Identification of Modulators of the C. elegans Aryl Hydrocarbon Receptor and Characterization of Transcriptomic and Metabolic AhR-1 Profiles.

The Aryl hydrocarbon Receptor (AhR) is a xenobiotic sensor in vertebrates, regulating the metabolism of its own ligands. However, no ligand has been identified to date for any AhR in invertebrates. In C. elegans, the AhR ortholog, AHR-1, displays physiological functions. Therefore, we compared the transcriptomic and metabolic profiles of worms expressing AHR-1 or not and investigated the putative panel of chemical AHR-1 modulators. The metabolomic profiling indicated a role for AHR-1 in amino acids, carbohydrates, and fatty acids metabolism. The transcriptional profiling in neurons expressing AHR-1, identified 95 down-regulated genes and 76 up-regulated genes associated with neuronal and metabolic functions in the nervous system. A gene reporter system allowed us to identify several AHR-1 modulators including bacterial, dietary, or environmental compounds. These results shed new light on the biological functions of AHR-1 in C. elegans and perspectives on the evolution of the AhR functions across species.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr-/- mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr-/- mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased ß-catenin levels and stimulated the binding of ß-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

AhR-deficiency as a cause of demyelinating disease and inflammation.

The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.

Regulation of Aquaporin 3 Expression by the AhR Pathway Is Critical to Cell Migration.

The regulation of cell migration is a key factor for the dissemination of metastatic cells during tumor progression. Aquaporins are membrane channels which allow transmembrane fluxes of water and glycerol in cells in a variety of mammalian tissues. Here, we show that AQP3, which has been incriminated in cancer progression, is regulated by the AhR, or dioxin receptor. AhR is a transcription factor which is triggered in response to environmental pollutants and it has been shown to regulate several cellular processes including cell migration and plasticity. In vivo, upon exposure to the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the expression of AQP3 is increased significantly in several murine tissues including the liver. In vitro, treatment of human HepG2 cells with TCDD also increased the expression of AQP3 mRNA and protein. These effects resulted from the activation of AhR as shown by RNA interference, chromatin immunoprecipitation and the use of several AhR ligands. Immunofluorescence and real-time analysis of cell migration (XCelligence) demonstrated that knockdown of AQP3 mRNA using small interfering RNA impairs the remodeling of cell shape and the triggering of cell migration that is induced by TCDD. Our work reveals, for the first time, a link between exposure to pollutant and the induction of an aquaporin which has been suspected to play a role during metastasis.

Aryl hydrocarbon receptor-dependent induction of liver fibrosis by dioxin.

The contribution of environmental pollutants to liver fibrosis is an important and poorly explored issue. In vitro studies suggest that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands induce several genes that are known to be upregulated during liver fibrosis. Our aim was to determine whether exposure to such pollutants can lead to liver fibrosis and to characterize the mechanisms of action. Mice were treated for 2, 14, or 42 days, once a week with 25 µg/kg of TCDD. Gene and protein expression, in vitro and in vivo, as well as liver histology were investigated for each treatment. Treatment of mice with TCDD for 2 weeks modified the hepatic expression of markers of fibrosis such as collagen 1A1 and α-smooth muscle actin. This is not observed in AhR knockout mice. Following 6 weeks of treatment, histological features of murine hepatic fibrosis became apparent. In parallel, the levels of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor α) and of markers of activated fibroblasts(fibroblast-specific protein 1) were found to be upregulated. Interestingly, we also found increased expression of genes of the TGF-ß pathway and a concomitant decrease of miR-200a levels. Because the transcription factors of the Snail family were shown to be involved in liver fibrosis, we studied their regulation by TCDD. Two members of the Snail family were increased, whereas their negative targets, the epithelial marker E-cadherin and Claudin 1, were decreased. Further, the expression of mesenchymal markers was increased. Finally, we confirmed that Snai2 is a direct transcriptional target of TCDD in the human hepatocarcinoma cell line, HepG2. The AhR ligand, TCDD, induces hepatic fibrosis by directly regulating profibrotic pathways.

Oculomotor deficits in aryl hydrocarbon receptor null mouse.

The Aryl hydrocarbon Receptor or AhR, a ligand-activated transcription factor, is known to mediate the toxic and carcinogenic effects of various environmental pollutants such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Recent studies in Caenorhabditis elegans and Drosophila melanogaster show that the orthologs of the AhR are expressed exclusively in certain types of neurons and are implicated in the development and the homeostasis of the central nervous system. While physiological roles of the AhR were demonstrated in the mammalian heart, liver and gametogenesis, its ontogenic expression and putative neural functions remain elusive. Here, we report that the constitutive absence of the AhR in adult mice (AhR-/-) leads to abnormal eye movements in the form of a spontaneous pendular horizontal nystagmus. To determine if the nystagmus is of vestibular, visual, or cerebellar origin, gaze stabilizing reflexes, namely vestibulo-ocular and optokinetic reflexes (VOR and OKR), were investigated. The OKR is less effective in the AhR-/- mice suggesting a deficit in the visuo-motor circuitry, while the VOR is mildly affected. Furthermore, the AhR is expressed in the retinal ganglion cells during the development, however electroretinograms revealed no impairment of retinal cell function. The structure of the cerebellum of the AhR-/- mice is normal which is compatible with the preserved VOR adaptation, a plastic process dependent on cerebellar integrity. Finally, intoxication with TCDD of control adults did not lead to any abnormality of the oculomotor control. These results demonstrate that the absence of the AhR leads to acquired central nervous system deficits in the adults. Given the many common features between both AhR mouse and human infantile nystagmus syndromes, the AhR-/- mice might give insights into the developmental mechanisms which lead to congenital eye disorders.

The AhR twist: ligand-dependent AhR signaling and pharmaco-toxicological implications.

The aryl hydrocarbon receptor (AhR) is a transcription factor which is activated by diverse compounds and regulates the expression of xenobiotic metabolism genes. Recent studies have unraveled unsuspected physiological roles and novel alternative ligand-specific pathways for this receptor. In this review, we discuss these novel aspects and focus on the different responses elicited by the diverse endogenous and/or exogenous AhR ligands. In addition to challenging the relevance of the 'agonist/antagonist' classification of ligands, we introduce the concept of AhR plasticity as a primordial factor in the generation of these pathways. Finally, we suggest several promising perspectives for the pharmacological modulation of these responses.

Inflammatory pathway genes belong to major targets of persistent organic pollutants in adipose cells.

BACKGROUND: Epidemiological studies emphasize the possible role of persistent organic pollutants (POPs) in obesity and the metabolic syndrome. These pollutants are stored in adipose tissue (AT). OBJECTIVES: Our aim was to study the effects of POPs on human adipose cells and rodent AT. METHODS: Using human multipotent adipose-derived stem cells, we carried out large-scale gene expression analysis to identify the major pathways modified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl (PCB) congener 126 (PCB-126), and PCB-153 and to evaluate their toxic effects. The effects of TCDD on gene expression and AT histology were also assessed in mice. RESULTS: The most significantly regulated genes in both precursor cells and adipocytes were those involved in the inflammatory/immune response, cancer, and metabolism pathways. Interestingly, the fold induction and the number of modulated genes were higher in precursors than in adipocytes, suggesting that the former could be more sensitive to the effect of pollutants. When cells were treated with combinations of pollutants, the effects of the AhR ligands TCDD and PCB-126 were dominant compared with those of the non-dioxin-like PCB-153. The effects of AhR ligands were reduced by the AhR antagonist α-naphthoflavone. The regulation of inflammatory pathway was observed in wild-type AT but not in AhR-knockout mice. CONCLUSIONS: Both in vitro and in vivo studies showed that adipose cells were targets of AhR ligands and suggest that inflammation is one of the main regulated pathways. These observations suggest a possible contribution of pollutants to low-grade AT inflammation that accompanies the pathogenesis of metabolic diseases.

Induction of the Ras activator Son of Sevenless 1 by environmental pollutants mediates their effects on cellular proliferation.

TCDD (2,3,7,8-tetrachlorodibenzodioxin), a highly persistent environmental pollutant and a human carcinogen, is the ligand with the highest affinity for the Aryl Hydrocarbon Receptor (AhR) that induces via the AhR, xenobiotic metabolizing enzyme genes as well as several other genes. This pollutant elicits a variety of systemic toxic effects, which include cancer promotion and diverse cellular alterations that modify cell cycle progression and cell proliferation. Large-scale studies have shown that the expression of Son of Sevenless 1 (SOS1), the main mediator of Ras activation, is one of the targets of dioxin in human cultured cells. In this study, we investigated the regulation of the previously uncharacterized SOS1 gene promoter by the AhR and its ligands in the human hepatocarcinoma cell line, HepG2. We found that several environmental pollutants (AhR ligands) induce SOS1 gene expression by increasing its transcription. Chromatin immunoprecipitation experiments demonstrated that the AhR binds directly and activates the SOS1 gene promoter. We also showed that dioxin treatment leads to an activated Ras-GTP state, to ERK activation and to accelerated cellular proliferation. All these effects were mediated by SOS1 induction as shown by knock down experiments. Our data indicate that dioxin-induced cellular proliferation is mediated, at least partially, by SOS1 induction. Remarkably, our studies also suggest that SOS1 induction leads to functional effects similar to those elicited by the well-characterized oncogenic Ras mutations.

2,3,7,8-tetrachlorodibenzo-p-dioxin counteracts the p53 response to a genotoxicant by upregulating expression of the metastasis marker agr2 in the hepatocarcinoma cell line HepG2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that binds the aryl hydrocarbon receptor (AhR), a transcription factor that triggers various biological responses. In this study, we show that TCDD treatment counteracts the p53 activation (phosphorylation and acetylation) elicited by a genotoxic compound, etoposide, in the human hepatocarcinoma cell line HepG2 and we delineated the mechanisms of this interaction. Using small interfering RNA knockdown experiments, we found that the newly described metastasis marker, anterior gradient-2 (AGR2), is involved in this effect. Both AGR2 messenger RNA (mRNA) and protein levels were increased (sixfold and fourfold, respectively) by TCDD treatment, and this effect was mediated by the AhR receptor. The half-life of AGR2 mRNA was unchanged by TCDD treatment. Analysis of the promoter of the AGR2 gene revealed three putative xenobiotic-responsive elements (XREs) in the proximal 3.5-kb promoter. Transient transfection of HepG2 cells by the Gaussia luciferase reporter gene driven by various deleted and mutated fragments of the promoter indicated that only the most proximal XRE was active. Binding of the AhR to the endogenous AGR2 promoter was also triggered by TCDD treatment. These results suggest that AhR ligands such as TCDD might contribute to tumor progression by inhibiting p53 regulation (phosphorylation and acetylation) triggered by genotoxicants via the increased expression of the metastasis marker AGR2.