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TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Empathy, crucial for social interaction, is impaired across various neuropsychiatric conditions. However, the genetic and neural underpinnings of empathy variability remain elusive. By combining forward genetic mapping with transcriptome analysis, we discover that aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a key driver modulating observational fear, a basic form of affective empathy. Disrupted ARNT2 expression in the anterior cingulate cortex (ACC) reduces affect sharing in mice. Specifically, selective ARNT2 ablation in somatostatin (SST)-expressing interneurons leads to decreased pyramidal cell excitability, increased spontaneous firing, aberrant Ca 2+ dynamics, and disrupted theta oscillations in the ACC, resulting in reduced vicarious freezing. We further demonstrate that ARNT2-expressing SST interneurons govern affective state discrimination, uncovering a potential mechanism by which ARNT2 polymorphisms associate with emotion recognition in humans. Our findings advance our understanding of the molecular mechanism controlling empathic capacity and highlight the neural substrates underlying social affective dysfunctions in psychiatric disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Scope: Chalcones are widely present in most plants and have various health beneficial functions. This study investigates the suppressive effect of 13 natural and synthetic chalcones on transformation of aryl hydrocarbon receptor (AhR) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC) in a cell-free system, Hepa-1c1c7 cells, and liver of ICR mice., Methods and Results: In the cell-free system, cardamonin dose-dependently inhibits AhR transformation. Chalcones with substitution on 2' and/or 6' position is important for the suppressive effect, while the substitution on 4' position is negatively for the effect. Moreover, cardamonin and 2'-hydroxychalcone competitively inhibit the binding of [ 3 H]-3-MC to the AhR. In Hepa-1c1c7 cells, cardamonin inhibits AhR transformation and expression of cytochrome P4501A1 (CYP1A1) in a dose-dependent manner through suppressing TCDD-induced phosphorylation of both AhR and AhR nuclear translocator, heterodimerization of them, and nuclear translocation of AhR. In the liver of mice, oral administered cardamonin also inhibits 3-MC-induced AhR translocation and expression of CYP1A1., Conclusion: Among used chalcones, a natural chalcone cardamonin competitively binds to AhR and suppresses its transformation. Thus, cardamonin is an effective food factor for suppression of the dioxin-caused biochemical alterations and toxicities., (© 2024 Wiley‐VCH GmbH.)

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor associated with adaptive responses to cellular stress. Its role in cutaneous squamous cell carcinoma (cSCC) remains poorly understood. The aim of this study was to investigate the role of ARNT in cSCC. Immunohistochemistry revealed downregulation of ARNT in cSCC, precancerous lesions (actinic keratosis), and cells. Knockdown of ARNT in A431 and SCL-1 cells significantly enhanced cell growth and metastasis. Microarray analysis and Ingenuity Pathway Analysis confirmed that loss of ARNT in A431 cells was highly correlated with cell growth and movement and upregulated CXCL3 expression. Cellular and xenograft experiments further confirmed that ARNT regulates cSCC proliferation and invasiveness in a CXCL3-dependent manner. ARNT may regulate CXCL3 expression through ROS-STAT3 pathway. In conclusion, this study demonstrates that ARNT plays a critical role in the development of cSCC and significantly affects the proliferation and metastatic ability of cSCC cells. It has the potential to serve as an ideal treatment target for cSCC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dake Dong reports financial support was provided by The National Natural Science Foundation of China. Zhanyan Pan reports financial support was provided by The National Natural Science Foundation of China. Dake Dong reports financial support was provided by Wuxi Science and Technology Plan Project. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cargo translocation across membranes is a crucial aspect of secretion. In conventional secretion signal peptide-equipped proteins enter the endoplasmic reticulum (ER), whereas a subset of cargo lacking signal peptides translocate into the ER-Golgi intermediate compartment (ERGIC) in a process called unconventional protein secretion (UcPS). The regulatory events at the ERGIC in UcPS are unclear. Here we reveal the involvement of ERGIC-localized small GTPases, Rab1 (Rab1A and Rab1B) and Rab2A, in regulating UcPS cargo transport via TMED10 on the ERGIC. Rab1 enhances TMED10 translocator activity, promoting cargo translocation into the ERGIC, whereas Rab2A, in collaboration with KIF5B, regulates ERGIC compartmentalization, establishing a UcPS-specific compartment. This study highlights the pivotal role of ERGIC-localized Rabs in governing cargo translocation and specifying the ERGIC's function in UcPS., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2α as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2α knockout and overexpression mice verified the protective effect of HIF-2α on NASH progression. Importantly, the HIF-2α stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2α is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2α in liver macrophages by suppressing the interaction of HIF-2α with ARNT, suggesting that macrophage HIF-2α may be a potential target for the treatment of NASH., (© 2024. The Author(s).)

Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD - 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes., Competing Interests: Declaration of competing interest During the course of this study, A.R.B, M.B.B. and M.E.A. were employed by ScitoVation LLC. J.A.H. is employed by the EPA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to β-naphthoflavone (BNF) significantly increased the expression of BoAhR , BoARNT , CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR / BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1 .

Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown. To this end, we generated HIF3A gene-disrupted mice by means of genome editing technology to explore the pleiotropic role of HIF-3α in development and physiology. We obtained adult mice carrying homozygous HIF3A gene mutations with comparable body weight and height to wild-type mice. However, the number of litters and ratio of homozygous mutation carriers born from the mating between homozygous mutant mice was lower than expected due to sporadic deaths on postnatal day 1. HIF3A gene-disrupted mice exhibited abnormal configuration of the lung such as a reduced number of alveoli and thickened alveolar walls. Transcriptome analysis showed, as well as genes associated with lung development, an upregulation of stearoyl-Coenzyme A desaturase 1, a pivotal enzyme for fatty acid metabolism. Analysis of fatty acid composition in the lung employing gas chromatography indicated an elevation in palmitoleic acid and a reduction in oleic acid, suggesting an imbalance in distribution of fatty acid, a constituent of lung surfactant. Accordingly, administration of glucocorticoid injections during pregnancy resulted in a restoration of normal alveolar counts and a decrease in neonatal mortality. In conclusion, these observations provide novel insights into a pivotal role of HIF-3α in the preservation of critically important structure and function of alveoli beyond the regulation of hypoxia-mediated gene expression., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kawahata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Breast cancer is one of the main causes of malignancy-related deaths globally and has a significant impact on women's quality of life. Despite significant therapeutic advances, there is a medical need for targeted therapies in breast cancer. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor mediates responses to environment stimuli, is emerging as a unique pleiotropic target. Herein, a combined molecular simulation and in vitro investigations identified 3-(3-fluorophenyl)-1H-pyrazolo[3,4-b]pyridine (3FPP) as a novel AhR ligand in T47D and MDA-MB-231 breast cancer cells. Its agonistic effects induced formation of the AhR-AhR nuclear translocator (Arnt) heterodimer and prompted its binding to the penta-nucleotide sequence, called xenobiotic-responsive element (XRE) motif. Moreover, 3FPP augmented the promoter-driven luciferase activities and expression of AhR-regulated genes encoding cytochrome P450 1A1 (CYP1A1) and microRNA (miR)-212/132 cluster. It reduced cell viability, migration, and invasion of both cell lines through AhR signaling. These anticancer properties were concomitant with reduced levels of B-cell lymphoma 2 (BCL-2), SRY-related HMG-box4 (SOX4), snail family zinc finger 2 (SNAI2), and cadherin 2 (CDH2). In vivo, 3FPP suppressed tumor growth and activated AhR signaling in an orthotopic mouse model. In conclusion, our results introduce the fused pyrazolopyridine 3FPP as a novel AhR agonist with AhR-specific anti-breast cancer potential in vitro and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates biological signals to control various complicated cellular functions. It plays a crucial role in environmental sensing and xenobiotic metabolism. Dysregulation of AhR is associated with health concerns, including cancer and immune system disorders. Upon binding to AhR ligands, AhR, along with heat shock protein 90 and other partner proteins undergoes a transformation in the nucleus, heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), and mediates numerous biological functions by inducing the transcription of various AhR-responsive genes. In this manuscript, the 3-dimensional structure of the entire human AhR is obtained using an artificial intelligence tool, and molecular dynamics (MD) simulations are performed to study different structural conformations. These conformations provide insights into the protein's function and movement in response to ligand binding. Understanding the dynamic behavior of AhR will contribute to the development of targeted therapies for associated health conditions. Therefore, we employ well-tempered metadynamics (WTE-metaD) simulations to explore the conformational landscape of AhR and obtain a better understanding of its functional behavior. Our computational results are in excellent agreement with previous experimental findings, revealing the closed and open states of helix α1 in the basic helix-loop-helix (bHLH domain) in the cytoplasm at the atomic level. We also predict the inactive form of AhR and identify Arginine 42 as a key residue that regulates switching between closed and open conformations in existing AhR modulators.

The aryl hydrocarbon receptor (AHR) signaling pathway is a complex regulatory network that plays a critical role in various biological processes, including cellular metabolism, development, and immune responses. The complexity of AHR signaling arises from multiple factors, including the diverse ligands that activate the receptor, the expression level of AHR itself, and its interaction with the AHR nuclear translocator (ARNT). Additionally, the AHR crosstalks with the AHR repressor (AHRR) or other transcription factors and signaling pathways and it can also mediate non-genomic effects. Finally, posttranslational modifications of the AHR and its interaction partners, epigenetic regulation of AHR and its target genes, as well as AHR-mediated induction of enzymes that degrade AHR-activating ligands may contribute to the context-specificity of AHR activation. Understanding the complexity of AHR signaling is crucial for deciphering its physiological and pathological roles and developing therapeutic strategies targeting this pathway. Ongoing research continues to unravel the intricacies of AHR signaling, shedding light on the regulatory mechanisms controlling its diverse functions., Competing Interests: Declaration of Competing Interest ST and CO are founders and CO is managing director of cAHRmeleon Bioscience GmbH. Authors of this manuscript have patents on AHR inhibitors in cancer (WO2013034685, CO); A method to multiplex tryptophan and its metabolites (WO2017072368, CO); A transcriptional signature to determine AHR activity (WO2020201825, ST, CO); Interleukin-4-induced gene 1 (IL4I1) as a biomarker (WO2020208190, ST, MTP, CO); Interleukin-4-induced gene 1 (IL4I1) and its metabolites as biomarkers for cancer (WO2021116357, ST, CO)., (Copyright © 2023. Published by Elsevier Inc.)

Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites. Many of these compounds are also linked to the translocator protein (TSPO), an outer mitochondrial membrane protein. Given a portion of the cellular pool of the AHR has also been localized to mitochondria and the overlap in putative ligands, we tested the hypothesis that crosstalk exists between the two proteins. CRISPR/Cas9 was used to create knockouts for AHR and TSPO in a mouse lung epithelial cell line (MLE-12). WT, AHR-/-, and TSPO-/- cells were then exposed to AHR ligand (TCDD), TSPO ligand (PK11195), or both and RNA-seq was performed. More mitochondrial-related genes were altered by loss of both AHR and TSPO than would have been expected just by chance. Some of the genes altered included those that encode for components of the electron transport system and the mitochondrial calcium uniporter. Both proteins altered the activity of the other as AHR loss caused the increase of TSPO at both the mRNA and protein level and loss of TSPO significantly increased the expression of classic AHR battery genes after TCDD treatment. This research provides evidence that AHR and TSPO participate in similar pathways that contribute to mitochondrial homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Preeclampsia (PE) is a common and serious complication of pregnancy with no cure except premature delivery. The root cause of PE is improper development of the placenta-the temporary organ supporting fetal growth and development. Continuous formation of the multinucleated syncytiotrophoblast (STB) layer via differentiation and fusion of cytotrophoblasts (CTBs) is vital for healthy placentation and is impaired in preeclamptic pregnancies. In PE, there is reduced/intermittent placental perfusion, likely resulting in a persistently low O 2 environment. Low O 2 inhibits differentiation and fusion of CTBs into STB and may thus contribute to PE pathogenesis; however, the underlying mechanisms are unknown. Because low O 2 activates a transcription factor complex in cells known as the hypoxia-inducible factor (HIF), the objective of this study was to investigate whether HIF signaling inhibits STB formation by regulating genes required for this process. Culture of primary CTBs, the CTB-like cell line BeWo, and human trophoblast stem cells under low O 2 reduced cell fusion and differentiation into STB. Knockdown of aryl hydrocarbon receptor nuclear translocator (a key component of the HIF complex) in BeWo cells restored syncytialization and expression of STB-associated genes under different O 2 levels. Chromatin immunoprecipitation sequencing facilitated the identification of global aryl hydrocarbon receptor nuclear translocator/HIF binding sites, including several near genes implicated in STB development, such as ERVH48-1 and BHLHE40, providing new insights into mechanisms underlying pregnancy diseases linked to poor placental O 2 supply., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in N-TERT (N-TERT1) immortalized human keratinocytes, and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1β), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl 2 and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1. As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR., (© 2023. The Author(s).)

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Osteosarcoma is the most common malignant tumor of bone, with rapid progressive growth, early distant metastases, and frequent recurrence after surgical treatment. Osteosarcoma is characterized by changes in the ratio and expression of different cytochrome P450 (CYP) isoforms that can affect the effectiveness of anticancer therapies. The inducible expression of CYP1 genes depends on the ligand-dependent functionality of the aryl hydrocarbon receptor (AHR). In this study, we examined the AHR/CYP1 signaling pathway in four osteosarcoma cell lines (MG63, HOS, SAOS2, and U2OS) induced by the known AHR ligands: indirubin, indole-3-carbinol, and beta-naphthoflavone. Using qPCR and Western blot analysis, we explored the effects of these ligands on the expression of the CYP1 genes and studied the correlation between these responses and the changes in the mRNA and protein levels of AHR and the AHR nuclear translocator (ARNT) in these osteosarcoma cell lines. The results show that the AHR/CYP1 signaling pathway retains its function only in MG63 and HOS cells, and is impaired in SAOS2 and U2OS cells. Our data should be taken into account when recommending new strategies for the treatment of osteosarcoma and when evaluating new drugs against osteosarcoma in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor regulating adaptive and maladaptive responses toward exogenous and endogenous signals. Research from various biomedical disciplines has provided compelling evidence that the AHR is critically involved in the pathogenesis of a variety of diseases and disorders, including autoimmunity, inflammatory diseases, endocrine disruption, premature aging and cancer. Accordingly, AHR is considered an attractive target for the development of novel preventive and therapeutic measures. However, the ligand-based targeting of AHR is considerably complicated by the fact that the receptor does not always follow the beaten track, i.e. the canonical AHR/ARNT signaling pathway. Instead, AHR might team up with other transcription factors and signaling molecules to shape gene expression patterns and associated physiological or pathophysiological functions in a ligand-, cell- and micromilieu-dependent manner. Herein, we provide an overview about some of the most important non-canonical functions of AHR, including crosstalk with major signaling pathways involved in controlling cell fate and function, immune responses, adaptation to low oxygen levels and oxidative stress, ubiquitination and proteasomal degradation. Further research on these diverse and exciting yet often ambivalent facets of AHR biology is urgently needed in order to exploit the full potential of AHR modulation for disease prevention and treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Angiogenesis is involved in the malignant transformation of cancers. Vascular endothelial growth factor (VEGF) is important in inducing angiogenesis. Cultured cells play an important role in analyzing the regulation of VEGF expression, and it is revealed that VEGF expression is induced under hypoxia. However, it has been shown that there are differences in the pathway for gene expression between two-dimensional (2D) cells and in vivo cells. Three-dimensional (3D) spheroids constructed in 3D culture with a gene expression pattern more similar to that of in vivo cells than 2D cells have been used to solve this problem. This study analyzed the VEGF gene expression pathway in 3D spheroids of human lung cancer cells, A549 and H1703. Hypoxia-inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) regulated VEGF gene expression in 3D spheroids. However, VEGF gene expression was not regulated by HIF-1α in 2D cells. To conclude, we found that the regulatory pathway of VEGF gene expression is different between 2D cells and 3D spheroids in human lung cancer cells. These results suggest the possibility of a new VEGF gene expression regulation pathway in vivo. In addition, they show useful knowledge for the analysis of angiogenesis induction mechanisms and also demonstrate the usefulness of 3D spheroids.

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the bHLH-PAS (basic Helix-Loop-Helix-Period/ARNT/Single-minded) family of transcription factors, which participate in the sensing and transmitting stimuli of exogenous and endogenous chemical substances, and subsequently activates genes transcription involved in various detoxification and physiological functions. However, they have not been identified in Dendroctonus armandi, and their roles in the detoxification metabolism are unclear. In the present study, AhR and ARNT of D. armandi were characterized. Spatiotemporal expression profiling indicated that DaAhR and DaARNT were highly expressed in the adult and larval stages of D. armandi and mainly expressed in the midgut and Malpighian tubules of adults. Additionally, the expression of DaAhR and DaARNT significantly increased after exposure to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene. Silencing DaAhR and DaARNT increased the susceptibility of D. armandi to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene, and the activities of detoxification enzyme were also remarkably reduced. Moreover, DaCYP6DF1 and DaGSTs2 were significantly down-regulated after injections of dsAhR and dsARNT in the male and female adults, with the expression of DaCYP6DF1 decreasing by higher than 70%. The present study revealed that the transcription factors AhR and ARNT of D. armandi were induced by terpenoids and participated in the regulation of DaCYP6DF 1 expression, which was associated with D. armandi 's susceptibility to (-)-𝛽-pinene and (±)-limonene. These results may provide a theoretical basis for the integrated control of D. armandi and improve our comprehension of insect toxicology.

Background: The circadian clock gene brain and muscle Arnt-like protein-1 (BMAL1) regulates energy metabolism, adipocyte proliferation and differentiation, glucose metabolism, and other functions. This study aimed to examine the association of potential polymorphisms in BMAL1 with obesity among Chinese youth., Methods: A total of 2973 participants were included in this study. According to the body mass index obesity standard of China, 208 subjects were defined as experiencing general obesity. According to the waist-to-hip ratio obesity standard, 335 participants were defined as experiencing central obesity. Four single nucleotide polymorphisms (SNPs) (rs9633835, rs6486121, rs7107287, and rs12364562) were genotyped using TaqMan probe techniques., Results: There was no significant difference in the either genotypic or allelic frequencies between the non-general and general obesity groups, while a positive association was observed between BMAL1 rs6486121 variant and central obesity risk (CC＋CT vs. TT: OR:2.139, 95% CI:1.164-3.930; P = 0.014) after adjusting for covariates. Stratification analyses revealed significant associations with central obesity risk for rs6486121 polymorphism in women according to the additive model (CC vs. CT vs. TT: OR：1.409; 95 % CI: 1.029-1.930; P = 0.032). Haplotype analysis showed that only paired haplotypes, including rs9633835G with rs6486121T, had a significant effect on central obesity with OR (95%CI) was 1.035 (1.011-1.060) and P = 0.004., Conclusion: our findings suggest that BMAL1 polymorphisms are significantly associated with central obesity and sex-specific genetic effects on BMAL1-mediated genetic susceptibility to obesity., Competing Interests: Declaration of Competing Interest Authors declare that there is no conflict of interest., (Copyright © 2022. Published by Elsevier Ltd.)

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular responses to hypoxic stress, is essential for tumor progression. It is a heterodimer comprising HIF1α and HIF1β, with multiple interfaces among their PAS-A, PAS-B, and bHLH domains. HIF1β is also known as aryl hydrocarbon receptor nuclear translocator (ARNT). Casein kinase 1δ-dependent phosphorylation of the solvent-front residue S247 on the HIF1α PAS-B domain interrupts HIF1α-ARNT complex formation and reduces HIF-1 transcription activity. However, S247 is involved in neither HIF1α-ARNT complex formation nor stabilization of the relative orientation between the HIF1α PAS-A and PAS-B domains. To uncover the underlying allosteric mechanism, we conducted Gaussian accelerated molecular dynamics simulations and identified two distinct conformations of the pS247-carrying HIF1α PAS-B domain: H291-in and H291-out. The H291-in structure can associate with the HIF1α PAS-A domain and form a V-shaped pouch to accommodate the ARNT PAS-A domain, but it cannot associate with the ARNT PAS-B domain. By contrast, the H291-out structure can bind to the ARNT PAS-B domain, but its association with the HIF1α PAS-A domain leads to an unsuitable relative orientation to accommodate the ARNT PAS-A domain. Both conformations were also collected in parallel simulations of the unphosphorylated PAS-B domain. Both structures manage to associate with the ARNT PAS-B and HIF1α PAS-A domains; thus, they are adequate for HIF1α-ARNT complex formation. The domain-domain contact pattern in a phosphorylated variant is shuffled by an order-to-disorder structural switch, triggered by the newly formed K251-pS247 interaction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Currently, the benefits of nasopharyngeal carcinoma (NPC) therapy are limited, and it is necessary to further explore possible therapeutic targets. Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) has been extensively studied in other cancer species, but little has been explored in NPC. The aim of this study was to verify the expression level of ARNT2 and its underlying mechanism in NPC., Methods: Datasets containing ARNT2 mRNA expression levels were retrieved and collected from various databases to explore the expression status of ARNT2 in NPC. ARNT2-related coexpressed genes, differential expressed genes, and target genes were obtained for functional enrichment analysis. The potential target gene of ARNT2 and their regulatory relationship were studied through ChIP-seq data. CIBERSORTx was used to assess the immune infiltration of NPC, and the association with ARNT2 expression was calculated through correlation analysis., Results: ARNT2 was upregulated and possessed an excellent discriminatory capability in NPC samples. ARNT2 positively correlated target genes were clustered in pathways in cancer, while negatively correlated target genes were enriched in immune-related pathway. The ChIP-seq information of ARNT2 and histone showed that prostaglandin-endoperoxide synthase 2 (PTGS2) was a potential target gene of ARNT2. CIBERSORTx revealed the immunity status in NPC, and ARNT2 expression was correlated with infiltration of five immune cells., Conclusions: ARNT2 is overexpressed in NPC and may regulate PTGS2 to participate in the cancer process. ARNT2 serves as a key oncogenic target in NPC patients., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Si-Wei Huang et al.)

Single-minded 2 (SIM2) is a neuron-enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology., (© 2022 The Author(s).)

Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans -regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT -specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.

Background: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer., Methods: This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed., Results: BaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl 2 . However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl 2 ., Conclusion: It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway., (© 2022. The Author(s).)

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform–specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform–specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

Transport and Golgi Organization protein 2 Homolog (TANGO2)-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2-related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and β-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition., (© 2022. The Author(s).)

Acute promyelocyte leukaemia (APL) is a subgroup of acute myeloid leukaemia. Dysregulation of clock genes has been revealed to be involved in APL progression. Herein, the mechanism of clock gene aryl hydrocarbon receptor nuclear translocator- like (ARNTL) in APL was explored. The expression of ARNTL, period circadian regulator 1 and 2 (PER1 and PER2) in APL tissue samples and normal samples was analysed by bioinformatic analysis. Gene expression in APL cells was detected by reverse transcription quantitative polymerase chain reaction. Acute promyelocyte leukaemia cell viability and cell cycle progression were assessed by cell counting kit 8 (CCK-8) assays and flow cytometry analyses, respectively. The protein levels of ARNTL and cell cycle markers were examined by western blotting. Interaction between ARNTL and miR-320a/b was confirmed by luciferase reporter assays. Aryl hydrocarbon receptor nuclear translocator-like was overexpressed in marrow tissues of patients with acute myeloid leukaemia and predicted poor outcome. Aryl hydrocarbon receptor nuclear translocator-like knockdown inhibited APL cell viability and arrested APL cells in the G1 phase. Mechanically, ARNTL was targeted by miR-320a/b. Moreover, miR-320a/b upregulation promoted cell cycle arrest in the G1 phase and suppressed the viability of APL cells, and the impacts were reversed by ARNTL overexpression. In conclusion, miR-320a/b suppresses cell viability and leads to cell cycle arrest by suppressing ARNTL in APL.

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1β under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1β and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1β decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Placental hypoxia and increased levels of maternal blood anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT1), are associated with the pathogenesis of pre-eclampsia. We have demonstrated that hypoxia-inducible factor (HIF)-2α mediates the upregulation of the hypoxia-induced FLT1 gene in trophoblasts and their cell lines. Here, we investigated the involvement of HIF-1β, which acts as a dimerization partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1β and HIF-2α in the nuclei of BeWo, JAR and JEG-3 cells under hypoxia via co-immunoprecipitation. We found that hypoxia-induced upregulation of the FLT1 gene in BeWo cells and secretion of sFLT1 in human primary trophoblasts were significantly reduced by siRNAs targeting HIF-1β. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxaloylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1β mRNA. It was recently shown that DNA demethylation increases both basal and hypoxia-induced expression levels of the FLT1 gene in three trophoblast-derived cell lines. In the demethylated BeWo cells, siRNAs targeting HIF-2α and HIF-1β suppressed the further increase in the expression levels of the FLT1 gene due to hypoxia or treatment with DMOG. However, luciferase reporter assays and bisulfite sequencing revealed that a hypoxia response element (-966 to -962) of the FLT1 gene is not involved in hypoxia or DMOG-induced upregulation of the FLT1 gene. These findings suggest that HIF-1β is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1β complex may be a crucial therapeutic target for pre-eclampsia., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 ( Tango10 ) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and excessive accumulation of dysfunctional PVAT are hallmarks of pathogenesis after angioplasty. Recent genome-wide association studies reveal that single-nucleotide polymorphism (SNP) in MIA3 is associated with atherosclerosis-relevant VSMC phenotypes. However, the role of MIA3 in the vascular remodeling response to injury remains unknown. Here, we found that expression of MIA3 is increased in proliferative VSMCs and knockdown of MIA3 reduces VSMCs proliferation, migration, and inflammation, whereas MIA3 overexpression promoted VSMC migration and proliferation. Moreover, knockdown of MIA3 ameliorates femoral artery wire injury-induced neointimal hyperplasia and increases brown-like perivascular adipocytes. Collectively, the data suggest that MIA3 deficiency prevents neointimal formation by decreasing VSMC proliferation, migration, and inflammation and maintaining BAT-like perivascular adipocytes in PVAT during injury-induced vascular remodeling, which provide a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lei, Xu, Li, Meng, Chen, Yang, Li, Zhuang and Zuo.)

Hypoxia-inducible factors (HIFs) activate gene transcription in response to reduced O 2 availability and play critical roles in development, physiology, and disease pathogenesis. Mutations that dysregulate HIF activity are the genetic basis for tumor predisposition in the von Hippel-Lindau syndrome and excess red blood cell production in hereditary erythrocytosis., (© 2021 The Author. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Nasopharyngeal carcinoma (NPC), a subclass of cancers of the neck and head, is a predominant cause of cancer-associated death worldwide. Hence, there is a critical need for research into NPC-related treatment strategies. Cisplatin is a promising therapy option for NPCs and other cancers that is frequently utilized. Some patients acquire resistance to cisplatin therapy, which complicates the successful use of cisplatin treatment in NPCs. Although exosomal transfer of oncogenic miRNAs has been shown to improve recipient cell proliferation, metastasis and chemoresistance, the molecular mechanism behind this effect on NPC has yet to be fully understood. Exosomal microRNAs (miRNAs) from cisplatin-resistant cells were identified as significant mediators of chemoresistance in NPC cells in this investigation. Initially, we found that exosomal miR-106a-5p levels in the serum of chemoresistant and last-cycle patients were greater than in that of non-resistant and first-cycle patients. Also, exosomal miR-106a-5p enhanced the proliferative ability of NPC cells. Mechanistically, exosomal miR-106a-5p targets ARNT2, which further activates AKT phosphorylation, and thus promotes NPC cell proliferation, decreases apoptosis and in turn regulates tumorigenesis. We found similar results using in vivo NPC models, where exosomal miR-106a-5p through regulation of ARNT2 (aryl hydrocarbon receptor nuclear translocator 2) promoted tumorigenesis. Taken together, these findings indicate that exosomal miR-106a-5p could be a promising diagnostic biomarker and drug target for patients with NPC., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

We investigated a hereditary syndrome in Cane Corso dogs. Affected dogs developed dental-skeletal-retinal anomaly (DSRA), clinically characterized by brittle, discolored, translucent teeth, disproportionate growth and progressive retinal degeneration resulting in vision loss. Combined linkage and homozygosity mapping delineated a 5.8 Mb critical interval. The comparison of whole genome sequence data of an affected dog to 789 control genomes revealed a private homozygous splice region variant in the critical interval. It affected the MIA3 gene encoding the MIA SH3 domain ER export factor 3, which has an essential role in the export of collagen and other secreted proteins. The identified variant, XM_005640835.3:c.3822+3_3822+4del, leads to skipping of two exons from the wild type transcript, XM_005640835.3:r.3712_3822del. Genotypes at the variant were consistent with monogenic autosomal recessive mode of inheritance in a complete family and showed perfect genotype-phenotype association in 18 affected and 22 unaffected Cane Corso dogs. MIA3 variants had previously been shown to cause related phenotypes in humans and mice. Our data in dogs together with the existing functional knowledge of MIA3 variants in other mammalian species suggest the MIA3 splice defect and a near complete loss of gene function as causative molecular pathomechanism for the DSRA phenotype in the investigated dogs.

Diabetic macular edema (DME) is a leading cause of blindness in diabetic retinopathy. Prolonged hyperglycemia plus hypoxia contributes to DME pathogenesis. Retinal pigmented epithelial cells comprise the outer blood-retinal barrier and are essential for maintaining physiological functioning of the retina. Melatonin acts as an antioxidant and regulator of mitochondrial bioenergetics and has a protective effect against ocular diseases. However, the role of mitochondrial dysfunction and the therapeutic potential of melatonin in DME remain largely unexplored. Here, we used an in vitro model of DME to investigate blood-retinal barrier integrity and permeability, angiogenesis, mitochondrial dynamics, and apoptosis signaling to evaluate the potential protective efficacy of melatonin in DME. We found that melatonin prevents cell hyper-permeability and outer barrier breakdown by reducing HIF-1α, HIF-1β and VEGF and VEGF receptor gene expression. In addition, melatonin reduced the expression of genes involved in mitochondrial fission (DRP1, hFis1, MIEF2, MFF), mitophagy (PINK, BNip3, NIX), and increased the expression of genes involved in mitochondrial biogenesis (PGC-1α, NRF2, PPAR-γ) to maintain mitochondrial homeostasis. Moreover, melatonin prevented apoptosis of retinal pigmented epithelial cells. Our results suggest that mitochondrial dysfunction may be involved in DME pathology, and melatonin may have therapeutic value in DME, by targeting signaling in mitochondria., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a man-made chemical compound contaminating the environment. An exposure of organisms to TCDD results in numerous disorders. The main mechanism of TCDD action involves the induction of the aryl hydrocarbon receptor (AhR) pathway followed by the increase in the expression and activity of cytochrome P450 family 1 (CYP1) enzymes. The main aim of the present study was to identify, by means of RNA sequencing, transcripts involved in the mechanism of TCDD action in Chinese hamster ovary (CHO) cells, known to not express CYP1A1 enzyme. The CHO cells were treated with TCDD for 3, 12 or 24 h, and total RNA was isolated and sequenced. Thirty six (p adjusted  < 0.05) or six (p adjusted  < 0.05, log2FC ≥ 1.0/log2FC≤-1.0) differentially expressed genes (DEGs) were identified in TCDD-treated cells depending on the assumed statistical criteria. The dioxin up- and downregulated the expression of genes associated with ovarian follicle functions, development, cardiovascular system, signal transduction, inflammation and carcinogenesis. TCDD did not affect the expression of any of 522 miRNAs which were identified in the cells. The expression of CYP1A1, CYP1A2 and CYP1B1 was demonstrated neither in control nor in TCDD-treated CHO cells, although the respective genes were found in the cell genome. Twenty two other CYP enzymes were identified in CHO cells, however their expression was also not affected by TCDD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is a member of the PER-ARNT-SIM superfamily of environmental sensors. This receptor has been a molecule of interest for many years in the field of toxicology, as it was originally discovered to mediate the toxic effects of certain environmental pollutants like benzo( a )pyrene and 2,3,7,8-tetrachlorodibenzo- p -dioxin. While all animals express this protein, there is naturally occurring variability in receptor size and responsiveness to ligand. This naturally occurring variation, particularly in mice, has been an essential tool in the discovery and early characterization of the AHR. Genetic models including congenic mice and induced mutations at the Ahr locus have proven invaluable in further understanding the role of the AHR in adaptive metabolism and TCDD-induced toxicity. The creation and examination of Ahr null mice revealed an important physiological role for the AHR in vascular and hepatic development and mediation of the immune system. In this review, we attempt to provide an overview to many of the AHR models that have aided in the understanding of AHR biology thus far. We describe the naturally occurring polymorphisms, congenic models, induced mutations at the Ahr locus and at the binding partner Ah Receptor Nuclear Translocator and chaperone, Ah receptor associated 9 loci in mice, with a brief description of naturally occurring and induced mutations in rats.

In the present study, the aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) of Nilaparvata lugens were cloned and identified. The NlAhR and NlARNT expression levels significantly increased after imidacloprid, etofenprox and isoprocarb treatments. Knockdowns of NlAhR and NlARNT increased the susceptibility of N. lugens to imidacloprid, etofenprox and isoprocarb, and the detoxification enzyme activities were also significantly decreased. In addition, NlCYP301A1, NlGSTt1 and NlCarE7 were significantly down-regulated after injections of dsNlAhR and dsNlARNT, with the NlCarE7 expression decreasing by greater than 80%. Moreover, after knocking down NlCarE7, the susceptibility of N. lugens to etofenprox and isoprocarb significantly increased. Both NlAhR and NlARNT bound the NlCarE7 promoter and significantly enhanced the transcriptional activity. Our research revealed the functional roles of transcription factors NlAhR and NlARNT in the detoxification metabolism of N. lugens. The results provide a theoretical basis for the pest management and comprehensive control of N. lugens and increase our knowledge of insect toxicology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

Lung cancer is the leading cause of cancer-related death worldwide. Glypican-5 (GPC5) is a member of heparan sulfate proteoglycans, and its biological importance in initiation and progression of lung cancer remains controversial. In the present study, we revealed that GPC5 transcriptionally enhanced the expression of CTDSP1 (miR-26b host gene) via AhR-ARNT pathway, and such up-regulation of CTDSP1 intracellularly contributed to the inhibited proliferation of lung cancer cells. Moreover, exosomes derived from GPC5-overexpressing human lung cancer cells (GPC5-OE-derived exosomes) had an extracellular repressive effect on human lymphatic endothelial cells (hLECs), leading to decreased tube formation and migration. Comparison between GPC5-WT- and GPC5-OE-derived exosomes showed that miR-26b (embedded within introns of CTDSP1 gene) was significantly up-regulated in GPC5-OE-derived exosomes and critical to the influence on hLECs. On the mechanism, we demonstrated that miR-26b transferred into hLECs directly targeted to PTK2 3'-UTR and led to PTK2 down-regulation, resulting in defects in tube formation and migration of hLECs. By uncovering the regulation network among GPC5, miR-26b, miR-26b host gene (CTDSP1), and target gene (PTK2), our findings demonstrated that GPC5 functioned as a tumor suppressor in human lung cancer.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim) family of proteins. The AhR was cloned and characterized for its role in mediating the toxicity of dioxins. Subsequent research has identified the role of AhR in suppression of cancer cell growth. We hypothesized that the AhR is a molecular target for therapeutic intervention in cancer, and that activation of the AhR by unique AhR ligands in cancer cells could have anti-cancer effects including induction of cell death. This study describes the discovery and characterization of a new class of anti-cancer agents targeting the AhR, that we designate as Select Modulators of AhR-regulated Transcription (SMAhRTs). We employed two independent small molecule screening approaches to identify potential SMAhRTs. We report the identification of CGS-15943 that activates AhR signaling and induces apoptosis in an AhR-dependent manner in liver and breast cancer cells. Investigation of the downstream signaling pathway of this newly identified SMAhRT revealed upregulation of Fas-ligand (FasL), which is required for AhR-mediated apoptosis. Our results provide a basis for further development of a new class of anti-cancer therapeutics targeting an underappreciated molecular target, the AhR.

Hypoxia-inducible factor1 (HIF1) plays a pivotal role in ensuring cells adapt to low-oxygen conditions. Depletion of oxygen, a co-substrate during hydroxylation of prolyl (P402 and P564) residues of HIF1⍺, evades HIF1⍺ ubiquitination and enables its dimerization with HIF1β to mediate global transcriptional response to hypoxia. Though HIF1 is largely considered eliciting a protective role during physiological or pathological hypoxia or ischemia, elevated HIF1 during chronic hypoxia contributes to glomerular diseases' pathology and proteinuria. The glomerulus is responsible for renal permselectivity and excretion of ultra-filtrated urine. Podocytes are the glomerulus' major cell types and are instrumental for glomerular filtration, permselectivity, and glomerular basement membrane maintenance. Podocyte injury is expected to impair the efficiency of glomerular filtration and manifestation of glomerulosclerosis and proteinuria. Accumulated evidence suggests that podocytes are susceptible to various insults during chronic hypoxia, including podocyte EMT, slit-diaphragm dysfunction, foot process effacement, and cytoskeletal derangement due to accumulation of HIF1. This review discusses how hypoxia/HIF1 signaling regulates various features and function of podocytes during exposure to chronic hypoxia or inducing HIF1 by various chemical modulators.

Recent genome-wide association studies (GWAS) highlighted the importance of genetic variations on SLC22A3 and MIA3 genes in developing coronary heart disease (CHD) among different ethnicities. However, the influence of these variations is not recognized within the Iranian population. Hence, in the present study, we aim to investigate two key single nucleotide polymorphisms (SNPs) on CHD incidence in this population. For this purpose, from Tehran Cardiometabolic Genetic Study (TCGS), 453 individuals with CHD were selected as a case and 453 individuals as a control that matched their age and gender. After quality control of two selected SNPs, rs2048327 (SLC22A3) and rs17465637 (MIA3), we used genotyps resulted from chip-typing technology and conducted the logistic regression analysis adjusted for non-genetic risk factors to detect the possible association of these SNPs with the CHD development. Our findings demonstrated the rs2048327-G and rs17465637-C can significantly increase the risk of CHD development about two times in only males and females, respectively. Interestingly, in the male carriers of the risk allele (G) of rs2048327, the low high-density lipoprotein (HDL) level can significantly predispose them to develop coronary heart disease in the future. According to our results, paying more attention to gender and genetic markers can help more efficient coronary heart disease screening and diagnosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a member of the PER-ARNT-SIM (PAS) superfamily of environmental sensors. The AHR is involved in a series of biological processes including adaptive metabolism of xenobiotics, toxicity of certain environmental pollutants, vascular development, fertility, and immune function. Mouse models, including the Ahr null and Ahr conditional null (Ahrfx) mice, are widely used for the study of AHR-mediated biology and toxicity. The Ahr conditional null mouse harbors the low-affinity Ahrd allele that exhibits approximately a 10-fold lower binding affinity for certain xenobiotic AHR ligands than the widely used C57BL/6 mouse that harbors the higher affinity Ahrb1 allele. Here, we report a novel mouse model that introduces a V375A polymorphism that converts the low-affinity allele into a high-affinity allele, offering a more sensitive conditional model. In the generation of this novel conditional allele, two additional mutants arose, including a 3-bp deletion in the PAS-B domain (AhrNG367R) and an early termination codon in the PAS-B domain (AhrTer383). The AhrNG367R allele presents as a phenocopy of the null and the AhrTer383 allele presents as an antimorph when assessing for the ductus venosus and liver lobe weight endpoints. These new models represent a series of tools that will be useful in further characterizing AHR biology., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The chicken (Gallus gallus), which has three aryl hydrocarbon receptor (AHR) isoforms (ckAHR1, ckAHR2, and ckAHR1β) and two AHR nuclear translocator (ARNT) isoforms (ckARNT1 and ckARNT2), is highly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and can serve as an avian model to gain an understanding of the mechanism underlying dioxin toxicity. To elucidate the mechanism of TCDD-induced immunotoxicity in avian species, we treated chicken embryos in ovo with graded concentrations of TCDD (1.5, 2.5, 3.0, 3.3, 3.5, and 4.0 μM). Initially, we measured mRNA expression levels of ckAHR and ckARNT isoforms and analyzed the T cell populations and transcriptome in the thymuses of TCDD-treated chicken embryos. Quantitative polymerase chain reaction analysis revealed that mRNA expressions of ckAHR1 and ckARNT2 were dominant in the thymus. Severe weight loss and thymus atrophy were observed in the TCDD-treated embryos. Immunophenotyping analyses demonstrated significant increases in CD4 + CD8 - CD25 + and CD4 + CD8 + CD25 + regulatory T cells (Tregs) populations following TCDD exposure, suggesting that TCDD suppresses T cell-mediated immune responses in chicken embryos. In addition, thymic transcriptome analyses intimated that alteration of the signaling pathways related to erb-b2 receptor tyrosine kinase 4 (ERBB4) and wnt family member 5A (WNT5A), and bone morphogenetic protein (BMP) may be associated with the TCDD-induced thymus atrophy. We also observed significantly altered expression levels of genes including interleukine 13 receptor subunit alpha 2 (IL13RA2), transforming growth factor beta 1 (TGFβ1), collagen type III alpha 1 chain (COL3A1), and collagen type IX alpha 3 chain (COL9A3), implying immunosuppression, fibrosis development, and collagen deposition. Collectively, these findings suggest that TCDD exposure activates the ckAHR1-ckARNT2 signaling pathway and suppresses immune responses through the prompted differentiation to CD4 + CD8 - CD25 + and CD4 + CD8 + CD25 + Tregs and altered expressions of immune-related genes in the thymus of chicken embryos., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.