Aline Chevallier

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.

Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity

Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.

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-β 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.

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.

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.

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 expressedin 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.

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

Significance Aryl hydrocarbon receptor (AHR) is well known to mediate xenobiotic metabolism in vertebrates. Growing evidence reveals that AHR seems to have endogenous roles in the development and functioning of different organs. In our current study, we describe a role of AHR in peripheral myelination and in nerve sheath tumors. We show that the AHR pathway is dysregulated in human biopsies of nerve tumors. The blockade of AHR provokes cell death in nerve tumors, suggesting a therapeutic avenue in the treatment of this invasive cancer. Furthermore, the inhibition of Ahr in mice provokes locomotor defects and alteration of myelin structure. This work unravels an endogenous role of Ahr in peripheral myelination and a potential treatment of nerve tumours. 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 β-catenin levels and stimulated the binding of β-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.