Aryl Hydrocarbon Receptor Knockdown Research Articles

A Tryptophan-derived Uremic Metabolite-Ahr-Pdk4 Axis Promotes Skeletal Muscle Mitochondriopathy in Chronic Kidney Disease

Introduction: Chronic kidney disease (CKD) and the subsequent accumulation of tryptophan-derived uremic metabolites (UMs) in circulation negatively impact skeletal muscle (SkM) health and function. Specifically, indoles and kynurenines are strongly associated with SkM atrophy, weakness, and fatigue found in CKD patients. Notably, these tryptophan-derived UMs are known agonist to the ubiquitously expressed aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, which has been shown to be activated in the blood of CKD patients and whose chronic activation has been proven toxic to multiple tissue types. Purpose: The purpose of this study was to determine whether chronic activation of the AHR by tryptophan-derived UMs contributes to CKD associated SkM atrophy and metabolic dysfunction. Methods: Muscle biopsies from CKD patients and adult controls with normal renal function were used to examine AHR signaling and mitochondrial function. The mechanistic role of the AHR was explored using SkM-specific AHR knockout mice (AHRmKO) and SkM-specific AHR knockdown via adeno-associated virus in mice with adenine-induced CKD, as well as ectopic expression of a constitutively active AHR (CAAHR) in mice with normal kidney function. Additional mechanistic experiments were carried out in cultured muscle cells. Outcome measures included gene and protein expression, mitochondrial bioenergetic testing, and robust muscle functional phenotyping. Results: Compared to controls with normal kidney function, AHR-dependent gene expression ( CYP1A1 and CYP1B1) was significantly upregulated in gastrocnemius SkM of CKD patients ( P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration ( P<0.001). In mice with CKD, SkM mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the level of tryptophan-derived UMs and AHR activation. AHRmKO significantly improved mitochondrial OXPHOS in mice with CKD (~28% increase, P=0.045) and abolished the relationship between UMs and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in SkM was further confirmed using SkM-specific AHR knockdown in C57BL6J that express a high-affnity AHR allele, as well as ectopic viral expression of CAAHR in mice with normal renal function. AHR activation led to impairments in pyruvate dehydrogenase (PDH) enzyme function (~29% decrease, P<0.05), significant increases in Pdk4 expression ( P<0.05) and phosphorylation of PDH enzyme ( P<0.05), mechanistically contributing to impaired pyruvate-supported OXPHOS function. Conclusion: These findings establish a uremic metabolite-AHR-Pdk4 axis in SkM that governs mitochondrial deficits in CKD. Supported by grants from the National Institutes of Health R01-HL149704 (TER) and F31-DK128920 (TT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A tryptophan-derived uremic metabolite/Ahr/Pdk4 axis governs skeletal muscle mitochondrial energetics in chronic kidney disease.

Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.

Abstract 5837: Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth

Abstract Therapy resistance is a ubiquitous and major challenge for curing cancer including prostate cancer. As the most prevalent non-skin cancer for men, prostate cancer is responsible for more than 30,000 deaths annually in the U.S. Despite a favorable prognosis at early stages, prostate cancer progressively develop into an incurable castration-resistant stage (CRPC) where prostate cancer grows in a low-androgen environment. Enzalutamide (ENZ) is the first drug approved by FDA for treating CRPC by inhibiting the residual androgen signaling. While ENZ significantly improves overall survival and quality of life, resistance inevitably develops and greatly limits its benefit. To understand ENZ resistance and identify novel therapeutic targets with the hope of overcoming resistance, we performed RNA-seq to analyze mRNA expression profiles from three ENZ-resistant human CRPC cell lines (C4-2BENZR, CWR-R1ENZR and VCaPENZR) and identified the aryl hydrocarbon receptor (AHR) among the 114 commonly upregulated genes. AHR is a ligand-dependent transcription factor that has been shown to drive disease progression in multiple types of cancer. Stable shRNA-mediated knockdown of AHR substantially decreased growth of ENZ-resistant CRPC cells. Our pilot study also showed that AHR knockdown resulted in fewer and smaller CWR-R1ENZR tumors in a xenograft mouse model. To elucidate the underlying mechanism of AHR-mediated cancer growth, gene set enrichment analysis (GSEA) revealed a declining trend of enrichment of the 114 commonly ENZ-upregulated genes among differentially altered genes upon AHR knockdown in ENZ-resistant cells, suggesting that AHR may act as a master regulator of ENZ resistance. Moreover, we found that the Hallmark androgen response gene set is downregulated in AHR-silenced ENZ-resistant cells, implying a role of AHR in sustaining androgen response for ENZ-resistance. Consistently, we performed AHR ChIP-seq and confirmed that AHR bound to promoter regions of several androgen-regulated genes in CWR-R1ENZR cells. In addition, three peroxisome-related Hallmark gene sets are also negatively enriched in AHR-knockdown cells, which provides a rationale for further exploring a potential AHR-dependent connection of peroxisomal function to ferroptosis as a newly emerging mechanism of action for ENZ. In summary, our study suggests that AHR plays a critical role in maintaining ENZ-resistant CRPC growth with the likely involvement of modulating androgen signaling and ferroptosis susceptibility. Citation Format: Chia-Hui Chen, Boyang Jason Wu. Aryl hydrocarbon receptor is critical for enzalutamide-resistant prostate cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5837.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1–37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Non-Toxicological Role of Aryl Hydrocarbon Receptor in Obesity-Associated Multiple Myeloma Cell Growth and Survival.

Obesity is not only a risk factor for multiple myeloma (MM) incidence, but it is also associated with an increased risk of progression from myeloma precursors-monoclonal gammopathy of undetermined significance-and smoldering myeloma. Adipocytes in the bone marrow (BMAs) microenvironment have been shown to facilitate MM cell growth via secreted factors, but the nature of these secreted factors and their mechanism of action have not been fully elucidated. The elevated expression of aryl hydrocarbon receptor (AhR) is associated with a variety of different cancers, including MM; however, the role of AhR activity in obesity-associated MM cell growth and survival has not been explored. Indeed, this is of particular interest as it has been recently shown that bone marrow adipocytes are a source of endogenous AhR ligands. Using multiple in vitro models of tumor-adipocyte crosstalk to mimic the bone microenvironment, we identified a novel, non-toxicological role of the adipocyte-secreted factors in the suppression of AhR activity in MM cells. A panel of six MM cell lines were cultured in the presence of bone marrow adipocytes in (1) a direct co-culture, (2) a transwell co-culture, or (3) an adipocyte-conditioned media to interrogate the effects of the secreted factors on MM cell AhR activity. Nuclear localization and the transcriptional activity of the AhR, as measured by CYP1A1 and CYP1B1 gene induction, were suppressed by exposure to BMA-derived factors. Additionally, decreased AhR target gene expression was associated with worse clinical outcomes. The knockdown of AhR resulted in reduced CYP1B1 expression and increased cellular growth. This tumor-suppressing role of CYP1A1 and CYP1B1 was supported by patient data which demonstrated an association between reduced target gene expression and worse overall survival. These data demonstrated a novel mechanism by which bone marrow adipocytes promote MM progression.

IFNγ-mediated IL-33 production is dependent on the aryl hydrocarbon receptor in human bronchial epithelial cells

IL-33 is an alarmin produced by stromal cells and is known to promote airway inflammation. IL-33 is a critical mediator of steroid-unresponsiveness in severe asthma. We have previously shown that IFNγ, a cytokine known to be elevated in airway inflammation and severe asthma, enhances the abundance of IL-33 in bronchial epithelial cells. Previous studies have shown that environmental insults such as particulate matter results in activation of the aryl hydrocarbon receptor (AhR) and IL-33 production. However, the role of AhR in cytokine-mediated IL-33 production is unknown. In this study, we demonstrate that the knockdown of AhR results in significant decrease in IFNγ-mediated IL-33 production and phosphorylation of STAT1 (Y701), in human bronchial epithelial cells. The findings of this report suggest that AhR may be an essential component in IFNγ-mediated IL-33 production in the lungs.

Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells.

Bone marrow mesenchymal stromal cells (BMSCs) are the commonly used seed cells in tissue engineering. Aryl hydrocarbon receptor (AhR) is a transcription factor involved in various cellular processes. However, the function of constitutive AhR in BMSCs remains unclear. To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs (mBMSCs) and the underlying mechanism. Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs. The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid. The osteogenic potential was observed by alkaline phosphatase and alizarin red staining. The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction (qPCR) and western blot. After coculture with different mBMSCs, the cluster of differentiation (CD) 86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry. To explore the underlying molecular mechanism, the interaction of AhR with signal transducer and activator of transcription 3 (STAT3) was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot. AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected. AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it. The ratio of CD86+ RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+ cells was increased. AhR directly interacted with STAT3. AhR overexpression increased the phosphorylation of STAT3. After inhibition of STAT3 via stattic, the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted. AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.

Inhibition of TLR4 Signaling by Isorhapontigenin Targeting of the AHR Alleviates Cerebral Ischemia/Reperfusion Injury.

Ischemic stroke is a major risk factor in human health, yet there are no drugs to cure cerebral ischemia/reperfusion injury (CIRI). Inflammation plays a fundamental role in the consequences of CIRI. Isorhapontigenin (ISOR) exhibits great anti-inflammatory activity; however, it is unclear whether ISOR can treat ischemic stroke through an anti-inflammation effect. Here, middle cerebral artery occlusion/reperfusion (MCAO/R) was used to investigate the effects of ISOR on CIRI. The in vitro activity was measured in BV-2 cells exposed to oxygen-glucose deprivation/reperfusion. As measured by neurological scores, brain water content, and infarction, neurological dysfunction was improved in the ISOR group. The neuronal death and microglial activation in the ipsilateral cortex were reduced by ISOR. TLR4 signaling was significantly inhibited by ISOR in vivo and in vitro. By reverse molecular docking, cellular thermal shift, and drug affinity-responsive target stability assays, an aryl hydrocarbon receptor (AHR) was found to be a target of ISOR. Furthermore, AHR knockdown blocked the effect of ISOR on TLR4 signaling, suggesting that ISOR may regulate TLR4-mediated inflammation through AHR, thereby protecting neurons from CIRI. This study demonstrated that ISOR is a promising drug candidate for the treatment of ischemic stroke and provided a theoretical basis for the development of the medicinal value of ISOR-derived foods, such as grapes.

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Anti-hormone therapies are not efficacious for reducing the incidence of triple negative breast cancer (TNBC), which lacks both estrogen and progesterone receptors. While the etiology of this aggressive breast cancer subtype is unclear, visceral obesity is a strong risk factor for both pre- and post-menopausal cases. The mechanism by which excessive deposition of visceral adipose tissue (VAT) promotes the malignant transformation of hormone receptor-negative mammary epithelial cells is currently unknown. We developed a novel in vitro system of malignant transformation in which non-tumorigenic human breast epithelial cells (MCF-10A) grow in soft agar when cultured with factors released from VAT. These cells, which acquire the capacity for 3D growth, show elevated aryl hydrocarbon receptor (AhR) protein and AhR target genes, suggesting that AhR activity may drive malignant transformation by VAT. AhR is a ligand-dependent transcription factor that generates biological responses to exogenous carcinogens and to the endogenous tryptophan pathway metabolite, kynurenine. The serum kynurenine to tryptophan ratio has been shown to be elevated in patients with obesity. Herein, we demonstrate that AhR inhibitors or knockdown of AhR in MCF-10A cells prevents VAT-induced malignant transformation. Specifically, VAT-induced transformation is inhibited by Kyn-101, an inhibitor for the endogenous ligand binding site of AhR. Mass spectrometry analysis demonstrates that adipocytes metabolize tryptophan and release kynurenine, which is taken up by MCF-10A cells and activates the AhR to induce CYP1B1 and promote malignant transformation. This novel hormone receptor-independent mechanism of malignant transformation suggests targeting AhR for TNBC prevention in the context of visceral adiposity.

#3048 INDOXYL SULFATE INHIBITS OSTEOGENESIS OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH AHR/HES1/BMP2 PATHWAY

Abstract Background and Aims There is a high global burden of chronic kidney disease (CKD). The increased risk of bone fracture in patients with CKD suggests that CKD-associated factors, such as uremic toxins, might be the cause. Recent studies demonstrated that low turnover osteodystrophy and bone formation defect develop in early CKD, which indicates that uremic toxins, particularly in relatively low concentrations, can potentially be a cause. Thus far, studies about defected osteogenesis adopted high concentrations of uremic toxins (for example, indoxyl sulfate (ISx) >100 μM). Furthermore, the pathology is not yet fully investigated. Uremic toxin induced aryl hydrocarbon receptor (AHR) has been linked to bone formation defect, however, the mechanisms are incompletely understood. AHR inhibit osteogenesis during early processes of differentiation of early cells (stem cells), but whether uremic toxins induced AHR to affect stem cell in osteogenesis lineage has yet to be studied. The present study aims to clarify whether ISx at concentrations approximate to average in CKD patients (20 μM in free form) affect osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), and further survey the underline signal transduction of AHR-induced defected osteogenesis. Method Mineralization of D1 cells (mouse BMSCs) was evaluated by alizarin red S staining (ARS). The mRNA expressions and protein levels of osteogenic markers including Runx2, BMP2, ALP, and osteocalcin were determined by quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. AHR translocation into the nucleus was evaluated by an immunofluorescence confocal microscope. Reporter gene assays were adopted to study the transcriptional regulation of Runx2 and BMP2. Results ISx at concentrations ≤50 μM significantly downregulated mineralization of BMSCs with the most influence in the early stage of osteogenesis. ISx decreased the mRNA expressions and protein levels of osteogenic markers Runx2, BMP2, ALP, and osteocalcin. ISx of 50 μM induced AHR translocation into the nucleus and upregulated AHR target genes CYP1a1 and CYP1b1. The ISx-induced mineralization defects of BMSCs were counteracted by AHR antagonist CH223191, while downregulated gene expressions of osteogenic markers were recovered by AHR antagonist and knockdown (KD) of AHR. In mechanistic studies, ISx upregulated mRNA levels of hairy and enhancer of split (HES1), which is a primary target of NOTCH signaling. Although there were no significant differences in expressions of Notch1∼4, the upregulated Hes1 was attenuated by the antagonist and KD of AHR, which indicated that Hes1 was directly regulated by AHR. We identified putative binding sites of transcription repressor Hes1 in the promoter area of BMP2 and Runx2. Following experimental treatment of ISx, expression levels of BMP2 and Runx2 monitored by reporter assays were downregulated, which indicated that Hes1 plays as a transcription repressor to downregulated BMP2 and Runx2. Conclusion ISx under clinically relevant concentrations attenuated mineralization of BMSCs, particularly in the early stage of osteogenesis. ISx-induced AHR upregulated Hes1, which inhibited expressions of not only Runx2, but also BMP2, which has not been published before. A study of the underline signal transduction pathways relevant to the AHR genomic control system may provide potential therapy for low bone formation in early CKD.

Astragaloside IV attenuates indoxyl sulfate-induced injury of renal tubular epithelial cells by inhibiting the aryl hydrocarbon receptor pathway.

Astragalus membranaceus Fisch. ex Bunge has long been used to treat chronic kidney disease (CKD) in China. However, the mechanism of action requires further study. Indoxyl sulfate accumulation is the key cause of CKD progression. The aryl hydrocarbon receptor (AhR) plays an essential role in the renal tubular injury induced by indoxyl sulfate (IS). We explored the effects of Astragaloside IV (AS-IV), a minor component of the flowering perennial Astragalus membranaceus Fisch. ex Bunge, on AhR activity during IS-induced injury of renal tubular epithelial cells. C57BL/6 mice fed a 0.2% adenine diet (adenine+IS) and intraperitoneally injected with IS were used to study the protective effects of AS-IV, and specifically the effect on the AhR. In addition, apoptosis (annexin/PI), oxidative stress and the AhR pathway were investigated in IS-stimulated HK-2cells treated with AS-IV. The binding of AS-IV to the AhR was assessed in a molecular docking analysis. AhR knockdown using AhR siRNA allowed determination of the effects of AS-IV in IS-stimulated HK-2cells. AS-IV inhibited tubulointerstitial injury in adenine+IS mice. While AS-IV did not reduce serum IS levels, it did inhibit AhR expression in the kidney. In IS-stimulated HK-2cells, AS-IV also dramatically reduced apoptosis, decreased oxidative stress responses and inhibited the expression of the AhR pathway. The molecular docking analysis showed surface binding of AS-IV to the AhR. Following AhR knockdown in HK-2cells, IS-induced apoptosis was reduced and could not be further reduced by AS-IV. By targeting the AhR, AS-IV may alleviate IS-induced renal tubular injury, thus offering a novel therapeutic approach to the treatment of chronic renal failure.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Apigenin improves cytotoxicity of antiretroviral drugs against HTLV-1 infected cells through the modulation of AhR signaling.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory autoimmune disease characterized by high levels of infected immortalized T cells in circulation, which makes it difficult for antiretroviral (ART) drugs to work effectively. In previous studies, we established that Apigenin, a flavonoid, can exert immunomodulatory effects to reduce neuroinflammation. Flavonoids are natural ligands for the aryl hydrocarbon receptor (AhR), which is a ligand activated endogenous receptor involved in the xenobiotic response. Consequently, we tested Apigenin's synergy in combination with ART against the survival of HTLV-1-infected cells. First, we established a direct protein-protein interaction between Apigenin and AhR. We then demonstrated that Apigenin and its derivative VY-3-68 enter activated T cells, drive nuclear shuttling of AhR, and modulate its signaling both at RNA and protein level. In HTLV-1 producing cells with high AhR expression, Apigenin cooperates with ARTs such as Lopinavir (LPN) and Zidovudine (AZT), to impart cytotoxicity by exhibiting a major shift in IC50 that was reversed upon AhR knockdown. Mechanistically, Apigenin treatment led to an overall downregulation of NF-κB and several other pro-cancer genes involved in survival. This study suggest the potential combinatorial use of Apigenin with current first-line antiretrovirals for the benefit of patients affected by HTLV-1 associated pathologies.

Indole-3-propionic acid alleviates chondrocytes inflammation and osteoarthritis via the AhR/NF-κB axis

BackgroundOsteoarthritis (OA) is a common chronic disease characterized by chronic inflammation and extracellular matrix degradation. Indole-3-propionic acid (IPA) is a tryptophan metabolite secreted by intestinal flora, which can exert anti-inflammatory effects in a variety of diseases. In this study, we further investigated the potential therapeutic role of IPA in OA and the underlying mechanism.MethodsIL-1β was utilized to induce chondrocyte inflammation. Then, the cytotoxicity of IPA on rat chondrocytes was assessed. Meanwhile, RT-qPCR, Griess reaction, ELISA, Western blot and immunofluorescence were performed to evaluate the expression of inflammatory factors and stromal proteins, and the NF-κB pathway in chondrocytes treated with IL-1β alone, with IPA or with aryl hydrocarbon receptor (AhR) knockdown. An OA rat model was established by anterior cruciate ligament transection, and hematoxylin-eosin staining, Safranin-O/Fast Green staining and immunochemistry were applied to estimate OA severity.ResultsIPA did not affect cellular viability at concentrations up to 80 µM. IPA significantly inhibited the IL-1β-induced expression of inflammatory factors (Nitric oxide, PGE2, TNF-α, IL-6, iNOS and COX-2) and matrix-degrading enzymes (MMP-3, MMP-13 and ADAMTS-5), upregulated the expression of anabolic markers (aggrecan and collagen-II) and inactivated the NF-κB pathway. However, AhR knockdown could abolish the above protection capabilities and the suppression of the NF-κB pathway induced by IPA. Furthermore, IPA significantly reduced serum inflammatory cytokines expression, cartilage destruction and synovitis in vivo, demonstrating its protective role in OA progression.ConclusionIPA improved IL-1β-induced chondrocyte inflammation and extracellular matrix degradation through the AhR/NF-κB axis, which provides an innovative therapeutic strategy for OA.

Benvitimod inhibits MCM6-meditated proliferation of keratinocytes by regulating the JAK/STAT3 pathway

BackgroundBenvitimod (Tapinarof), as a small-molecule topical therapeutical aryl hydrocarbon receptor (AHR)-modulating agent, is in clinical development for treating psoriasis and atopic dermatitis. Benvitimod reduces proinflammatory cytokines in psoriasis by specifically binding and activation of AHR. However, whether benvitimod can inhibit keratinocyte proliferation remains unclear. Minichromosome maintenance protein 6 (MCM6) is a key element of the prereplication complex (pre-RC) assembly which is one of the essential steps in the initiation of DNA replication for cell proliferation. ObjectivesThis study aimed to determine whether benvitimod could reduce the excessive proliferation of psoriatic keratinocytes by inhibiting MCM6. MethodsWe examined the inhibitory effect of benvitimod on MCM6-mediated proliferation of keratinocytes by HaCaT cells in vitro and an IMQ-induced psoriatic model of mice in vivo. ResultsEpidermal MCM6 expression was enhanced in the skin lesions of psoriatic patients. The experiments further revealed that MCM6 was required for the proliferation of keratinocytes and governed by the IL-22/STAT3 pathway. In addition, the antiproliferation effect of benvitimod is achieved by the inhibition of p-JAK1 and p-JAK2, which further restrained the activation of STAT3 in keratinocytes. Lastly, benvitimod could repressed imiquimod-induced skin lesions and the expression of epidermal MCM6 and p-STAT3 in mice. Moreover, knockdown of AHR in keratinocytes enhanced the activation of JAK1 and JAK2. ConclusionThe findings reveal that benvitimod could decrease MCM6-mediated proliferation of keratinocytes by affecting the JAK/STAT3 pathway, thereby serving as a new treatment modality for psoriasis.

Cannabidiol mediates epidermal terminal differentiation and redox homeostasis through aryl hydrocarbon receptor (AhR)-dependent signaling

BackgroundCannabidiol, a non-psychoactive phytocannabinoid, has antioxidant and anti-inflammatory activity in keratinocytes. However, the signaling pathway through which cannabidiol exerts its effect on keratinocytes or whether it can modulate keratinocyte differentiation has not been fully elucidated yet. ObjectiveWe investigated whether cannabidiol modulates epidermal differentiation and scavenges reactive oxygen species through the aryl hydrocarbon receptor (AhR) in keratinocytes and epidermal equivalents. MethodsWe investigated the cannabidiol-induced activation of AhR using AhR luciferase reporter assay, qRT-PCR, western blot, and immunofluorescence assays. We also analyzed whether keratinocyte differentiation and antioxidant activity are regulated by cannabidiol-induced AhR activation. ResultsIn both keratinocytes and epidermal equivalents, cannabidiol increased both the mRNA and protein expression of filaggrin, involucrin, NRF2, and NQO1 and the mRNA expression of the AhR target genes, including CYP1A1 and aryl hydrocarbon receptor repressor. Additionally, cannabidiol showed antioxidant activity that was attenuated by AhR knockdown or co-administration with an AhR antagonist. Moreover, cannabidiol increased the ratio of OVOL1/OVOL2 mRNA expression, which is a downstream regulator of AhR that mediates epidermal differentiation. In addition to increased expression of barrier-related proteins, cannabidiol-treated epidermal equivalent showed a more prominent granular layer than the control epidermis. The increased granular layer by cannabidiol was suppressed by the AhR antagonist. ConclusionCannabidiol can be a modulator of the AhR-OVOL1-filaggrin axis and AhR-NRF2-NQO1 signaling, thus indicating a potential use of cannabidiol in skin barrier enhancement and reducing oxidative stress.

AhR Promotes the Development of Non-small cell lung cancer by Inducing SLC7A11-dependent Antioxidant Function.

Objective: Aryl hydrocarbon receptor (AhR) is a transcription factor. It is reported that AhR is associated with non-small cell lung cancer (NSCLC), but the mechanisms underlying this relationship remain unclear. Therefore, we investigated the role of AhR in NSCLC to elucidate the underlying mechanisms. Methods: We collected clinical lung cancer samples and constructed AhR overexpression and knockdown cell lines to investigate the tumorigenicity of AhR in vivo and in vitro. Furthermore, we performed a ferroptosis induction experiment and chromatin immunoprecipitation experiment. Results: AhR was highly expressed in NSCLC tissue. AhR knockdown cells showed ferroptosis related phenomenon. Furthermore, Chromatin immunoprecipitation confirmed the correlation between AhR and solute carrier family 7 member 11 (SLC7A11) and ferroptosis induction experiment confirmed that AhR affects ferroptosis via SLC7A11. Specifically, AhR regulates ferroptosis-related SLC7A11, which affects ferroptosis and promotes NSCLC progression. Conclusions: AhR promoted NSCLC development and positively correlated with SLC7A11, affecting its actions. AhR bound to the promoter region of SLC7A11 promotes NSCLC by activating SLC7A11 expression, improving the oxidative sensitivity of cells, and inhibiting ferroptosis. Thus, AhR affects ferroptosis in NSCLC by regulating SLC7A11, providing foundational evidence for novel ferroptosis-related treatments.

Possible Involvement of the Upregulation of ΔNp63 Expression Mediated by HER2-Activated Aryl Hydrocarbon Receptor in Mammosphere Maintenance.

Cancer stem cells (CSCs) contribute to the drug resistance, recurrence, and metastasis of breast cancers. Recently, we demonstrated that HER2 overexpression increases mammosphere formation via the activation of aryl hydrocarbon receptor (AHR). In this study, the objective was to identify the mechanism underlying mammosphere maintenance mediated by HER2 signaling-activated AHR. We compared the chromatin structure of AHR-knockout (AHRKO) HER2-overexpressing MCF-7 (HER2-5) cells with that of wild-type HER2-5 cells; subsequently, we identified TP63, a stemness factor, as a potential target gene of AHR. ΔNp63 mRNA and protein levels were higher in HER2-5 cells than in HER2-5/AHRKO cells. Activation of HER2/HER3 signaling by heregulin treatment increased ΔNp63 mRNA levels, and its induction was decreased by AHR knockdown in HER2-5 cells. The results of the chromatin immunoprecipitation assay revealed an interaction between AHR and the intronic region of TP63, which encodes ΔNp63. A luciferase reporter gene assay with the intronic region of TP63 showed that AHR expression increased reporter activity. Collectively, our findings suggest that HER2-activated AHR upregulates ΔNp63 expression and that this signaling cascade is involved in CSC maintenance in HER2-expressing breast cancers.

Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

Natural Compounds Tapinarof and Galactomyces Ferment Filtrate Downregulate IL-33 Expression via the AHR/IL-37 Axis in Human Keratinocytes.

Interleukin (IL)-37 suppresses systemic and local inflammation. It is expressed in the epidermis, the external layer of the skin, and is decreased in inflammatory skin diseases including atopic dermatitis (AD) and psoriasis. Therefore, an agent applied topically on the skin that can increase IL-37 could be promising for treating AD and psoriasis; however, the mechanism regulating IL-37 remains largely unknown. Given that IL-37 expression is induced in differentiated keratinocytes, a major component of the epidermis, and that activation of aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, promotes keratinocyte differentiation, we hypothesized that AHR might be involved in the IL-37 expression in human keratinocytes. We analyzed normal epidermal human keratinocytes (NHEKs) treated with tapinarof and Galactomyces ferment filtrate (GFF), which are potent AHR modulators. We found that tapinarof and GFF upregulated IL-37 in NHEKs, which was canceled by the knockdown of AHR using siRNA transfection, indicating that AHR mediates IL-37 expression in NHEKs. Furthermore, we found that the knockdown of IL-37 resulted in the upregulation of IL-33, an alarmin cytokine with crucial roles in the pathogenesis of AD and psoriasis. These findings suggest that IL-37 negatively regulates IL-33 expression in NHEKs. Finally, we examined whether tapinarof and GFF treatment modulates IL-33 expression in NHEKs. Such treatment inhibited IL-33 expression, which was partially reversed by the knockdown of either AHR or IL-37. Taken together, our findings provide the first evidence that tapinarof and GFF could have potential to prevent IL-33-overexpressing disorders such as AD and psoriasis via the AHR/IL-37 axis.