Aryl Hydrocarbon Receptor Target Research Articles

Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation.

Sphingolipids play vital roles in metabolism and regulation. Previously, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, was reported to directly regulate ceramide synthesis genes by binding to their promoters. Herein, sphingosine kinase 2 (SPHK2), responsible for producing sphingosine-1-phosphate (S1P), was found to interact with AHR through LXXLL motifs, influencing AHR nuclear localization. Through mutagenesis and co-transfection studies, AHR activation and subsequent nuclear translocation was hindered by SPHK2 LXXLL mutants or SPHK2 lacking a nuclear localization signal (NLS). Similarly, an NLS-deficient AHR mutant impaired SPHK2 nuclear translocation. Silencing SPHK2 reduced AHR expression and its target gene CYP1A1, while SPHK2 overexpression enhanced AHR activity. SPHK2 was found enriched on the CYP1A1 promoter, underscoring its role in AHR target gene activation. Additionally, S1P rapidly increased AHR expression at both the mRNA and protein levels and promoted AHR recruitment to the CYP1A1 promoter. Using mouse models, AHR deficiency compromised SPHK2 nuclear translocation, illustrating a critical interaction where SPHK2 facilitates AHR nuclear localization and supports a positive feedback loop between AHR and sphingolipid enzyme activity in the nucleus. These findings highlight a novel function of SPHK2 in regulating AHR activity and gene expression.

Modulation of Ceramide-Induced Apoptosis in Enteric Neurons by Aryl Hydrocarbon Receptor Signaling: Unveiling a New Pathway beyond ER Stress.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant and a potent aryl hydrocarbon receptor (AHR) ligand, causes delayed intestinal motility and affects the survival of enteric neurons. In this study, we investigated the specific signaling pathways and molecular targets involved in TCDD-induced enteric neurotoxicity. Immortalized fetal enteric neuronal (IM-FEN) cells treated with 10 nM TCDD exhibited cytotoxicity and caspase 3/7 activation, indicating apoptosis. Increased cleaved caspase-3 expression with TCDD treatment, as assessed by immunostaining in enteric neuronal cells isolated from WT mice but not in neural crest cell-specific Ahr deletion mutant mice (Wnt1Cre+/-/Ahrb(fl/fl)), emphasized the pivotal role of AHR in this process. Importantly, the apoptosis in IM-FEN cells treated with TCDD was mediated through a ceramide-dependent pathway, independent of endoplasmic reticulum stress, as evidenced by increased ceramide synthesis and the reversal of cytotoxic effects with myriocin, a potent inhibitor of ceramide biosynthesis. We identified Sptlc2 and Smpd2 as potential gene targets of AHR in ceramide regulation by a chromatin immunoprecipitation (ChIP) assay in IM-FEN cells. Additionally, TCDD downregulated phosphorylated Akt and phosphorylated Ser9-GSK-3β levels, implicating the PI3 kinase/AKT pathway in TCDD-induced neurotoxicity. Overall, this study provides important insights into the mechanisms underlying TCDD-induced enteric neurotoxicity and identifies potential targets for the development of therapeutic interventions.

The Aryl Hydrocarbon Receptor Regulates Invasiveness and Motility in Acute Myeloid Leukemia Cells through Expressional Regulation of Non-Muscle Myosin Heavy Chain IIA.

Acute myeloid leukemia (AML) is the most prevalent type of hematopoietic malignancy. Despite recent therapeutic advancements, the high relapse rate associated with extramedullary involvement remains a challenging issue. Moreover, therapeutic targets that regulate the extramedullary infiltration of AML cells are still not fully elucidated. The Aryl Hydrocarbon Receptor (AHR) is known to influence the progression and migration of solid tumors; however, its role in AML is largely unknown. This study explored the roles of AHR in the invasion and migration of AML cells. We found that suppressed expression of AHR target genes correlated with an elevated relapse rate in AML. Treatment with an AHR agonist on patient-derived AML cells significantly decreased genes associated with leukocyte trans-endothelial migration, cell adhesion, and regulation of the actin cytoskeleton. These results were further confirmed in THP-1 and U937 AML cell lines using AHR agonists (TCDD and FICZ) and inhibitors (SR1 and CH-223191). Treatment with AHR agonists significantly reduced Matrigel invasion, while inhibitors enhanced it, regardless of the Matrigel's stiffness. AHR agonists significantly reduced the migration rate and chemokinesis of both cell lines, but AHR inhibitors enhanced them. Finally, we found that the activity of AHR and the expression of NMIIA are negatively correlated. These findings suggest that AHR activity regulates the invasiveness and motility of AML cells, making AHR a potential therapeutic target for preventing extramedullary infiltration in AML.

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

The modern human aryl hydrocarbon receptor is more active when ancestralized by genome editing

The aryl hydrocarbon receptor (AHR) is a transcription factor that has many functions in mammals. Its best known function is that it binds aromatic hydrocarbons and induces the expression of cytochrome P450 genes, which encode enzymes that metabolize aromatic hydrocarbons and other substrates. All present-day humans carry an amino acid substitution at position 381 in the AHR that occurred after the divergence of modern humans from Neandertals and Denisovans. Previous studies that have expressed the ancestral and modern versions of AHR from expression vectors have yielded conflicting results with regard to their activities. Here, we use genome editing to modify the endogenous AHR gene so that it encodes to the ancestral, Neandertal-like AHR protein in human cells. In the absence of exogenous ligands, the expression of AHR target genes is higher in cells expressing the ancestral AHR than in cells expressing the modern AHR, and similar to the expression in chimpanzee cells. Furthermore, the modern human AHR needs higher doses of three ligands than the ancestral AHR to induce the expression of target genes. Thus, the ability of AHR to induce the expression of many of its target genes is reduced in modern humans.

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

Differential eigengene network analysis reveals benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin consensus regulatory network in human liver cell line HepG2

Aryl hydrocarbon receptor (AHR) is one of the main mediators of the toxic effects of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, a vast number of BaP- and TCDD-affected genes may suggest a more complex transcriptional regulatory network driving common adverse effects of these two chemicals. Unlike TCDD, BaP is rapidly metabolized in the liver, yielding products with a questionable ability to bind and activate AHR. In this study, we used transcriptomics data from the BaP- and TCCD-exposed human liver cell line HepG2, and performed differential eigengene network analysis to understand the correlation among genes and to untangle the common regulatory mechanism in the action of BaP and TCDD. The genes were grouped into 11 meta-modules with an overall preservation of 0.72 and were also segregated into three consensus time clusters: 12, 24, and 48 h. The analysis showed that the consensus genes in each time cluster were either directly regulated by the AHR or the AHR-TF interactions. Some TFs form a direct physical interaction with AHR such as ESR1, FOXA1, and E2F1, whereas others, including CTCF, RXRA, FOXO1, CEBPA, CEBPB, and TP53 show an indirect interaction with AHR. The analysis of biological processes (BPs) identified unique and common BPs in BaP and TCDD samples, with DNA damage response detected in all three time points. In summary, we identified a consensus transcriptional regulatory network common for BaP and TCDD consisting of direct AHR targets and AHR-TF targets. This analysis sheds new light on the common mechanism of action of a genotoxic (BaP) and non-genotoxic (TCDD) chemical in liver cells.

Aryl hydrocarbon receptor and IL-13 signaling crosstalk in human keratinocytes and atopic dermatitis.

Atopic dermatitis (AD) is an allergic skin disease mediated by skin barrier impairment and IL-13-driven immune response. Activation of the aryl hydrocarbon receptor (AHR) has shown promise in early clinical trials for AD; however, the mechanism by which AHR partially ameliorates AD is not well known. Gene expression data from human biopsies were analyzed, and compared to gene expression from RNA-sequencing in our in-vitro HaCaT cell model system. Western blot, ELISA qRT-PCR were used to further explore the relationship between AHR and IL-13 signaling in HaCaT cells. The AHR target gene CYP1A1 was decreased in lesional skin compared with healthy control skin (p = 4.30 × 10-9). Single-cell RNA sequencing (scRNAseq) demonstrated increased AHR expression (p < 1.0 × 10-4) and decreased CYP1A1 expression in lesional AD keratinocytes compared with healthy control keratinocytes (p < 0.001). Activation of AHR by AHR agonists in HaCaT cells reversed IL-13-dependent gene expression of several key genes in AD pathogenesis, most notably the eosinophil chemoattractant CCL26 (eotaxin-3). Differentially expressed genes in keratinocytes of patients with AD substantially overlapped with genes regulated by AHR agonists from HaCaT cells by RNAseq, but in reverse direction. Mechanistically, there was evidence for direct transcriptional effects of AHR; AHR binding motifs were identified in the differentially expressed genes from lesional AD keratinocytes compared to control keratinocytes, and AHR activation did not modify IL-13-dependent signal transducer and activator of transcription 6 (STAT6) translocation to the nucleus. Together, these data suggest that the AHR pathway is dysregulated in AD and that AHR modulates IL-13 downstream signaling in keratinocytes through genome-wide, transcriptional regulatory effects.

Expression of AhR-regulated miRNAs in non-small cell lung cancer in smokers and never smokers.

Smoking is a risk factor for non-small cell lung cancer (NSCLC). The most common subtypes of NSCLC are lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC). The cigarette smoke contains aryl hydrocarbon receptor (AhR) ligands, such as benzo(a)pyrene (BaP). By activating the AhR, BaP can change the expression of many genes, including miRNA-encoding genes. In this study, we have evaluated the expression of few miRNAs potentially regulated by AhR (miR-21, -342, -93, -181a, -146a), as well as CYP1A1, a known AhR target gene, in lung tumor samples from smoking (n=40) and non-smoking (n=30) patients with LAC and from smoking patients with SCC (n=40). We have also collected macroscopically normal lung tissue >5 cm from the tumor margin. We compared the obtained data on the miRNA expression in tumors with data from The Cancer Genome Atlas (TCGA). We found that in 76.7% of non-smoking LAC patients, CYP1A1 mRNA was not detected in tumor and normal lung tissues, while in smoking patients, CYP1A1 expression was detected in tumors in almost half of the cases (47.5% for SCC and 42.5% for LAC). The expression profile of AhR-regulated miRNAs differed between LAC and SCC and depended on the smoking status. In LAC patients, the expression of oncogenic miRNA-21 and miRNA-93 in tumors was higher than in normal lung tissue from the same patients. However, in SCC patients from our sample, the levels of these miRNAs in tumor and non-transformed lung tissue did not differ significantly. The results of our studies and TCGA data indicate that the expression levels of miRNA-181a and miRNA-146a in LAC are associated with smoking: expression of these miRNAs was significantly lower in tumors of smokers. It is possible that their expression is regulated by AhR and AhRR (AhR repressor), and inhibition of AhR by AhRR leads to a decrease in miRNA expression in tumors of smoking patients. Overall, these results confirm that smoking has an effect on the miRNA expression profile. This should be taken into account when searching for new diagnostic and therapeutic targets for NSCLC.

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400μM) in the presence or absence of the AHR antagonist CH223191 (1μM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

Impact of IDO activation and alterations in the kynurenine pathway on hyperserotonemia, NAD+ production, and AhR activation in autism spectrum disorder

Hyperserotonemia is the most replicated biochemical anomaly associated with autism spectrum disorder (ASD) and has been reported in 35–46% of individuals with ASD. Serotonin is synthesised from the essential amino acid tryptophan (TRP). However, the main catabolic route of TRP is the kynurenine pathway (KP), which competes with serotonin synthesis when indoleamine dioxygenase (IDO) is activated. Using the same cohort of individuals with ASD, we used to report extensive studies of the serotonin/melatonin pathway, and found increased kynurenine (KYN), suggesting IDO activation in 58.7% of individuals with ASD (159/271), supported by a strong negative correlation between KYN/TRP ratio and miR-153-3p plasma levels, which negatively regulates IDO. IDO activation was associated with normoserotonemia, suggesting that IDO activation could mask hyperserotonemia which meant that hyperserotonemia, if not masked by IDO activation, could be present in ~94% of individuals with ASD. We also identified several KP alterations, independent of IDO status. We observed a decrease in the activity of 3-hydroxyanthranilate dioxygenase which translated into the accumulation of the aryl hydrocarbon receptor (AhR) selective ligand cinnabarinic acid, itself strongly positively correlated with the AhR target stanniocalcin 2. We also found a deficit in NAD+ production, the end-product of the KP, which was strongly correlated with plasma levels of oxytocin used as a stereotypical neuropeptide, indicating that regulated neuropeptide secretion could be limiting. These results strongly suggest that individuals with ASD exhibit low-grade chronic inflammation that is mediated in most cases by chronic AhR activation that could be associated with the highly prevalent gastrointestinal disorders observed in ASD, and explained IDO activation in ~58% of the cases. Taken together, these results extend biochemical anomalies of TRP catabolism to KP and posit TRP catabolism as a possible major component of ASD pathophysiology.

Tumor suppressive activity of AHR in environmental arsenic-induced carcinogenesis

The aryl hydrocarbon receptor (AHR) is a highly conserved pleiotropic transcription factor that senses environmental pollutants, microbial products, and endogenous ligands. The transcriptional targets of AHR include phase I and phase II detoxification enzymes, as well as numerous signaling molecules that affect a wide spectrum of biological and biochemical processes in a manner of cellular context-dependent. In this review, we systematically assess the latest discoveries of AHR in carcinogenesis with an emphasis on its tumor suppressor-like property that represses the expression of genes in oncogenic signaling pathways. Additionally, we outline recent progress in our studies on the interaction among AHR, TGFb and NRF2 in cellular responses to arsenic and malignant transformation. Our findings indicate that AHR antagonized TGFb and NRF2, suggesting that AHR could serve as a potential tumor suppressor in arsenic-induced carcinogenesis. Notably, while AHR can exhibit both oncogenic and tumor-suppressive properties in cancer development and the generation of the cancer stem-like cells (CSCs), the tumor suppressor-like effect of AHR warrants further extensive exploration for the prevention and clinical treatment of cancers.

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.

Activation of Chaperone-Mediated Autophagy Inhibits the Aryl Hydrocarbon Receptor Function by Degrading This Receptor in Human Lung Epithelial Carcinoma A549 Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a substrate protein of a Cullin 4B E3 ligase complex responsible for diverse cellular processes. In the lung, this receptor is responsible for the bioactivation of benzo[a]pyrene during tumorigenesis. Realizing that the AHR function is affected by its expression level, we are interested in the degradation mechanism of AHR in the lung. Here, we have investigated the mechanism responsible for AHR degradation using human lung epithelial A549 cells. We have observed that the AHR protein levels increase in the presence of chloroquine (CQ), an autophagy inhibitor, in a dose-dependent manner. Treatment with 6-aminonicotinamide (6-AN), a chaperone-mediated autophagy (CMA) activator, decreases AHR protein levels in a concentration-dependent and time-dependent manner. This decrease suppresses the ligand-dependent activation of the AHR target gene transcription, and can be reversed by CQ but not MG132. Knockdown of lysosome-associated membrane protein 2 (LAMP2), but not autophagy-related 5 (ATG5), suppresses the chloroquine-mediated increase in the AHR protein. AHR is resistant to CMA when its CMA motif is mutated. Suppression of the epithelial-to-mesenchymal transition in A549 cells is observed when the AHR gene is knocked out or the AHR protein level is reduced by 6-AN. Collectively, we have provided evidence supporting that AHR is continuously undergoing CMA and activation of CMA suppresses the AHR function in A549 cells.

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.

Bojungikgi-tang improves skin barrier function and immune response in atopic dermatitis mice fed a low aryl hydrocarbon receptor ligand diet

BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor that plays a crucial role in regulating the immune system and maintaining skin barrier function. AhR signaling is pivotal in the pathogenesis of inflammatory diseases such as atopic dermatitis (AD), and the absence of AhR ligands further contributes to the progression or worsening of AD symptoms.MethodsAD was induced with 2,4-dinitrochlorobenzene (DNCB), and Bojungikgi-tang (BJIKT) was administered orally daily for 10 weeks. Serum IgE, splenocyte IL-4, and IFN-γ levels, skin barrier genes, and AhR target gene expressions were analyzed using RNA-sequencing analysis. Spleen tissues were extracted for fluorescence-activated cell sorting (FACS) analysis to analyze the effect of BJIKT on immune responses. A correlation analysis was conducted to analyze the correlation between immune markers and skin barrier genes and AhR target genes.ResultsBJIKT effectively improved AD symptoms in AD mice fed a low AhR ligand diet by reducing neutrophil and eosinophil counts, lowering IgE levels in the blood, and decreasing IL-4 and IFN-γ levels in the splenocytes. Additionally, BJIKT significantly reduced epithelial skin thickness and transepidermal water loss (TEWL) values and reversed the decreased expression of skin barrier genes. BJIKT also considerably altered the expression of AhR target genes, including Ahr, Ahrr, cytochrome P450 1A1 (CYP1A1), and CYP1B1. Furthermore, AhR target pathway genes were negatively correlated with immune cell subtypes, including CD4 + and CD8 + T cells and macrophages (CD11b + F4/80 +) at the systemic level.ConclusionsBJIKT can regulate AhR activation and may help reduce inflammation in AD by regulating the expression of skin barrier genes and immune responses.

Role of ERK1/2 Signaling in Cinnabarinic Acid-Driven Stanniocalcin 2-Mediated Protection against Alcohol-Induced Apoptosis.

We have previously shown that a bona fide aryl hydrocarbon receptor (AhR) agonist, cinnabarinic acid (CA), protects against alcohol-induced hepatocyte apoptosis via activation of a novel AhR target gene, stanniocalcin 2 (Stc2). Stc2 translates to a secreted disulfide-linked hormone, STC2, known to function in cell development, calcium and phosphate regulation, angiogenesis, and antiapoptosis-albeit the comprehensive mechanism by which the CA-AhR-STC2 axis confers antiapoptosis is yet to be characterized. In this study, using RNA interference library screening, downstream antiapoptotic molecular signaling components involved in CA-induced STC2-mediated protection against ethanol-induced apoptosis were investigated. RNA interference library screening of kinases and phosphatases in Hepa1 cells and subsequent pathway analysis identified mitogen-activated protein kinase (MAPK) signaling as a critical molecular pathway involved in CA-mediated protection. Specifically, phosphorylation of ERK1/2 was induced in response to CA treatment without alterations in p38 and JNK signaling pathways. Silencing Stc2 in Hepa1 cells and in vivo experiments performed in Stc2-/- (Stc2 knockout) mice, which failed to confer CA-mediated protection against ethanol-induced apoptosis, showed abrogation of ERK1/2 activation, underlining the significance of ERK1/2 signaling in CA-STC2-mediated protection. In conclusion, activation of ERK1/2 signaling in CA-driven AhR-dependent Stc2-mediated protection represents a novel mechanism of protection against acute alcohol-induced apoptosis. SIGNIFICANCE STATEMENT: Previous studies have shown the role of stanniocalcin 2 (Stc2) in cinnabarinic acid (CA)-mediated protection against alcohol-induced apoptosis. Here, using RNA interference library screening and subsequent in vivo studies, the functional significance of ERK1/2 activation in CA-induced Stc2-mediated protection against acute ethanol-induced apoptosis was identified. This study is thus significant as it illustrates a comprehensive downstream mechanism by which CA-induced Stc2 protects against alcoholic liver disease.

#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) &amp;gt;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.

Ethyl Caffeate Can Inhibit Aryl Hydrocarbon Receptor (AhR) Signaling and AhR-Mediated Potentiation of Mast Cell Activation.

Ethyl caffeate (EC) is a natural phenolic compound that is present in several medicinal plants used to treat inflammatory disorders. However, its anti-inflammatory mechanisms are not fully understood. Here, we report that EC inhibits aryl hydrocarbon receptor (AhR) signaling and that this is associated with its anti-allergic activity. EC inhibited AhR activation, induced by the AhR ligands FICZ and DHNA in AhR signaling-reporter cells and mouse bone marrow-derived mast cells (BMMCs), as assessed by AhR target gene expressions such as CYP1A1. EC also inhibited the FICZ-induced downregulation of AhR expression and DHNA-induced IL-6 production in BMMCs. Furthermore, the pretreatment of mice with orally administered EC inhibited DHNA-induced CYP1A1 expression in the intestine. Notably, both EC and CH-223191, a well-established AhR antagonist, inhibited IgE-mediated degranulation in BMMCs grown in a cell culture medium containing significant amounts of AhR ligands. Furthermore, oral administration of EC or CH-223191 to mice inhibited the PCA reaction associated with the suppression of constitutive CYP1A1 expression within the skin. Collectively, EC inhibited AhR signaling and AhR-mediated potentiation of mast cell activation due to the intrinsic AhR activity in both the culture medium and normal mouse skin. Given the AhR control of inflammation, these findings suggest a novel mechanism for the anti-inflammatory activity of EC.

Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes

BackgroundIndoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated...