Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the ectopic expression of defined factors. iPSCs hold great promise for pharmaceutical screening and regenerative medicine but the mechanism of reprogramming is not well understood. This work examines a component process of reprogramming that is the mesenchymal-to-epithelial transition (MET), an important step in the generation of iPS cells. In this thesis I demonstrate a connection between BMP signaling and the reprogramming factor Klf4 in the activation of the MET expression program. Using ChIP-Seq I mapped the binding of Klf4 and BMP Smads across the genome and linked their co-binding to a MET expression program determined by RNA-Seq. My work uncovers a thus-far unreported interaction between Klf4 and BMP signaling in cellular epithelialization that can directly improve the technical methods of reprogramming and have important implications for the induction of epithelial tissues in general.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the ectopic expression of defined factors. iPSCs hold great promise for pharmaceutical screening and regenerative medicine but the mechanism of reprogramming is not well understood. This work examines a component process of reprogramming that is the mesenchymal-to-epithelial transition (MET), an important step in the generation of iPS cells. In this thesis I demonstrate a connection between BMP signaling and the reprogramming factor Klf4 in the activation of the MET expression program. Using ChIP-Seq I mapped the binding of Klf4 and BMP Smads across the genome and linked their co-binding to a MET expression program determined by RNA-Seq. My work uncovers a thus-far unreported interaction between Klf4 and BMP signaling in cellular epithelialization that can directly improve the technical methods of reprogramming and have important implications for the induction of epithelial tissues in general.

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No description available.

Chez la souris, le Leukemia Inhibitory Factor (LIF) joue un rôle clé dans le maintien des cellules souches embryonnaires (ES) à l’état pluripotent. Le LIF agit en activant le facteur de transcription STAT3 via les kinases Jak. Cette activation est nécessaire et suffisante au maintien des cellules ES en autorenouvellement en présence de sérum. Une étude du transcriptome de STAT3 réalisée au laboratoire a permis d’identifier plusieurs gènes cibles de ce facteur, parmi lesquels plusieurs gènes inconnus. L’un d’eux, le gène 1456160_at, est fortement exprimé dans les cellules ES de souris et son expression diminue après induction de la différenciation. Ce gène a été appelé Asgard pour Another Self-renewal GuARDian. La caractérisation et le séquençage de ce gène ont permis de mettre en évidence qu'Asgard code pour un microARN. De nombreux microARNs jouent un rôle clé dans le maintien de l'autorenouvellement des cellules ES et dans le contrôle de la différenciation. Des expériences d’inhibition et de surexpression ont permis de montrer que Asgard est impliqué dans la régulation de la différenciation endoderme versus mésoderme. Des analyses préliminaires ont permis d’identifier Pbx3, FoxA2 et Sox17 comme cibles potentielles. Bien que les mécanismes d’action du microARN Asgard restent à confirmer, ce travail a permis d’identifier un nouveau gène clé de l'autorenouvellement des cellules ES de souris / The Leukemia Inhibitory Factor (LIF) activates the transcription factor STAT3, which results in the maintenance of mouse embryonic stem cells in the undifferentiated state by inhibiting mesodermal and endodermal differentiation. We identified several target genes of STAT3 by transcriptomic analysis. Among them, we focused on an unknown gene referred as 1456160_at on Affymetrix array. This gene is highly expressed in embryonic stem cells and its expression level decreases during differentiation. We named this gene Asgard for Another Self-renewal GuARDian. Its characterization and sequencing revealed that Asgard encodes for a microRNA sequence. Several microRNAs have been shown to play key role in the maintenance of self-renewal of mouse ES cells and in the control of differentiation. Inhibition and overexpression assays showed that Asgard inhibits endodermal differentiation in order to maintain self-renewal. Through preliminary analysis, we identified Pbx3, FoxA2 and Sox17 as potential targets of the microRNA Asgard. Our work enables us to identify a new key gene of self-renewal of mouse ES cells

Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2-/- embryos die earlier, by E11.5. E9.5 FVB/N KLF2-/- hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2-/- AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2-/- hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2-/- AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2-/- embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2-/- hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2-/- embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (UGDH), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and UGDH promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. Thus KLF2 plays a role in EMT, through its regulation of important cardiovascular genes. E10.5 FVB/N KLF2-/- embryos show gaps in the endothelial lining at the dorsal aorta and a number of blood cells localized outside the aorta suggesting either hemorrhaging or inability of the hematopoietic progenitors to reach the aortic endothelium and enter circulation. This is not observed in KLF2-/- embryos in a mixed genetic background. In conclusion, KLF2-/- cardiovascular phenotypes are genetic background-dependent. KLF4 is another member of the Krüppel-like transcription factor family phylogenetically close to KLF2. It is known to play an important role in vascular regulation. Our studies show that in vascular development KLF4 plays a complementary role to KLF2, indicated by cranial hemorrhaging in E9.5 KLF2-/-KLF4-/- embryos in an undefined mixed background. This phenotype is absent in either of the single knockouts. The role of KLF2 and KLF4 in vascular development has not been studied as much as adult vascular regulation. This study begins to define the roles of these two transcription factors in development of blood vessels. Congenital heart and valve defects are a common cause of infant mortality. KLF2 has never been studied in this context. Thus this work is important for a better understanding of the biology of valve development.

La sclérodermie systémique (ScS) est une maladie auto-immune caractérisée par une fibrose cutanée et viscérale ainsi que par des anomalies microcirculatoires. Son origine multifactorielle et ses manifestations cliniques variées en font une maladie à la physiopathologie complexe. Cette maladie rare demeure une affection dont l’étiologie est encore inconnue et pour laquelle il n’existe aucun traitement curatif. Notre laboratoire a mis en évidence le rôle des formes réactives de l’oxygène en mettant au point un modèle animal induit par l’acide hypochloreux. Ce modèle nous a permis d’explorer différentes voies de signalisation intracellulaire, impliquées dans la formation de FRO, favorisant la fibrose. Dans ce travail, nous avons choisi d’explorer dans la sclérodermie systémique différentes voies de signalisation impliquées dans l’inflammation à l’aide d’un modèle de réaction du greffon contre l’hôte sclérodermiforme (GVH-Scl) et d’un modèle de sclérodermie induit par les formes réactives de l’oxygène. De plus, nous avons étudié l’impact de molécules ciblant ces voies afin de fournir de nouvelles données permettent d’enrichir l’arsenal thérapeutique de cette maladie, actuellement pauvre. Les souris obtenues à partir du premier modèle présentent une fibrose cutanée et pulmonaire, une alopécie, une diarrhée et une inflammation hépatique. Une quantité importante d’auto-anticorps ainsi qu’une activation du système immunitaire sont retrouvées. Nous avons observés une activation de la voie de l’EGFR, de STAT3, de Wnt/β-caténine, d’AKT, d’ERK1/2 et de Notch dans la GVH-Scl. L’inhibition de la voie de l’EGFR par l’erlotinib a montré une amélioration clinique associée à une réduction de la fibrose cutanée et de l’inflammation cutanée et hépatique. De plus, l’erlotinib agit sur le système immunitaire et restaure la proportion de LT CD4+ naïfs chez les souris malades ainsi que le taux d’auto-anticorps anti-topoisomérase 1. La co-inhibition des voies de signalisation STAT3, Wnt/β-caténine, AKT, ERK1/2 et Notch par le niclosamide améliore les symptômes de la GVH-Scl chez la souris. Nous avons noté une diminution de la fibrose cutanée et pulmonaire, une diminution de l’inflammation cutanée, hépatique et gastro-intestinale et une diminution de la production d’auto-anticorps. La proportion de cellules naïves parmi les LT CD4+ et CD8+ est plus élevée chez les souris malades traitées que chez les malades non traitées. Les souris du second modèle présentent une fibrose cutanée et pulmonaire et une activation du système immunitaire accompagnée d’une production d’autoanticorps anti-topoisomérase. Nous avons retrouvé une activation des voies de signalisation de STAT3, de Wnt/β-caténine et d’AKT chez les souris sclérodermiques. Nous avons également observés une activation de la voie de signalisation de STAT6 et une surexpression de KLF4 chez ces souris malades. La co-inhibition des voies de signalisation STAT3, Wnt/β-caténine et AKT par le niclosamide améliore la fibrose cutanée et pulmonaire chez les souris sclérodermiques. Cette molécule diminue le nombre et l’activation des LB et des LT CD4+ et CD8+ ainsi que la production des auto-anticorps chez les souris malades. Le traitement de souris sclérodermique par le léflunomide, inhibiteur de STAT6, a aussi montré une amélioration de la fibrose cutanée et pulmonaire et des anomalies immunitaires présentées par les souris sclérodermiques. De plus l’inhibition de STAT6 et de KLF4 par le léflunomide inhibe la polarisation des macrophages en macrophages M2. Ainsi nous avons mis en évidence le rôle des voies de signalisation de l’EGFR, de STAT3, de Wnt/β-caténine, d’AKT, de STAT6 et de KLF4 dans la physiopathologie de la sclérodermie systémique. (...) / Systemic sclerosis (SSc) is a connective tissue disorder that results in skin and inner organs fibrosis, microvascular injuries and auto-immunity. This rare disease has a complex physiopathology which is due to its multifactorial origin and its various clinical manifestations. Its etiology remains unknown and no curative treatment exists at present. Our team highlighted the role of reactive oxygen species (ROS) by developing a hypochlorous acid (HOCl)-induced mouse model of SSc. This model allowed the exploration of several intracellular signalizing pathways which were involved in ROS production and promote fibrosis. In this work, we choose to investigate inflammatory signalizing pathways in SSc with a sclerodermatous graft versus host disease (Scl-GVHD) mouse model and a ROS-induced mouse model. Moreover, we studied the effects of drugs targeting these pathways in order to reinforce data providing new therapeutics. Mice from Scl-GVHD model developed a diffuse cutaneous SSc with pulmonary fibrosis, alopecia, diarrhea and liver inflammation. Production of anti-DNA topoisomerase 1 auto-antibodies and immunological activation was also found. We observed an activation of EGFR, STAT3, Wnt/β-catenin, AKT, ERK1/2 and Notch signaling pathways in Scl-GVHD. Inhibition of EGFR by Erlotinib showed clinical amelioration with a decreased skin fibrosis and decreased skin and liver inflammation. Moreover, Erlotinib decreased production of activated/memory CD4+ T cells and of auto-antibody anti-topoisomerase1. Co-inhibition of STAT3, Wnt/β-catenin, AKT, ERK1/2 and Notch pathways by Niclosamide reversed clinical symptoms of Scl-GVHD in mice. We observed an improvement of skin and lung fibrosis, of cutaneous, hepatic and intestinal inflammation, and a reduced production of auto-antibodies. The ratio of CD4 and CD8 naive T cells was higher in Niclosamide-treated GVHD mice than in untreated GVHD mice. Mice from HOCl-model present skin and lung fibrosis and an immune activation along with the production of auto-antibodies anti-topoisomerase. We found an activation of STAT3, Wnt/β-catenin and AKT signaling pathways in HOCl-mice. We also observed an activation of STAT6 signaling pathway and an overexpression of KLF4 in these sicked mice. Co-inhibition of STAT3, Wnt/β-catenin and AKT pathways by Niclosamide improves skin and lung fibrosis in HOCl-mice. This drug decreased number and activation of B cells and CD4+ and CD8+ T cells and auto-antibodies production in mice. Treatment of HOCl-mice with Leflunomide, STAT6 inhibitor, also showed an improvement of skin and lung fibrosis and of immunological abnormalities in HOCl-mice. Moreover, inhibition of STAT6 and KLF4 by Leflunomide inhibits M2 polarization of macrophages. Thus, we highlighted the role of EGFR, STAT3, Wnt/β-catenin, AKT, STAT6 and KLF4 signaling pathways in physiopathology of SSc. Use of inhibitors of these pathways such as Etrlotinib, Niclosamide and Leflunomide, indicate a clinical and biological efficacity in mouse. These drugs allow the control of the 3 characteristic features of SSc reproduced in these animal models: fibrosis, inflammation and autoimmunity and could thus be effective in fighting the development of clinical-biological abnormalities of SSc.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Durant la grossesse, les défenses immunitaires maternelles sont considérablement réduites, ce qui augmente la vulnérabilité aux pathogènes. Cette modulation immunitaire est principalement due à l’involution aiguë du thymus, qui réduit la production de lymphocytes T essentiels à une réponse immunitaire efficace. L’involution thymique associée à la grossesse est un processus naturel et hautement conservé chez les vertébrés. Elle est cruciale pour augmenter la fertilité maternelle et la tolérance immunitaire au fœtus. Bien que sévère, cette involution est temporaire. La régénération thymique commence rapidement après l'accouchement, rétablissant ainsi rapidement la fonction thymique. Le rôle du thymus dans la sélection des lymphocytes T est principalement attribué aux cellules épithéliales thymiques (TECs). L’involution et la régénération thymique associées à la grossesse sont médiées par les TECs sous l’influence de la progestérone. La suppression des récepteurs de la progestérone exprimés par les TECs permet de prévenir l’involution thymique, mais elle altère également la fertilité. Ces éléments font de ce processus un modèle idéal pour étudier les régulateurs de la fonction et de l'homéostasie des TECs. Bien que des études antérieures aient identifié des acteurs clés dans la régénération thymique à l'aide de divers modèles d’involution, les mécanismes spécifiques à l’atrophie et à la régénération thymique liée à la grossesse restent encore mal compris. La première étude de cette thèse analyse les modifications transcriptomiques des TECs et la régulation de leur homéostasie pendant la régénération post-partum. Ces résultats ont révélé que l’involution thymique associée à la grossesse n’entraînait pas de perte de TECs, tandis que la régénération post-partum était principalement guidée par des changements transcriptomiques et phénotypiques dans les cTECs. De plus, cette étude a mis en évidence le rôle central du facteur de transcription FOXN1 dans la régulation de la fonction thymique durant la régénération. Dans l’objectif d’identifier les facteurs de transcription régulant l’homéostasie des TECs pendant la grossesse, la seconde étude présente le rôle protecteur de KLF4 pour l’intégrité thymique. La suppression spécifique de Klf4 dans les TECs a montré un impact négatif sur la cellularité thymique pendant la grossesse, entraînant une survie réduite des cTECs et l’acquisition de caractéristiques mésenchymateuses. Cette involution exacerbée a des répercussions durables sur la régénération thymique post-partum, soulignant ainsi le rôle critique de Klf4 dans le maintien de l'intégrité des cTECs pendant la grossesse. Enfin, la dernière étude, basée sur des études transcriptomiques et épigénétiques, révèle que KLF4 protège les cTECs en activant la réponse au stress, dont l'autophagie, tout en diminuant l’inflammation, l'apoptose et la transition épithélio-mésenchymateuse. De plus, nous avons développé une technique qui permet, pour la première fois, d’analyser des interactions ADN-protéine dans des TECs primaires isolées de thymus. Ces travaux apportent une meilleure compréhension des mécanismes cellulaires et moléculaires responsables de la régulation de la fonction thymique pendant et après la grossesse. La découverte du rôle protecteur de l’intégrité des cTECs par KLF4 ouvre une voie prometteuse pour prévenir ou ralentir l’involution thymique liée à l’âge. En effet, le vieillissement du thymus précède celui des autres organes, rendant les personnes âgées plus à risque d'infections, de cancer et d'une efficacité vaccinale réduite. / During pregnancy, maternal immune defenses are significantly reduced, increasing susceptibility to pathogens. This immune modulation is primarily due to acute thymic involution, which reduces the production of T lymphocytes essential for an effective immune response. Pregnancy-associated thymic involution is a natural and highly conserved process among vertebrates. It is critical for enhancing maternal fertility and fetal immune tolerance. Although severe, this involution is transient. Thymic regeneration begins immediately after birth, rapidly restoring thymic function. The role of the thymus in T lymphocyte selection is primarily attributed to thymic epithelial cells (TECs). Thymic involution and regeneration associated with pregnancy are mediated by TECs under the influence of progesterone. Suppression of progesterone receptors expressed by TECs prevents thymic involution but also impairs fertility. These aspects make this process an ideal model to study the regulators of TEC function and homeostasis. While previous studies have identified key factors in thymic regeneration using different models of involution, the specific mechanisms of thymic atrophy and regeneration associated with pregnancy remain poorly understood. The first study presented in this thesis analyzes the transcriptomic changes in TECs and the regulation of their homeostasis during postpartum regeneration. These results showed that pregnancy-associated thymic involution does not lead to TEC loss, while postpartum regeneration is mainly controlled by transcriptomic and phenotypic changes in cortical TECs (cTECs). In addition, this study highlights the central role of the transcription factor FOXN1 in regulating thymic function during regeneration. To identify the transcription factors that regulate TEC homeostasis during pregnancy, the second study presents the protective role of KLF4 for thymic integrity. Specific deletion of Klf4 in TECs showed a negative impact on thymic cellularity during pregnancy, leading to reduced cTEC survival and acquisition of mesenchymal characteristics. This exacerbated involution has lasting effects on postpartum thymic regeneration, underscoring the critical role of Klf4 in maintaining cTEC integrity during pregnancy. Finally, the last study, based on transcriptomic and epigenetic analyses, shows that KLF4 protects cTECs by activating the stress response, including autophagy, while reducing inflammation, apoptosis and epithelial-to-mesenchymal transition. In addition, we have developed a technique that allows, for the first time, the analysis of DNA-protein interactions in primary thymic TECs. These findings provide a better understanding of the cellular and molecular mechanisms regulating thymic function during and after pregnancy. The discovery of KLF4’s protective role in maintaining cTEC integrity opens promising avenues for preventing or slowing age-related thymic involution. Indeed, thymic aging precedes that of other organs, making older individuals more susceptible to infections, cancer, and reduced vaccine efficacy.