Xenobiotic-sensing nuclear receptors involved in drug metabolism: a structural perspective

Xenobiotic compounds undergo a critical range of biotransformations performed by the phase I, II, and III drug-metabolizing enzymes. The oxidation, conjugation, and transportation of potentially harmful xenobiotic and endobiotic compounds achieved by these catalytic systems are significantly regulated, at the gene expression level, by members of the nuclear receptor (NR) family of ligand-modulated transcription factors. Activation of NRs by a variety of endo- and exogenous chemicals are elemental to induction and repression of drug-metabolism pathways. The master xenobiotic sensing NRs, the promiscuous pregnane X receptor and less-promiscuous constitutive androstane receptor are crucial to initial ligand recognition, jump-starting the metabolic process. Other receptors, including farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4 alpha, peroxisome proliferator activated receptor, glucocorticoid receptor, liver X receptor, and RAR-related orphan receptor, are not directly linked to promiscuous xenobiotic binding, but clearly play important roles in the modulation of metabolic gene expression. Crystallographic studies of the ligand-binding domains of nine NRs involved in drug metabolism provide key insights into ligand-based and constitutive activity, coregulator recruitment, and gene regulation. Structures of other, noncanonical transcription factors also shed light on secondary, but important, pathways of control. Pharmacological targeting of some of these nuclear and atypical receptors has been instituted as a means to treat metabolic and developmental disorders and provides a future avenue to be explored for other members of the xenobiotic-sensing NRs

Impacts de concentrations supraphysiologiques d'acides biliaires sur la physiologie testiculaire et les fonctions de reproduction

Clinical data describe an association between liver diseases and disorders of male fertility. Several experimental models of cholestasis have confirmed this link and highlight an impact on testicular physiology. Interestingly, such correlation exists in adult as well as in during pre-pubertal animals. However, the molecular links have not been explored yet. The increase of plasma bile acids levels is a common feature of liver diseases. In this context, the hypothesis of the deleterious impact of bile acids on reproductive function remains to be defined. For that purpose, we used a mouse model of liver injury induced by a diet supplemented with cholic acid. Main results show that: 1) supra-physiological activation of Fxra, during pubertal period, alters endocrine function of the testis and then sexual maturation. 2) during adult age excessive activation of membrane receptor TGR5 by bile acids leads to subfertility. This is associated with impaired spermatogenesis due to a detachment of the seminiferous epithelium and specific apoptosis of spermatids. 3) Finally, we show for the first time the transgenerational impact of bile acid exposure. Two generations of progenies from males exposed to bile acid-diet show developmental and metabolic abnormalities. These effects, mediated by TGR5, are correlated with alterations of the spermatozoa epigenome. In conclusion, our data demonstrate that bile acids affect reproductive functions with impacts on testicular functions. In line with the increasing number of people with liver diseases, the deleterious effects of bile acids may contribute to the incidence of male infertility. Interestingly, agonists of FXRα and TGR5 are now considered in the treatment of several diseases. In this context, our study might alert health authorities regarding the potential consequences of these treatments on fertility and health futures generations.Chez l’homme, des données cliniques décrivent une association entre des pathologies hépatiques et des désordres de la fertilité masculine. Plusieurs modèles expérimentaux de cholestase ont permis de confirmer ce lien et de souligner un impact sur la physiologie testiculaire. De manière intéressante, une telle corrélation existe aussi bien à l’âge adulte que dans des modèles animaux en période pré-pubertaire. Pour autant, le lien moléculaire pouvant expliquer cette association physiopathologique n’a pas été exploré. L’ensemble des hépatopathies a pour dénominateur commun une augmentation des taux plasmatiques d’acides biliaires et ce dès les stades les plus précoces de la maladie. Dans ce contexte, l’hypothèse de l’impact délétère des acides biliaires sur la fonction reproductrice reste à définir. Notre projet de recherche s’articule autour de l’analyse d’un modèle murin d’atteinte hépatique induite par un régime supplémenté en acide cholique. Nos résultats principaux montrent que : 1) lors d’une exposition pubertaire, l’activation supra-physiologique des signalisations Fxrα conduit à un défaut de maturation sexuelle associé à une altération de la fonction endocrine du testicule ; 2) dans un contexte d’exposition à l’âge adulte, l’activation excessive du récepteur membranaire Tgr5 par les acides biliaires est associées à une hypofertilité. Celle-ci s’accompagne d’une altération de la spermatogenèse consécutive à un détachement progressif de l’épithélium séminifère et à une apoptose spécifique des spermatides ; 3) enfin, nos conclusions démontrent pour la première fois l’impact transgénérationnel de l’exposition aux acides biliaires. Sur deux générations successives, les descendants des mâles adultes nourris par un régime supplémenté en acide cholique présentent des anomalies développementales et métaboliques. Dépendantes de l’action de Tgr5, ces dernières sont attribuées à des altérations de l’épigénome des spermatozoïdes issus des mâles exposés aux acides biliaires. En conclusion, nos données démontrent que, dans des conditions cholestatiques, les acides biliaires altèrent les fonctions de reproduction notamment par leurs impacts sur les fonctions testiculaires. Au regard du nombre croissant de personnes souffrant de troubles hépatiques, ces effets délétères des acides biliaires pourraient contribuer à l’augmentation de l’incidence de l’infertilité masculine. Des molécules agonistes des signalisations FXRα et TGR5 sont aujourd’hui envisagées dans le cadre du traitement de pathologies courantes de notre société. Dans ce contexte, notre étude permettra d’alerter les instances sanitaires quant aux conséquences de l’accès à de tels traitements sur la fertilité et la santé des générations futures

Modulation of hepatic lipoprotein metabolism by dietary procyanidins

INTRODUCTIONDuring the past decade, Nutrition research has been subjected to a shift of focus, from epidemiology and physiology to the comprensión of the molecular basis of nutrients actions.Thus, the new "-omics" disciplines transcriptomics, proteomics or metabolomics, provide the tools to understand the molecular mechanisms involved in the modulation of gene expression by nutrients. The study of the beneficial properties of wine procyanidins has not avoided this shift of focus. Thus, from the initial studies which defined the "French paradox", to nowadays, a wide array of studies have been focused in defining the properties of the non-alcoholic components of red wine, mainly flavonoids, a family of polyphenolic compounds. The objectives of this thesis have been to define the molecular mechanisms by which grape procyanidins modulate the hepatic metabolism of lipoproteins, reducing the risk of cardiovascular disease and other pathologies which basis is found in the dysregulation of the lipoprotein metabolism.SUMMARYIn the present thesis, the effect of procyanidins in the hepatic lipoprotein metabolism has been studied. With this objective, HepG2, HeLa and CV-1 cells have been used as invitro models. In vivo studies have been performed in Wistar rats and C57BL6 mice, wild-type and transgenic mice lacking SHP (NR0B2) and FXR (NR5H1).RESULTS1. Procyanidins improve plasma lipid profile in the postprandial phase in rats. A single oral dose of procyanidins decreases plasma triglycerides and ApoB levels to 50% of control values. In addition LDL-Cholesterol is significantly reduced, thus improving the atherosclerotic risk index.2. Procyanidins display a triglyceride-lowering effect both in vivo and in vitro. In rat and mouse, procyanidin treatment triggers a hypotriglyceridemic response. In HepG2 cultures, procyanidins down-regulate the secretion of triglycerides and ApoB, thus showing that these flavonoids act directly on hepatic cells. This fact strongly suggests that, in vivo, a direct action of procyanidins on the liver contributes to their hypotriglyceridemic response.3. Nuclear receptor Small Heterodimer Partner (SHP) is a target of procyanidins in hepatic cells. Procyanidins modulate the expression of SHP, rapidly increasing its expression in rat liver as well as in HepG2 cultured cells.4. SHP mediates the triglyceride-lowering activity of procyanidins in vitro and in vivo. When SHP expression is silenced in HepG2 or abolished in SHP-null mice, procyanidins lose their hypotriglyceridemic activity. In contrast, in SHP-silenced HepG2 cells, procyanidins are still able to reduce apoB secretion. Hence, procyanidins reduce triglyceride via a SHP-dependent mechanism, whereas they reduce apoB in a SHPindependent manner.5. Nuclear receptor Farnesoid X Receptor (FXR) is an essential mediator of the hypotriglyceridemic action of procyanidins upstream SHP. Oral gavage of procyanidins to FXR-null mice have not a hypotriglyceridemic effect. Moreover, luciferase based in vitro assays showed that procyanidins increase the transcriptional activity of FXR. Thus, FXR is an essential component of the signalling pathway used by procyanidins to elicit the triglyceride lowering effect.6. Key genes of the inflammation process are targets of procyanidins in liver, in the postprandial phase. Oral administration of procyanidins to rats rapidly downregulates the expression, in liver, of transcription factor Egr1, a mediator of the hepatic inflammatory response, and several acute-phase proteins, namely haptoglobin, fibrinogen B and alpha-1 antitrypsin. In addition, expression of DUSP6, a component of the ERK1/2 subfamily of MAPK, is repressed by this treatment. Nfkbia, a repressor of NF-kB activity, is overexpressed upon procyanidin treatment. This expression pattern strongly suggests that procyanidins attenuate the pro-inflammatory state associated to the postprandial phase.INTRODUCCIÓNDurante la pasada década, la investigación en nutrición se ha visto sujeta a un cambio en sus objetivos, pasando de los estudios basados en la fisiología y la epidemiología a la comprensión de las bases moleculares implicadas en las acciones biológicas de los nutrientes. Así, las nuevas disciplinas, como la biología molecular o las "-omics", transcriptómica, proteómica o metabolómica, proporcionan las herramientas para el estudio de los mecanismos moleculares implicados en la modulación génica por nutrientes.El estudio de las propiedades beneficiosas del vino no ha evitado este cambio de foco. Así, desde los primeros estudios que definieron la "paradoja francesa", hasta la actualidad, una ámplia gama de estudios se han dedicado a definir las propiedades de los componentes no alcohólicos del vino, mayoritariamente, los Flavonoides, una familia de compuetos polifenólicos. El objetivo de esta tesis ha sido definir los mecanismos moleculares mediante los cuales las procianidinas de uva modulan el metabolismo de lipoproteínas en el hígado, disminuyendo así el riesgo cardiovascular y diferentes patologías cuya base se encuentra en la desregulación del metabolismo lipoproteico.MEMORIADurante esta tesis se ha estudiado el efecto de las procianidinas sobre el metabolismo lipoproteico en el hígado. Con este objetivo se han usado líneas celulares como modelo in vitro, tanto hepatocitos (HepG2) como líneas accesorias (HeLa y CV-1). Como modelos para el estudio de las procianidinas in vivo se han usado ratas de la cepa Wistar y ratones de la cepa C57BL6, tanto wild-type como dos líneas de transgénicos, Knockout para SHP (NR0B2) y FXR (NR5H1).RESULTADOSSe han obtenido los siguientes resultados:Las procianidinas de uva disminuyen los niveles de lipoproteínas ricas en triglicéridos, así como mejoran los índices de riesgo cardiovascular en ratas.Estos efectos se deben a la modulación de la expresión génica en el hígado, tejido adiposo y músculo entre otras acciones.El mecanismo por el cual las procianidinas disminuyen las lipoproteínas ricas en triglicéridos ha sido estudiado in Vitro (HepG2) e in vivo (C57BL6 wild-type y knockout para SHP). Se han definido dos mecanismos principales. El primero implica la señalización de las procianidinas por una vía dependiente de SHP (Small heterodimer partner, NR0B2), un receptor nuclear. El segundo mecanismo es independiente de SHP e inhibe la expresión de MTP (enzima controlador de la síntesis de lipoproteínas) y consecuente secreción de un menor número de lipoproteínas de muy baja densidad (VLDL).Por encima de SHP, se ha definido FXR (Farnesoid X receptor) como sensor de las procianidinas mediante el uso de ratones C57BL6 KO para FXR y sistemas reporter basados en luciferasa. Estableciendo que el mecanismo de señalización de las procianidinas pasa por FXR, que a su vez induce la expresión de SHP y este inhibe la expresión de SREBP1, factor de transcripción clave para la síntesis de lípidos, disminuyendo así la cantidad de lípidos hepáticos y, consecuentemente, la secreción de lipoproteínas.DISCUSIÓNLa modulación del metabolismo de lipoproteínas es el principal objetivo para el tratamiento de las diferentes patologías relacionadas con dislipemias. Así, la definición de las procianidinas de uva como agentes hipolipidémicos, las convierte en un componente de la dieta de alta importancia para prevenir y mejorar una ámplia gama de patologías, desde la aterogénesis hasta otros estados metabólicos alterados, causantes de la resistencia a la insulina o el síndrome metabólico.Por otro lado, el establecimiento de los mecanismos moleculares implicados en los efectos de las procianidinas de uva, aumenta el conocimiento sobre estos compuestos, así como su aplicabilidad en diferentes estados metabólicos alterados. De esta manera, se ha propuesto que la activación de FXR podría usarse como una estrategia en el tratamiento de la hiperlipidemia o la resistencia a la insulina. Así, las procianidinas emergen como un importante agente terapéutico, cuya importancia radica en la amplia presencia de estos compuestos en la dieta

Caractérisation de l’activité Nur77 et de ses complexes en essais BRET par complémentation

Dans le système nerveux central, la dopamine joue un rôle crucial dans de nombreuses fonctions physiologiques telles que : l’apprentissage, le mouvement volontaire, la motivation, la cognition et la production hormonale. Il a été aussi démontré que le système de signalisation dopaminergique est altéré dans plusieurs maladies neurologiques et psychiatriques comme la maladie de Parkinson et la schizophrénie. Des études, effectuées dans le laboratoire du Dr.Daniel Lévesque (laboratoire d’accueil), ont montré que les récepteurs nucléaires Nur77 (NR4A1, NGFI-B) et RXRγ (retinoid X receptors γ) sont impliqués dans la régulation des effets de la dopamine dans le système nerveux central. De plus, ces données suggèrent que le complexe Nur77 et RXR joueraient un rôle crucial dans l’effet des médicaments antipsychotiques et antiparkinsoniens. Toutefois, très peu de médicaments ciblant Nur77 ont été identifiés à ce jour et les médicaments agissant sur RXRγ restent mal caractérisés. En outre, les analyses actuellement disponibles ne peuvent pas résumer la complexité des activités des NRs et génèrent des mesures indirectes des activités des drogues. Afin de mieux comprendre comment est régulée l’interaction Nur77/RXRγ dans ces processus, mon projet a été de mettre au point un essai BRET (Bioluminescence Resonance Energy Transfer) et PCA-BRET (Protein Complementation Assay-BRET) basé sur le recrutement d'un motif mimant un co-activateur fusionné avec la YFP. Nos différents essais ont été validés par courbes dose-réponse en utilisant différents composés RXR . Les EC50 (concentration efficace médiane, qui permet de mesurer l'efficacité d'un composé) obtenues étaient très semblables aux valeurs précédemment rapportées dans la littérature. Nous avons aussi pu identifier un composé le SR11237 (BMS649) qui semble posséder une sélectivité pour le complexe Nur77/RXRγ par rapport aux complexes Nurr1/RXRγ et RXRγ /RXRγ. Nos résultats indiquent que ces essais de BRET peuvent être utilisés pour évaluer la sélectivité de nouveaux composés pour les complexes Nur77/RXRγ, Nurr1/RXRγ et RXRγ /RXRγ. Un autre aspect de mon projet de doctorat a été de mettre en évidence par BRET l’importance de la SUMOylation dans la régulation de l'activité de Nur77 dans sa forme monomèrique, homodimèrique et hétérodimèrique. Nous avons ainsi identifié que Nur77 recrute principalement SUMO2 sur sa lysine 577. Il est intéressant de noté que le recrutement de la SUMO2 à Nur77 est potentialisé en présence de la SUMO E3 Ligase PIASγ. Aussi, la perte de la SUMOylation sur la lysine 577 entraîne l'incapacité de Nur77 de recruter divers motifs de co-activation mais pas pour ses formes homo- et hétérodimèrique. Cependant, la présence de PIASγ ne potentialise pas le recrutement du co-activateur, suggérant que cette SUMO E3 Ligase est seulement impliqué dans le processus de recrutement de la SUMO mais pas dans celui du co-activateur. Nous avons ainsi déterminé une nouvelle modification post-traductionnelle sur Nur77 régulant spécifiquement son activité monomérique Ces projets pourraient donc apporter de nouvelles données cruciales pour l’amélioration du traitement de la maladie de Parkinson ou de la schizophrénie, ainsi que d'obtenir une meilleure compréhension sur les mécanismes permettant la régulation de la fonction de Nur77In the central nervous system, dopamine plays a critical role in many physiological functions such as learning, voluntary movement, motivation, cognition and hormone production. It was also shown that the dopamine signaling system is altered in many neurological and psychiatric disorders like Parkinson's disease and schizophrenia. Studies conducted in the laboratory of Dr. Daniel Lévesque (host laboratory) have shown that nuclear receptors Nur77 (NR4A1, NGFI-B) and RXRγ (retinoid X receptor γ isoform) are involved in the regulation of dopamine effects. These data suggest that the Nur77/RXR complex plays a crucial role in the effect of antipsychotic and anti-parkinsonian drugs. However, very few drugs targeting Nur77 have been identified to date and drugs acting at RXRγ remain poorly characterized. Furthermore, currently available tests cannot recapitulate the complexity of nuclear receptor activities and generate indirect measures of drug activities. To better understand Nur77/RXRγ complex activity we developed a new and original Bioluminescence Resonance Energy Transfer (BRET)-luciferase Protein Complementation Assay (PCA) based on the recruitment of a co-activator motif fused with YFP. The assays have been validated by dose-response curves using different RXR compounds. EC50 obtained were very similar to the values previously reported in the literature. We were also able to identify a compound, SR11237 (BMS649), which appears to have specificity for the complex Nur77/RXRγ compared to Nurr1/RXRγ and RXRγ/RXRγ. Our results indicate that these BRET assays can be used to evaluate the selectivity of compounds at Nur77/RXRγ, Nurr1/RXRγ or RXRγ/RXRγ complexes. Another aspect of my PhD project was to better understand how Nur77 activity is regulated by post-translational modifications. We were able to highlight the importance of Nur77 SUMOylation in the activity of monomeric, homodimer and heterodimer forms of Nur77 using various BRET assays. We identified that Nur77 preferentially recruits SUMO2 mainly on its lysine 577 residue. It is interesting to note that SUMO2 recruitment by Nur77 is potentiated in the presence of SUMO E3 ligase PIAS4 (PIASγ). Also, the loss of SUMOylation on lysine 577 strongly reduced co-activator motif recruitment by monomeric form of Nur77, but not for homo- and hetero-dimer complexes. However, PIAS4 by itself does not potentiate co-activator recruitment, suggesting that this SUMO E3 ligase is only involved in the recruitment process of SUMO, but not in the co-activator recruitment. Thus, we identified a new post-translational modification on Nur77 that specifically regulates its monomeric activity. These results provide critical new data that will help to identify new compounds targeting selective nuclear receptor complexes (Nur77/RXR) that may improve the treatment of Parkinson's disease and schizophrenia, as well as to get a better understanding of the mechanisms regulating the activity of Nur77

LRH-1 as a Key Regulator of Estrogen Responses in Breast Cancer Cells

Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). LRH- 1 has been linked to a number of key developmental, metabolic and proliferative processes and is known to play an important role in the regulation of cholesterol biosynthesis, lipid homeostasis and the control of steroid aromatisation. In this respect, LRH-1 is recognised to play an important role in breast cancer, where it acts to regulate aromatase activity, leading to the paracrine production of estrogen. Recent findings have also suggested a direct role for LRH-1 in cancer, where LRH-1 is found to be involved in the induction of intestinal tumours and LRH-1 has been found by immunohistochemistry in tumour cells of human mammary ductal carcinomas. Recently, a gene expression microarray analysis of estrogen responses in an engineered breast cancer cell line, where Estrogen Receptor-α (ERα) activity can be conditionally repressed, provisionally identified LRH-1 as an estrogen responsive gene that may be important in the estrogen-regulated growth of breast cancer cells. Based on this initial observation, I have gone on to study the role of LRH-1 in the estrogen response in breast cancer cells. Using ERα-positive breast cancer cell lines, I have confirmed that LRH-1 levels increase in response to estrogen and are inhibited by anti-estrogens (tamoxifen and ICI 182,780). Using 5`RNA Ligase Mediated Rapid Amplification of cDNA Ends (5`RLM-RACE) to characterise the 5’ end of the LRH-1 mRNA, I have found that the estrogen regulation of LRH-1 is mediated through a previously undescribed gene promoter, which results in the production of a variant LRH-1 mRNA species that initiates transcription just upstream of exon 2 of the LRH-1 gene. Further, reverse transcriptase polymerase chain reaction (RT-PCR) showed that the newly identified variant LRH-1 transcript is expressed in tissues in which LRH-1 expression has previously been described. Moreover, this variant was seen to be the major form of LRH-1 expressed in breast cancer cell lines. Having established the estrogen regulation of LRH-1, studies were carried out to investigate the role of LRH-1 in growth and gene expression in breast cancer cells. siRNA-mediated inhibition of LRH-1 expression inhibited proliferation of MCF-7, ZR-75-1 and T47-D breast cancer cell lines but did not inhibit BT474 and MDAMB- 231 breast cancer cells, in which LRH-1 is not expressed. Further, a group of recently described synthetic agonists for LRH-1 stimulated the growth of breast cancer cell lines expressing LRH-1 in a dose dependent manner, but did not stimulate growth in those breast cancer cell lines which do not express LRH-1. Finally, RNA interference experiments directed against LRH-1 identified ERα as an important LRH-1 regulated gene. These results show that LRH-1 potentially plays a key role in regulating estrogen responses in ERα–positive breast cancer cells, primarily through the direct regulation of ERα gene expression. These new findings, taken together with the previously described role for LRH-1 in regulating aromatase gene expression identify LRH-1 as a potentially important target for the development of new therapies for the treatment of breast cancer

Nur77 and FHL2: Novel players in vascular and immune disease

The nuclear receptor Nur77 is an early response gene that is induced by diverse extracellular signals in a wide range of tissues and cultured cells. It has been implicated in the regulation of genes involved in metabolic disease, adipogenesis, inflammation, and vascular disease. FHL2/DRAL/SLIM3 is a LIM-only protein that has been shown to interact with many proteins and acts as a co-activator or co- repressor depending on the cell-type and cellular context. It is a crucial adaptor protein and plays a pivotal role in a range of physiological and pathological processes, including proliferation, migration, differentiation and apoptosis. The aim of this thesis is to increase our understanding of fundamental pathways critical in vascular diseases including atherosclerosis, restenosis, coagulation, and immune diseases including asthma, airway inflammation and schistosomiasis. To achieve this goal, we performed numerous distinct studies on the role of nuclear receptor Nur77 and LIM-only protein FHL2 using mouse models and several cell types. In this thesis, functional properties of Nur77 and FHL2, as well as their impact on multiple signaling pathways in vascular and immune disease were studied. These research efforts are ultimately directed at designing novel therapeutic strategies that may aid in mitigating the development of vascular and immune disease

Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells

The CAD gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. CAD gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 17b-estradiol (E2) resulted in a 3-5 fold increase in CAD mRNA levels in both cell lines. E2 induced reporter gene activity in MCF-7 and ZR-75 cells transfected with a construct containing the growth-responsive -90/+115 (pCAD1) region of the CAD gene promoter, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the CAD gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2-responsiveness. Results of gel shift and chromatin immunoprecipitation (CHIP) assays show that both Sp1 and estrogen receptor a (ERa) interact with the GC-rich region of the CAD gene promoter. Moreover, hormone-induced transactivation of pCAD1 was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the CAD gene is also regulated by a nonclassical ERa/Sp1-mediated pathway. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. TCDD inhibited hormone-induced activation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 cells transfected with E2-responsive pCAD promoter constructs. E2-mediated transactivation of pCAD constructs with a mutant inhibitory dioxin responsive element DRE (iDRE) were also inhibited by TCDD suggesting that inhibitory AhR-ERa/Sp1 crosstalk was iDRE-independent. It was not possible to determine whether the levels of ERa in cells cotreated with E2 plus TCDD were limiting since the proteasome inhibitor MG132 itself directly decreased CAD mRNA levels. Using fluorescence resonance energy transfer (FRET), it was shown that both E2 and TCDD enhanced AhR-ERa interactions. E2 also induced interactions between ERa and Sp1. However cotreatment with TCDD abrogated this effect. Results of this study demonstrate a unique model of AhR-ERa crosstalk where the liganded AhR inhibits ERa-Sp1 interactions and also recruits ERa to Ahresponsive gene promoters such as CYP1A1

Etude des interactions bidirectionnelles entre le microbiote intestinal et les récepteurs aux xénobiotiques CAR et PXR

Le pregnane X receptor (PXR, NR1I2) et le récepteur constitutif aux androstanes (CAR, NR1I3) sont deux récepteurs nucléaires hépatiques et intestinaux qui régulent la transcription d'enzymes de détoxification des xénobiotiques. Des travaux antérieurs ont montré que l'expression des gènes cibles de CAR et PXR est significativement réduite dans le foie des souris axéniques. Dans ce projet de thèse, nous avions pour objectif de mieux comprendre les interactions bidirectionnelles entre le microbiote intestinal et ces xénosenseurs. Nous avons d'abord utilisé une approche pharmacologique chez les souris mâles WT vs Pxr-/- et comparé la signature transcriptomique des gènes régulés par PXR dans le foie lors de l'activation via le PCN. L'activation de PXR a augmenté l'accumulation de triglycérides hépatiques. Nous avons observé un chevauchement significatif entre les gènes régulés négativement lors de l'activation de PXR et une liste de gènes cibles de PPARδ induits par le jeûne. Parmi ceux-ci, nous avons identifié le facteur de croissance de fibroblastes 21 (Fgf21) comme un nouveau gène régulé par PXR. L'activation de PXR a aboli les taux plasmatiques de FGF21. Ces premiers résultats ont fourni une signature complète de l'activation de PXR dans le foie et ont identifié de nouveaux gènes cibles potentiellement impliqués dans les effets stéatogènes et pléiotropes de PXR. Ensuite, nous avons comparé la signature hépatique à la signature intestinale de l'activation pharmacologique de PXR, ce qui nous a permis d'identifier les gènes cibles communs de PXR dans ces 2 organes. Enfin, nous avons utilisé des souris Pxr+/+ et Pxr-/- littermate et supprimé le microbiote intestinal au moyen d'antibiotiques (ATB). En utilisant les gènes cibles de PXR identifiés précédemment, nous avons confirmé que les ATB réduisaient de manière significative l'activité de PXR dans le foie et l'iléon. Des analyses transcriptomiques hépatiques ont montré que les ATB diminuaient un nombre beaucoup plus élevé de gènes PXR-dépendants dans le foie des souris mâles que chez les femelles. Chez les mâles, l'axe microbiote intestinal-PXR contrôlait le métabolisme des xénobiotiques et le remodelage des lipides hépatiques. À l'inverse, le séquençage 16S et la métabolomique par RMN du contenu caecal ont révélé des différences subtiles mais significatives dans la composition du microbiote intestinal des souris Pxr-/- par rapport aux souris Pxr+/+, uniquement chez les mâles. Nos résultats démontrent donc que, dans le foie, PXR est un senseur majeur du microbiote intestinal qui contrôle les capacités de détoxication de l'hôte et le métabolisme des lipides de manière sexuellement dimorphique. Dans le dernier chapitre, nous avons étudié les interactions microbiote-CAR. Chez les souris Car+/+ et Car-/- littermates, la suppression du microbiote par les antibiotiques a diminué l'activité de CAR dans le foie et l'iléon des mâles, mais uniquement dans le foie des femelles. Dans le contenu caecal, le séquençage 16S et la metabolomique ont montré une différence significative dans la composition et l'activité métabolique du microbiote intestinal chez les souris Car+/+ vs Car-/- mâles, mais pas chez les femelles. Nous avons cherché les conséquences potentielles de cette dysbiose CAR dépendante et avons observé que la délétion de CAR augmentait l'accumulation de tissu adipeux chez les souris mâles à 37 semaines. Cependant, l'implication du microbiote CAR-dépendant dans ce phénotype reste à vérifier. Ainsi, nos résultats montrent pour la première fois que l'interaction CAR-microbiote est sexuellement dimorphique et pourrait contrôler le dépôt adipeux chez les souris mâles. Dans l'ensemble, nos résultats montrent que le dialogue entre le microbiote intestinal et les récepteurs aux xénobiotiques CAR et PXR est impliqué de façon sexuellement dimorphique dans le contrôle des capacités de détoxification de l'hôte, et joue un rôle dans l'homéostasie lipidique.The pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two liver and intestine-enriched nuclear receptors that act as transcriptional regulators of enzymes critical for the detoxification of xenobiotics and endogenous metabolites. Previous works have shown that the expression of CAR and PXR target genes is significantly reduced in the liver of germ-free mice. In this PhD project, we aimed to gain insights into the bidirectional interactions between the gut microbiota and these xenosensors. We first used a pharmacological approach in WT vs Pxr-/- male mice and performed a transcriptomic comparison of the PXR-regulated genes in the liver upon activation via the rodent activator PCN. We confirmed that PXR activation increased liver triglycerideaccumulation and significantly regulated the expression of genes, mostly involved inxenobiotic metabolism. We also highlighted a significant overlap between the genes downregulated upon PXR activation and a list of fasting-induced PPARδ target genes. Among these, we identified the well-described PPARδ target fibroblast growth factor 21 (Fgf21) as a new PXR-regulated gene. PXR activation abolished plasmatic levels of FGF21. This first set of results provided a comprehensive signature of PXR activation in the liver and identified new PXR target genes that might be involved in the steatogenic and pleiotropic effects of PXR. Next, we compared the hepatic vs. intestinal signature of the pharmacological activation of PXR. This allowed us to unravel the strongest PXR target genes in both organs. Finally, we used Pxr+/+ and Pxr-/- littermate mice and suppressed the gut microbiota using antibiotics (ATB). Using the previously identified PXR targets, we confirmed that ATB significantly decreased Pxr activity in the liver and ileum. Liver transcriptomic analyses showed that ATB decreased a much higher number of PXR-dependent genes in the liver of male mice than in females. In males, this gut microbiota-PXR axis controlled xenobiotic metabolism and lipid remodelling. Conversely, 16S sequencing and 1H-NMR-based metabolic profiling of caecal content revealed subtle but significant differences in the gut microbiota composition of male Pxr-/- vs. Pxr+/+ mice, while no difference was observed in females. Our results therefore demonstrate that hepatic PXR is a major sensor of the gut microbiota that controls the host detoxifying capacities and lipid metabolism in a sexually dimorphic way. In the final chapter, we investigated the microbiota-CAR interactions. In Car+/+ and Car-/- littermate mice. Microbiota suppression by antibiotics decreased CAR activity in the liver and ileum of males but only in the liver of females. In caecal content, male-specific and CAR-dependant metabolites were also detected through 1H-NMR-based metabolomics. Furthermore, 16S sequencing confirmed a significant difference in gut microbiota composition of Car-/- vs Car+/+ male mice but not in females. We investigated the potential consequences of this sexually dimorphic CAR-dependent dysbiosis and observed that long-term Car deletion increased adipose tissue accumulation in male mice (at 37 weeks old). Whether the Car-dependent dysbiosis is responsible for this phenotype remains to be determined. In 37-week-old females, Car deletion induced a significant increase in spleen weight and a decrease in colon length, therefore suggesting a role for Car in systemic and intestinal inflammation. Thus, our result show for the first time that the CAR-gut microbiota interaction is sexually dimorphic and might control adipose deposition in male mice. Overall, our results shed new light into the crosstalk between the gut microbiota and the host's xenobiotic receptors CAR and PXR, demonstrating that this cross-talk might be involved in the control the host's hepatic lipid and xenobiotic metabolism