Aryl Hydrocarbon Receptor Nuclear Translocator Research Articles

Aryl hydrocarbon receptor: current perspectives on key signaling partners and immunoregulatory role in inflammatory diseases.

The aryl hydrocarbon receptor (AhR) is a versatile environmental sensor and transcription factor found throughout the body, responding to a wide range of small molecules originating from the environment, our diets, host microbiomes, and internal metabolic processes. Increasing evidence highlights AhR's role as a critical regulator of numerous biological functions, such as cellular differentiation, immune response, metabolism, and even tumor formation. Typically located in the cytoplasm, AhR moves to the nucleus upon activation by an agonist where it partners with either the aryl hydrocarbon receptor nuclear translocator (ARNT) or hypoxia-inducible factor 1β (HIF-1β). This complex then interacts with xenobiotic response elements (XREs) to control the expression of key genes. AhR is notably present in various crucial immune cells, and recent research underscores its significant impact on both innate and adaptive immunity. This review delves into the latest insights on AhR's structure, activating ligands, and its multifaceted roles. We explore the sophisticated molecular pathways through which AhR influences immune and lymphoid cells, emphasizing its emerging importance in managing inflammatory diseases. Furthermore, we discuss the exciting potential of developing targeted therapies that modulate AhR activity, opening new avenues for medical intervention in immune-related conditions.

Gene expression and trace elements in Greenlandic ringed seals (Pusa hispida)

Marine top predators such as ringed seals biomagnify environmental contaminants; and with the increasing human activities in the Arctic, ringed seals are exposed to biologically significant concentrations of trace elements resulting in reproductive impairment, immunosuppression, and neurological damages. Little is known about the molecular effects of heavy metals on these vulnerable apex predators suffering from a rapidly changing Arctic with significant loss of sea-ice. In the present study, concentrations of cadmium (Cd), mercury (Hg) and selenium (Se) were measured in liver of sixteen Greenlandic ringed seals (nine adults and seven subadults) together with molecular biomarkers involved in bio-transformation, oxidative stress, endocrine disruption and immune activity in blood and blubber. The concentrations of trace elements increased in the following order: Hg > Se > Cd with levels of mercury and selenium being highest in adults. Aryl hydrocarbon receptor nuclear translocator (ARNT), peroxisome proliferator activated receptor alpha (PPARα, estrogen receptor alpha (ESR1), thyroid hormone receptor alpha (TRα) and interleukin – 2 (IL-2) mRNA transcript levels were highest in blubber, while heat shock protein 70 (HSP70) and interleukin – 10 (IL-10) were significantly higher in blood. There were no significant correlations between the concentrations of trace elements and mRNA transcript levels suggesting that stressors other than the trace elements investigated are responsible for the changes in gene expression levels. Since Hg seems to increase in Greenlandic ringed seals, there is a need to re-enforce health monitoring of this ringed seal population.

Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine β synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

Abstract 16879: Endothelial Prolyl Hydroxylase 3 Regulates Post-Ischemic Kidney Injury Through HIF Dependent Mechanisms

Introduction: Acute kidney injury (AKI) is highly prevalent clinical condition after cardiac surgeries and is associated with high morbidity and mortality in hospitalized patients. Ischemia reperfusion injury (IRI) is one of the major causes of AKI. Previously, we have reported a critical role for the endothelial oxygen sensor prolyl hydroxylase (PHD2) in ischemic AKI. Recently, we found that prolyl hydroxylase 3 (PHD3) shows robust increase in kidney endothelium after renal IRI. However, its role in ischemic AKI is unknown. Hypothesis: We hypothesized that endothelial PHD3 regulates post-ischemic kidney injury. Methods: To delete endothelial PHD3, VECadherin (Cdh5)-CreER transgenic mice were crossed to mice carrying conditional PHD3 allele, generating PHD3 iEC mice. To test the HIF-dependent role of PHD3, we generated Cdh5CreER; Phd3 flox/flox Arnt flox/flox mice ( PHD3ARNT iEC ). Inactivating ARNT (aryl hydrocarbon receptor nuclear translocator), also known as Hif-1β, blocks HIF signaling. Unilateral IRI was induced by renal artery clamping, and tamoxifen treatment was started at day 1 post IRI, followed by analysis at day 14. Results: Compared to Cre - controls, inactivation of Phd3 in endothelial cells (ECs) following IRI exacerbated kidney damage and inflammation, as indicated by increased expression of kidney injury molecule 1 ( Kim1 ) and profibrotic genes lysyl oxidase-like 2 ( Loxl2 ), transforming growth factor-beta 1 ( Tgfb1 ) , smooth muscle actin ( Acta2 ) and collagen deposition assessed by Picro Sirius red staining (n=6-7; p< 0.05). Post-ischemic inactivation of ARNT along with PHD3 ameliorated the effects of only PHD3 inactivation and normalized levels of Kim1 , Loxl2 , Tgfb1 and collagen deposition ( n=7; p< 0.05) . In vitro suppression of PHD3 in human primary EC using PHD3-siRNA showed significant upregulation of inflammatory molecules such as ICAM1 (Intercellular Adhesion Molecule 1), VCAM1 (Vascular Cell Adhesion Molecule 1), C-X-C motif chemokine ligand 9 and 10 ( CXCL9 and CXCL10 ), which were again blunted by ARNT- siRNA treatment (n= 3; p< 0.05). Conclusions: In summary, our data support a critical role for endothelial PHD3 in regulating post-ischemic kidney inflammation and fibrosis through HIF-dependent mechanisms.

Jasmone Is a Ligand-Selective Allosteric Antagonist of Aryl Hydrocarbon Receptor (AhR).

Herbal extracts represent a wide spectrum of biologically active ingredients with potential medical applications. By screening minor constituents of jasmine essential oil towards aryl hydrocarbon receptor (AhR) activity using a gene reporter assay (GRA), we found the antagonist effects of jasmone (3-methyl-2-[(2Z)-pent-2-en-1-yl]cyclopent-2-en-1-one). It inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-, benzo[a]pyrene (BaP)-, and 6-formylindolo[3,2-b]carbazole (FICZ)-triggered AhR-dependent luciferase activity in a concentration-dependent manner. However, the inhibition differed markedly between TCDD, BaP, and FICZ, with the latter being significantly less inhibited. The dose-response analysis confirmed an allosteric type of AhR antagonism. Furthermore, jasmone efficiently inhibited AhR activation by AhR agonists and microbial catabolites of tryptophan (MICTs). TCDD- and FICZ-inducible CYP1A1 expression in primary human hepatocytes was inhibited by jasmone, whereas in the human HepG2 and LS180 cells, jasmone antagonized only TCDD-activated AhR. Jasmone only partially displaced radiolabeled TCDD from its binding to mouse Ahr, suggesting it is not a typical orthosteric ligand of AhR. TCDD-elicited AhR nuclear translocation was not affected by jasmone, whereas downstream signaling events, including the formation of the AhR:ARNT complex and enrichment of the CYP1A1 promoter, were inhibited by jasmone. In conclusion, we show that jasmone is a potent allosteric antagonist of AhR. Such discovery may help to find and/or clarify the use of jasmone in pharmaco- and phytotherapy for conditions where AhR plays a key role.

Quercetin ameliorates ulcerative colitis by activating aryl hydrocarbon receptor to improve intestinal barrier integrity.

Ulcerative colitis (UC) pathogenesis is largely associated with intestinal epithelial barrier dysfunction. A therapeutic approach to UC involves the repair of damaged intestinal barrier. Our study aimed to investigate whether aryl hydrocarbon receptor (AhR) mediated the intestinal barrier repair effects of quercetin to ameliorate UC. 3% dextran sulfate sodium was used to induce colitic mice, and quercetin (25, 50, and 100 mg/kg) was administered orally for 10 days to assess the therapeutic effects. In vitro, Caco-2 cells were used to explore the effect of quercetin on tight junction protein expression and AhR activation. The results showed that quercetin alleviated colitic mice by restoring tight junctions (TJs) integrity via an AhR-dependent manner (p < 0.05). In vitro, quercetin dose-dependently elevated the expressions of TJs protein ZO-1 and Claudin1, and activated AhR by enhancing the expression of CYP1A1 and facilitating AhR nuclear translocation in Caco-2 cells (p < 0.05). While AhR antagonist CH223191 reversed the therapeutic effects of quercetin (p < 0.05) and blocked quercetin-induced AhR activation and enhancement of TJs protein (p < 0.05). In conclusion, quercetin repaired intestinal barrier dysfunction by activating AhR-mediated enhancement of TJs to alleviate UC. Our research offered new perspectives on how quercetin enhanced intestinal barrier function.

THU625 Med12 Mutation Activates Tryptophan-Kynurenine-ahr Pathway To Promote Growth Of Uterine Leiomyoma

Abstract Disclosure: A. Zuberi: None. Y. Huang: None. A. Dotts: None. H. Wei: None. J. Coon V: None. T. Lizuka: None. O. Wu: None. O. Sotos: None. D. Chakravarti: None. Y. Dai: None. S.E. Bulun: None. P. Yin: None. A.Z. and Y.H. contributed equally to this work.† Y.D., S.E.B., and P.Y. are co-senior authors. ‡To whom correspondence should be addressed: Serdar E. Bulun, M.D.Division of Reproductive Science in MedicineDepartment of Obstetrics and Gynecology Feinberg School of Medicine at Northwestern University 250 E. Superior Street, Suite 03-2306 Chicago, Illinois 60611-2914 Tel: 312-472-3636 Fax: 312-472-3740 Email: s-bulun@northwestern.edu Ping Yin, M.D., Ph.D. Division of Reproductive Science in Medicine Department of Obstetrics and Gynecology Feinberg School of Medicine at Northwestern University 303 E. Superior Street, Suite 10-111 Chicago, Illinois 60611 Tel: 312-503-1831 Fax: 312-503-0095 Email: p-yin@northwestern.edu. Conflict of interest: The authors have declared that no conflict of interest exists. Uterine leiomyomas (LM) cause heavy menstrual bleeding, anemia and pregnancy loss in approximately 10 million US women. Driver mutations in the mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% LM tumors that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 and a shift of binding sites from proximal promoters to intronic/intergenic regions in mutant (mut-MED12) vs wild-type LM tissues. Integration of cistrome and transcriptome data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene, which was massively upregulated in mut-MED12 LM. TDO2 catalyzes the conversion of tryptophan to kynurenine, which is an aryl hydrocarbon receptor (AHR) ligand and significantly elevated in mut-MED12 LM. Incubation of primary mut-MED12 LM cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis and induced AHR-target gene expression, whereas blocking the TDO2-kynurenine-AHR pathway by siRNA or pharmacologically abolished these effects. Progesterone receptor regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for LM growth. In summary, activation of the TDO2-kynurenine-AHR pathway selectively in mut-MED12 LM induces tumor growth and may inform the development of targeted treatments and precision medicine. Presentation: Thursday, June 15, 2023

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

2,2',4,4'-Tetrabromodiphenyl ether and cadmium co-exposure activates aryl hydrocarbon receptor pathway to induce ROS and GSDME-dependent pyroptosis in renal tubular epithelial cells.

We have previously found that a mixture exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and cadmium (Cd) causes kidney damage; however, the mechanism was not fully understood. The aryl hydrocarbon receptor (AhR) is a ligand-receptor transcription factor that plays an important role in the adaptive response or metabolic detoxification of environmental toxins. Thus, this study aimed to examine the role of AhR in kidney toxicity. BDE-47 (50 μM) or Cd (5 μM) exposure reduced cell viability in renal tubular epithelial cells (HKC), with a larger effect observed in co-treatment. The cell morphology presented pyroptotic changes, including swollen cells, large bubbles, and plasma membrane pore formation. The gene expressions of AhR, heat shock protein 90 (Hsp90), AhR nuclear translocator (ARNT), and cytochrome P450 1B1 (CYP1B1) were increased, while CYP1A1 was decreased. Reactive oxygen species (ROS) were generated, which was reduced by the AhR antagonist CH223191. The apoptosis, necrosis, and intracellular lactated hydrogenase (LDH) release was elevated, and this was attenuated by N-acetylcysteine (NAC). Furthermore, the pyroptosis pathway was activated with increased protein levels of cleaved-caspase-3 and gasdermin E N-terminal (GSDME-NT), while caspase-8, caspase-3, and GSDME were decreased. These effects were alleviated by NAC and CH223191. Our data demonstrate a combined effect of BDE-47 and Cd on nephrotoxicity by activating AhR to induce ROS contributing to GSDME-dependent pyroptosis, and retardation of the AhR pathway could reduce this toxicity.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes

ABSTRACT Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-β-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

Bio-based material-edible rosemary induced biodegradation of aflatoxin B1 via altering endogenous protective enzymes signatures in animal-derived foods

Aflatoxin B1 (AFB1) is the most hazardous mycotoxin, posing risks to public health. Utilization of bio-based materials to biodegrade AFB1 is a green strategy to overcome this issue. The investigation aimed to screen for endogenous protective enzymes in bio-based material-edible rosemary based on ultra-high performance liquid chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS)-proteomics and ascertain their impacts on the biodegradation and biotransformation of AFB1, and the trade-offs of multilevel metabolism of the animal-derived foods through untargeted metabolomics. The proteomics results verified that bio-based material-edible rosemary (0.20%, w/w) significantly up-regulated glutathione S-transferase and stimulated the down-regulation of cytochrome P450 1A2 levels via activating AhR nuclear translocation in rosemary-pickled AFB1-contaminated goat meat. Metabolomics results demonstrated that edible rosemary substantially increased histidine and glutathione implicated in the antioxidant status of goat meat. More importantly, edible rosemary with high endogenous protective enzyme content could efficiently biodegrade AFB1 in goat meat. We first unveiled that rosemary could not only efficiently biodegrade AFB1 up to 90.20% (20.00–1.96 μg kg−1) but also elevate the bio-ingestion quality of goat meat. These findings suggest that the bio-based material-rosemary is an efficient and environmentally friendly approach for biodegrading AFB1 and elevating the bio-ingestion composition of goat meat.

High-Altitude Andean H194R HIF2A Allele Is a Hypomorphic Allele.

For over 10,000 years, Andeans have resided at high altitude where the partial pressure of oxygen challenges human survival. Recent studies have provided evidence for positive selection acting in Andeans on the HIF2A (also known as EPAS1) locus, which encodes for a central transcription factor of the hypoxia-inducible factor pathway. However, the precise mechanism by which this allele might lead to altitude-adaptive phenotypes, if any, is unknown. By analyzing whole genome sequencing data from 46 high-coverage Peruvian Andean genomes, we confirm evidence for positive selection acting on HIF2A and a unique pattern of variation surrounding the Andean-specific single nucleotide variant (SNV), rs570553380, which encodes for an H194R amino acid substitution in HIF-2α. Genotyping the Andean-associated SNV rs570553380 in a group of 299 Peruvian Andeans from Cerro de Pasco, Peru (4,338 m), reveals a positive association with increased fraction of exhaled nitric oxide, a marker of nitric oxide biosynthesis. In vitro assays show that the H194R mutation impairs binding of HIF-2α to its heterodimeric partner, aryl hydrocarbon receptor nuclear translocator. A knockin mouse model bearing the H194R mutation in the Hif2a gene displays decreased levels of hypoxia-induced pulmonary Endothelin-1 transcripts and protection against hypoxia-induced pulmonary hypertension. We conclude the Andean H194R HIF2A allele is a hypomorphic (partial loss of function) allele.

Particulate matter-induced skin inflammation is suppressed by polyphenol-enriched dietary supplement via inhibition of the AhR/ARNT signaling pathway

Environmental factors that cause skin diseases cause an inflammatory response and cause various diseases. In this study, the ability of polyphenol-enriched dietary supplement (Zeropollution, ZP), a mixture of four types of natural extracts, to inhibit skin inflammation caused by particulate matter (PM) and ultraviolet B (UVB), was evaluated using an experimental mouse model (HR-1 or SKH-1 mice) and human keratinocytes (HaCaT cells). Treatment with ZP significantly inhibited the levels of pro-inflammatory markers, such as cyclooxygenase-2 (COX-2) and cytochrome P450 family 1 subfamily A1 (CYP1A1). In addition, the pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), were decreased by ZP pretreatment before exposure to PM10 or UVB. In particular, ZP significantly inhibited the expression of the aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT), which initiate inflammatory responses by sensing PM10 and UVB. Therefore, these results suggest that ZP has the potential to be used as a material for the development of a naturally derived functional compound that can prevent skin aging triggered by PM10 and UVB.

#6635 ARNT AND ALK3 MEDIATE RENO PROTECTION AFTER ISCHEMIC PRECONDITIONING

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

Discovery of a highly potent NPAS3 heterodimer inhibitor by covalently modifying ARNT

Neuronal PAS domain protein 3 (NPAS3), a basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) family member, is a pivotal transcription factor in neuronal regeneration, development, and related diseases, regulating the expression of downstream genes. Despite several modulators of certain bHLH-PAS family proteins being identified, the NPAS3-targeted compound has yet to be reported. Herein, we discovered a hit compound BI-78D3 that directly blocks the NPAS3-ARNT heterodimer formation by covalently binding to the aryl hydrocarbon receptor nuclear translocator (ARNT) subunit. Further optimization based on the hit scaffold yielded a highly potent Compound 6 with a biochemical EC50 value of 282 ± 61 nM and uncovered the 5-nitrothiazole-2-sulfydryl as a cysteine-targeting covalent warhead. Compound 6 effectively down-regulated NPAS3's transcriptional function by disrupting the interface of NPAS3-ARNT complexes at cellular level. In conclusion, our study identifies the 5-nitrothiazole-2-sulfydryl as a cysteine-modified warhead and provides a strategy that blocks the NPAS3-ARNT heterodimerization by covalently conjugating ARNT Cys336 residue. Compound 6 may serve as a promising chemical probe for exploring NPAS3-related physiological functions.

Molecular characterization of the unusual peptide CORO1C-47aa encoded by the circular RNA and docking simulations with its binding partner

Circular RNAs, which have covalently closed ends, are in the class of non-coding RNAs. Recent studies reveal that they are associated with various biochemical pathways. One such involvement of circular RNAs is in the onset of different types of cancers. Though the circular RNAs are known as non-coding RNAs, some of them are found to possess the capacities to code for proteins. One such circular RNA is hsa-circ-0000437 which is known to code for a short peptide referred to as CORO1C-47aa. The peptide has anti-angiogenic activity and is associated with the prevention of endometrial cancer. The peptide binds to the PAS-B domain of the Aryl hydrocarbon Receptor Nuclear Translocator (ARNT). However, till date only the amino acid sequence of the peptide is known and no structural details of the peptide are available. Therefore, in this work, our aim was to predict how the peptide would fold and what could be its possible ligand binding sites. We used computational tools to determine the structure of the peptide refined further by molecular dynamics simulations. We then performed molecular docking simulations of the peptide with its known binding partner ARNT to gain an insight into the modes of binding as the process is associated with endometrial cancer. The possible ligand binding sites along-with the natures of the possible other different ligands of the peptide were analyzed further. From this structure function analysis study, we tried to elucidate the plausible mechanism of the involvements of the peptide in the onset of endometrial cancer. This is the first report on the structural characterization of the peptide and its modes of interactions with the partner protein ARNT. This study may therefore be useful in determining the structures of new drug candidates for the treatment of endometrial cancer.

AhR and HIF-1α Signaling Pathways in Benign Meningioma under Hypoxia.

The role of hypoxia in benign meningiomas is less clear than that in the malignant meningiomas. Hypoxia-induced transcription factor 1 subunit alpha (HIF-1α) and its downstream signaling pathways play a central role in the mechanism of hypoxia. HIF-1α forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and can compete for ARNT with aryl hydrocarbon receptor (AhR). In this work, the status of HIF-1α- and AhR-dependent signaling pathways was investigated in World Health Organization (WHO) grade 1 meningioma and patient-derived tumor primary cell culture under hypoxic conditions. mRNA levels of HIF-1α, AhR, and of their target genes as well as of ARNT and nuclear receptor coactivator NCOA2 were determined in tumor tissues from patients in whom the tumor was promptly removed either with or without prior endovascular embolization. Using the patient-derived nonembolized tumor primary cell culture, the effects of a hypoxia mimetic cobalt chloride (CoCl2) and an activator of the AhR signaling pathway benzo(α)pyrene (B[a]P) on mRNA levels of HIF-1α, AhR, and their target genes were investigated. Our findings show active functioning of AhR signaling in meningioma tissue of patients with tumor embolization and crosstalk between HIF-1α and AhR signaling in meningeal cells under hypoxia.

Aryl-hydrocarbon receptor agonist: A novel topical therapeutic approach for inflammatory skin diseases.

The broadening of novel therapeutic options for chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis (PSO), known to be associated with dysregulation of the immune response, systemic comorbidities and a reduced quality of life for patients and caretakers, offers better chances for long-lasting disease control.1, 2 Recent advances in clarifying the mechanisms that lead to the immune imbalance in both diseases indicate a predominant IL-23/IL-17A/IL36 pathway in psoriasis, and a predominant Th2 IL-4/IL-13, with the participation of IL-17 and IL-22 axis in atopic dermatitis.1, 2 The development of targeted-oriented therapies such as biologics and small molecule inhibitors (SMIs) changed the traditional management of such inflammatory conditions, therefore optimizing the standard of care in both diseases.1, 2 One of the new foci of targeted therapies for inflammatory skin diseases is the aryl-hydrocarbon receptor (AhR)/AhR-nuclear translocator (ARNT) system, which works as a sensor for exogenous and endogenous molecules. It is expressed in the skin and has a relevant role in the skin barrier maintenance, upregulating filaggrin and loricrin and enhancing epidermal differentiation. Furthermore, the AhR complex modulates the T-helper 17/22 and T regulatory axis, and some AHR ligands also exert a role in the neutralization of oxidative stress through the activation of the nuclear factor-erythroid 2-related factor-2 (NRF2) transcription factor and a ROS-scavenging structure.3 Bissonnette et al.4 present a review article on tapinarof (3,5-dihydroxy-4-isopropylstilbene), an AhR agonist from a historical perspective. His group has been working with this component since 2007, and the initial studies focused on psoriasis.5 Tapinarof 1% cream is a topical medication applied once daily, and the review shows the main outcomes of its use in clinical trials in patients with psoriasis and AD. The key point of this review indicates the profile of tapinarof in psoriasis and AD, as follows4: Psoriasis: A phase 2, single-centre clinical trial, showed a rapid start of action in topically applied bid tapinarof 1% group than vehicle. There was a 67.5% of improvement in PGA (physician global assessment) score to clear or almost clear against 4.8% in vehicle group at week 12 in psoriasis patients. In phase 2b (multicentre trial), tapinarof 0.5% and 1%, once or twice a day compared to vehicle was tested and reached the primary endpoints (PGA0/1) with 1%. The relevant finding of phase 2b was the sustained improvement during the 4-week follow-up. Subsequentially, the studies with tapinarof 1% once a day versus placebo including 1366 psoriasis patients, demonstrated an improvement of 35.4% and 40.2% for tapinarof group, versus 6% and 6.3% for vehicle in PGA score, and a PASI (Psoriasis Area Index Severity) 75 achievement in 36.1% in tapinarof group versus 10.2% for vehicle (at week 12). In the 1-year open-label extension study, the mean duration of effect remission (PGA0/1) off-therapy was 130.1 days. Atopic dermatitis: In one single-centre study, 34 AD patients randomized (1:1:1) to tapinarof 0.5%, 1% or vehicle were treated bid for 4 weeks, showing that 50% of AD patients in both treatment groups achieved an IGA of clear or almost clear compared to 8.3% for the vehicle group (at week 5). These findings were confirmed in a phase 2 study with AD patients. A randomized phase 2b study with 247 AD patients under bid or once daily application of tapinarof 0.5%, 1% or vehicle (12 weeks), assessing safety and efficacy in adolescents 12 years and older, indicated that at week 12, 53% of the AD patients with tapinarof 1% BID and 46% with once daily application achieved the primary endpoint (IGA of clear/almost clear with a 2-point decrease proportion) versus 24% and 28% for vehicle respectively. Tapinarof is currently undergoing phase 3 trials for AD patients in >2 years of age. The present study with topical tapinarof 1% cream in both psoriasis and AD opens unlimited field for more research and expansion of its use. One advantage for such medication is the long-lasting clearing of the skin lesions despite discontinuation, which could be very beneficial to patients with chronic conditions, impacting on the need for prolonged systemic therapies and therefore impacting on the patients' safety regarding adverse events and alleviating the economic impact of the disease. There are still more points to be addressed that may be clarified after more studies are conducted, such as whether the response to treatment will be effective and homogeneous depending on: stages of the disease (acute × chronic), age and ethnic/racial immune phenotypes (folliculitis5 is a clinical finding that may be more present in patients with AD of African descent, and was one of the reported initial adverse events of tapinarof). None declared. VA has participated in clinical trials for Lilli and Sanofi and as a consultant for Abbvie, Galderma and Leopharma. RLO has participated in clinical trials for Lilli, Sanofi and Amgen and as consultant for Bayer and Abbvie. Data sharing not applicable.

Discovery of Norisoboldine Analogue III11 as a Novel and Potent Aryl Hydrocarbon Receptor Agonist for the Treatment of Ulcerative Colitis.

The aryl hydrocarbon receptor (AhR) is a transcript factor, belonging to the basic helix-loop-helix-Per-ARNT-SIM family, is closely associated with health and diseases. Targeting AhR is an emerging therapeutic strategy for various diseases. Norisoboldine (NOR), which is the main alkaloid of Linderae Radix, has been known to activate AhR. Unfortunately, the oral bioavailability (F) of NOR is only 2.49%. To improve the chemical efficacy and bioavailability, we designed and synthesized NOR analogues. Using various in vitro assays, 2-methoxy-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-9-ol (III11) was discovered as a potent AhR agonist. Compound III11 enhanced the expression of AhR downstream target genes, triggered AhR nuclear translocation, and promoted differentiation of regulatory T cells. More importantly, III11 exhibited good bioavailability (F = 87.40%) and remarkable therapeutic effects in a mouse model of ulcerative colitis at a dosage of 10 mg/kg. These findings may serve as a reference for the design of novel AhR agonists against immune and inflammatory diseases.

Genome-Wide Analysis of Hypoxia-Inducible Factor Binding Reveals Targets Implicated in Impaired Human Placental Syncytiotrophoblast Formation under Low Oxygen

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 O2 environment. Low O2 inhibits differentiation and fusion of CTBs into STB and may thus contribute to PE pathogenesis; however, the underlying mechanisms are unknown. Because low O2 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 O2 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 O2 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 O2 supply.