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Orientadores: Karin Spat Albino Barcellos Silveira, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:35:28Z (GMT). No. of bitstreams: 1 Bernusso_VanessaAline_M.pdf: 2366309 bytes, checksum: d89f227d922fcfaba6696c0e1af0fdae (MD5) Previous issue date: 2013 / Resumo: VASP (Vasodilator-stimulated phosphoprotein) e Zyxin são proteínas reguladoras de actina que controlam a adesão célula-célula. Zyxin dirige a montagem da actina através da interação e recrutamento da VASP a sítios específicos da adesão. A fosforilação da VASP ou da Zyxin altera suas atividades nas junções aderentes. PKA fosforila VASP em serina 157, regulando, assim, importantes funções celulares de VASP. VASP interage com ABL e é substrato da oncoproteína BCR-ABL. A presença da proteína BCR-ABL promove a oncogênese em pacientes com leucemia mieloide crônica (LMC) devido à ativação constitutiva da atividade tirosina quinase. Apesar de já descrita alteração da expressão de VASP e Zyxin em diferentes tumores epiteliais, o papel de VASP e Zyxin na LMC, na via de sinalização BCR-ABL e a participação destas proteínas na hematopoiese são desconhecidos. Desta maneira, demonstramos aqui ausência de p-VASP ser157 em células de medula óssea de pacientes com LMC, em contraste com a presença de p-VASP ser157 em doadores saudáveis. Pacientes com LMC em remissão, responsivos a inibidores de tirosina quinase, apresentam p-VASP ser157, enquanto os pacientes resistentes não expressam p-VASP ser157. Utilizando células K562 inibidas para VASP ou Zyxin, observamos que VASP e Zyxin modulam as proteínas anti-apoptóticas BCL-2 e BCL-XL da via de sinalização do BCR-ABL. Em adição, células K562 silenciadas para a VASP apresentam diminuição na atividade de FAK y925 e demonstramos que VASP interage com FAK. A expressão de VASP e Zyxin e de suas formas ativas aumenta durante a diferenciação megacariocítica e a inibição de VASP implica em diminuição na expressão do marcador CD61. Identificamos no presente estudo a participação de VASP e Zyxin na via do BCR-ABL, regulando a expressão de proteínas efetoras anti-apoptóticas e, também, na diferenciação megacariocítica. Desta maneira, a expressão alterada da atividade de VASP nos pacientes com LMC pode contribuir para a patogênese da doença, seja afetando a diferenciação celular ou a adesão das células leucêmicas / Abstract: VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP and Zyxin modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP interacts with ABL and VASP is a substrate of BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. It has been described an altered expression of VASP and Zyxin in different types of tumor; however the function of VASP and Zyxin in CML, in BCR-ABL pathway and in hematopoiesis remains unknown. We describe here the absence of p-VASP ser157 in CML bone marrow cells, in contrast to p-VASP ser157 expression in healthy donors. Patients responsive to tyrosine kinase inhibitors present p-VASP ser157, while resistant patients do not have p-VASP ser157. In K562 cells we observed that VASP and Zyxin modulate anti-apoptotic proteins BCL-2 and BCL-XL. VASP depletion in K562 cells decreases FAK y925 activity and VASP interacts with FAK. Expression of VASP, p-VASP, Zyxin and p-Zyxin increases during megakaryocyte differentiation and VASP inhibition affects this differentiation through reduced CD61 expression in VASP depleted cells. We identify here the participation of VASP and Zyxin in BCR-ABL pathway affecting anti-apoptotic proteins and, also, in megakaryocyte differentiation. Then, the altered expression of VASP activity in CML patients may contribute to CML pathogenesis, affecting cellular differentiation or leukemic cell adhesion / Mestrado / Fisiopatologia Médica / Mestra em Ciências

La thrombopoïétine (TPO) favorise successivement la prolifération et la maturation des progéniteurs mégacaryocytaires, soulevant la question du mécanisme expliquant cette dualité d'action. La signalisation SCF/ c-Kit est essentielle pour la prolifération de tous les progéniteurs hématopoïétiques, alors que l'extinction de l'expression du récepteur c-Kit est requise pour l'engagement en différenciation terminale. Réciproquement, l'équipe a montré que la stimulation de la voie Notch affecte une sous-population de progéniteurs bipotents érythro-mégacaryocytaires exprimant fortement CD9 (tétraspanine induite durant la maturation mégacaryocytaire) et favorise la reprise de leurs divisions au détriment de leur différenciation mégacaryocytaire terminale. Cet effet de la voie Notch s'accompagne d'une augmentation de l'expression de c-Kit. Ces observations m'ont conduite à m'intéresser aux mécanismes de régulation de c-Kit par la TPO en m'appuyant sur un modèle de progéniteurs bipotents immortalisés et dont la prolifération est strictement dépendante de la TPO (cellules G1ME). Les travaux réalisés durant ma thèse m'ont permis d'établir que (i) La stimulation des cellules G1ME par le ligand de Notch DLL1 favorise l'expression de c-Kit et réprime celle de CD9 (ii) L'activation inattendue de c-Kit par la TPO contribue à la prolifération (iii) c-Kit contribue activement à restreindre la polyploïdisation des cellules G1ME en présence de TPO (iv) La tétraspanine CD9 elle-même réprime l'expression de c-Kit à la membrane. Sur la base de ces résultats, nous proposons le modèle selon lequel, la TPO participerait à la fois à la prolifération des progéniteurs du fait de sa capacité à activer c-Kit, mais contribue aussi à l'augmentation de l'expression de CD9 qui en atteignant un seuil suffisant conduit à l'extinction de l'expression de c-Kit à la surface, entrainant alors l'arrêt des divisions et la différenciation mégacaryocytaire terminale / The Thrombopoietin (TPO) favors both the proliferation and the maturation of megakaryocytic progenitors, raising the question of the molecular mechanism explaining its dual function. SCF/ c-Kit signaling is essential for all hematopoietic progenitors amplification, whereas terminal differentiation requires the extinction of c-Kit receptor expression. Reciprocally, we evidenced in our team that Notch stimulation enables the induction of c-Kit expression and act on a particular subpopulation of bipotent erythro-megakaryocytic progenitors highly expressing the tetraspanin CD9 (induced during megakaryocytic maturation) and favors their re-entry in a cycling state by blocking their megakaryocytic maturation. These observations lead to the investigation of the molecular mechanism of c-Kit regulation by TPO in a cellular model of bipotent progenitors immortalized and dependent on TPO, the G1ME cells. During my thesis, I evidenced that: i) Notch stimulation induces the expression of c-Kit while repressing CD9 expression; ii) Surprisingly TPO is able to activate c-Kit allowing its contribution to cell proliferation; iii) c-Kit also represses megakaryocytic polyploidization (endomitosis characterizing megakaryocytic maturation) of G1ME cells; iv) The tetraspanin CD9 represses the expression of c-Kit. The ensemble of these data allows us to propose the following model wherein TPO activates c-Kit allowing the proliferation of megakaryocytic progenitors, while concomitantly induces the expression of the tetraspanin CD9 that will reach a sufficient level to provoke the extinction of c-Kit expression at the cell surface, thus enabling the arrest of cell cycling progress and the engagement into terminal megakaryocytic maturation