Previous research in our laboratory found that inhibiting expression of vimentin, a marker of epithelial-to mesenchymal transition, inhibited cell growth and motility in vitro and in vivo. Tumors derived from vimentin knockdown cells showed features of epithelial redifferentiation and increased expression of differentiation-specific keratins. It is unknown what causes re-expression of keratins when vimentin is inhibited. Although, canonical Wnt signaling may activate NF-κB and repress of keratin and/or induce vimentin expression through β-catenin. We hypothesize that downregulation of differentiation-specific keratins contributes to tumor progression, mediated directly or indirectly by expression of vimentin. Vimentin-negative HN4 cells were transfected with plasmids encoding wild-type, PKCε-phosphomimetic, or unphosphorylatable versions of vimentin. Expression of vimentin was confirmed by western blot and immunofluorescence. Effects on cell growth and motility were determined using MTT, cell proliferation, and wound-closure assays. These results indicate that mutation of vimentin PKCε-phosphorylation sites cause changes in proliferation and filament assembly. Treatment of cells with an NF-κB inhibitor or 5-Aza-C, which allows re-expression of the Wnt inhibitor DKK3, led to a decrease in proliferation. These results suggest that inhibiting Wnt signaling removes the inhibition on GSK-3β and prevents activation of NF-κB, which decreases proliferation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Wnt signalling represents an important mechanism participating in control of cellular and developmental processes, including establishment of cell polarity, cell fate specification, stem cell self-renewal, tissue patterning and organogenesis, homeostasis maintenance and regeneration. Misregulation of the Wnt signalling during embryogenesis leads to developmental defects while aberrant activation later in development is associated with degenerative diseases and a number of cancers. The presented PhD thesis is based on four original publications that deal with the post-translational modifications of Wnt ligands and molecular mechanisms contributing to the regulation of a transcriptional profile of the so-called canonical Wnt pathway. Wnt signalling pathway is used repetitively both in time and different cellular contexts throughout development of multicellular organisms. Inevitably, in each single situation -catenin/TCF complexes, the downstream effectors, induce only subsets of all potential target genes. How this differential tissue- and stage-specific control over various subsets of target genes is achieved with such a limited number of nuclear effectors is not fully understood. Along with the expression of specific LEF/TCF family members or their variants containing different functional domains...

A via canônica Wnt/beta-catenina regula várias funções em vertebrados, incluindo diferenciação de células T, bem como a proliferação, sobrevivência, morfogênese e migração de vários tipos celulares. As células T CD4+ é fundamental para a competência imunológica. Foi observado pelo nosso grupo que células T CD4+ humanas apresentam ativação da via Wnt/beta-catenina após tratamento com sais de lítio ou outros agonistas da via. A ativação desta via induziu a proliferação de células T CD4+ naive e de memória central. Em conjunto, estes dados sugerem um importante papel da via Wnt/beta-catenina na homeostase de células T CD4+ humanas. Seria importante avaliar o papel da via Wnt/beta-catenina nas células do sistema imune no modelo murino, já que pouco se sabe sobre seu efeito na homeostase de células T CD4+ murinas. A ativação da via Wnt/beta-catenina pode ser induzida com inibidores da proteína Glicogênio sintase quinase 3beta (GSK3beta), por exemplo, os sais de lítio (LiCl e Li2CO3) e inibidores específicos (SB, CHIR) em vários tipos celulares. Neste trabalho, avaliamos o efeito de inibidores de GSK3? na ativação da via Wnt/beta-catenina canônica em esplenócitos e células T CD4+, através da realização de experimentos in vivo e in vitro, avaliando a expressão de seus genes alvo HIG2, Bcl-xL, Ciclina D1 e c-myc. Verificou-se que o tratamento in vivo agudo (2-12 h após a administração) ou crônico (administração diária por 30 dias) de camundongos não é capaz de ativar a via Wnt/beta-catenina in vivo em células esplênicas e células T CD4+, embora o mesmo tratamento induza a expressão dos genes alvo da via no tecido cerebral (córtex e hipocampo). Além disso, também não foi possível verificar ativação da via em esplenócitos e células T CD4+ após tratamento in vitro das mesmas com LiCl ou os inibidores específicos de GSK3beta testados(CHIR99021, SB-216763), embora essa ativação tenha sido observada na linhagem celular HEK293. Nossos resultados sugerem que a via Wnt/beta-catenina (canônica) não é induzível em células T CD4+ murinas maduras, com os agonistas testados. Isso pode ter implicações fisiológicas, por exemplo sobre a homeostase de células T CD4+, já que a proliferação homeostática de células T, influenciada em humanos pela via Wnt/beta-catenina, é menos importante em camundongos / The Wnt/beta-catenin pathway regulates many functions in vertebrates, including T cell differentiation, as well as proliferation, morphogenesis and migration in different cell types. CD4+ T cells play is fundamental for immunological competence. Our group has observed that human CD4+ T cells present activation of the Wnt/beta-catenin pathway after treatment with lithium salts or other pathway agonists. The activation of this pathway induced proliferation in naive and central memory CD4+ T cells. Together, these results suggest an important role for the Wnt/beta-catenin pathway in the homeostasis of human CD4+ T cells. It would be very important to evaluate the role of the Wnt/beta-catenin pathway in T cells in the mouse model, since little is known about its effect in mice CD4+ T cell homeostasis. The activation of the Wnt/beta-catenin pathway may be induced with Glycogen Synthase Kinase 3B (GSK3beta) inhibitors, i.e., lithium salts as mentioned above, and specific GSK3beta inhibitors (SB, CHIR) in different cell types. In this work, we evaluated the effect of GSK3beta inhibitors in the activation of the canonical Wnt/beta-catenin in splenocytes and CD4+ T cells, by conducting experiments in vivo and in vitro, evaluating the expression of its target genes HIG2, Bcl-xL, Cyclin D1 and c-myc. We verified that acute (2-12 hours after administration) or chronic (daily administration for 30 days) treatment of mice with lithium salts is not able to activate the Wnt/beta-catenin pathway in splenocytes and CD4+ T cells, although we could observe activation in brain tissues (cortex and hypothalamus). Besides, no activation of the Wnt/beta-catenin pathway was observed in these cell types after in vitro treatment with LiCl or the specific inhibitors of GSK3beta (CHIR99021, SB-216763), while the pathway was activated by the same treatments in HEK293 cells. Our results suggest that the Wnt/beta-catenin pathway is not inducible in murine mature CD4+ T cells with the tested agonists. This may have physiological implications, for instance on the homeostasis of CD4+ T cells, where homeostatic proliferation - influenced the Wnt/beta-catenin pathway in human T cells - is less important in the maintenance of the murine peripheral T cell pool

Le muscle squelettique adulte a une importante capacité à se régénérer après une lésion. La régénération musculaire dépend de divers signaux moléculaires tels que l’activation de la signalisation Wnt dans les cellules souches musculaires, appelées cellules satellites. Les protéines R-spondins (Rspo) composent une famille de quatre protéines qui ont un rôle d’activateurs/potentialisateurs sur les voies Wnt dans les cellules souches de différents tissus. Bien qu’il soit connu que ces protéines sont importantes pour la régénération de ces tissus, leur rôle dans la myogenèse régénérative n’a pas été étudié à ce jour. L’expression génique de R-spondin1 étant sur-régulée par Pax7, le marqueur des cellules satellites, nous avons émis l’hypothèse que R-spondin1 participe à la régénération musculaire. Nous avons, tout d’abord, isolé les cellules souches musculaires des modèles murins d’invalidation constitutive pour Rspo1 et avons observé qu’une déficience de R-spondin1 n’altère pas le cycle cellulaire de ces cellules. Cependant, une altération de l’expression de Rspo1 induit un défaut global de la cinétique de différenciation myogénique. Nous montrons que R-spondin1 inhibe la fusion des cellules musculaires puisque les myotubes déficients pour R-spondin1 possèdent un plus grand nombre de noyaux. Nous avons ensuite induit la régénération du muscle squelettique Tibalis Antérieur par une injection de Cardiotoxine et nous avons analysé les muscles à différents temps de régénération. Nos données prouvent qu’en l’absence de R-spondin1, les cellules souches présentent un retard de différenciation alors qu’elles possèdent une plus grande capacité de fusion, ayant pour conséquence une hypertrophie des myofibres dans le muscle. Concordant au rôle de R-spondin dans les cellules souches intestinales ou dans le follicule pileux, la protéine R-spondin1 stimule l’expression des gènes cibles de la voie Wnt canonique dans les cellules souches musculaires. Nous avons mis en évidence que R-spondin1 potentialise la voie Wnt canonique et régule négativement l’activation de la voie non-canonique dans les cellules. Nos résultats démontrent que la protéine R-spondin1 contribue à la régénération du muscle squelettique adulte par la régulation de l’activation des voies Wnt. / Adult mammalian skeletal muscles have the remarkable ability to repair after injury. Muscle regeneration depends on various cellular and molecular responses, such as activation of Wnt signaling pathways in muscle stem cells called satellite cells. R-spondin (Rspo) proteins are able to potentiate Wnt signaling pathways in vivo in many stem cells and play important role for regeneration of several tissues. The role of R-spondin in injury-induced myogenesis has not been studied. Given that R-spondin1 gene expression is up-regulated by Pax7, the satellite cell-specific transcription factor, we explored the hypothesis that R-spondin1 plays a role during skeletal muscle regeneration. We firstly isolated primary myoblasts from Rspo1 constitutive knock-out mice and observed that a depletion of Rspo1 did not alter cell cycle of these cells. However, a lack of R-spondin1 on cells resulted in global alteration of differentiation kinetics. We found that R-spondin1 inhibits muscle cell fusion, as Rspo1 knock-out myotubes contain an higher number of myonuclei. Then, we injured the Tibialis Anterior (TA) muscle of Rspo1-null mice and littermates controls by Cardiotoxin injection and analyzed muscle regeneration at different time points following injury. Our data show that R-spondin1 removal results in a delay of stem cell differenciation. In contrast, a R-spondin1 deficiency leads to better cell capacity to fuse to dommaged myofibers, giving rise to myofiber hypertrophy. As with other tissue-specific stem cells, such as hair follicle or intestinal crypt stem cells, R-spondin1 potentiates canonical Wnt signaling target genes expression in muscle stem cells. We proved that R-spondin1 potentiates canonical Wnt signaling target genes expression and negatively regulates non-canonical signaling in muscle stem cells. Our results demonstrate that R-spondin1 is crucial for adult muscle regeneration through a tighly cross-talk regulation between Wnt signalings.

La voie Wnt est une voie de signalisation importante pour l’embryogenèse, la morphogenèse et l’homéostasie des tissus au stade adulte. Des mutations dans cette voie de signalisation sont souvent associées à une létalité embryonnaire ou à des pathologies sévères.Une caractéristique singulière de la signalisation Wnt est sa grande complexité génétique. Chez les vertébrés, ce sont 19 ligands Wnt différents qui peuvent potentiellement lier les 10 membres de la famille des récepteurs Frizzled (Fz). De plus, la liaison d’un ligand Wnt à un récepteur Fz peut conduire à l’activation d’au moins trois voies de transduction distinctes. Pourtant, l’interaction Wnt/Fz est incompatible avec une reconnaissance monospécifique étant donné que Wnt et Fz interagissent via des résidus conservés dans les deux familles. Les patrons d’expression des différents Wnt et des différents Fz sont complexes et souvent chevauchant. Malgré cela, la délétion sélective d’un Wnt peut conduire à des phénotypes spécifiques non-observés lors de la délétion d’un autre ligand Wnt co-exprimé. C’est notamment le cas des ligands Wnt7a et Wnt7b. Seules leurs expressions par les progéniteurs neuronaux permettent d’activer la voie de signalisation Wnt/β-caténine dans les cellules endothéliales malgré l’expression simultanée d’autres ligands Wnt. Dès lors, comment les cellules des vertébrés sont-elles capables de discriminer les différents ligands Wnt ?L’étude de l’activation des signalisations induites par les ligands Wnt7a et Wnt7b permet d’illustrer par quel mécanisme moléculaire des co-récepteurs, tels que Gpr124 et Reck, médient la reconnaissance spécifique d’un ligand Wnt et permettent la formation d’un signalosome spécifique. Reck est un récepteur spécifique des ligands Wnt7a et Wnt7b qui permet de les discriminer de tous les autres ligands Wnt. Il interagit avec ceux-ci via une région intrinsèquement désordonnée et divergente dans la famille Wnt appelée « le peptide linker ». Etant donné que cette région est exposée au solvant et qu’elle ne comprend pas les sites d’interaction avec le récepteur Fz, elle pourrait jouer un rôle critique dans la discrimination des différents ligands Wnt. Gpr124, quant à lui, interagit avec Reck via son domaine extracellulaire et permet la co-localisation de ce dernier et des récepteurs Fz dans le même signalosome. Pour ce faire, Gpr124 interagit avec la protéine adaptatrice Dvl via son domaine intracellulaire. Le recrutement membranaire et la polymérisation de Dvl permettent la formation d’une plateforme d’ancrage facilitant la formation du complexe Reck/Gpr124/Fz/Lrp5/6 qui active alors sélectivement la voie la signalisation Wnt/β-caténine en réponse aux ligands Wnt7a et Wnt7b. L’identification d’un tel mécanisme de décodage laisse supposer qu’il pourrait exister plusieurs modules de reconnaissance spécifique adaptés à d’autres ligands Wnt assurant ainsi un réglage précis de la signalisation Wnt en fonction du contexte moléculaire de la membrane plasmique. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.

No description available.

The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle 1 (Nkd), a Dvl-interacting protein. The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects all the way up to vertebrates. To test the role of the Nkd EF-hand in Wnt signal integration, we created two different mutations in the zebrafish Nkd: one with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like antagonizes Wnt/β-catenin more robustly than zebrafish Nkd. The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT, but not the two mutant forms, shows localization changes in the calcium fluxing cells that also host converging Wnt signals versus calcium quiescent cells. Our functional data suggests that the Nkd EF-hand is important for Wnt signal integration. Interestingly, Nkd only contains one EF-hand, and proteins that bind calcium tend to have multiple. Calcium binding can also be influenced by binding partners. Because of this, we investigate the role of the Nkd binding protein Dvl and their possible calcium affinity. Dvl is a pivotal point in the Wnt signaling network, leading to the output decision of a cell. EF-hand of Nkd binds to the PDZ domain of Dvl. Interestingly, the Dvl PDZ domain contains a region rich in negatively charged amino acids that could aid in binding calcium. In the same manner as Nkd, we generated a Dvl with neutralized putative EF-hand and tested its function and localization relative to wildtype Dvl. This work elucidates the elegant mechanism by which a cell receiving multiple Wnt signals integrates the information into a specific response. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.