Canonical Wnt signaling is involved in many aspects of development including axis specification and anterior-posterior neuroectoderm formation during vertebrate embryogenesis. Axin2, a homologue of Axin1, is thought to have a similar regulatory role within the cell, but differences in their expression and binding partners suggest Axin2 is not completely redundant with Axin1. To better understand Axin2 in canonical Wnt signaling, I utilized several approaches to explore its expression and function. In the zebrafish embryo, I found Axin2 is expressed in known active domains of Wnt signaling, suggesting an inducible regulatory role. Additionally, canonical Wnt signaling was sufficient and necessary to induce Axin2 expression and Axin2 was sufficient and necessary to inhibit Wnt signaling. As Wnt signaling is important in development and its dysregulation has been implicated in diseases such as colorectal cancer, this study helps advance our understanding of how Wnt signaling regulates itself through the use of negative feedback inhibitors, such as Axin2.

Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape

Des mutations du gène STK11 furent initialement décrites comme responsable du syndrome Peutz-Jeghers, dont la gravité est lliée à une incidence accrue d’apparition de tumeurs. Le produit de ce gène, la sérine/thréonine kinase LKB1, a une expression ou une activité catalytique réduite, voir perdue, consécutivement à des mutations somatiques dans plusieurs types de cancer mais principalement du poumon (30% des NSCLC). Cette kinase est considérée de ce fait comme un suppresseur de tumeur d’importance. Les mécanismes moléculaires responsables de sa propriété suppresseur de tumeur restent à identifier. En effet, alors que sa fonction dans le métabolisme cellulaire, au travers de l’activation de la kinase AMPK, fut longtemps privilégiée, elle est actuellement remise en cause au profit de sa fonction de régulatrice de la signalisation Wnt canonique. Mes travaux de thèse confortent cette éventualité dans le cas des tumeurs pulmonaires (NSCLC). En effet, parmi les deux complexes fonctionnels que forme LKB1 avec les pseudokinases STRADα ou β, mes résultats démontrent que seul celui impliquant STRADβ intervient dans la régulation de la voie Wnt. Pour cela, le complexe LKB1/STRADβ se localise au niveau du cil primaire et participe à l’activation de la kinase MARK3. Ces résultats, étayés par un modèle murin invalidé pour STRADβ ainsi que l’analyse, a posteriori, de bases de données transcriptomiques adossées aux données cliniques de patients atteints de NSCLC, suggèrent que l’activité suppresseur de tumeur de LKB1 est associée à sa localisation et à sa fonction au niveau du cil primaire en participant à l’activation de MARK3 et à la régulation de la signalisation Wnt canonique. / STK11 gene mutations were originally identified as responsible for the Peutz-Jeghers syndrome of which severity is mainly related to an increase incidence of tumor development. The product of this gene the serine/threonine kinase LKB1 gets its activity or its expression reduced, sometimes even lost, following somatic mutations in several types of cancer such as pancreas, liver but mainly from lung. Indeed, almost 30% of non-small cell lung carcinoma (NSCLC) does not express anymore or only an inactive form, has led to consider this kinase as tumor suppressor of importance. While there is no doubt of the involvement of its catalytic activity molecular mechanisms responsible for its tumor suppressor properties remain to be identified. Indeed, whereas its function as regulator of cellular metabolism through AMPK has been favor for a while, it is currently re-assess to benefit to its regulator function on canonical Wnt signaling. My thesis work, reinforce this eventuality in NSCLC. Indeed, among the two functional complexes formed by LKB1 through its association with STRADα or β pseudokinases, my results show that only the complex related to STRADβ is involved in the canonical Wnt pathway regulation. For that, LKB1/STRADβ complex localizes at primary cilia and participates to MARK3 kinase activation. These results strengthened by a STRADβ knockout mouse model and an a posteriori transcriptomic analysis of lung adenocarcinoma patient datasets related to their clinical records, suggest that LKB1 tumour suppressor activity is associated with its localization and its function at primary cilia participating in the activation of MARK3 and thus regulation of canonical Wnt signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22133号 / 生博第420号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 千坂 修, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23383号 / 医博第4752号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 髙折 晃史, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

For more than 20 years, T-cell specific factor 4 (Tcf4) is the most intensively studied member of the conserved Tcf/Lymphoid enhancer-binding factor (Lef) family of transcription factors. Together with β-catenin coactivator, Tcf4 represents the prominent nuclear effector of canonical Wnt signaling in the intestinal epithelium. Regulation of Wnt-β-catenin signaling in intestinal stem cells is crucial for tissue homeostasis and tumor formation initiation. Up to date, several mouse models were generated to manipulate Tcf4 abundance or activity in vivo and dissect its function. Moreover, mutational screens and expression profiling of human colorectal tumors were carried out to disclose a contribution of TCF4 to tumor progression. However, subsequent studies brought conflicting results in relation to the potential of Tcf4 to activate or repress Wnt target genes and drive or inhibit cell proliferation. Here in this study, we analyze publicly available datasets for global expression of TCF4 and its paralogs in human tissues and colorectal cancer (CRC) samples. Notably, we present newly generated Tcf4flox5 mouse with a conditional Tcf4 allele that can be used to eliminate expression of Tcf4 from two alternative promoters of the gene. Using this mouse strain we documented that Tcf4 loss led to the demise of...

Final publication is available at http://www.plosgenetics.org/article/related/info%3Adoi%2F10.1371%2Fjournal.pgen.1005802 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19627号 / 医博第4134号 / 新制||医||1016(附属図書館) / 32663 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 松田 秀一, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Wnt/ β-Catenin-Signaltransduktion nimmt eine exponierte Stellung in der kolorektalen Karzinogenere ein. Niclosamid ist ein Derivat der Salicylsäure, das bei Bandwurm- infektionen eingesetzt wird. Es konnte gezeigt werden, dass Niclosamid den Wnt/ β-Catenin-Signalweg moduliert. Ziel der vorliegenden Arbeit war es, den therapeutischen Einsatz des Niclosamids beim kolorektalen Karzinom zu evaluieren. Die Zellproliferation von kolorektalen Karzinomzelllinien (humane SW480 und SW620 Zellen sowie CC531 Zellen einer Ratte) und von Rattenfibroblasten wurde nach 12 und 24 Stunden Inkubation mit Niclosamid durch lichtmikroskopische Zellzahlbestimmungen beurteilt. Die Apoptoseraten wurden mit einem Zelltod-Assay ermittelt. Eine Immunfluoreszenzfärbung gab Aufschluss über das Expressionsmuster von aktivem β-Catenin. Die Promotoraktivität des LEF/ TCF-Transkriptionsfaktors wurde nach Transfektion mit TOPflash mithilfe eines Luciferase Assays analysiert. Die Genexpression von Wnt-modulierenden Faktoren (Bcl-9 und Wif1), von Komponenten des ß-Catenin- Degradationskomplexes (Axin2 und GSK 3β), von kanonischen Zielgenen (Met, MMP7 und Cyclin D1) und von c-jun als Schlüsselprotein des nicht-kanonischen Wnt/ JNK-Signalweges wurde in der RT-PCR untersucht. Auf Proteinebene wurden zur Bestätigung zusätzlich Western Blots mit Antikörpern gegen aktives β-Catenin und c-jun durchgeführt. Die Zellproliferation kolorektaler Karzinomzelllinien wurde dosisabhängig inhibiert, und Niclosamid führte zu Apoptose. Nach Inkubation mit Niclosamid kam es nicht zur Umverteilung von aktivem β-Catenin von der nukleären in die zytosolische Fraktion. Die Wnt-Promotor-Aktivität von LEF/ TCF wurde nach 12 Stunden Inkubation mit 10 und 50 μM Niclosamid jedoch signifikant gesenkt. Kanonische Wnt-Zielgene (Met, MMP7 und Cyclin D1) sowie der Koaktivator Bcl-9 wurden auf Transkriptionsebene gehemmt, während das nicht-kanonische Schlüsselprotein c-jun aktiviert wurde. Fasst man zusammen, so führt die Inkubation mit Niclosamid zu inhibitorischen Effekten auf kolorektale Karzinomzelllinien und zu einer reduzierten kanonischen Wnt-Aktivität. Diese Effekte können durch eine gestörte Formation des Triple-Komplexes aus Bcl-9, β- Catenin und LEF/ TCF und einer Aktivierung von c-jun und damit des nicht-kanonischen Wnt/ JNK-Signalweges bedingt sein. In in vivo-Untersuchungen beabsichtigen wir, in einem Tiermodell die Daten zu verifizieren und so den Einsatz des Niclosamids als Option für Patienten mit metastasiertem kolorektalem Karzinom weiterführend zu beurteilen.

Les études cliniques et in vitro suggèrent que la sclérose de l’os sous-chondral due aux ostéoblastes (Ob) anormaux est impliquée dans la progression de l’ostéoarthrose (OA). Les Ob OA humains isolés à partir d’os sous-chondral sclérosé montrent un phénotype altéré, un niveau réduit de signalisation Wnt/β-caténine canonique et une minéralisation in vitro réduite. Il existe également deux voies non-canoniques, Wnt/PKC et Wnt/PCP qui ont étés décrites dans la littérature. Cependant, il n’existe aucune étude qui traite de ces deux voies dans les Ob OA. Ces voies sont activées après qu’un ligand Wnt non-canonique tel que Wnt-5a se lie à un récepteur Wnt couplé à des corécepteurs de la voie non-canonique. Ceci enclenche, respectivement pour la voie Wnt/PKC-Ca2+ et Wnt/PCP, la phosphorylation de PKC (p-PKC) et la phosphorylation de JNK (p-JNK) et agit sur les cibles en aval. Nous avons voulu déterminer s’il était possible de constater des altérations dans les voies Wnt non-canoniques dans les Ob OA. Nous avons préparé des cultures primaires d’ostéoblastes sous-chondral humains à partir de plateaux tibiaux de patients OA subissant une arthroplastie totale du genou, ainsi qu’à partir de plateaux tibiaux recueillis à l’autopsie de patients « normaux ». L’expression des gènes impliqués dans les voies Wnt/PKC et Wnt/PCP a été évaluée par RT-qPCR et la production par Western Blot des protéines, ainsi que celle de p-PKC et p-JNK et que l’activité des facteurs NFAT et AP-1 utilisés par ces deux voies. L’activité phosphatase alcaline (ALPase) et la quantité d’ostéocalcine (OC) ont étés évaluées respectivement à l’aide d’hydrolyse de substrat et d’ELISA. Le niveau de minéralisation a été évalué par la coloration au rouge Alizarine. Nos résultats montrent que l’expression et la production de Wnt-5a étaient augmentées dans les Ob OA comparées aux Ob N et LGR5 était significativement plus élevée. De plus, l’expression de LGR5 est directement régulée via la stimulation ou la diminution de Wnt-5a, à la fois au niveau de l’ARNm et des protéines. Par ailleurs, Wnt-5a a stimulé la phosphorylation de JNK et de PKC ainsi que l’activité NFAT et AP-1. Les niveaux de minéralisation ainsi que d’activité ALPase et de sécrétion d’OC ont aussi été affectés par les changements du niveau de Wnt-5a. Ces résultats suggèrent que Wnt-5a, qui est augmentée dans les OA Ob, peut stimuler les voies Wnt non-canoniques et affecter le phénotype et la minéralisation des OA Ob humains. / Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. Besides the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. These pathways are activated when a non-canonical Wnt ligand like Wnt-5a binds to a Wnt receptor coupled with non-canonical co-receptors. This activates, respectively for Wnt/PKC-Ca2+ and Wnt/PCP, the phosphorylation of PKC (pPKC) and the phosphorylation of JNK (pJNK) and their effect on downstream targets. However, there are no reports of either pathway in OA Ob. Here, we studied if alterations of the non-canonical pathways could be observed in OA Ob. We prepared primary human subchondral Ob using the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals at autopsy. The expression of genes involved in Wnt/PKC and Wnt/PCP was evaluated by RT-qPCR and their protein production by Western blot analysis, in addition to p-PKC and p-PCP and NFAT and AP-1 activity with luciferase. Alkaline phosphatase activity and osteocalcin levels were evaluated respectively by substrate hydrolysis and ELISA respectively, and mineralization levels were evaluated with Alizarin red staining. OA Ob showed an increased alkaline phosphatase activity and osteocalcin release. The expression of Wnt5a was increased in OA Ob compared to normal. The expression of LGR5 was significantly increased in these cells. Moreover, the expression and production of LGR5 is directly modulated via the stimulation or inhibition of Wnt5a. However, Wnt5a did not stimulate the expression of LGR4. Wnt5a increased the phosphorylation of PKC and JNK as well as NFAT and AP-1 activity. Mineralization levels as well as alkaline phosphatase activity and osteocalcin secretion levels were also linked with changes in Wnt-5a levels. These data indicate that Wnt5a, which is increased in OA Ob, can directly stimulate the Wnt/PKC and Wnt/PCP pathways and this can affect the phenotype and mineralization observed in human OA Ob.

No description available.