Células Y-1 apresentam o gene K-ras amplificado, o que resulta em altos níveis de expressão da proteína codificada por este gene. Este fato faz com que células Y-1 apresentem níveis cronicamente altos de K-Ras-GTP. Além disso, estas células apresentam uma relativa desregulação da transição G0→Gl→S, a qual é caracterizada por uma porcentagem de células entrando na fase S do ciclo celular na condição carenciada; e também, por um afrouxamento na regulação de Myc, o qual apresenta níveis basais significantes de mRNA e proteína. Para verificar se existe uma relação entre K-Ras-GTP elevado e os níveis basais de Myc e a desregulação na transição G0→Gl→S, células Y-1 foram transfectadas com uma forma dominante negativa de H-ras, H-ras Asn-17 (RasN 17). Os transfectantes resultantes também foram utilizados para verificar o papel de Ras na transdução do sinal iniciado por FGF-2 e ACTH. Com estes clones foi possível verificar uma redução nos níveis de ativação de K-Ras na condição carenciada, e com isso ficou claro que FGF-2 e ACTH são capazes de induzir a ativação de K-Ras, porém com cinética diferentes: uma ativação tardia e lenta para FGF-2, e rápida e transiente para ACTH. Com a redução nos níveis de Ras-GTP, verificamos uma concomitante redução no basal da proteína c-Myc e também no basal de entrada em S, indicando que existe uma correlação entre estes fatores. Além disso, os clones Yl-RasN17 foram determinantes para mostrar que em células Y-1 a presença de Akt/PKB constitutivamente ativada é conseqüência dos níveis cronicamente elevados de K-Ras-GTP (Forti et al, 2002). / Abstract not available.

Células Y-1 apresentam o gene K-ras amplificado, o que resulta em altos níveis de expressão da proteína codificada por este gene. Este fato faz com que células Y-1 apresentem níveis cronicamente altos de K-Ras-GTP. Além disso, estas células apresentam uma relativa desregulação da transição G0→Gl→S, a qual é caracterizada por uma porcentagem de células entrando na fase S do ciclo celular na condição carenciada; e também, por um afrouxamento na regulação de Myc, o qual apresenta níveis basais significantes de mRNA e proteína. Para verificar se existe uma relação entre K-Ras-GTP elevado e os níveis basais de Myc e a desregulação na transição G0→Gl→S, células Y-1 foram transfectadas com uma forma dominante negativa de H-ras, H-ras Asn-17 (RasN 17). Os transfectantes resultantes também foram utilizados para verificar o papel de Ras na transdução do sinal iniciado por FGF-2 e ACTH. Com estes clones foi possível verificar uma redução nos níveis de ativação de K-Ras na condição carenciada, e com isso ficou claro que FGF-2 e ACTH são capazes de induzir a ativação de K-Ras, porém com cinética diferentes: uma ativação tardia e lenta para FGF-2, e rápida e transiente para ACTH. Com a redução nos níveis de Ras-GTP, verificamos uma concomitante redução no basal da proteína c-Myc e também no basal de entrada em S, indicando que existe uma correlação entre estes fatores. Além disso, os clones Yl-RasN17 foram determinantes para mostrar que em células Y-1 a presença de Akt/PKB constitutivamente ativada é conseqüência dos níveis cronicamente elevados de K-Ras-GTP (Forti et al, 2002). / Abstract not available.

As células Y-1 pertencem a uma linhagem clonal de células funcionais de córtex adrenal de camundongo, que respondem a ACTH. Em células Y-1, ACTH promove a esteroidogênese (função) e tem efeitos regulatórios complexos na transição G0→G1→S do ciclo celular. ACTH promove a transição G0→G1, mas inibe a transição G1→S. É possível que a regulação do ciclo celular por ACTH seja mediada pelo controle da expressão dos proto-oncogenes das famílias fos, jun e myc. Nosso laboratório mostrou, anteriormente, que ACTH induz a expressão dos genes fos e jun, mas inibe c-myc. O objetivo deste trabalho foi identificar pontos de controle na expressão dos genes fos, jun e myc e na atividade dos fatores de transcrição AP-1 (dímeros da proteínas Fos e Jun) por ACTH, derivados de cAMP (ativadores de PKA), PMA (ativador de PKC) e FCS (soro fetal bovino). ACTH, PMA e dcAMP aumentam a atividade de ligação de AP-1 a DNA, independentemente de síntese protéica. Ensaios de elongação de cadeia nascente de RNA (run off transcription) mostram que ACTH, PMA e FCS são fortes indutores de c-fos, c-jun e junB, enquanto dcAMP induz apenas c-fos e junB. Hibridizações Northern permitiram estimar a meia-vida dos mRNAs de c-fos e c-jun em 30 min, independentemente do tratamento com ACTH ou PMA. Diferentemente de c-fos, o mRNA de fosB é superinduzido por ActinomicinaD em células Y-1 tratadas com ACTH e PMA. / The Y-1 cells belong to a clonal lineage of functional mouse adrenocortical cells, which are responsive to ACTH. In Y-1 cells, ACTH promotes esteroidogenesis (function) and has complex effects on the G0→G1→S transition of the Y-1 cell cycle. ACTH induces the G0→G1 transition but inhibits the G1+S transition. Probably, the cell cycle regulation by ACTH is mediated by the expression control of the proto-oncogenes from the fos, jun and myc families. Our laboratory has previously shown that ACTH induces the fos and jun genes expression, but inhibits c-myc expression. The target of this work was to identify control points in the fos, jun and myc genes expression and in the AP-1 transcription factors (Fos and Jun proteins dimers) by ACTH, cAMP derivatives (PKA activators), PMA (PKC activator) and FCS (Fetal Calf Serum). ACTH, PMA and dcAMP raise the AP-1 DNA binding activity, independently of protein synthesis. Run off transcription assays show that ACTH, PMA and FCS are strong c-fos, c-jun and junB inducers, while dcAMP induces only c-fos and junB. Northern hybridisations allowed us to estimate the half life of the fos and jun mRNAs in about 30 min, independently of ACTH or PMA treatment. Differently of c-fos, fosB mRNA is superinduced by ActinomicinD treatment in Y-1 cells treated with ACTH or PMA.

No description available.

As células Y-1 pertencem a uma linhagem clonal de células funcionais de córtex adrenal de camundongo, que respondem a ACTH. Em células Y-1, ACTH promove a esteroidogênese (função) e tem efeitos regulatórios complexos na transição G0→G1→S do ciclo celular. ACTH promove a transição G0→G1, mas inibe a transição G1→S. É possível que a regulação do ciclo celular por ACTH seja mediada pelo controle da expressão dos proto-oncogenes das famílias fos, jun e myc. Nosso laboratório mostrou, anteriormente, que ACTH induz a expressão dos genes fos e jun, mas inibe c-myc. O objetivo deste trabalho foi identificar pontos de controle na expressão dos genes fos, jun e myc e na atividade dos fatores de transcrição AP-1 (dímeros da proteínas Fos e Jun) por ACTH, derivados de cAMP (ativadores de PKA), PMA (ativador de PKC) e FCS (soro fetal bovino). ACTH, PMA e dcAMP aumentam a atividade de ligação de AP-1 a DNA, independentemente de síntese protéica. Ensaios de elongação de cadeia nascente de RNA (run off transcription) mostram que ACTH, PMA e FCS são fortes indutores de c-fos, c-jun e junB, enquanto dcAMP induz apenas c-fos e junB. Hibridizações Northern permitiram estimar a meia-vida dos mRNAs de c-fos e c-jun em 30 min, independentemente do tratamento com ACTH ou PMA. Diferentemente de c-fos, o mRNA de fosB é superinduzido por ActinomicinaD em células Y-1 tratadas com ACTH e PMA. / The Y-1 cells belong to a clonal lineage of functional mouse adrenocortical cells, which are responsive to ACTH. In Y-1 cells, ACTH promotes esteroidogenesis (function) and has complex effects on the G0→G1→S transition of the Y-1 cell cycle. ACTH induces the G0→G1 transition but inhibits the G1+S transition. Probably, the cell cycle regulation by ACTH is mediated by the expression control of the proto-oncogenes from the fos, jun and myc families. Our laboratory has previously shown that ACTH induces the fos and jun genes expression, but inhibits c-myc expression. The target of this work was to identify control points in the fos, jun and myc genes expression and in the AP-1 transcription factors (Fos and Jun proteins dimers) by ACTH, cAMP derivatives (PKA activators), PMA (PKC activator) and FCS (Fetal Calf Serum). ACTH, PMA and dcAMP raise the AP-1 DNA binding activity, independently of protein synthesis. Run off transcription assays show that ACTH, PMA and FCS are strong c-fos, c-jun and junB inducers, while dcAMP induces only c-fos and junB. Northern hybridisations allowed us to estimate the half life of the fos and jun mRNAs in about 30 min, independently of ACTH or PMA treatment. Differently of c-fos, fosB mRNA is superinduced by ActinomicinD treatment in Y-1 cells treated with ACTH or PMA.

La voie de signalisation Hippo est composée classiquement des kinases mammalian sterile twenty-like 1 et 2 (MST1/2) et des kinases large tumor suppressor 1 et 2 (LATS1/2) qui régulent les effecteurs transcriptionnels Yes-associated protein (YAP) et transcriptional coactivator with PDZ-binding motif (TAZ). Elle est impliquée dans plusieurs mécanismes cellulaires tels que la prolifération, la différenciation et l’apoptose ainsi que dans le maintien de l’homéostasie de différents tissus. Cependant, bien qu’il ait été démontré que YAP/TAZ contribuent au maintien de la glande surrénale, le rôle joué par les principales kinases de la voie Hippo dans ce tissu n’a jamais encore été évalué. L’objectif de ce projet de thèse est donc d’identifier les rôles des différentes kinases de la voie Hippo dans le développement et le maintien du cortex surrénalien par la création de cinq modèles de souris transgéniques. Grâce au modèle d’inactivation des kinases Mst1/2 dans le cortex surrénalien (Mst1flox/flox;Mst2flox/flox;Nr5a1-cre), nous avons démontré que Mst1/2 empêchent l’apparition prématurée de cellules ressemblant aux cellules du primordium adrénogonadique (AGP-like) dans la couche sous-capsulaire. Les analyses du transcriptome de ce modèle ont de plus démontré que les kinases MST1/2 régulent finement l’activité de YAP et des cellules de la zone glomérulée (zG) du cortex surrénalien à l’âge adulte mais pas l’activité des cellules de la zone fasciculée (zF). Ceci suggère que MST1/2 sont surtout impliqués dans la régulation de la fonction et de la différenciation des cellules de la zG. Contrairement à l’inactivation de Mst1/2, l’inactivation des kinases Lats1/2 dans tout le cortex en utilisant la même lignée Cre (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre), a démontré que LATS1/2 empêchent la transdifférenciation des cellules du cortex en cellules ressemblant à des myofibroblastes et à la formation de fibrose durant le développement embryonnaire. De plus, l’inactivation de LATS1/2 spécifiquement dans les cellules de la zG (Lats1flox/flox;Lats2flox/flox;AS-cre) a démontré que LATS1/2 empêchent la transdifférenciation des cellules de la zG en cellules partageant des caractéristiques avec des ostéoblastes et des cellules de Sertoli. Cette inactivation est également responsable de l’induction d’une importante réponse immuno-inflammatoire chez les animaux juvéniles. De plus, l’inactivation de Yap/Taz simultanément à l’inactivation de Lats1/2 dans les cellules de la zG (Yapflox/flox;Tazflox/flox;Lats1flox/flox; Lats2flox/flox;AS-cre) a permis de confirmer que l’augmentation de l’activité transcriptionnelle de YAP et de TAZ était responsable du phénotype observé à la suite de l’inactivation de Lats1/2. Finalement, l’utilisation d’un modèle exprimant une forme constitutivement active de YAP humain dans les cellules de la zG (Rosa26YAP5SA/+;AScre/+) nous a permis de démontrer que l’augmentation de l’activité transcriptionnelle de YAP seule ne pouvait reproduire pleinement le phénotype observé suite à l’inactivation de Lats1/2 mais conduit plutôt à l’hyperplasie de la glande surrénale. Ensemble, nos modèles murins démontrent un rôle important pour la voie Hippo, et particulièrement les kinases LATS1/2 et les effecteurs YAP/TAZ, dans la différenciation des cellules stéroïdogéniques du cortex surrénalien et le maintien de l’homéostasie de la glande surrénale chez la souris. Ces travaux ouvrent non seulement la voie à l’étude plus poussée de cette voie dans des pathologies de la glande surrénale, comme les tumeurs surrénaliennes, mais mettent également en évidence la voie Hippo en tant que cible thérapeutique pour les troubles surrénaliens. / The Hippo pathway canonical signaling is composed of the mammalian kinases sterile twenty-like 1 and 2 (MST1/2) and large tumor suppressor 1 and 2 (LATS1/2) which ultimately regulate the transcriptional co-activators Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ). This pathway plays critical roles in maintaining the homeostasis of various tissues and regulating biological processes such as cellular proliferation, differentiation, and apoptosis in a tissue-specific manner. Although previous studies have shown that the effectors YAP/TAZ are involved in the maintenance of the adrenal gland, the roles played by their main upstream kinases in this tissue have not yet been evaluated. The objective of this thesis project is therefore to identify the importance of these distinct Hippo pathway kinases in the development and maintenance of the adrenal cortex by generating five different conditional transgenic mouse models. Using a Mst1/2 inactivation model (Mst1flox/flox;Mst2flox/flox;Nr5a1-cre), we first demonstrated that loss of Mst1/2 in the entire cortex leads to premature appearance of spindle-shaped cells resembling adrenogonadal primordium “AGP-like” cells in the sub-capsular region of the adrenal cortex. Further transcriptomic analyses performed on this model showed that MST1/2 can regulate the activity of YAP and of cells forming the zona glomerulosa (zG) in adult mice but not the activity of cells of the zona fasciculata (zF). This finding suggested that kinases MST1/2 are mainly involved in the regulation of zG cell function or differentiation. The inactivation of Lats1/2 using the same Cre line (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre) demonstrated that LATS1/2 prevent the transdifferentiation of cortical cells into myofibroblast-like cells and the onset of fibrosis during embryonic development. Moreover, the inactivation of Lats1/2 specifically in the zG cells (Lats1flox/flox;Lats2flox/flox;AS-cre) demonstrated that LATS1/2 prevent the transdifferentiation of zG cells into cells sharing characteristics with osteoblast and Sertoli cells. This inactivation is also responsible for the induction of an immune-inflammatory response in juvenile mice. Most importantly, the concomitant inactivation of Yap/Taz with Lats1/2 in zG cells (Yapflox/flox;Tazflox/flox;Lats1flox/flox;Lats2flox/flox;AS-cre) demonstrated that the increase in the transcriptional activity of YAP/TAZ was responsible for the phenotype observed following the loss of Lats1/2. To complete our studies, we employed a mouse model expressing a constitutively active human form of YAP in zG cells (Rosa26YAP5SA/+;AScre/+) which demonstrated that increased transcriptional activity of YAP alone is not sufficient to recapitulate the phenotype observed following the loss of Lats1/2 but induces adrenal gland hyperplasia. Together, these findings indicate an important role for the Hippo signaling pathway, particularly LATS1/2 kinases and YAP/TAZ effectors, in the differentiation of steroidogenic cells of the adrenal cortex and the maintenance of adrenal gland homeostasis in mice. These findings not only pave the way for further studies of this pathway in important adrenal gland pathologies, such as adrenal tumors, but also highlight the Hippo pathway as a potential target for therapeutic treatments of adrenal disorders.

La voie de signalisation Hippo est une cascade de kinases hautement conservée entre les espèces qui contrôle des processus cellulaires fondamentaux comme la prolifération, l’apoptose et la différenciation. Dans la cascade, les kinases Large tumor suppressor 1 et 2 (LATS1/2) phosphorylent et inactivent les co-activateurs transcriptionnels Yes-associated protein (YAP) et Transcriptional coactivator with PDZ-binding motif (TAZ) pour réguler finement le développement, la taille et l’homéostasie de nombreux tissus. Bien qu’il ait été démontré que YAP/TAZ étaient essentiels au maintien du cortex surrénalien durant la vie adulte, le rôle des kinases en amont n’a jamais été étudié. L’objectif de ce projet était donc d’élucider le rôle de LATS1 et de LATS2 dans le développement corticosurrénalien. Pour ce faire, un modèle de souris Lats1flox/flox;Lats2flox/flox;Nr5a1-cre, permettant l’inactivation conditionnelle de Lats1/2 dans les tissus stéroïdiens, a été créé. La caractérisation de ce modèle a révélé que Lats1/2 étaient indispensables pour maintenir l’identité des cellules adrénocorticales durant le développement embryonnaire. En l’absence de Lats1/2, les cellules du cortex acquièrent des propriétés myofibroblastiques et perdent progressivement leur capacité de stéroïdogenèse, causant une insuffisance surrénalienne fatale vers 2 à 3 semaines d’âge chez les deux sexes. Cette transdifférenciation est probablement causée, du moins en partie, par une augmentation de l’activité transcriptionnelle de YAP/TAZ puisqu’une augmentation de l’expression nucléaire de YAP/TAZ et de l’expression de certains gènes cibles de la voie Hippo ont été observées dans les cellules myofibroblastiques. Ces résultats révèlent donc un tout nouveau rôle de la voie Hippo dans le développement du cortex surrénalien. / The Hippo signaling pathway is a highly conserved kinase cascade that controls fundamental cellular processes such as proliferation, apoptosis and differentiation. In this cascade, the Large tumor suppressor kinase 1 and 2 (LATS1/2) phosphorylate and inactivate the main downstream effectors, Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), to finely regulate the development, size and homeostasis of several organs. It was recently demonstrated that YAP and TAZ were essential for the maintenance of the adrenal cortex during adult life, but the role of the upstream kinases has never been studied. The objective of the present project was therefore to elucidate the role of LATS1 and LATS2 in adrenocortical development. We generated a Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mouse model in which Lats1/2 were conditionally inactivated in steroidogenic cells. The characterization of this model revealed that Lats1/2 were required to maintain proper adrenocortical cell identity during embryonic development. In the absence of Lats1/2, adrenocortical cells acquire myofibroblastic properties and lose their steroidogenic capacity, leading to adrenal deficiency and death at 2 to 3 weeks of age. This transdifferentiation is probably caused, at least in part, by an increased transcriptional activity of YAP/TAZ since a stronger nuclear expression of YAP/YAZ and higher expression levels of Hippo target genes were observed in the myofibroblastic population. These results suggest an important novel role of Hippo signaling in adrenal cortex development.