Biomarker?

AbstractAbstract 2903 Background:Despite improvements in chemotherapy protocols and bone marrow transplantation techniques, AML remains a lethal disease. Although patients with AML typically respond well to initial therapy, the majority relapse and eventually succumb to the disease. This relapse is thought to be the result of the resistance of leukaemic stem cells to the effects of chemotherapy. Some mechanisms have been discovered which contribute to resistance, but this phenomenon remains incompletely understood and is a significant barrier to improving patient outcomes. TEL is a member of the ETS transcription factor gene family and is essential for developmental processes such as haematopoiesis. TEL is frequently targeted by chromosomal translocations in human malignancies, resulting in the expression of oncogenic TEL gene fusions. A TEL-ARNT gene fusion was reported in a single case of AML, FAB subtype M2, in a patient whose leukaemic cells contained the balanced translocation t(1;12)(q21;p13). ARNT (also known as HIF-1β) is a nuclear protein and functions as part of a heterodimer, whose partners include HIF-1α and the aryl hydrocarbon receptor (AhR). HIF-1 expression is commonly deregulated in cancer and AhR signalling has been linked to leukaemogenesis. Materials and Results:Immunoblot analysis of patient bone marrow mononuclear cell lysates revealed that 5 of 29 AML specimens (∼17%) had elevated levels of ARNT protein, whereas 0 of 19 MDS specimens showed ARNT expression. In addition, AML specimens that had elevated ARNT protein levels also had elevated levels of phosphorylated Akt, while AML specimens that did not have elevated ARNT protein expression did not have activation of Akt signaling. Intriguingly, ARNT expression was noted in one patient who had progressed from MDS to AML. We then studied the expression of ARNT in HL60 cells, which are highly sensitive to induction of apoptosis by troglitazone (TG), and in U937 cells, which are TG resistant. In HL60 cells, ARNT mRNA levels remained constant following TG treatment and ARNT protein levels markedly decreased, while in U937 cells, ARNT mRNA levels increased and ARNT protein levels remained constant. We then tested the effect of exogenous expression of ARNT on the sensitivity of HL-60 cells to apoptosis induced by TG, daunorubin and hydrogen peroxide. HL-60 cells transduced with a retrovirus expressing ARNT became resistant to the induction of apoptosis by all these agents. These cells also had constitutive activation of Akt signaling, and treatment of these cells with a specific inhibitor of Akt signaling reversed their resistance to TG-induced apoptosis. Conclusions:The expression of ARNT confers resistance to apoptosis in AML cell lines and activates Akt signalling. In addition, ARNT is overexpressed in a significant subset of AML patients, in whom it activated Akt signaling, and ARNT expression correlates with progression of MDS to AML. ARNT may play an important role in chemoresistance and may be useful as a predictive or prognostic biomarker. Disclosures:No relevant conflicts of interest to declare.

Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein ( Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip(+/-) mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas.