L'agressivité des cancers est souvent liée au pouvoir métastatique des cellules tumorales et la dissémination de ces dernières peut survenir suite à un phénomène appelé la transition épithéliomésenchymateuse. Une analyse de l'expression de la Cycline A2 conduite sur des échantillons humains de tumeurs primaires colorectales et de leurs métastases correspondantes révèle que cette protéine est moins abondante dans ces dernières. Le travail décrit dans cette thèse a permis de relier la Cycline A2 au remodelage du cytosquelette d'Actine dans les fibroblastes. Cette régulation requiert la localisation cytoplasmique de la molécule ainsi que son domaine N-terminal qui ne lie pas les CDKs. Nos expériences suggèrent que cette nouvelle activité est la conséquence d'une liaison directe entre la GTPase RhoA et la Cycline A2. La présence de cette dernière augmente l'activation de RhoA par sa GEF in vitro. L'utilisation de cellules épithéliales mammaires normales a permis l'identification d'un autre partenaire, RhoC. Dans ce contexte cellulaire, l'invalidation de la Cycline A2 diminue l'activation de RhoA et, renforce celle de RhoC ce qui conduit à une augmentation de l'invasion cellulaire en matrice de collagène. Ces cellules acquièrent aussi des propriétés mésenchymateuses caractéristiques de l'EMT, et ce phénotype est exacerbé par la présence de RasV12. Ce travail établit donc l'existence de nouvelles fonctions pour la Cycline A2 qui viennent compléter le tableau de régulation de la motilité par les protéines du cycle cellulaire et contribuent à une meilleure compréhension de son rôle dans le cancer. / Cancer aggressiveness is often associated with metastases occurrence and their dissemination can arise following an epithelial to mesenchymal transition (EMT). Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma of matched human tumors. This manuscript describes new links between Cyclin A2 and Actin cytoskeleton remodeling in fibroblasts. This regulation requires a cytoplasmic localization of the protein and its N-terminal domain, which is unable to bind CDKs. This new Cyclin A2 activity appears to be mediated by its binding to RhoA. Accordingly, the activity of its GEF is potentiated when Cyclin A2 is present, in vitro. Furthermore, we used a normal mammary epithelial cell line and identified another Cyclin A2 partner, RhoC. Cyclin A2 depletion in this context leads to a reciprocal RhoGTPase activation where RhoA activation is impaired and that of RhoC is increased. Moreover, cell invasiveness is increased in a collagen matrix following Cyclin A2 knockdown in these cells. In addition, the epithelial cells acquire mesenchymal properties, which are exarcerbated by the expression of RasV12 and are characteristic of an EMT. Our work completes the network involving cell cycle proteins in motility. These novel functions of Cyclin A2 will hopefully help to understand the impact of its deregulation in cancer.

O cristalino de vertebrados se origina a partir da invaginação do ectoderme que recobre a vesícula óptica. A invaginação epitelial em diversos modelos é causada pela constrição celular apical, mediada pela contração apical de actina e miosina II e regulada pela GTPase RhoA. Neste trabalho nós investigamos se a invaginação do cristalino em embriões de galinha ocorre devido à constrição celular apical e se este evento é controlado por RhoA. Actina filamentosa e miosina II são expressas na porção apical do cristalino durante a invaginação. Quando a polimerização de actina é inibida por Citocalasina D, o cristalino não invagina, sugerindo que a constrição celular apical poderia contribuir para a invaginação do cristalino. RhoA também é expressa durante o desenvolvimento do cristalino, mas a inibição de RhoA, por eletroporação da forma dominante-negativo, não impediu a invaginação do placóide do cristalino, não alterou a distribuição de miosina II na porção apical do cristalino nem sua ativação, indicando que a invaginação do cristalino independe de RhoA. / Vertebrate lens derives from invagination of the ectoderm that overlies optic vesicles. Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase RhoA. Here we investigate the possibility that chick lens placode invagination could also be driven by apical cell constriction and controlled by RhoA. We show that actin and myosin II are expressed at lens apical side during lens invagination. Actin polymerization inhibition by in ovo Cytochalasin D treatment prevents lens placode invagination, suggesting that lens placode invagination could be driven by apical cell constriction. RhoA GTPase is also expressed at apical portion of lens placode and during lens invagination. However, when we overexpressed by electroporation the dominant-negative RhoA in the pre-lens ectoderm invagination was not affected. Furthermore, dominant-negative RhoA didnt affect myosin II apical localization nor myosin II phosphorilation, indicating that in lens invagination this process is not regulated by GTPase RhoA.

LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo. / LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.

Neisseria gonorrhoeae verursacht die sexuell übertragbare Krankheit Gonorrhoe und ist ein Typ-IV-Pili (Tfp) exprimierendes Bakterium, das den Urogenitaltrakt besiedelt. Frühe Infektionsstadien piliierter N. gonorrhoeae (P+GC) sind durch die Tfp-vermittelte Adhärenz an Wirtszellen gekennzeichnet, dann erfolgt die Bildung von Mikrokolonien auf Wirtszellepithelien. Hier wird gezeigt, dass die Wirtszellen an der effizienten Bildung der extrazellulären Mikrokolonien beteiligt sind. P+GC die fixierte Wirtszellen infizieren weisen eine verzögerte Mikrokoloniebildung gegenüber einer Infektion lebender Wirtszellen auf. Kortikales Aktin wird zusammen mit Signalproteinen innerhalb der Wirtszellen zu den adhärierten Bakterien rekrutiert, darunter das Hauptstrukturprotein von Caveolae-Membrandomänen, Caveolin-1 (Cav1). Eine Reduzierung der Expression von Cav1 führt zu einer verstärkten Aufnahme von P+GC in die Wirtszellen, wohingegen die Expression von Cav1 in Cav1-negativen Zellen eine Internalisierung verhindert. Internalisierte Bakterien weisen dabei geringere Überlebensraten auf je länger sie in den Wirtszellen verbleiben. Die Rekrutierung von Cav1 ist eine unmittelbare und kontinuierliche zelluläre Antwort auf eine Infektion mit P+GC, welche die Phosphorylierung von Cav1 an Tyrosin 14 bedingt. Zusätzlich erforderte die Cav1-vermittelte Blockierung der Internalisierung der Bakterien und die Verankerung von Cav1 mit dem Zytoskelett eine Tyrosinphosphorylierung von Cav1. Eine Analyse möglicher Interaktionspartner von phosphoryliertem Cav1 zeigte eine direkte Interaktion mit Vav2. Sowohl Vav2 als auch sein Substrat, die kleine GTPase RhoA, blockieren die Aufnahme von Bakterien in die in Wirtszellen. Die Aktivierung von RhoA nach P+GC Infektion erfordert die Expression von Cav1, was auf einen Cav1-Vav2-RhoA Signalweg hindeutet. Darüber hinaus wurden in dieser Arbeit sechs neue, eine SH2-Domäne-beinhaltende Interaktionspartner von phosphoryliertem Cav1 identifiziert. / Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea and colonizes mucosal epithelia of the human urogenital tract. The early stages of infection with piliated N. gonorrhoeae (P+GC) are characterized by Tfp-mediated adherence to host cells, followed by formation of bacterial microcolonies on the surface of host cells. This study provides evidence that host cell participation is required for the efficient formation of extracellular microcolonies during Neisseria infection. P+GC infecting fixed host cells demonstrate altered motility and delayed microcolony formation compared to infecting living host cells. Cortical actin and various signal transducing proteins are recruited to the site of bacterial attachment within host cells, one of them being the major structural protein of plasma membrane caveolae, Caveolin-1 (Cav1). Down-regulation of Cav1 results in increased uptake of P+GC into host cells whereas expression of the protein in Cav1-negative cells blocks bacterial internalization. Host cell entry results in decreased viability of internalized bacteria over time. Cav1 recruitment is demonstrated to be an immediate and continuous cellular response to P+GC infection that involves Cav1 phosphorylation on its tyrosine 14 residue. Prevention of bacterial uptake mediated by Cav1 as well as tight association of Cav1 with the cytoskeleton also requires tyrosine phosphorylation. A broad analysis of interaction partners of phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, are involved in preventing bacterial uptake and RhoA activation after P+GC infection requires Cav1 expression, thus providing evidence for a Cav1-Vav2-RhoA signaling cascade. Moreover, six novel SH2-domain containing interaction partners of tyrosine phosphorylated Cav1 have been identified, all of which have been implicated in modulating the cytoskeleton.

Chronisch entzündliche Darmerkrankungen stellen eine Gruppe von chronischen, häufig in Schüben verlaufenden Erkrankungen mit rezidivierenden Entzündungen des Gastrointestinaltraktes dar. Es konnte gezeigt werden, dass eine gestörte Barrierefunktion einen wichtigen Schritt für die Pathogenese darstellt und dass das Zytokin Tumornekrosefaktor alpha (TNF) eine entscheidende Rolle dabei spielt. Die Rolle der kleinen GTPase RhoA bei der TNF-induzierten Barrieredysfunktion ist aufgrund der Komplexität der Signalwege nicht vollständig verstanden. Daher sollte der Einfluss von RhoA und der RhoA Effektorkinase PKN auf diese Prozesse in vitro mit Hilfe eines induzierbaren Expressionssystems untersucht werden, welches die kontrollierte Expression einer konstitutiv aktiven (KA) RhoA- und PKN-Mutante sowie einer dominant negativen (DN) PKN-Mutante ermöglichte. Die Induktion der KA RhoA Expression führte zu einer Störung der epithelialen Barriere. Eine simultane Interferon-gamma und TNF-Behandlung resultierte ebenfalls in einer gestörten Barrierefunktion, welche in KA RhoA Zellen weniger stark ausgeprägt war. Die TNF-Behandlung führte zu einer Aktivierung von PKN, weshalb dieses Protein ein Kandidat für die Vermittlung dieser Effekte darstellte. Inhibition von PKN mit Inhibitoren oder der Expression der DN Mutante führten zu einer Aggravierung der TNF-induzierten Barrieredysfunktion, welche durch eine Verringerung des transepithelialen elektrischen Widerstandes und eine erhöhte Ionenpermeabilität charakterisiert war. Diese Veränderungen wurden von einer Erhöhung des Myosin Leichtketten und NF-kappaB p65-Phosphorylierungsniveaus sowie von morphologischen Veränderungen begleitet. Im Gegensatz dazu konnten diese Veränderungen durch die Expression der KA PKN Variante abgeschwächt bzw. verhindert werden. Diese Ergebnisse liefern Hinweise auf eine potenzielle Rolle der RhoA Effektorkinase PKN bei der Modulation der TNF-induzierten Barrieredysfunktion in intestinalen Epithelzellen. / Inflammatory bowel diseases are relapsing systemic inflammatory diseases of the gastrointestinal tract associated with high morbidity and costs. A plethora of studies demonstrated that impaired intestinal barrier function is a key step in the pathogenesis of inflammatory bowel diseases and that the cytokine tumor necrosis factor alphpa (TNF) is of pivotal importance for this effect. Although the small GTPase RhoA has been implicated in the control of tight junction function, its role in TNF induced barrier dysfunction is not entirely understood due to the complexity of its downstream signaling pathways. Therefore, the contribution of RhoA and its effector kinase PKN on TNF induced barrier dysfunction was investigated in vitro. An inducible expression system that allowed the doxycyline controlled expression of a constitutively active (CA) RhoA and PKN mutant as well as a dominant negative (DN) PKN mutant was generated. Induction of CA RhoA expression led to an impaired epithelial barrier. Simultaneous Interferon-gamma and TNF treatment also resulted in barrier perturbation, but this defect was attenuated when CA RhoA was expressed. As treatment with TNF resulted in activation of the RhoA effector kinase PKN, this protein constitutes a candidate molecule for the mediation of these effects. Inhibition of PKN by inhibitory compounds as well as expression of a dominant negative PKN mutant aggravated TNF-induced barrier dysfunction, characterized by a decline in transepithelial electrical resistance and increased ion permeability. These alterations were accompanied by an increase in myosin light-chain and NF-kappaB p65 subunit phosphorylation level as well as morphological changes of the tight junctions. Conversely, expression of a CA PKN mutant attenuated or prevented these changes. These results provide support for a potential role of the RhoA effector kinase PKN in modulating the barrier disrupting effects of TNF in the intestinal epithelium.

La motilité cellulaire est un processus intégré essentiel à de nombreux phénomènes physiologiques tels que la formation du cône de croissance et la plasticité synaptique. Des dérégulations de la motilité cellulaire peuvent être à l’origine de la formation de métastases ou de pathologies neuropsychiatriques comme la schizophrénie et l'autisme. La compréhension des mécanismes régulant la migration cellulaire est donc un enjeu majeur. La motilité cellulaire repose sur la formation de diverses structures constituées de réseaux d’actine branchés telles que le lamellipode. La formation du lamellipode nécessite l’intervention de protéines régulatrices de l’actine telles que Rac1 et les complexes Wave et Arp2/3. Grâce à l’utilisation de suivi de protéine unique, nous avons pu comprendre comment la coordination spatio-temporelle de ces régulateurs contrôle la formation et la morphologie des lamellipodes de cellules migrantes. Nous avons ainsi découvert que l’activation et la localisation du complexe Wave étaient régulées de manière enzymatique mais également mécanique. Dans une première étude, nous avons montré que la RhoGTPase Rac1 active le complexe Wave spécifiquement à l’extrémité du lamellipode. Dans une seconde étude, nous avons révélé que la localisation du complexe Wave est régulée par la dynamique des filaments des réseaux branchés d’actine. Ces données soulignent l’importance du complexe Wave dans la formation du lamellipode et révèlent l’existence d’une régulation mécanique de la localisation du complexe Wave. / Cell motility is an integrated process involved in critical phenomena such as axonal pathfinding and synaptic plasticity. Dysregulation of cell motility can induce metastasis and abnormal spine shapes observed in neuropsychiatric disorders like autism and schizophrenia. Therefore it is essential to understand how cell motility is regulated. Cell motility requires the formation of branched actin networks propelled by actin polymerization that lead to the formation of membrane protrusions such as the lamellipodium. Several actin regulatory proteins are involved in this process, such as Rac1 and the WAVE and ARP2/3 complexes. Using single protein tracking, we revealed key phenomena concerning the spatio-temporal regulation of lamellipodium formation by actin regulatory proteins. We found that the localization and activation of the WAVE complex was enzymatically regulated, but also mechanically. First, we showed that the Rac1 RhoGTPase activates the WAVE complex specifically at the tip of the lamellipodium. We also showed that WAVE complex localization is regulated by the dynamics of branched-network actin filaments. This study confirms the crucial role of the WAVE complex in lamellipodium formation and reveals the existence of a mechanical regulation of the localization of this complex in the cell.

LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo. / LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

No description available.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.