Le TGF-β (Transforming growth factor-β) régule de nombreux processus cellulaires et la dérégulation de la signalisation du TGF-β est associée à divers troubles vasculaires. Dans le laboratoire du Dr Génot, le TGF-β a été découvert comme étant le facteur capable d’induire la formation de podosomes organisés en superstructures, appelées rosettes, dans les cellules endothéliales aortiques. Les podosomes sont des structures à base d’actine, formées de façon transitoire, capables de dégrader la matrice extracellulaire (MEC). Dans ce projet, nous avons étudié les récepteurs du TGF-β et les mécanismes moléculaires associés, impliqués dans la formation des podosomes en réponse au TGF-β dans le modèle des cellules endothéliales aortiques bovines primaires (BAEc). Deux types de récepteurs du TGF-β de type I (TβRI), ALK5 et ALK1, régulent les réponses au TGF-β dans les cellules endothéliales, ALK5 étant un récepteur ubiquitaire et ALK1 étant un récepteur dont l’expression est restreinte aux cellules endothéliales. Ces deux récepteurs contrôlent l'activation de protéines Smads distinctes et réagissent à la stimulation par le TGF-β. ALK5 active Smad2/3 et ALK1 active Smad1/5/8. BMP9 est un autre ligand d’ALK1. ALK1 n'active pas Smad2/3 dans les BAEc et BMP9 inhibe la formation des podosomes induite par le TGF-β. La stimulation de Smad1/5/8 par le traitement au TGF-β est induite par la signalisation du complexe ALK1/ALK5. En utilisant une approche à base de siRNA ciblant l’un ou l’autre des TβRI, l’induction des podosomes par le TGF-β est supprimée. Cependant, la transfection des TβRI constitutivement actifs (CA) a montré que l’expression du CA-ALK5 se substitue au TGF-β pour induire des podosomes alors que l'expression du CA-ALK1 est inefficace. Concernant la signalisation en aval des TβRI, l'implication des protéines Smads a également été étudiée dans la régulation du processus. La diminution d’expression de Smad3 abolit complètement la formation des podosomes induite par le TGF-β alors que la déplétion des protéines Smad1 ou Smad5 augmente leur formation. La surexpression des protéines Smad2 ou Smad3, elles aussi, dans une certaine mesure, se substituent aux signaux du TGF-β, alors que la surexpression de Smad1 diminue la formation des podosomes en réponse au TGF-β. Le TGF-β est également capable de moduler la formation d'un autre type de structure d'actine appelée étoiles d’actine. Le nombre de cellules présentant des étoiles d'actine diminue avec le traitement au TGF-β. Cependant, dans les cellules déficientes en Smad3, la formation de ces étoiles d’actine semble être stimulée par le TGF-β. Dans les BAEc, la rigidité ainsi que les protéines de la MEC semblent aussi moduler la formation des podosomes et des étoiles d'actine. Ces travaux démontrent que, bien que le TGF-β stimule à la fois ALK5 et ALK1, la signalisation d’ALK5 induit la formation des podosomes et la signalisation d’ALK1 atténue ce signal. Les voies canoniques, par l’intermédiaire de la régulation des protéines Smads, contribuent à la formation des podosomes induits par le TGF-β dans les BAEc. / Transforming growth factor-β (TGF-β) regulates a wide array of cellular processes and deregulation of TGF-β signalling is associated with various vascular disorders. In the Lab of Dr. Génot it was discovered that TGF-β induces the formation of podosomes organised in superstructures called rosettes, in aortic endothelial cells. Podosomes are transient actin-based structures able to degrade the extracellular matrix (ECM). In this project we have studied TGF-β receptors and associated molecular mechanisms underlying podosome formation in response to TGF-β in primary bovine aortic endothelial cells (BAEc). Two types of TGF-β type I receptors (TβRI), ALK5 and ALK1, regulate TGF-β responses in endothelial cells. ALK5 being an ubiquitous receptor and ALK1 being endothelial cell specific. Both ALK5 and ALK1 receptors control the activation of distinct Smad proteins, and both are responsive to TGF-β stimulation. ALK5 activates Smad 2/3 and ALK1 activates Smad 1/5/8. BMP9 is another ligand for ALK1. ALK1 doesn’t activate Smad2/3 in BAEc and ALK1 inhibits TGFβ-induced podosome formation. Smad1/5/8 stimulation by TGF-β treatment is induced through ALK1/ALK5 complex signalling. Using a knockdown approach, at the TβRI level, TGF-β induction of podosomes was inhibited. However, transfection of constitutively active (CA) TβRI showed that CA-ALK5 expression bypassed the TGF-β requirement for podosome induction whereas CA-ALK1 expression was ineffective. Looking downstream of TβRI signalling, the involvement of Smad proteins was also analysed in terms of podosome formation. Smad3 depletion completely abolished TGFβ-induced podosome formation whereas depletion of Smad1 or Smad5 proteins enhanced the TGFβ-induced podosome response. When overexpressed, Smad2 or Smad3, to some extent, bypassed TGFβ signals, whereas Smad1 overexpression diminished the TGFβ-induced podosome response. TGF-β also modulated the formation of another type of actin structure named actin-stars. The number of cells presenting actin-stars decreased with TGF-β treatment. However, in Smad3 depleted cells the formation of these actin-stars seemed to be stimulated by TGF-β. In BAEc stiffness and ECM proteins also seemed to modulate podosome and actin star formation. This project establishes that although TGF-β stimulates both ALK5 and ALK1, ALK5 signalling triggers podosome formation and ALK1 mitigates this signal. The canonical pathways through Smad protein regulation are important for TGF-β induced podosome in BAEc.

Yes / Aberrant transforming growth factor-β (TGF-β) signaling activation is linked to pulmonary arterial hypertension (PAH). BMPR2 mutations perturb the balance between bone morphogenetic protein (BMP) and TGF-β pathways, leading to vascular remodeling, narrowing of the lumen of pulmonary vasculature, and clinical symptoms. This forum highlights the association of the TGF-β pathway with pathogenesis and therapeutic approaches. / Research carried out at Nasim laboratories is funded by GrowMedtech, the Royal Society, the Commonwealth Scholarship Commission (CSC) and the University of Bradford (UoB). N.S. is funded by the CSC and C.C.N. is partly funded by the UoB.

Orientadora: Profa. Dra. Káthia Maria Honório / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência & Tecnologia - Química, 2014. / Este trabalho propoe o uso de tecnicas de modelagem molecular para entender os principais fatores relacionados com a interacao entre um conjunto de ligantes bioativos e o receptor TGF-¿À tipo 1 (ALK5), alvo biologico envolvido no desenvolvimento de doencas como cancer e fibrose. Para entender essas interacoes, foram utilizadas tecnicas computacionais de modelagem molecular que sao utilizadas para quantificar essas interacoes. Dentre as metodologias utilizadas neste trabalho, foram empregadas tecnicas de acoplamento molecular e analises das relacoes quantitativas tridimensionais entre estrutura quimica e atividade biologica (QSAR 3D). Dessa forma, foi estudado um conjunto de dados contendo 70 moleculas empregando tecnicas de QSAR 3D (CoMFA e CoMSIA). Os modelos CoMFA obtidos apresentaram significativos valores de validacao interna (r2 = 0,979 e q2LOO = 0,882) e externa (conjunto de predicao - r2pred = 0,998 e potencial preditivo externo . r2m= 0,998) e os modelos obtidos da analise CoMSIA tambem apresentaram valores significativos de validacao interna (r2 = 0,913 e q2LOO = 0,822) e validacao externa (conjunto de predicao - r2pred = 0,975 e potencial preditivo externo . r2m = 0,975. A analise dos mapas de contribuicoes estereoquimicas e eletrostaticas (CoMFA) e dos mapas de contribuicoes eletrostaticas e dos grupos doadores de ligacao de hidrogenio (CoMSIA) para o composto mais ativo e menos ativo foi capaz de indicar caracteristicas importantes para a inibicao do alvo biologico em estudo. Portanto, o emprego de metodologias computacionais foi de fundamental importancia para o entendimento sobre os principais fatores envolvidos na interacao entre os ligantes estudados e o receptor ALK5. / This study proposes the use of molecular modeling techniques to understand the main factors related to the interaction between a set of ligands and bioactive TGF-â receptor type 1 (ALK5), biological target involved in the development of diseases such as cancer and fibrosis. To understand these interactions, computational molecular modeling techniques were used, which are used to quantify these interactions. Among the methodologies used in this study, techniques of molecular docking and analyses of three-dimensional quantitative relations between chemical structure and biological activity (3D QSAR) were performed with the goal of understanding the main physico-chemical features related to the biological activity. Thus, we studied a data set containing 70 molecules and 3D QSAR analyses (CoMFA and CoMSIA) were carried out. The CoMFA models have good statistical parameters (r2 =0.979 and q2LOO =0.882) and a significant predictive power (r2pred=0.998 and r2m = 0.998). The CoMSIA models also showed significant values for the internal validation (r2=0.913 and q2LOO =0.822) and the external validation (r2pred=0.975 and r2m=0.975). The analysis of the steric and electrostatic contributions (CoMFA), as well as the electrostatic contributions and donor groups for hydrogen bonding (CoMSIA) for the most active and least active compounds was able to indicate important characteristics of inhibiting the biological target under study. Therefore, the use of computational methodologies was of fundamental importance to understand the main factors involved in the interaction between the studied ligands and the ALK5 receptor.

Orientadora: Profa. Dra. Káthia Maria Honório / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2015. / A proteina quinase TGFBRI/ALK5 esta envolvida em uma variedade de processos patologicos, tais como fibroses e cancer. Essa proteina propaga uma sinalizacao intracelular capaz de atingir o nucleo e modular a transcricao de genes. Neste trabalho, os metodos do holograma QSAR (HQSAR) e da analise comparativa de campos de interacao molecular (CoMFA), foram utilizados para uma serie de inibidores da proteina TGF¿ÒRI/ALK5. Os modelos obtidos apresentaram valores significativos de validacao interna (CoMFA, r2calibracao=0,99 e q2cv=0,85; HQSAR, r2calibracao=0,92 e q2cv=0,0,72) e externa (CoMFA, r2teste=0,85 e r2m=0,64, HQSAR, r2teste= 0,79 e r2m= 0,69), indicando capacidade preditiva dos modelos 2D e 3D para os compostos testados. Os modelos foram usados para analisar a capacidade preditiva de um conjunto de compostos-teste e os valores preditos a partir dos modelos de HQSAR e CoMFA apresentaram boa concordancia com os resultados experimentais. Os modelos finais juntamente com as informacoes obtidas a partir dos mapas 2D e 3D (estereoquimica e eletrostatica) para os compostos mais ativos e menos ativos da serie foram capazes de indicar caracteristicas importantes para a inibicao do alvo biologico em estudo. Com isso, os resultados obtidos neste trabalho podem guiar futuros projetos para o desenvolvimento de novos candidatos a farmacos para o tratamento de doencas como fibrose e cancer. / TGFBRI/ALK5 protein is a biological receptor involved in a variety of pathological processes such as cancer and fibrosis. TGF¿ÒRI/ALK5 receptor propagates an intracellular signaling that forms a protein complex capable of reaching the nucleus and modulating the gene transcription. In the present study, comparative molecular field analysis (CoMFA) and hologram quantitative structure-activity (HQSAR) studies were conducted on a series of potent TGF¿ÒRI/ALK5 inhibitors. Significant correlation indexes from the internal (CoMFA, r2calibration=0,99 e q2cv=0,85; HQSAR, r2calibration=0,92 e q2cv=0,0,72) and external (CoMFA, r2teste=0,85 e r2m=0,64, HQSAR, r2teste= 0,79 e r2m= 0,69) validations indicated the predictive power of the 2D and 3D models for untested compounds. The models were then used to investigate the predictive ability of a test-set, and the predicted values from the HQSAR and CoMFA models were in good agreement with the experimental results. The final QSAR models, along with the information obtained from 3D (steric and electrostatic) contour maps and 2D contribution maps, can be useful for the design of novel bioactive ligands.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13502号 / 論医博第2261号 / 新制||医||1061(附属図書館) / (主査)教授 村川 泰裕, 教授 柳田 素子, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

With the dawn of metazoans, the ability of cells to communicate with each other became of paramount importance in maintaining tissue homeostasis. The transforming growth factor β (TGF-β) signaling pathway, which plays important roles during embryogenesis and in the adult organism, signals via a heterodimeric receptor complex consisting of two type II and two type I receptors. After receptor activation through ligand binding, Smads mediate the signal from the receptor complex to the nucleus, where they orchestrate transcription. Depending on the context of activation, TGF-β can mediate a plethora of cellular responses, including proliferation, growth arrest, apoptosis and differentiation. In cancer, TGF-β can act as both as a tumor suppressor and promoter. During early stages of tumorigenesis, TGF-β prevents proliferation. However, TGF-β is also known to promote tumor progression during later stages of the disease, where it can induce differentiation of cancer cells towards a migratory phenotype. The aim of this thesis was to investigate how cells can differentiate their response upon TGF-β pathway activation. The first paper describes the role of Notch signaling in TGF-β induced growth arrest, demonstrating that TGF-β promotes Notch activity and that Notch signaling is required for prolonged TGF-β induced cell cycle arrest. In the second and third paper, we investigate the role of SIK, a member of the AMPK family of kinases, mediating signaling strength of TGF-β through degradation of the TGF-β type I receptor ALK5. While the second paper focuses on the effect of SIK on ALK5 stability and subsequent alterations in TGF-β signaling, the third paper emphasizes cooperation between SIK, Smad7 and the E3 ligase Smurf in degradation of ALK5. Finally, the fourth paper explores a novel role of SIK during TGF-β induced epithelial to mesenchymal transition (EMT). SIK binds to and degrades the polarity protein Par3, leading to enhanced EMT.

Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.