The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of ß-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.

Baicalein ameliorates ulcerative colitis by improving intestinal epithelial barrier via AhR/IL-22 pathway in ILC3s.

Ulcerative colitis (UC) is a chronic inflammatory disease of the gastrointestinal tract, which is closely related to gut barrier dysfunction. Emerging evidence shows that interleukin-22 (IL-22) derived from group 3 innate lymphoid cells (ILC3s) confers benefits on intestinal barrier, and IL-22 expression is controlled by aryl hydrocarbon receptor (AhR). Previous studies show that baicalein protects the colon from inflammatory damage. In this study we elucidated the molecular mechanisms underlying the protective effect of baicalein on intestinal barrier function in colitis mice. Mice were administered baicalein (10, 20, 40 mg·kg-1·d-1, i.g.) for 10 days; the mice freely drank 3% dextran sulfate sodium (DSS) on D1-D7 to induce colitis. We showed that baicalein administration simultaneously ameliorated gut inflammation, decreased intestinal permeability, restored tight junctions of colons possibly via promoting AhR/IL-22 pathway. Co-administration of AhR antagonist CH223191 (10 mg/kg, i.p.) partially blocked the therapeutic effects of baicalein in colitis mice, whereas AhR agonist FICZ (1 µg, i.p.) ameliorated symptoms and gut barrier function in colitis mice. In a murine lymphocyte line MNK-3, baicalein (5-20 µM) dose-dependently increased the expression of AhR downstream target protein CYP1A1, and enhanced IL-22 production through facilitating AhR nuclear translocation, these effects were greatly diminished in shAhR-MNK3 cells, suggesting that baicalein induced IL-22 production in AhR-dependent manner. To further clarify that, we constructed an in vitro system consisting of MNK-3 and Caco-2 cells, in which MNK-3 cell supernatant treated with baicalein could decrease FITC-dextran permeability and promoted the expression of tight junction proteins ZO-1 and occluding in Caco-2 cells. In conclusion, this study demonstrates that baicalein ameliorates colitis by improving intestinal epithelial barrier via AhR/IL-22 pathway in ILC3s, thus providing a potential therapy for UC.

Endogenous AhR agonist FICZ accumulates in rainbow trout (Oncorhynchus mykiss) alevins exposed to a mixture of two PAHs, retene and fluoranthene.

Multiple studies have reported synergized toxicity of PAH mixtures in developing fish larvae relative to the additive effect of the components. From a toxicological perspective, multiple mechanisms are known to contribute to synergism, such as altered toxicodynamics and kinetics, as well as increased oxidative stress. An understudied contributor to synergism is the accumulation of endogenous metabolites, for example: the aryl hydrocarbon receptor 2 (AhR2) agonist and tryptophan metabolite 6-Formylindolo(3,2-b)carbazole (FICZ). Fish larvae exposed to FICZ, alongside knock-down of cytochrome p450 (cyp1a), has been reported to induced symptoms of toxicity similar to those observed following exposure to PAHs or the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we explored if FICZ accumulates in newly hatched rainbow trout alevins (Oncorhynchus mykiss) exposed to two PAHs with different properties: retene (potent AhR2 agonist) and fluoranthene (weak AhR2 agonist and Cyp1a inhibitor), either alone or as a binary mixture for 3 and 7 days. We found that exposure to the mixture resulted in accumulation of endogenous FICZ, synergized the blue sac disease index (BSD), and altered the body burden profiles of the PAHs, when compared to the alevins exposed to the individual components. It is thus very plausible that accumulation of endogenously derived FICZ contributed to the synergized BSD index and toxicity in exposed alevins. Accumulation of endogenously derived FICZ is a novel finding that extends our general understanding on PAHs toxicity in developing fish larvae, while at the same time highlighting why environmental risk assessment of PAHs should not be based solely results from the assessment of individual compounds.

The Aryl Hydrocarbon Receptor Ligand FICZ Improves Left Ventricular Remodeling and Cardiac Function at the Onset of Pressure Overload-Induced Heart Failure in Mice.

Adverse ventricular remodeling is the heart's response to damaging stimuli and is linked to heart failure and poor prognosis. Formyl-indolo [3,2-b] carbazole (FICZ) is an endogenous ligand for the aryl hydrocarbon receptor (AhR), through which it exerts pleiotropic effects including protection against inflammation, fibrosis, and oxidative stress. We evaluated the effect of AhR activation by FICZ on the adverse ventricular remodeling that occurs in the early phase of pressure overload in the murine heart induced by transverse aortic constriction (TAC). Cardiac structure and function were evaluated by cardiac magnetic resonance imaging (CMRI) before and 3 days after Sham or TAC surgery in mice treated with FICZ or with vehicle, and cardiac tissue was used for biochemical studies. CMRI analysis revealed that FICZ improved cardiac function and attenuated cardiac hypertrophy. These beneficial effects involved the inhibition of the hypertrophic calcineurin/NFAT pathway, transcriptional reduction in pro-fibrotic genes, and antioxidant effects mediated by the NRF2/NQO1 pathway. Overall, our findings provide new insight into the role of cardiac AhR signaling in the injured heart.

Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4',7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.

Aryl hydrocarbon receptor engagement during redox alteration determines the fate of CD4+ T cells in C57BL/6 mice.

The preservation of the redox homeostasis is critical for cell survival and functionality. Redox imbalance is an essential inducer of several pathological states. CD4+ /helper T cells are highly dependent on the redox state of their surrounding milieu. The potential of the aryl hydrocarbon receptor (AhR) engagement in controlling CD4+ T-cell fate during redox alteration is still challenging. C57BL/6 mice were treated with AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ), AhR antagonist CH223191, an inhibitor of glutathione biosynthesis buthionine sulfoximine (BSO), and the antioxidant N-acetylcysteine (NAC) alone or in combination. Six days later, splenocytes were evaluated for the expression of the redox-related genes and the possible changes in T-cell subsets. FICZ like BSO significantly elevated the expression of HMOX1, GCLC, and GCLM genes but it failed to increase the expression of the Nrf2 gene. Moreover, FICZ + BSO increased while FICZ + CH223191 or NAC decreased the expression of these genes. FICZ also significantly increased Th1 cell numbers but decreased Tregs in a dose-dependent manner. Furthermore, a high dose of FICZ + CH223191 + NAC significantly enhanced Th1, Th17, and Treg cells but its low dose in such a situation increased Th2 and Th17 while decreased Treg cells. AhR engagement during redox alteration can determine the fate of CD4 + T cells, so, AhR agonists or antagonists might be useful in assessing immune responses. However, these results need further verifications in vitro and in animal models of various diseases.

A New Insight into the Potential Role of Tryptophan-Derived AhR Ligands in Skin Physiological and Pathological Processes.

The aryl hydrocarbon receptor (AhR) plays a crucial role in environmental responses and xenobiotic metabolism, as it controls the transcription profiles of several genes in a ligand-specific and cell-type-specific manner. Various barrier tissues, including skin, display the expression of AhR. Recent studies revealed multiple roles of AhR in skin physiology and disease, including melanogenesis, inflammation and cancer. Tryptophan metabolites are distinguished among the groups of natural and synthetic AhR ligands, and these include kynurenine, kynurenic acid and 6-formylindolo[3,2-b]carbazole (FICZ). Tryptophan derivatives can affect and regulate a variety of signaling pathways. Thus, the interest in how these substances influence physiological and pathological processes in the skin is expanding rapidly. The widespread presence of these substances and potential continuous exposure of the skin to their biological effects indicate the important role of AhR and its ligands in the prevention, pathogenesis and progression of skin diseases. In this review, we summarize the current knowledge of AhR in skin physiology. Moreover, we discuss the role of AhR in skin pathological processes, including inflammatory skin diseases, pigmentation disorders and cancer. Finally, the impact of FICZ, kynurenic acid, and kynurenine on physiological and pathological processes in the skin is considered. However, the mechanisms of how AhR regulates skin function require further investigation.

Synthesis and biological evaluation of FICZ analogues as agonists of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor involved in multiple biological processes including immune cell differentiation, intestinal function and inflammation. Based on the scaffold of naturally occurring AhR ligand 6-formylindolo (3,2-b) carbazole (FICZ, 2), a series of analogues has been designed, synthesized and evaluated by cell-based assays. The structure-activity relationships study has successfully led to the discovery of compound 11e with extremely potent activity.

Selective cytochrome P450 1A1 but not 1B1 promoterCpG island DNA methylation by 6-formylindolo[3,2-b]carbazole (FICZ).

Epigenetic alterations are essential for normal mammalian development and regulation of gene expression. In this study, we aimed to determine if an enigmatic endogenous ligand of the aryl hydrocarbon receptor (AHR), 6-formylindolo[3,2-b]carbazole (FICZ), and methionine (Meth) have an epigenetic impact on AHR-regulated cytochrome P450 1A1 and B1 (CYP1A1 and CYP1B1) gene expression. Human hepatoma (HepG2-XRE-Luc and huh7) cells were exposed to FICZ in a medium with and without Meth supplementation. Selective and transient silencing of CYP1A1 but not CYP1B1 were seen by FICZ. Here we found that FICZ transiently represses CYP1A1 by targeting DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and concomitant DNA methylation of the CYP1A1 promoter gene. Treatments with 5-aza-dC augmented CYP1A1 transcription activity. Our results reveal a new mechanism for transient activation of AHR by FICZ that can negatively and positively influence gene expression, and highlight the regulatory role of Meth on the CYP1A1 gene expression.

The AHR1-ARNT1 dimerization pair is a major regulator of the response to natural ligands, but not to TCDD, in the chicken.

The activation of the aryl hydrocarbon receptor (AHR) occurs through the binding of dioxin-like compounds (DLCs) or natural ligands. In this pathway, the AHR-ARNT (AHR nuclear translocator) heterodimer serves to regulate critical physiological functions, such as immune responses and the metabolism of xenobiotics. Birds have three AHR isoforms (AHR1, AHR1ß, and AHR2) and two ARNT isoforms (ARNT1 and ARNT2). However, how AHR and ARNT dimerization pair in birds regulates the AHR signaling pathway in an isoform-specific manner remains unknown. In this study, we initially sought to clarify the major chicken AHR-ARNT (ckAHR-ckARNT) pairs by estimating the mRNA tissue distributions of various ckAHR and ckARNT isoforms. Our results indicated that the ckAHR1-ckARNT1 represented the major dimerization pair in most tissues except the brain. We then measured the transactivation potencies of various ckAHR-ckARNT pairs by natural ligands and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in in vitro reporter gene assays using COS-7 and LMH cell lines. Our results from the in vitro assays demonstrated that the ckAHR1-ckARNT1 pair was strongly activated by the five natural ligands, namely, 6-formylindolo [3,2-b]carbazole, L-kynurenin, kynurenic acid, indoxyl-3-sulfate, and 1,3,7-tribromodibenzo-p-dioxin, but not by TCDD. In in silico ligand docking simulations with ckAHR1 homology models, all the natural ligands showed a interaction pattern that was distinct from that observed with anthropogenic DLCs, including TCDD. In conclusion, our findings indicate that the ckAHR1-ckARNT1 may be the most important dimerization pair in most tissues for regulating the physiological functions driven by natural ligands, although it was less reactive to TCDD.

Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells-In Vitro Studies.

Tryptophan metabolites: kynurenine (KYN), kynurenic acid (KYNA) and 6-formylindolo[3,2-b]carbazole (FICZ) are considered aryl hydrocarbon receptor (AhR) ligands. AhR is mainly expressed in barrier tissues, including skin, and is involved in various physiological and pathological processes in skin. We studied the effect of KYN, KYNA and FICZ on melanocyte and melanoma A375 and RPMI7951 cell toxicity, proliferation and cell death. KYN and FICZ inhibited DNA synthesis in both melanoma cell lines, but RPMI7951 cells were more resistant to pharmacological treatment. Tested compounds were toxic to melanoma cells but not to normal human adult melanocytes. Changes in the protein level of cyclin D1, CDK4 and retinoblastoma tumor suppressor protein (Rb) phosphorylation revealed different mechanisms of action of individual AhR ligands. Importantly, all tryptophan metabolites induced necrosis, but only KYNA and FICZ promoted apoptosis in melanoma A375 cells. This effect was not observed in RPMI7951 cells. KYN, KYNA and FICZ in higher concentrations inhibited the protein level of AhR but did not affect the gene expression. To conclude, despite belonging to the group of AhR ligands, KYN, KYNA and FICZ exerted different effects on proliferation, toxicity and induction of cell death in melanoma cells in vitro.

Aryl hydrocarbon receptor activation alleviates dextran sodium sulfate-induced colitis through enhancing the differentiation of goblet cells.

BACKGROUND: The intestinal inflammation induces disruption of the intestinal barrier function and leads to bacteria invasion. Accumulating evidences revealed that the aryl hydrocarbon receptor (AhR) plays a vital role in maintaining the intestinal barrier function. However, the precise mechanism remains to be unclear. METHODS: Adult C57BL/6J mice were randomly divided into three groups: Sham, DSS and DSS + 6-formylindolo (3, 2-b) carbazole (FICZ)group. The colons and epithelial cell were harvested for histological examination, pro-inflammatory cytokines detection, bacterial load analysis, immunohistochemistry and Muc2 protein analysis. Under physiological condition, AhRKO model and FICZ treatment were used to evaluate the roles of AhR in the differentiation of goblet cells and the expression of Muc2 in mice. In vitro, we used HT29 mol to research the signaling pathway. RESULTS: AhR activation by FICZ could increase the Muc2 expression and the number of goblet cells and reduce bacterial infiltration to ameliorate DSS-induced Colitis. Under physiological conditions, the treatment of FICZ promote the differentiation of goblet cell and the expression of Muc2 and inhibit the notch-signaling. Genetic deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression and enhance the notch-signaling. In HT29 cells, the differentiation of goblet cell meditated by AhR can be abolished by the inhibitor of AhR, pErk1/2 and knocking-down AhR. CONCLUSION: FICZ promoted the differentiation of goblet cell through AhR-pErk1/2 signaling pathway and ameliorate DSS-induced Colitis.

Protective roles of FICZ and aryl hydrocarbon receptor axis on alveolar bone loss and inflammation in experimental periodontitis.

AIM: The aryl hydrocarbon receptor (AhR)-ligand axis has been shown to be involved in inflammatory diseases and bone homeostasis. However, the activation of AhR signalling pathway and the possible functions of AhR ligands in periodontitis are underexplored. This study investigated the expression of the AhR target gene cytochrome P450 subfamily B member 1 (CYP1B1) and the functions and mechanisms of the AhR ligand 6 formylindolo[3,2-b]carbazole (FICZ) in periodontitis. MATERIALS AND METHODS: CYP1B1 expression was detected in human periodontitis samples, mice with ligature-induced periodontitis and lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs) in vitro. FICZ was administered topically or systemically. The therapeutic functions of FICZ were detected via qPCR, micro-computed tomography and immunohistochemistry. Finally, the mechanisms of AhR signalling in periodontitis were investigated by cell assays. RESULTS: CYP1B1 expression was downregulated in periodontitis. FICZ rescued the alveolar bone loss and mitigated the inflammatory cytokines in periodontitis mice. In vitro, FICZ pre-treatment reduced the LPS-induced inflammation in PDLCs via the increased phosphorylation of STAT3. Additionally, FICZ prompted the mineralization of PDLCs via activation of the Wnt/ß-catenin signalling pathway. CONCLUSION: AhR signalling pathway is suppressed in periodontitis and the AhR ligand FICZ can prevent periodontitis.

Direct activation of aryl hydrocarbon receptor by benzo[a]pyrene elicits T-helper 2-driven proinflammatory responses in a mouse model of allergic dermatitis.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that binds to various environmental chemicals and contributes to numerous toxicological effects. However, the direct effects of AhR on the development of allergic diseases are not fully understood. The main aim of this study was to elucidate the action of AhR in the development of cutaneous allergies. Initially, the potential for a direct relationship between AhR and the immune cells was investigated in vitro, using murine bone marrow-derived dendritic cells, human epidermal keratinocytes, and the mixed leukocyte reaction assay. Benzo[a]pyrene (BaP) and 6-formylindolo[3,2-b]carbazole were used as selective ligands for the AhR. Pretreatment with BaP and/or 6-formylindolo[3,2-b]carbazole significantly induced cytokine release by activated keratinocytes and T-cell proliferation, whereas interleukin-12 production in bone marrow-derived dendritic cells was reduced by AhR activation. To confirm the in vitro results, in vivo experiments were also performed in T-helper (Th)2-type hapten toluene-2,4-diisocyanate- and Th1-type hapten dinitrochlorobenzene-induced mouse models of allergic dermatitis. Mice were orally administered BaP at 48, 24 and 4 hours before the final allergen challenge. In the Th2 model, ear-swelling response and scratching behavior were promoted by BaP exposure, which supported the observed significant increases in local cytokine secretion. The infiltration of helper T cells, B cells and dendritic cells into the auricular lymph node was significantly enhanced by BaP administration, although Th1-type immune responses were not influenced by AhR activation. Our findings demonstrate that AhR activation directly activates keratinocytes and T cells, which leads to the exacerbation of Th2-type cutaneous allergy.

GSH/GSSG redox couple plays central role in aryl hydrocarbon receptor-dependent modulation of cytochrome P450 1A1.

The redox regulation of aryl hydrocarbon receptor (AHR) target genes such as the best characterized, cytochrome P450 1A1 (CYP1A1) has not been known. Therefore the aim of this study was to explore how cellular redox state can influence on AHR-dependent modulation of CYP1A1 transcription and enzyme activities. Male BALB/c albino mice, HepG2 cells, and human hepatoma cell line (HepG2-XRE-Luc) carrying CYP1A1 response elements were exposed to suggested endogenous ligand of AHR,6-formylindolo[3,2-b] carbazole (FICZ) alone or in combination with, buthionine-(S,R)-sulfoximine (BSO) or N-acetyl-l-cysteine (NAC). A clear link between CYP1A1 transcription and enzyme activity and changes in the glutathione/oxidised glutathione (GSH/GSSG) redox couple was shown. In vivo and in vitro findings demonstrated that the time course of AHR activation/inhibition is characterized by an increase/decrease in the GSH/GSSG ratio. Based on these findings, we propose that many environmental pollutants and oxidants by alteration in the intracellular redox potential may interfere with the normal function of AHR target genes.

The highly bioactive molecule and signal substance 6-formylindolo[3,2-b]carbazole (FICZ) plays bi-functional roles in cell growth and apoptosis in vitro.

The maintenance of cellular homeostasis is a complex process that is governed by the receipt of prototypical growth and death signals. The endogenous functions of aryl hydrocarbon receptor (AHR) in cellular homeostasis are not well understood. We aimed to establish whether the disturbance of endogenously activated AHR can influence cell growth, and if so, what mechanism(s) are involved. Cell growth was measured in mouse hepatoma Hepa-1 wild-type and cytochrome P4501A1 (CYP1A1)-deficient c37 cells. In other sets of experiments, HepG2 cells were exposed to different doses of FICZ (0.01nM-1 µM) alone or in combination with 50 nM of the CYP1A1 inhibitor 3'methoxy-4'nitro-flavone (MNF). CYP1A1 enzyme activity, cell viability, oxidative stress, and several endpoints of apoptosis were measured. FICZ treatment at a high concentration or in combination with MNF induced sustained CYP1A1 activity and led to oxidative stress and activation of apoptosis via a mitochondrial-dependent pathway. In comparison with the wild-type Hepa-1 cells, c37 cells lacking CYP1A1 activity proliferated faster in normal medium which contains trace levels of FICZ. Besides, in HepG2 cells, FICZ stimulated cell growth at low concentrations but inhibited cell growth at high concentrations. Based on these findings, we propose that CYP1A1 inhibitors, by increasing the levels of the endogenous ligand FICZ, change the cell growth kinetics and trigger cell death and apoptosis through a mitochondrial-dependent pathway. Since AHR controls multiple cellular functions, a wide range of toxicity can be expected by disturbing its endogenous functions.

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation.

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.

Novel role of hnRNP-A2/B1 in modulating aryl hydrocarbon receptor ligand sensitivity.

The aryl hydrocarbon receptor (AHR) is responsible for susceptibility to its ligand-dependent responses. However, the effect of non-AHR factors is less clear. To explore the non-AHR factors, we used two mouse strains with different AHR genetic variants, namely C3H/lpr and MRL/lpr strains with Ala and Val as the 375th amino acid residue, respectively. To assess the contribution of AHR alone, COS-7 cells transiently expressing AHR from each strain were treated with 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and xenobiotic-responsive element (XRE)-driven reporter gene activities were measured. FICZ-EC50 values for the C3H/lpr and MRL/lpr AHR-mediated transactivation were 0.023 and 0.046 nM, respectively, indicating a similar susceptibility in both AHR genotypes. In contrast, C3H/lpr AHR was fourfold more sensitive to TCDD than MRL/lpr AHR. By a pull-down assay using a XRE-containing PCR product as bait and the hepatic nuclear extracts of both FICZ-treated mouse strains, we identified two interacting proteins as heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2) and its splicing variant (hnRNP-A2b). Immunoprecipitation assays demonstrated the AHR interaction with hnRNP-A2/B1. When hnRNP-A2 was co-expressed with the MRL/lpr or C3H/lpr AHR in COS-7, FICZ treatment decreased EC50 to about threefold in both AHR genotypes, compared with EC50 in AHR alone. Similarly, hnRNP-A2b co-expression also lowered the FICZ-EC50 values. In TCDD-treated COS-7, responses depended on the AHR genotype; while no change in TCDD-EC50 was observed for C3H/lpr AHR when hnRNP-A2 was co-expressed, the value was reduced to nearly tenfold for MRL/lpr AHR. Co-transfection with hnRNP-A2b attenuated the AHR sensitivity to TCDD. In conclusion, the hnRNP-A2/B1 interacting with AHR may be a modulator of the AHR ligand sensitivity.

Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte-derived dendritic cells.

Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1ß, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1ß, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases.