Endogenous Aryl Hydrocarbon Receptor Agonist Research Articles

Polycyclic aromatic hydrocarbon (PAH) activation of AhR inhibits glutathione S-transferase (GST) activity in primary human small airway epithelial cells (hSAECs)

Background: PAH represents the organic phase of particulate matter (PM) in air pollution emitted via the incomplete combustion of various compounds (i.e. diesel fuel, wood, and plastics). It is widely regarded that PAH activation of aryl hydrocarbon receptor (AhR) plays an important role in pro-inflammatory signaling, however the mechanisms remain unknown. In contrast- endogenous AhR agonists, such as 6-Formylindolo[3,2-b]carbazole (FICZ), exert pleiotropic protective effects against inflammation, fibrosis, and oxidative stress. The objective of our research program is to delineate the differences between activation of AhR by PAH (with adverse effects) compared to its beneficial homeostatic activation by endogenous FICZ. PM may induce oxidative stress in part through inhibition of antioxidant defense mechanisms. GSTs are a superfamily of enzymes that plays an important role in protecting cells from oxidative damage (by conjugating reduced glutathione to substrates under pro-oxidizing conditions). We hypothesize that PAH activation of AhR contributes to oxidative stress via inhibition of GST activity in primary hSAECs. Methods: Primary hSAECs were subjected to varying dosages of PAH [0, 250, 500, 1000, 5000 ng/mL] to model airborne PM exposure levels, and to increasing doses of FICZ [0, 0.50, 10, 100, 1000 nM] to model normal and excessively high endogenous ligand activation of AhR. We determined the relative potencies of PAH and FICZ by measuring CYP1A1 and CYP1B1 gene expression. Oxidative stress was measured using DHE-labeling and GST activity was measured with enzymatic kits (Sigma Aldrich). AhR activity was pharmacologically inhibited using 10μM CH223191. Statistical analysis was conducted using SigmaPlot 14.0 (Systat Software). Results: PAH and FICZ maximally stimulate hSAEC CYP1A1 and CYP1B1 expression at 1000ng/mL and 100nM, respectively, and are therefore defined as equipotent concentrations used in our studies. In a representative experiment in hSAECs, PAH (1000ng/mL) significantly increased hSAEC oxidative stress vs FICZ (100nM) n=6, p<0.01), while significantly decreasing GST specific activity vs equipotent FICZ activation of AhR in primary hSAECs from a 66-year-old female without lung disease (26.6±0.874 vs 28.3±.611 nmol/mL/min); n=6 from 2 independent assays; p=0.032. Similar differential effects of PAH vs FICZ activation of AhR on GST were seen in additional experiments with male hSAECs and Calu-3 cells (male). Importantly, CH223191 inhibition of GST enzymatic activity suggests that AhR plays an important role in protecting cells from oxidative stress. Conclusions: Our studies indicate that airborne PAH exposure can have detrimental health effects on the lung via aberrant AhR signaling. Significance: Understanding activity of AhR signaling can leads to novel therapeutic options for redox disorders in the lung. Funding: VA 5I01BX001777-05 (RP) and R01HL137033 (MH). 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.

The Role of Aryl Hydrocarbon Receptor in the Pathogenesis and Treatment of Psoriasis.

The pathogenesis of psoriasis is complex. Aryl hydrocarbon receptor (AhR) is a transcription factor that can be bound and activated by structurally diverse ligands and plays an important role in a range of biological processes and in the pathogenesis of different diseases. Recently, the role of AhR in psoriasis has attracted attention. AhR has toxicological functions and physiological functions. The overexpression and activation of AhR induced by the environmental pollutant and exogenous AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can drive the development of psoriasis. This TCDD-mediated toxicological response disrupts the physiological functions of AhR resulting in skin barrier disorders and the release of inflammatory cytokines, 2 of the pivotal factors of psoriasis. In addition, highly upregulated kynureninase in psoriasis decreases endogenous AhR agonists, thereby weakening the physiological functions of AhR. Activating AhR physiological signalling should be useful in the treatment of psoriasis. Studies have demonstrated that physiological activation of AhR can dampen the severity of psoriasis. The oldest and effective treatment for psoriasis coal tar works by activating AhR, and both new anti-psoriasis drugs tapinarof and benvitimod are formulations of AhR agonist, supporting that activation of AhR can be used as a new strategy for the treatment of psoriasis. Preclinical and preliminary clinical studies have revealed the anti-psoriasis effects of a number of AhR agonists, providing potential candidates for the development of new drugs for the treatment of psoriasis.

Etoricoxib enhances aryl hydrocarbon receptor activity

Etoricoxib is a nonsteroidal anti-inflammatory drug (NSAID) that possesses properties that include reducing inflammation and relieving pain and fever. Etoricoxib is an oral medication that selectively inhibits cyclooxygenase-2 with high efficacy. Controversies about its cardiovascular side effects have long existed. The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor that plays a key role in the metabolism of xenobiotics and many physiological functions. 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a tryptophan metabolite and endogenous AhR agonist. Activation of AhR by its ligand induces upregulation of AhR-targeted cytochrome P450 (CYP) 1A1 expression. We found that etoricoxib (10–60 μM) induced CYP1A1 mRNA and protein expressions and the transcriptional activity of AhR mediated by the aryl hydrocarbon response element (AHRE) in both mouse Hepa-1c1c7 and human HepG2 cells. Its induction did not appear in AhR signaling-deficient cells, and was inhibited by the AhR antagonist, CH-223191. Etoricoxib was able to induced the translocalization of AhR from cytosol into nucleus. Etoricoxib also worked synergistically with ITE to further increase the expression of CYP1A1 mRNA and protein in human cells. The synergistic effect was higher in cells with than cells without overexpression of AhR. In summary, etoricoxib is an agonist of AhR in both mouse and human cells. Etoricoxib has a synergistic effect on ITE-induced CYP1A1 expression in human cells. The effect of etoricoxib on AhR and ITE on endothelial cells and cardiomyocytes should be further elucidated to in hope to clarify the mechanism of increased cardiovascular events in COX-2 inhibitors and etoricoxib.

AhR activation promotes Treg cell generation by enhancing Lkb1-mediated fatty acid oxidation via the Skp2/K63-ubiquitination pathway.

Several aryl hydrocarbon receptor (AhR) agonists have been reported to promote the generation of regulatory T cells (Treg cells), and the action mechanisms need to be identified. In this study, we addressed the underlying mechanism of AhR activation to induce the generation of Treg cells in the view of cellular metabolism. Naïve CD4+ T cells were purified with mouse CD4+ CD62L+ T Cells Isolation Kits. The proportions of Treg cells were detected by flow cytometry. The value of oxygen consumption rate (OCR) of CD4+ T cells was detected by the Seahorse XFe 96 analyzer. The activation of fatty acid oxidation (FAO)-related metabolic pathways was detected by Western blotting. Intracellular localization of Lkb1 was detected by immunofluorescence. The Strad-Mo25-Lkb1 complex formation and K63 chain ubiquitination modification of Lkb1 were detected by co-immunoprecipitation. The binding of AhR to the Skp2 promoter was detected by constructing luciferase reporter gene. AhR or carnitine palmitoyltransferases 1 was knockdown in dextran sulphate sodium (DSS)-induced colitis or collagen-induced arthritis (CIA) mice by infecting mice with adeno-associated virus via the tail vein injection. Compared to the control group, exogenous and endogenous AhR agonists 3,3'-diindolylmethane (DIM) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) were shown to preferentially upregulate the mRNA expression of FAO-related enzymes and the value of OCR. Consistently, pharmacological or genetic inhibition of FAO markedly diminished the induction of DIM and ITE on the differentiation of Treg cells. DIM and ITE functioned mainly through activating the liver kinase B1 (Lkb1)-AMPK pathway via promotion of Lkb1-Strad-Mo25 complex formation and Lkb1 K63 ubiquitination. DIM and ITE were also shown to upregulate the mRNA expression of Skp2, a ubiquitination-related enzyme, and facilitate the binding of AhR to the xenobiotic responsive element of Skp2 promoter region by luciferase reporter gene assay. Furthermore, the contribution of Skp2/K63 ubiquitination/Lkb1/FAO axis was verified in (DSS)-induced colitis or CIA mice. In summary, these findings indicate that AhR activation promotes Treg cell generation by enhancing Lkb1-mediated FAO via the Skp2/K63-ubiquitination pathway, and AhR agonists may be used as inducers of Treg cells to prevent and treat autoimmune diseases.

Cinnabarinic Acid Provides Hepatoprotection Against Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition in which excess lipids accumulate in the liver and can lead to a range of progressive liver disorders including non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. While lifestyle and diet modifications have proven to be effective as NAFLD treatments, they are not sustainable in the long-term, and currently no pharmacological therapies are approved to treat NAFLD. Our previous studies demonstrated that cinnabarinic acid (CA), a novel endogenous Aryl hydrocarbon Receptor (AhR) agonist, activates the AhR target gene, Stanniocalcin 2, and confers cytoprotection against a plethora of ER/oxidative stressors. In this study, the hepatoprotective and anti-steatotic properties of CA were examined against free fatty-acid-induced in vitro and high-fat-diet fed in vivo NAFLD models. The results demonstrated that CA treatment significantly lowered weight gain and attenuated hepatic lipotoxicity both before and after the established fatty liver, thereby protecting against steatosis, inflammation, and liver injury. CA mitigated intracellular free fatty acid uptake concomitant with the downregulation of CD36/fatty acid translocase. Genes involved in fatty acid and triglyceride synthesis were also downregulated in response to CA treatment. Additionally, suppressing AhR and Stc2 expression using RNA interference in vitro verified that the hepatoprotective effects of CA were absolutely dependent on both AhR and its target, Stc2. Collectively, our results demonstrate that the endogenous AhR agonist, CA, confers hepatoprotection against NAFLD by regulating hepatic fatty acid uptake and lipogenesis. SIGNIFICANCE STATEMENT: In this study using in vitro and in vivo models, we demonstrate that cinnabarinic acid (CA), an endogenous AhR agonist, provides protection against non-alcoholic fatty liver disease. CA bestows cytoprotection against steatosis and liver injury by controlling expression of several key genes associated with lipid metabolism pathways, limiting the hepatic lipid uptake, and controlling liver inflammation. Moreover, CA-induced hepatoprotection is absolutely dependent on AhR and Stc2 expression.

081 Modulation of the aryl hydrocarbon receptor by adipose tissue: Implications for skin carcinogenesis

The aryl hydrocarbon receptor(AhR) is a ligand-activated transcription factor that responds to chemical carcinogens. An endogenous AhR agonist, 6-Formylindolo[3,2-b]carbazole, is produced following ultraviolet light(UV) absorption by tryptophan. Epidemiological studies show a correlation between exposure to AhR agonists and non melanoma skin cancer(NMSC). As an example, AhR activation promotes metabolism of pro-carcinogens to carcinogens such as the metabolism of benzo(a)pyrene to benzo[a]pyrene diol epoxide(BPDE).

Cinnabarinic Acid-Induced Stanniocalcin 2 Confers Cytoprotection against Alcohol-Induced Liver Injury.

We recently identified upregulation of a novel aryl hydrocarbon receptor (AhR) target gene, stanniocalcin 2 (STC2), by an endogenous AhR agonist, cinnabarinic acid (CA). STC2 is a disulfide-linked homodimeric secreted glycoprotein that plays a role in various physiologic processes, including cell metabolism, inflammation, endoplasmic reticulum (ER) and oxidative stress, calcium regulation, cell proliferation, and apoptosis. Our previous studies have confirmed that CA-induced AhR-dependent STC2 expression was able to confer cytoprotection both in vitro and in vivo in response to injury induced by variety of ER/oxidative insults. Here, we used mouse models of chronic and acute ethanol feeding and demonstrated that upregulation of STC2 by CA was critical for cytoprotection. In STC2 knockout mice (STC2-/-), CA failed to protect against both acute as well as chronic-plus-binge ethanol-induced liver injury, whereas re-expression of STC2 in the liver using in vivo gene delivery restored cytoprotection against injury based on measures of apoptosis and serum levels of liver enzymes, underlining STC2's indispensable function in cell survival. In conclusion, the identification of STC2 as an AhR target gene receptive to CA-mediated endogenous AhR signaling and STC2's role in providing cytoprotection against liver injury represents a key finding with potentially significant therapeutic implications. SIGNIFICANCE STATEMENT: We recently identified stanniocalcin 2 (STC2) as a novel aryl hydrocarbon receptor (AhR) target gene regulated by endogenous AhR agonist and tryptophan metabolite, cinnabarinic acid (CA). Here, we showed that CA-induced STC2 expression conferred cytoprotection against apoptosis, steatosis, and liver injury in chronic as well as acute models of ethanol feeding. Therefore, this study will prove instrumental in developing CA as a promising lead compound for future drug development against hepatic diseases.

Involvement of the CYP1A1 inhibition-mediated activation of aryl hydrocarbon receptor in drug-induced hepatotoxicity.

Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.

Abstract 11010: Aryl Hydrocarbon Receptor Plays an Essential Role in the Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by arteriopathy in the small to medium-sized distal pulmonary arteries, often accompanied by infiltration of inflammatory cells. Aryl hydrocarbon receptor (AHR), a nuclear receptor/transcription factor, detoxifies xenobiotics and regulates the differentiation and function of various immune cells. However, the role of AHR in the pathogenesis of PAH is largely unknown. Hypothesis: We hypothesized that AHR has a role in PAH pathogenesis both in human and animal models. Methods: Wild type (WT) and Ahr -knockout ( Ahr -/- ) rats were either treated with AHR agonists or VEGFR2 inhibitors or fed a diet containing a Chinese herbal drug Qing-Dai, which may cause drug-induced PAH, in combination with hypoxia, and then subjected to hemodynamic analysis. Histological characteristics were analyzed in the lungs of these rats and patients with idiopathic PAH. RNA-seq and qRT-PCR were performed on specimens from SU5416/Hypoxia (SuHx) rats, including lungs and the peripheral blood mononuclear cells (PBMCs), and PBMCs of PAH patients. AHR agonistic activity in serum was judged by luciferase reporter assay. Results: WT rats treated with the potent endogenous AHR agonist (6-formylindolo[3,2-b]carbazole) in combination with hypoxia, developed severe pulmonary hypertension (PH) with plexiform-like lesions, whereas WT rats treated with the potent VEGFR2 inhibitors (KI8751 and TAK-593) did not. Ahr -/- rats did not develop PH in the SuHx rats. A diet containing Qing-Dai in combination with hypoxia led to development of PH in Ahr +/+ rats, but not in Ahr -/- rats. Several inflammatory signaling pathways were up-regulated in endothelial cells and PBMCs, which led to infiltration of CD4 + IL-21 + T cells and MRC1 + macrophages into vascular lesions in an AHR-dependent manner. AHR agonistic activity in serum was significantly higher in PAH patients than in healthy volunteers and was associated with poor prognosis of PAH. The expression of several AHR-related genes were changed in PBMCs of PAH patients. Conclusions: AHR plays crucial roles in the development and progression of PAH, indicating that this receptor is a potential target for both diagnosis and treatment of PAH.

Decoding Cinnabarinic Acid-Specific Stanniocalcin 2 Induction by Aryl Hydrocarbon Receptor.

Aryl hydrocarbon receptor (AhR) is a ligand-mediated transcription factor known for regulating response to xenobiotics, including prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the activation of CYP1A1 expression. Upon ligand-binding, AhR translocates to the nucleus, interacts with the AhR nuclear translocator, and binds to xenobiotic response elements (XREs; GCGTG) present in the promoter region of AhR-regulated genes. Recently, we identified a novel tryptophan catabolite, cinnabarinic acid (CA), as an endogenous AhR agonist capable of activating expression of AhR target gene stanniocalcin 2 (stc2). The CA-driven stc2 induction bestowed cytoprotection against hepatotoxicity in an AhR-dependent manner. Interestingly, only CA but not TCDD was able to induce stc2 expression in liver, and CA was unable to upregulate the TCDD responsive cyp1a1 gene. In this report, we identified CA-specific histone H4 lysine 5 acetylation and H3 lysine 79 methylation at the AhR-bound stc2 promoter. Moreover, histone H4 lysine 5 acetylation writer, activating transcription factor 2 (Atf2), and H3 lysine 79 methylation writer, disruptor of telomeric silencing 1-like histone lysine methyltransferase (Dot1l), were interacting with the AhR complex at the stc2 promoter exclusively in response to CA treatment concurrent with the histone epigenetic marks. Suppressing Atf2 and Dot1l expression using RNA interference confirmed their role in stc2 expression. CRISPR/Cas9-assisted replacement of cyp1a1 promoter-encompassing XREs with stc2 promoter XREs resulted in CA-dependent induction of cyp1a1, underlining a fundamental role of quaternary structure of XRE sequence in agonist-specific gene regulation. In conclusion, CA-driven recruitment of specific chromatin regulators to the AhR complex and resulting histone epigenetic modifications may serve as a molecular basis for agonist-specific stc2 regulation by AhR. SIGNIFICANCE STATEMENT: Results reported here provide a mechanistic explanation for the agonist-specific differential gene regulation by identifying interaction of aryl hydrogen receptor with specific chromatin regulators concomitant with unique histone epigenetic marks. This study also demonstrated that the agonist-specific target-gene expression can be transferred with the gene-specific promoter xenobiotic response element-sequence in the context of chromatin architecture.

Activation of the aryl hydrocarbon receptor by 3-methylcholanthrene, but not by indirubin, suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.

Endogenous ligands of Aryl hydrocarbon receptor may play an important role in DNA damage responses

Aryl hydrocarbon receptor (AhR) was initially discovered as a cellular protein involved in mediating the detoxification of xenobiotic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Subsequent studies have identified several families of metabolic products as endogenous AhR agonists such as 6-Formylindolo[3,2-b] carbazole (FICZ) and kynurenine and revealed important physiological roles of AhR in the regulation of metabolic homeostasis and immune responses. Recently, we found that treatment with DNA damage agents including etoposide, doxorubicin, and hexavalent chromium resulted in elevated levels of AhR and CYP1A2, the latter being an AhR transcriptional target. Moreover, we found that treatment with the oxidant hydrogen peroxide stimulated AhR expression, which was also accompanied by enhanced expression of CYP1A2. It has been shown that hydrogen peroxide is capable of converting tryptophan into FICZ in vitro. Thus, we speculated that oxidant-induced activation of the AhR axis was mediated through the generation of AhR ligand(s) in vivo. Supporting this, we showed that pre-incubation of tryptophan that was dissolved in PBS with hydrogen peroxide elicited a rapid induction of CYP1A2 expression with an kinetics similar to that of FICZ. Given that a common feature of treatment with etoposide, doxorubicin, and hexavalent chromium is the generation of reactive oxygen species in vivo, we propose that enhanced expression of AhR by these DNA damage agents is primarily due to an elevated level of tryptophan metabolic products including FICZ and that the AhR signaling axis may play a significant role in limiting the extent of DNA damage caused by these compounds.

The Endogenous Tryptophan-derived Photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo.

UV-chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390nm (ε=9180L mol-1 cm-1 ), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC-25, HaCaT-ras II-4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6Jcm-2 ) and FICZ (≥10nm), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg-sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 "high-risk" mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV-induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.

Targeting the aryl hydrocarbon receptor with a novel set of triarylmethanes

The aryl hydrocarbon receptor (AhR) is a chemical sensor upregulating the transcription of responsive genes associated with endocrine homeostasis, oxidative balance and diverse metabolic, immunological and inflammatory processes, which have raised the pharmacological interest on its modulation. Herein, a novel set of 32 unsymmetrical triarylmethane (TAM) class of structures has been synthesized, characterized and their AhR transcriptional activity evaluated using a cell-based assay. Eight of the assayed TAM compounds (14, 15, 18, 19, 21, 22, 25, 28) exhibited AhR agonism but none of them showed antagonist effects. TAMs bearing benzotrifluoride, naphthol or heteroaromatic (indole, quinoline or thiophene) rings seem to be prone to AhR activation unlike phenyl substituted or benzotriazole derivatives. A molecular docking analysis with the AhR ligand binding domain (LBD) showed similarities in the binding mode and in the interactions of the most potent TAM identified 4-(pyridin-2-yl (thiophen-2-yl)methyl)phenol (22) compared to the endogenous AhR agonist 5,11-dihydroindolo[3,2-b]carbazole-12-carbaldehyde (FICZ). Finally, in silico predictions of physicochemical and biopharmaceutical properties for the most potent agonistic compounds were performed and these exhibited acceptable druglikeness and good ADME profiles. To our knowledge, this is the first study assessing the AhR modulatory effects of unsymmetrical TAM class of compounds.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR’s role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

Concentration and Duration of Indoxyl Sulfate Exposure Affects Osteoclastogenesis by Regulating NFATc1 via Aryl Hydrocarbon Receptor.

Indoxyl sulfate (IS) is a chronic kidney disease (CKD)-specific renal osteodystrophy metabolite that affects the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a transcription factor promoting osteoclastogenesis. However, the mechanisms underlying the regulation of NFATc1 by IS remain unknown. It is intriguing that the Aryl hydrocarbon receptor (AhR) plays a key role in osteoclastogenesis, since IS is an endogenous AhR agonist. This study investigates the relationship between IS concentration and osteoclast differentiation in Raw 264.7 cells, and examines the effects of different IS concentrations on NFATc1 expression through AhR signaling. Our data suggest that both osteoclastogenesis and NFATc1 are affected by IS through AhR signaling in both dose- and time-dependent manners. Osteoclast differentiation increases with short-term, low-dose IS exposure and decreases with long-term, high-dose IS exposure. Different IS levels switch the role of AhR from that of a ligand-activated transcription factor to that of an E3 ubiquitin ligase. We found that the AhR nuclear translocator may play an important role in the regulation of these dual functions of AhR under IS treatment. Altogether, this study demonstrates that the IS/AhR/NFATc1 signaling axis plays a critical role in osteoclastogenesis, indicating a potential role of AhR in the pathology and abnormality of bone turnover in CKD patients.

PCB126 blocks the thermogenic beiging response of adipocytes.

Subcutaneous white adipose tissue is capable of becoming thermogenic in a process that is referred to as "beiging." Beiging is associated with activation of the uncoupling protein, UCP1, and is known to be important for preventing adipose hypertrophy and development of insulin resistance. Polychlorinated biphenyls (PCBs) accumulate in fat, and it is hypothesized that disruption of adipogenesis and adipocyte function by PCBs may be causative in the development of obesity and diabetes. We developed immortal human subcutaneous preadipocytes that, when differentiated, are capable of beiging. Preadipocytes that were treated with polychlorinated biphenyl congener 126 (PCB126), followed by differentiation, were suppressed for their ability to activate UCP1 upon β-adrenergic stimulation with norepinephrine (NE), demonstrating a block in the beiging response. Treatment of preadipocytes with another known endogenous AhR agonist, indoxyl sulfate (IS), followed by differentiation also blocked the NE-stimulated upregulation of UCP1. Knockdown of the aryl hydrocarbon receptor (AhR) caused the preadipocytes to be refractory to PCB126 and IS effects. The chemical AhR antagonist, CH223191, was effective at preventing the effects of PCB126 but not IS, indicating AhR ligand specificity of CH223191. Repression of NE-induced UCP1 upregulation was also observed when already-differentiated mature adipocytes were treated with PCB126 but not IS. These results indicate that exposure of preadipocytes to endogenous (IS) or exogenous (PCB126) AhR agonists is effective at blocking them from becoming functional adipocytes that are capable of the beiging response. Mature adipocytes may have differential responses. This finding suggests a mechanism by which dioxin-like PCBs such as PCB126 could lead to disruption in energy homeostasis, potentially leading to obesity and diabetes.

Cytochrome P4501-inhibiting chemicals amplify aryl hydrocarbon receptor activation and IL-22 production in T helper 17 cells

The aryl hydrocarbon receptor (AHR) controls interleukin 22 production by T helper 17 cells (Th17). IL-22 contributes to intestinal homeostasis but has also been implicated in chronic inflammatory disorders and colorectal cancer, highlighting the need for appropriate regulation of IL-22 production. Upon activation, the AHR induces expression of cytochrome P4501 (CYP1) enzymes which in turn play an important feedback role that curtails the duration of AHR signaling by metabolizing AHR ligands. Recently we described how agents that inhibit CYP1 function potentiate AHR signaling by disrupting metabolic clearance of the endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). In the present study, we investigated the immune-modulating effects of environmental pollutants such as polycyclic aromatic hydrocarbons on Th17 differentiation and IL-22 production. Using Th17 cells deficient in CYP1 enzymes (Cyp1a1/1a2/1b1−/−) we show that these chemicals potentiate AHR activation through inhibition of CYP1 enzymes which leads to increases in intracellular AHR agonists. Our findings demonstrate that IL-22 production by Th17 cells is profoundly enhanced by impaired CYP1-function and strongly suggest that chemicals able to modify CYP1 function or expression may disrupt AHR-mediated immune regulation by altering the levels of endogenous AHR agonist(s).

An endogenous tryptophan photo-product, FICZ, is potentially involved in photo-aging by reducing TGF-β-regulated collagen homeostasis

BackgroundPersistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-β transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-β signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. ObjectiveTo evaluate the effects of FICZ on the TGF-β-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). MethodsQuantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-β. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. ResultsWe found that FICZ significantly inhibited the TGF-β-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-β. The inhibitory actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-β-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. ConclusionsThese findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging.

Carbidopa is an activator of aryl hydrocarbon receptor with potential for cancer therapy.

Carbidopa is used with l-DOPA (l-3,4-dihydroxyphenylalanine) to treat Parkinson's disease (PD). PD patients exhibit lower incidence of most cancers including pancreatic cancer, but with the notable exception of melanoma. The decreased cancer incidence is not due to l-DOPA; however, the relevance of Carbidopa to this phenomenon has not been investigated. Here, we tested the hypothesis that Carbidopa, independent of l-DOPA, might elicit an anticancer effect. Carbidopa inhibited pancreatic cancer cell proliferation both in vitro and in vivo Based on structural similarity with phenylhydrazine, an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1), we predicted that Carbidopa might also inhibit IDO1, thus providing a molecular basis for its anticancer effect. The inhibitory effect was confirmed using human recombinant IDO1. To demonstrate the inhibition in intact cells, AhR (aryl hydrocarbon receptor) activity was monitored as readout for IDO1-mediated generation of the endogenous AhR agonist kynurenine in pancreatic and liver cancer cells. Surprisingly, Carbidopa did not inhibit but instead potentiated AhR signaling, evident from increased CYP1A1 (cytochrome P450 family 1 subfamily A member 1), CYP1A2, and CYP1B1 expression. In pancreatic and liver cancer cells, Carbidopa promoted AhR nuclear localization. AhR antagonists blocked Carbidopa-dependent activation of AhR signaling. The inhibitory effect on pancreatic cancer cells in vitro and in vivo and the activation of AhR occurred at therapeutic concentrations of Carbidopa. Chromatin immunoprecipitation assay further confirmed that Carbidopa promoted AhR binding to its target gene CYP1A1 leading to its induction. We conclude that Carbidopa is an AhR agonist and suppresses pancreatic cancer. Hence, Carbidopa could potentially be re-purposed to treat pancreatic cancer and possibly other cancers as well.