Un remodelage vasculaire anormal est à la base de la pathogenèse des maladies cardio-vasculaires (MCV) telles que l’athérosclérose et l’hypertension. Des dysfonctionnements au niveau de la migration, l’hypertrophie et la prolifération des cellules musculaires lisses vasculaires (CMLV) sont des évènements cellulaires qui jouent un rôle primordial dans le remodelage vasculaire. L’insulin-like growth factor 1 (IGF-1), puissant facteur mitogène, contribue au développement des MCV, notamment via l’activation des protéines MAPK et PI3-K/PKB, composantes clés impliquées dans les voies de croissance cellulaire. Ces molécules sont également impliquées dans la modulation de l’expression de nombreux facteurs de transcription, incluant le facteur Egr-1. Egr-1 est régulé à la hausse dans différents types de maladies vasculaires impliquant les voies de signalisation de croissance et de stress oxydant qui par ailleurs peuvent être déclenchées par l’IGF-1. Cependant, la question d’une possible modulation de l’expression d’Egr-1 dans les CMLV demeure inabordée; plus spécifiquement, la caractérisation de la voie de signalisation reliant l’action d’IGF-1 à l’expression d’Egr-1 reste à établir. Dans cette optique, l’objectif de cette étude a été d’examiner l’implication de MAPK, PKB et des dérivés réactifs de l’oxygène (DRO) dans l’expression d’Egr-1 induite par l’IGF-1 dans les CMLV. L’IGF-1 a induit une augmentation marquée du niveau protéique de l’Egr-1 en fonction du temps et de la concentration utilisés. Cette augmentation a été inhibée en fonction des doses d’agents pharmacologiques qui ciblent les voies de signalisation de MAPK, PKB et DRO. De plus, l’expression du facteur de transcription, Egr-1, en réponse de l’IGF-1, a été atténuée suite à un blocage pharmacologique des processus cellulaires responsables de la synthèse d’ARN et de synthèse protéique. Pour conclure, on a démontré que l’IGF-1 stimule l’expression d’Egr-1 via les voies de signalisation, impliquant ERK1/2/JNK, PI3K/PKB. On a également proposé que les DRO jouent un rôle important dans ce processus. Dans l’ensemble, nous avons suggéré un nouveau mécanisme par lequel l’IGF-1 promeut la prolifération et l’hypertrophie cellulaire, processus à la base des anomalies vasculaires. / Aberrant vascular remodelling underlies the pathogenesis of major cardiovascular diseases (CVD), such as atherosclerosis and hypertension. Abnormal growth, migration and proliferation of vascular smooth muscle cells (VSMC) are believed to play a critical role in vascular remodelling. IGF-1, potent mitogen, is believed to contribute to the development of CVD through the hyperactivation of proliferative and growth promoting pathways including mitogen-activated protein kinase (MAPK) and protein kinase B (PKB) pathways. It has also been implicated in modulating the expression of multiple transcription factors, including the early growth response protein-1 (Egr-1). Egr-1 upregulation has been observed in different models of vascular diseases implicating growth and redox signalling such as observed in response to IGF-1. However, modulation of Egr-1 expression by IGF-1 in VSMC, more specifically the signaling pathways involved in this process remain poorly characterized. Therefore, in the present studies, we investigated the implication of MAPK, PKB and ROS in IGF-1-induce Egr-1 expression in VSMC. IGF-1 induced a marked increase in Egr-1 protein level in a time and dose-dependent fashion. This increase was dose dependently inhibited by different pharmacological inhibitors targeting MAPK, PKB and reactive oxygen species (ROS) generation. Furthermore, pharmacological inhibitors of RNA and protein synthesis also attenuated IGF-1-induce response on Egr-1 expression. In conclusion, we showed that IGF-1 stimulates the expression of Egr-1 through ERK1/2/JNK and PI3K/PKB. We also propose that ROS generation plays an important role in this response. Overall, we propose a novel mechanism through which IGF-1 may exert its deleterious responses in vascular abnormalities.

O objetivo desse estudo foi avaliar o efeito do tratamento com a aldosterona (Aldo), por 21 dias, sobre a função e a morfologia renal de ratos, procurando correlacionar as alterações com a expressão do EGFR e da ERK1/2 fosforilados. A Aldo não alterou os parâmetros fisiológicos, a PA e a função renal dos ratos, mas verificou-se uma tendência para o aumento das concentrações plasmáticas de K+. No córtex e na medula renal, a Aldo aumentou a expressão do EGFR e da ERK1/2 fosforilados, sendo que este efeito foi abolido pelo tratamento concomitante com a espironolactona ou o RU 486 (antagonistas dos receptores da Aldo). O tratamento hormonal também induziu um aumento na marcação imuno-histoquímica para essas proteínas no córtex e medula; contudo, este efeito foi reduzido, principalmente, com o tratamento conjunto com a espironolactona. Observou-se um aumento na área glomerular e na porcentagem da área intersticial, após o tratamento com a Aldo. As alterações na morfometria renal foram parcialmente reduzidas pelo tratamento com a espironolactona ou o RU 486. Esses resultados indicam que a Aldo pode induzir alterações morfológicas no rim, aparentemente, de maneira dependente dos receptores MR e GR e da via do EGFR-ERK1/2, mas sem o comprometimento da função renal. / The purpose of this study was to evaluate the effect of 21 days of treatment with aldosterone (Aldo) in the renal function and morphology of rats, correlating changes with the expression of EGFR and ERK1/2 phosphorylated. The Aldo did not alter physiological parameters, blood pressure and renal function of rats, but there was a tendency to increased K+ plasma concentrations. In renal cortex and medulla, Aldo increased the expression of EGFR and ERK1/2 phosphorylated and this effect was abolished by treatment Aldo plus spironolactone or RU 486 (Aldo receptor antagonists). Hormonal treatment also induced an increase in immunohistochemical staining for these proteins in the cortex and medulla; however, this effect was limited mainly to treatment Aldo plus spironolactone. There was an increase in glomerular area and the percentage of interstitial area after treatment with Aldo. Changes in renal morphology were partially reduced by treatment Aldo plus spironolactone or RU 486. These results indicate that Aldo may induce morphological changes in the kidney of dependent manner of MR and GR receptors and EGFR-ERK1/2 signaling, but without the impairment of renal function.

No description available.

Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear. The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals. To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation. This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis. In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.

<p>Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the body. The secretion of this hormone is suppressed at high [Ca²⁺]_o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca²⁺ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca²⁺]_o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca²⁺]_o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca²⁺]_o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca²⁺]_o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca²⁺-independent. The magnitude of neomycin-induced release of Ca²⁺ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca²⁺ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca²⁺]_o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca²⁺ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.</p>

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

No description available.

Les maladies inflammatoires intestinales (MII) sont caractérisées par des périodes d’inflammation chronique entrecoupées de périodes de rémission. Les cellules épithéliales intestinales (CÉI) participent activement à l’élaboration de la réponse immune innée. Les nucléotides extracellulaires sont reconnus comme des molécules immunoactives. Cette thèse s’intéresse aux rôles de l’UDP et de son récepteur PDY[indice inférieur 6] dans l’expression et la sécrétion de CXCL8 par les CÉI et à leurs implications dans le recrutement des neutrophiles lors des maladies inflammatoires intestinales. Premièrement, la stimulation du récepteur PDY[indice inférieur 6] sur les CÉI, active une cascade de signalisation intracellulaire impliquant la phospholipase C (PLC), la protéine kinase Cð (PKCð) et la MAP kinase ERK1/2. La phosphorylation de c-fos par ERK1/2 induit la formation d’un dimère c-fos/c-jun. Le complexe AP-1 ainsi formé lie le promoteur de la chimiokine CXCL8 et stimule la transcription de celle-ci. La chimiokine est par la suite sécrétée dans le milieu extracellulaire. Deuxièmement, la présence d'UDP chez la souris, lors de l'induction d’une colite aigüe par le DSS aggrave les signes d'inflammation. L'UDP aggrave l'index de la sévérité de la maladie (DAI), le score histologique, la perméabilité de la muqueuse et le recrutement des intervenants cellulaire tel que les cellules T, les cellules dendritiques, les macrophages et les neutrophiles. Troisièmement, l'administration d'UDP aux souris en rémission dans un modèle de colite chronique est suffisante pour déclencher les symptômes d'inflammation. L'UDP aggrave le DAI, le score histologique et la perméabilité de la muqueuse. L'activation du récepteur P2Yð durant la colite chronique est responsable du recrutement différentiel des neutrophiles au détriment des cellules dendritiques tolérogéniques et des macrophages, ce qui somme toute aggrave la colite. Finalement, in cellulo, l'antagoniste du récepteur P2Y[indice inférieur 6], le MRS2578 réduit la relâche de la CXCL8 par les CÉI. In vivo, l'administration de MRS2578 dans le modèle aigu et chronique de colite réduit les signes d'inflammation. Cette thèse démontre l’importance du récepteur P2Y[indice inférieur 6] dans la réponse inflammatoire des cellules épithéliales intestinales, notamment dans la stimulation de la relâche de CXCL8 et dans le recrutement des cellules immunes tel que les neutrophiles. De plus, elle met en évidence le potentiel thérapeutique de l'administration d'antagoniste du récepteur P2Y[indice inférieur 6] lors des maladies inflammatoires intestinales.

O objetivo desse estudo foi avaliar o efeito do tratamento com a aldosterona (Aldo), por 21 dias, sobre a função e a morfologia renal de ratos, procurando correlacionar as alterações com a expressão do EGFR e da ERK1/2 fosforilados. A Aldo não alterou os parâmetros fisiológicos, a PA e a função renal dos ratos, mas verificou-se uma tendência para o aumento das concentrações plasmáticas de K+. No córtex e na medula renal, a Aldo aumentou a expressão do EGFR e da ERK1/2 fosforilados, sendo que este efeito foi abolido pelo tratamento concomitante com a espironolactona ou o RU 486 (antagonistas dos receptores da Aldo). O tratamento hormonal também induziu um aumento na marcação imuno-histoquímica para essas proteínas no córtex e medula; contudo, este efeito foi reduzido, principalmente, com o tratamento conjunto com a espironolactona. Observou-se um aumento na área glomerular e na porcentagem da área intersticial, após o tratamento com a Aldo. As alterações na morfometria renal foram parcialmente reduzidas pelo tratamento com a espironolactona ou o RU 486. Esses resultados indicam que a Aldo pode induzir alterações morfológicas no rim, aparentemente, de maneira dependente dos receptores MR e GR e da via do EGFR-ERK1/2, mas sem o comprometimento da função renal. / The purpose of this study was to evaluate the effect of 21 days of treatment with aldosterone (Aldo) in the renal function and morphology of rats, correlating changes with the expression of EGFR and ERK1/2 phosphorylated. The Aldo did not alter physiological parameters, blood pressure and renal function of rats, but there was a tendency to increased K+ plasma concentrations. In renal cortex and medulla, Aldo increased the expression of EGFR and ERK1/2 phosphorylated and this effect was abolished by treatment Aldo plus spironolactone or RU 486 (Aldo receptor antagonists). Hormonal treatment also induced an increase in immunohistochemical staining for these proteins in the cortex and medulla; however, this effect was limited mainly to treatment Aldo plus spironolactone. There was an increase in glomerular area and the percentage of interstitial area after treatment with Aldo. Changes in renal morphology were partially reduced by treatment Aldo plus spironolactone or RU 486. These results indicate that Aldo may induce morphological changes in the kidney of dependent manner of MR and GR receptors and EGFR-ERK1/2 signaling, but without the impairment of renal function.

La voie de signalisation des Récepteurs Tyrosine Kinase (RTK) occupe un rôle central dans la régulation de la croissance cellulaire, la prolifération, la différentiation et la motilité. Une régulation anormale des RTKs mène à plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalité chez les femmes à cause de l’amplification et la mutation fréquente de la protéine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une protéine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour réguler de multiples voies de signalisation. En réponse à la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recruté à la membrane plasmique où il potentialise l’activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d’occuper un rôle essentiel durant le développement du système hématopoïétique, Gab2 est souvent amplifié dans les cancers, notamment le cancer du sein et les mélanomes. Cependant, les mécanismes moléculaires qui régulent le fonctionnement de Gab2 sont peu connus. Il est établi que lors de l’activation des RTKs, Gab2 est phosphorylé au niveau de plusieurs résidus Tyrosine, menant à l’association de différentes protéines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d’autres groupes de recherche avons identifié que Gab2 est aussi phosphorylé au niveau de résidus Ser/Thr suite à l’activation de la voie de signalisation MAPK. Cependant, la régulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est régulé par la voie de signalisation MAPK, nous avons utilisé différentes approches. Dans la première partie de ma thèse, nous avons déterminé un nouveau mécanisme démontrant que la voie de signalisation Ras/MAPK, par le biais des protéines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce phénomène se produit à la fois in vivo et in vitro au niveau de trois résidus Ser/Thr conservés. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant à l’augmentation de la motilité cellulaire, ce qui suggère que les protéines RSK restreignent les fonctions dépendantes de Gab2. Ce phénomène est le résultat de la participation de RSK dans la boucle de rétroaction négative de la voie de signalisation MAPK. Dans la seconde partie de ma thèse, nous avons démontré que les protéines ERK1/2 phosphorylent Gab2 au niveau de plusieurs résidus pS/T-P à la fois in vitro et in vivo, entrainant l’inhibition du recrutement de p85. De plus, nous avons établi pour la première fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l’activation de la voie de signalisation MAPK. Par ailleurs, nous avons montré un nouveau domaine d’attache du module ERK1/2 sur Gab2. Des mutations sur les résidus essentiels de ce domaine d’attache n’entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent également la phosphorylation dépendante de ERK1/2 sur Gab2. Ainsi, nos données montrent que la voie de signalisation MAPK régule les fonctions de la protéine Gab2 par le biais des kinases RSK et ERK1/2. Cette thèse élabore par ailleurs un schéma complet des fonctions de Gab2 dépendantes de la voie de signalisation MAPK dans le développement de nombreux cancers. / Receptor tyrosine kinase (RTK) signaling plays an essential role in modulating cell growth, proliferation, differentiation and motility. Abnormal regulation of RTKs results in many human diseases, including breast cancer, the second leading cause of cancer mortality in women by the frequent amplification and mutation of the HER2 (ERBB2) tyrosine kinase. The Grb2-associated binder (Gab) 2 is an adaptor protein that acts downstream of several RTKs, including HER2, to regulate multiple signaling pathways. In response to the stimulation of a number of growth factors and cytokines, Gab2 is recruited to the plasma membrane, where it potentiates the activation of the Ras/mitogen-activated protein kinase (MAPK) and PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B) pathways. In addition to playing an important role in the hematopoietic system during development, GAB2 is often amplified in cancers including breast cancer and melanoma, however, little is known about the molecular mechanisms that regulate Gab2 function. It has been well established that upon RTKs activation, Gab2 becomes phosphorylated on several Tyr residues leading to diverse adaptor protein associations, such as p85 and Shp2. Aside from the tyrosine phosphorylation, our lab and other groups noticed that Gab2 is also phosphorylated on Ser/Thr residues upon activation of MAPK signaling. However, less is known about this post-translational modification in the regulation of Gab2 functions. In order to understand how Gab2 is regulated by the MAPK pathway, we used different approaches. In the first part of my thesis, we determined a new mechanism by which the Ras/MAPK pathway through RSK (p90 ribosomal S6 kinase) phosphorylated Gab2 on three conserved Ser/Thr residues, both in vivo and in vitro. Mutation of these phosphorylation sites promoted Shp2 recruitment leading to increased cell motility, suggesting that RSK restricts Gab2-dependent functions as a result of participation in the negative feedback loop of MAPK signaling. In the second part of the thesis, we found that ERK1/2 phosphorylated Gab2 on several potential pS/T-P residues, both in vivo and in vitro, resulting in inhibited p85 recruitment. In addition, to the best of our knowledge, we established for the first time that Gab2 physically interacted with ERK1/2 in cells upon activation of the MAPK pathway. Furthermore, we revealed a novel ERK1/2 docking domain in Gab2. Mutation of the essential residues in this docking domain not only disrupted ERK1/2-Gab2 interaction, but also diminished ERK1/2-dependent phosphorylation on Gab2. Taken together, our data showed that the MAPK pathway regulates Gab2 functions through both RSK- and ERK1/2-dependent manners. Given that Gab2 is overexpressed in several cancers, this thesis decodes a complete figure of modulating actions of Gab2 by MAPK signaling in cancer development.