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Colorectal tumors with mutations on Wnt family members show an overexpression of several Notch target genes, suggesting a link between Wnt and Notch signaling pathways. In this work, we have demonstrated that there is a direct relation between Wnt and Notch pathways that it is responsible for the maintenance of the intestinal proliferative compartment and crucial for intestinal tumorigenesis. We have identified two different mechanisms through β-catenin interacts with Notch to activate transcription: 1) β-catenin activates Notch through the transcriptional activation of Notch ligand Jagged1. Jagged1 expression leads the binding to the Notch receptor, promoting its cleavage and activation. This mechanism occurs in human colorectal tumors, with nuclear β-catenin, overexpressed Jagged1 and activated Notch. 2) β-catenin and Notch bind simultaneously to a specific group of gene promoters. We observed that this group of genes is overexpressed in tumours with mutations in Wnt signaling. Moreover, β-catenin and Notch physically interact in the nucleus of colorectal tumour cell lines. We have characterized a group of genes that are double target genes for β-catenin and Notch; in other words, these genes require the activation of both Wnt and Notch signaling pathways. These genes are expressed in the intestinal undifferentiated compartment. The loss of any of these signalling pathways reduced stem cell marker expression, reinforcing the importance of the interaction between both signalling pathways. The study of conditional knockouts mice for Jagged1 indicated that Jagged1 is not required for maintaining the intestinal homeostasis. However, Jagged1 is crucial during the tumorigenic process. Our data opens a new possibility for colorectal treatment, using inhibitors of Notch ligand Jagged1, avoiding side effects in the intestinal normal tissue.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Oct. 31, 2007). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

The canonical Wnt pathway is a vitally important signalling pathway that plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues. The twelve Armadillo (ARM) repeat-containing protein beta-catenin acts as the signal transducer in this pathway and is continuously degraded in the cytosol by the beta-catenin destruction complex (BDC). Upon receiving the Wnt signal the BDC is inactivated, allowing beta-catenin to accumulate in the cytosol and be transported to the nucleus where it binds to the TCF/LEF family of transcription factors, inducing the expression of cell cycle promotor genes. In this Thesis I describe investigations into the roles of leucine-rich repeat kinase 2 (LRRK2) and the transcription factor TCF7L2 within this signalling pathway. LRRK2 is a large multi-domain protein with strong links to Parkinson’s disease and suggested to play a role in inactivating the BDC in response to the Wnt signal. A recent paper proposed that the previously uncharacterised regions of LRRK2 contain a series of tandem repeat sub-domains. I began an investigation into these sub-domains but I was unable to produce soluble protein constructs despite the use of a range of common techniques, and so I was forced to conclude this project early. The main body of this thesis focuses on the interaction between the intrinsically disordered TCF7L2 and the repeat protein beta-catenin, a very long interface of approximately 4800 Å2 that spans from the third to the eleventh ARM repeat of beta-catenin and residues 12 to 50 of TCF7L2, as determined by X-ray crystal structures. First, a fluorescence reporter system for the binding interaction was developed and used to determine the kinetic rate constants for the association and dissociation of the wild-type construct using stopped-flow fluorescence spectroscopy and time-dependent fluorescence spectroscopy. It was found that association of TCF7L2 and beta-catenin was rapid (7.3 ± 0.1 x107 M-1s-1) with only a single phase was observed, whereas dissociation was biphasic and slow (5.7 ± 0.4 x10-4 s-1, 15.2 ± 2.8 x10-4 s-1). Using either of these two dissociation rate constants the calculated Kd value obtained is much lower than the values previously reported in the literature (8 ± 1 / 20 ± 2 pM compared with 16 nM). This reporter system was then used to investigate the striking variability between three crystal structures previously obtained for the TCF7L2-beta-catenin complex, in which different regions of TCF7L2 show different elements of secondary structure. Mutational analysis revealed that the interface residues on TCF7L2 identified in these structures make little or no contribution to the overall binding affinity, pointing to a transient nature of these contact in solution and suggesting that the observed differences between the structures are due to differences in crystal packing. Further experiments into the effect of osmolarity on the binding equilibrium and kinetics supported this conclusion and suggest a change in the association/dissociation mechanism as a function of ionic strength. Lastly, further mutational analysis of TCF7L2 revealed two regions that contribute particularly strongly to the binding kinetics, suggesting that TCF7L2-beta-catenin assembly proceeds via a two-site avidity mechanism. Some of the most destabilising variants display two additional dissociation phases, indicating the presence of an alternative dissociation pathway that is inaccessible to the wild-type. In summary, the results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an extended repeat protein surface, a process shown to involve multiple routes with multiple steps in each.

Orientador: Rebeca de Souza Azevedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T01:17:16Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_M.pdf: 2889311 bytes, checksum: 3530f20f2b5d10e200ba02ec5d6d58b2 (MD5) Previous issue date: 2012 / Resumo: O ameloblastoma (AME) é um dos tumores odontogênicos (TO) mais comuns e, apesar de ser uma lesão benigna, pode apresentar comportamento clínico agressivo, localmente destrutivo e invasivo, enquanto o tumor odontogênico cístico calcificante (TOCC) é um TO benigno cístico que apresenta similaridades microscópicas com o AME, mas, geralmente, exibe comportamento clínico de menor agressividade. A via de sinalização Wnt/ß-catenina está envolvida no desenvolvimento e progressão tumoral de neoplasias benignas e malignas, e a expressão alterada de suas proteínas já foi identificada em alguns TO, provavelmente contribuindo para suas biologias tumorais. Dessa forma, o objetivo deste estudo foi avaliar e comparar a expressão imunoistoquímica de marcadores associados a via de sinalização Wnt/ß-catenina - Wnt1, Wnt5a, ß--catenina e syndecan-1(SDC-1) - em 17 casos de AME sólido e de 6 casos de TOCC. Os resultados revelaram que nos AME, a maioria dos casos foram focalmente positivos para Wnt1 e Wnt5a, a ß--catenina foi positiva no citoplasma isoladamente ou em associação a positividade de membrana, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 40% dos casos. Nos casos de TOCC, a positividade para Wnt1 e Wnt5a foi de 100% e incluiu também as células fantasmas. A ß--catenina foi positiva no citoplasma e no núcleo das células epiteliais da maioria dos casos, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 16,7% dos casos. Em conclusão, este estudo evidenciou que a expressão de Wnt1 e Wnt5a é mais proeminente nos casos de TOCC, a expressão de ß--catenina em AME é citoplasmática e em TOCC é nuclear, e que a expressão estromal de SDC-1 é mais proeminente nos casos de AME, reforçando, assim, o papel de Wnt1 e Wnt5 no desenvolvimento do TOCC, de ß--catenina no desenvolvimento do TOCC e do AME, e de SDC-1 estromal no fenótipo invasivo do AME. Além disso, a expressão de Wnt1 e de Wnt5a nas células fantasmas do TOCC pode contribuir para esta histogênese. Por conseguinte, a via de sinalização Wnt/ß--catenina parece contribuir para o desenvolvimento das lesões de AME sólido e de TOCC / Abstract: Ameloblastoma (SMA) is one of the most common odontogenic tumors (OT), and although benign, it may have aggressive clinical behavior, be locally destructive and invasive, while calcifying cystic odontogenic tumor (CCOT) is a benign cystic OT that presents microscopic similarities with AME, but, in general, it has a less aggressive clinical behavior. Wnt/ß--catenin signaling pathway is involved in benign and malignant tumor development and progression, and changes in expression of its proteins have been identified in some OT, probably contributing to their tumoral biologies. Thus, the aim of this study was to evaluate and compare the immunohistochemical expression of markers associated with Wnt/ß--catenin signaling pathway - Wnt1, Wnt5a, ß--catenin and syndecan-1 (SDC-1) - in 17 cases of solid SMA and 6 cases of CCOT. The results showed that in AME, most fo the cases were focally positive for Wnt1 and Wnt5a, ß--catenin was positive in the cytoplasm only or in association with membrane positivity, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 40% of the cases. In CCOT cases, Wnt1 and Wnt5a was of 100% and also included ghost cells. ß--catenin was positive in the cytoplasm and in the nucleus of epithelial cells in most of the cases, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 16.7% of the cases. In conclusion, this study highlighted that Wnt1 and Wnt5a expression was most prominent in CCOT, ß--catenin expression was cytoplasmatic in AME and nuclear in CCOT, and that stromal expression of SDC-1 was most prominent in AME, so, reinforcing Wnt1 and Wnt5 role in CCOT development, ß--catenin role in CCOT and AME development, and stromal SDC-1 role in the AME invasive phenotype. In addition, Wnt1 and Wnt5a expression in ghost cells of CCOT may contribute to this histogenesis. Therefore, Wnt/ß--catenin signaling pathway seems to contribute to solid AME and CCOT development / Mestrado / Estomatologia / Mestre em Estomatopatologia

La voie de signalisation Wnt/β-caténine (Wnt canonique) est impliquée dans une grande variété de fonctions biologiques, entre autres dans l’établissement des axes embryonnaires, l’organogenèse et l’homéostasie de cellules souches adultes. En absence de signaux Wnt, un complexe multiprotéique comprenant le suppresseur de tumeur Adenomatous Polyposis Coli (APC) marque la β-caténine pour la dégradation protéasomique. L’activation de la voie Wnt canonique induit un arrêt de la dégradation de la β-caténine, qui s’accumule dans le noyau, où elle active l’expression de gènes cibles de Wnt. Au cours de la myogenèse embryonnaire, processus pendant lequel le muscle squelettique est formé, une partie des cellules pluripotentes du dermomyotome acquièrt l’identité musculaire, se différencie et forme les myofibres, les unités fonctionnelles du muscle squelettique. La myogenèse n’est pas confinée qu’à la période embryonnaire. En effet, elle peut être réactivée dans le muscle adulte suite à une lésion ou dans certaines conditions pathologiques. Dans ce contexte, les cellules souches du muscle squelettique, appelées cellules satellites, sortent de leur quiescence et génèrent des progéniteurs myogéniques qui prolifèrent et se différencient en formant de nouvelles myofibres pour réparer le tissu. De plus, une partie des progéniteurs myogéniques retournent à l’état quiescent, renouvelant ainsi la population de cellules souches résidentes. Le rôle de la voie de signalisation Wnt/β-caténine dans l’engagement des cellules pluripotentes du dermomyotome vers le destin myogénique demeure méconnu. De même, la fonction de la voie Wnt canonique dans les cellules satellites au cours de la régénération du muscle squelettique adulte reste à l’heure actuelle controversée, car différentes approches sont parvenues à des conclusions contradictoires. Grâce à des modèles génétiques murins, nous avons caractérisé le rôle précis de la voie Wnt canonique au cours de la myogenèse embryonnaire et adulte. Nous montrons in vivo que l’hyperactivation constitutive de la voie de signalisation Wnt/β-caténine induite par l’inactivation conditionnelle d’APC, le principal régulateur négatif de la cascade, se traduit par un défaut majeur de formation et de régénération du muscle squelettique. Nos résultats ex vivo et in vitro démontrent que l’hyperactivation de la voie Wnt canonique altère la progression du cycle cellulaire et entraîne la mort par apoptose. De plus, l’inactivation conditionnelle de la β-caténine n’affecte pas la prolifération des progéniteurs myogéniques mais perturbe leur différenciation. Globalement, nos résultats suggèrent deux rôles différents de la voie de signalisation Wnt/β-caténine dans le muscle squelettique. D’une part, l’inhibition de la voie Wnt canonique est nécessaire au cours de l’initiation de la myogenèse pour permettre l’engagement myogénique des cellules pluripotentes du dermomyotome et l’activation des cellules satellites. D’autre part, la voie de signalisation Wnt/β-caténine est requise à la fois dans les progéniteurs musculaires embryonnaires et adultes pour leur différenciation et la formation des myofibres. / The Wnt/β-catenin signaling pathway, also called canonical Wnt signaling, is implicated in a large variety of biological processes, including embryonic axis determination, organogenesis and adult stem cells homeostasis. Canonical Wnt signaling regulates the stability of β-catenin, a transcriptional co-activator that, in absence of Wnt ligands, is targeted to proteasomal degradation by a multiproteic complex comprising the Adenomatous Polyposis Coli (APC) tumor suppressor. Activation of canonical Wnt signaling blocks β-catenin degradation and results in its accumulation into the nucleus, where it induces the expression of Wnt target genes. During embryonic myogenesis, the process of skeletal muscle formation, a proportion of pluripotent dermomyotomal cells restrict their fate to acquire a myogenic identity and differentiate into contractile myofibers, the functional units of skeletal muscle. Myogenesis can take place also in adult skeletal muscle. Indeed, upon acute injury or in pathological conditions, quiescent muscle-specific stem cells, called satellite cells, become activated and give rise to myogenic progenitors that massively proliferate, differentiate and fuse to form new myofibers and restore tissue functionality. In addition, a proportion of proliferating progenitors returns back to quiescence and replenish the pool of satellite cells in order to maintain the regenerative potential of skeletal muscle. The role of canonical Wnt signaling in the cell fate choice that drives multipotent dermomyotomal cells toward the myogenic lineage remains elusive. Similarly, a possible involvement of the Wnt/β-catenin cascade has been hypothesized in satellite cells during adult skeletal muscle regeneration, but different approaches came to contradictory results. In this study, we use genetic mouse models to investigate the precise role of canonical Wnt signaling in embryonic and adult myogenesis. In vivo constitutive overactivation of Wnt/β-catenin signaling following conditional deletion of APC, the major intracellular negative regulator of the pathway, results in complete abrogation of skeletal muscle formation and regeneration. By combining ex vivo and in vitro approaches, we show that canonical Wnt signaling hyperactivation alters cell cycle progression and results in programmed cell death. Conversely, conditional inactivation of β-catenin does not perturb the proliferative ability of myogenic progenitors but rather affects their differentiation. Collectively, our results demonstrate at least two distinct roles of the Wnt/β-catenin cascade in skeletal muscle. First, during myogenic initiation, canonical Wnt signaling must be inhibited to allow proper activation of myogenesis, in particular to elicit myogenic commitment of dermomyotomal cells and activation of adult satellite cells. Second, in myogenic progression, the Wnt/β-catenin pathway is required in both embryonic and adult muscle progenitors for proper differentiation and myofibers formation.

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

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