INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Une sous population de cellules au sein des tumeurs mammaires présente une capacité accrue de se renouveler et de reproduire l’hétérogénéité du cancer du sein. Maintenant il est bien connu que les cellules souches présomptives du cancer du sein possèdent des programmes d’expression des gènes qui correspondent à leurs caractéristiques biologiques uniques. Notre groupe a été impliqué dans la caractérisation épigénétique des cellules souches présomptives du cancer du sein et l’importance de la dérégulation des mécanismes épigénétiques comme la méthylation de l’ADN et le microARN au cours de la carcinogenèse. Plus spécifiquement cette étude détaille l’idée que la survie des cellules souches du cancer du sein peut être due à une signalisation via des circuits spécifiques de régulation, y compris la voie d’inflammation IL6-JAK-STAT. Ces cellules présentent une activation constitutive de cette voie associée à une configuration particulière de la chromatine. Une autre part de cette étude est d’explorer l’idée que des changements dans l’expression des microARN sont fondamentaux pour la maintenance des principales caractéristiques de ces cellules, et leur ciblage peut représenter une nouvelle approche de thérapie contre le cancer du sein. De plus, en testant directement les conséquences in vivo de la régulation de miR30a nous ouvrons la voie pour la recherche et la validation de l’utilisation potentielle des microARN comme thérapie anti cancéreuse. Ensemble, nos résultats apportent une nouvelle compréhension du rôle des modifications épigénétiques dans la maintenance des cellules souches du cancer du sein. De façon importante ces découvertes intègrent l’idée que des mécanismes de régulations différents mais coordonnés ont un rôle dans la survie des cellules souches du cancer du sien et donnent une perspective élargie pour la découverte de nouvelles cibles thérapeutiques / A subpopulation of cells within breast tumors is known to display an increased ability to self-renew and reproduce breast cancer cell heterogeneity. It is now known that putative breast cancer stem cells (CSCs) display distinct programs of gene expression that correlate with their unique biological characteristics. Our group has been involved in the epigenetic characterization of putative breast CSCs and the importance of the deregulation of epigenetic mechanisms such as DNA methylation and microRNA during carcinogenesis. More specifically, this study is detailing the idea that the survival of breast CSC may be dependent on signaling through specific regulatory circuits, including the well known inflammatory IL6-JAK-STAT pathway. These cells display a constitutive activation of this pathway associated with a distinct chromatin configuration. Another part of the study is exploring the idea that changes in microRNA expression are fundamental in sustaining the main attributes of these cells, and their targeting may represent a novel approach for breast cancer therapy. In addition, by directly testing the in vivo consequences of miR30a regulation, we open a window of opportunity for testing and validating the potential use of microRNAs in anti-neoplastic therapy. Together our results bring a new understanding of the role of epigenetic modifications in the maintenance of breast CSC. Importantly, these findings integrate the idea that different but coordinated regulation mechanisms play a role in the survival of CSC and give a larger perspective for finding novel therapeutic targets

INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

La leucémie myéloïde chronique (LMC) est une pathologie de la cellule souche hématopoïétique caractérisée par la présence de la translocation chromosomique t(9 :22) conduisant à l’expression de la kinase BCR-ABL responsable de la maladie. Un inhibiteur de l’activité de BCR-ABL a été identifié, l’Imatinib (IM). L’IM a révolutionné la prise en charge de la LMC en bloquant sélectivement la croissance des cellules tumorales, conduisant à la rémission des patients. Cependant, une majorité d’entre eux subissent des récidives en cas d’arrêt du traitement, et environ 15% développent des résistances à l’inhibiteur. BCR-ABL active de multiples voies de signalisation parmi lesquelles figurent les facteurs de signalisation STAT5. Nous avons analysé les rôles respectives des deux facteurs STAT5, STAT5A et STAT5B, dans les cellules souches hématopoïétiques normales et de LMC, par une approche d’ARN interférence. Nos observations indiquent que l’activité des deux facteurs STAT5 permet la survie et le maintien à long terme des cellules souches de patients LMC au diagnostic. Nous avons de plus montré qu’indépendamment de son activité transcriptionnelle, STAT5A aide les cellules normales et leucémiques à limiter leur stress oxydatif. Nous avons aussi pu observer que les cellules de patients présentant des résistances secondaires à l’IM, sans mutations ni surexpression de BCR-ABL, manifestent une dépendance caractéristique vis-à-vis de l’activité STAT5A. Pour mieux comprendre les mécanismes d’action des facteurs STAT5, nous avons recherché les gènes cibles de STAT5 par une approche transcriptomique et avons identifié le récepteur tyrosine kinase Axl dont l’expression est augmentée par STAT5A. L’inhibition d’Axl dans les cellules LMC sensibles à l’IM n’a aucun effet sur leur survie, alors qu’elle diminue fortement la survie des cellules LMC résistantes à l’IM. De plus, Axl contrôle le niveau des réactifs oxygénés dans les cellules de patients LMC. Nous avons analysé l’expression d’un des activateurs d’Axl, le ligand Gas6, et avons observé que son expression diminue fortement dans les cellules primaires de LMC par rapport aux contrôles sains. Ces résultats suggèrent que le tandem Gas6/Axl pourrait participer au processus leucémique de la LMC à différents niveaux. De manière globale, nos travaux montrent que les facteurs STAT5 favorisent le maintien des cellules souches de LMC, leur résistance au stress oxydatif et aux traitements thérapeutiques Ces deux dernières activités sont au moins en partie liées à l’activité d’une nouvelle cible de STAT5, le récepteur Axl, par ailleurs déjà impliqué dans la résistance aux traitements thérapeutiques. Les facteurs STAT5 représentent donc des nouvelles cibles thérapeutiques potentielles dans l’éradication de la maladie résiduelle. / The Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the t(9:22) genetic translocation and expression of the oncogenic tyrosine kinase BCR-ABL . A first BCR-ABL Tyrosine Kinase Inhibitor (TKI), Imatinib (IM), was identified that inhibits proliferation of BCR-ABL expressing hematopoietic cells and leads to disease remission. However, BCR-ABL mRNA remains detectable in the most immature HSCs and discontinuation of IM results in clinical relapse. STAT5 factors play a crucial role in the CML pathogenesis of human primary CML cells. However, the contribution of the two related STAT5 genes, STAT5A and STAT5B, was unknown. We used an RNAinterference based strategy to analyze STAT5A or STAT5B roles in normal and CML cells. We showed that STAT5A/5B double knock-down (KD) triggers normal and CML cell apoptosis and suppressed long-term clonogenic potential of immature hematopoietic stem and progenitor cells known to be resistant to TKI treatment and responsible for residual disease. STAT5A loss alone was ineffective at impairing growth of both normal and CML cells under standard conditions. In contrast, STAT5A loss was sufficient to enhance Reactive Oxygen Species (ROS) which correlated with enhanced DNA damages in both normal and leukemic cells. We reported that STAT5A regulates oxidative stress through unconventional mechanisms, in a non-transcriptional-dependent manner. We further showed that, in contrast to primary cells at diagnosis, IM-resistant cells exhibited enhanced STAT5A dependence, by being sensitive to STAT5A single KD. To investigate the molecular basis of STAT5A activity in TKI-resistance and oxidative stress, we performed a transcriptomic analysis of STAT5 regulated genes. We identified Axl, which encodes a receptor tyrosine kinase, recently shown to be crucial in TKI-resistant CML cells. Specifically, Axl expression is enhanced by STAT5A. We investigated the role of Axl and we found that Axl KD did not affect survival of IM-sensitive CML cells. However, Axl KD decreased survival of IM-resistant cells, miming the activity of STAT5A. Moreover, Axl loss increased ROS levels in CML cells, promoting STAT5A anti-oxidant activity. We further sought to determine the expression of the Axl ligand, Gas6. Gas6 expression is dramatically reduced in CML primary cells at diagnosis compared to healthy cells. The strong and consistent down-regulation of Gas6 in CML cells suggested a possible role in the pathophysiology. Collectively, our findings highlight the pro-survival, stress protection and drug resistance roles of STAT5 factors, providing new understanding for medical treatment of CML patients. We suggest that STAT5A acts in synergy with Axl to face exogenous insults and propose a new mechanism by which CML cells increase their proliferation and reduce their motility by down-regulating Gas6 expression.

La voie JAK / STAT est une voie majeure de signalisation intracellulaire, activée par plusieurs cytokines, y compris par les interférons (IFNs). Mon laboratoire a précédemment établi que l'endocytose et le trafic du récepteur de l’IFN alpha/beta (IFNAR) jusqu’à l'endosome précoce, joue un rôle clé dans l’activation de la voie de signalisation JAK/STAT et dans les activités antivirales et antiprolifératives induites par les IFNs de type I. Le récepteur de l’IFN de type I se compose de deux sous-unités: IFNAR1 et IFNAR2. Durant le trafic du récepteur vers l'endosome précoce, les deux sous-unités prennent des itinéraires différents - IFNAR1 est ubiquitiné et dégradé au lysosome tandis que IFNAR2 est recyclé vers la membrane plasmique. Les mécanismes moléculaires permettant un trafic différent des IFNAR1 et IFNAR2 restent mal compris.J’ai tout d’abord étudié le trafic intracellulaire des sous-unités IFNAR1 et IFNAR2 et j’ai pu montrer que les protéines Rab4 et Rab11 sont nécessaires au recyclage d’IFNAR2 à la membrane plasmique. Par la suite, j’ai identifié le complexe rétromère endosomal - VPS26A/VPS29/VPS35 comme un partenaire d’interaction avec IFNAR2 nécessaire pour son recyclage. En outre, j’ai montré que le complexe rétromère contrôle la séparation spatiotemporelle des sous-unités IFNAR1 et IFNAR2 au niveau de l'endosome précoce. En effet, la déplétion du complexe rétromère entraine une prolongation de l’association endosomale de deux sous-unités et une prolongation de la signalisation JAK/STAT induite par l’IFN tant pour la réponse précoce (phosphorylation de STAT1) que la réponse à plus long terme (expression des gènes stimulés par l'IFN). Des résultats en cours de publication par l’équipe montrent un rôle d’ESCRT-0 dans l’initiation de la signalisation JAK/STAT. J’ai montré que le rôle de la machinerie ESCRT dans ce processus est limité au complexe ESCRT-0. Les sous-complexes en aval d’ESCRT-0, bien que nécessaires à la dégradation d’IFNAR1, ne sont pas impliqués dans l’activation de la voie JAK/STAT.En conclusion, ces résultats nous ont permis d’établir un modèle dans lequel le retromère joue un rôle clef dans la régulation spatio-temporelle du tri endosomal d’IFNAR et de la signalisation JAK/STAT au niveau de l’endosome précoce. Après la séparation rétromère-dépendante des sous-unités du récepteur et la terminaison de la signalisation JAK/STAT, la dégradation lysosomale d’IFNAR1 est assurée par la machinerie ESCRT en aval d’ESCRT-0. / The JAK/STAT pathway is a major intracellular signaling pathway that is activated by several cytokines including interferons (IFNs). My laboratory has previously established that endocytosis and trafficking of the IFN alpha/beta receptor (IFNAR) to the early endosome is key for the activation of JAK/STAT signaling and for the antiviral and antiproliferative activities induced by type I IFNs. The functional type I IFN receptor - IFNAR - consists of two subunits: IFNAR1 and IFNAR2. Upon endocytosis to the early endosome, both subunits take different trafficking routes – IFNAR1 is ubiquitinated and degraded in the lysosome, whereas IFNAR2 recycles back to the plasma membrane. The molecular mechanisms behind the separation of IFNAR1 and IFNAR2, as well as their distinct trafficking routes remain poorly understood.First, I studied the intracellular trafficking of IFNAR1 and IFNAR2 subunits and showed the requirement for Rab4 and Rab11 in IFNAR2 recycling to the plasma membrane. Next, I identified the endosomal retromer complex – VPS26A/VPS29/VPS35 as an IFNAR2 interacting partner, required for IFNAR2 recycling. Additionally, I was able to show that retromer controls the spatiotemporal separation of IFNAR1 and IFNAR2 subunits at the early endosome. Indeed, retromer depletion resulted in prolonged endosomal association of both subunits and prolonged activation of IFN-induced JAK/STAT signaling, at both early (STAT1 phosphorylation) and longer time response (IFN-stimulated gene expression). Unpublished results of our team indicate the role of ESCRT-0 in initiation of the IFN-mediated JAK/STAT signaling. I found that role of the ESCRT machinery in this process is limited to the ESCRT-0. ESCRT subcomplexes downstream of ESCRT-0, although required for IFNAR1 degradation, are not involved in activation of the JAK/STAT pathway.In conclusion, these results permit us to draw a model in which the retromer is a key spatiotemporal regulator of IFNAR endosomal sorting and the JAK/STAT signaling at level of the early endosome. Once retromer-mediated subunits separation is accomplished and JAK/STAT signaling is terminated, ESRCT machinery downstream to ESCRT-0 mediates IFNAR1 lysosomal degradation.

Die Entstehung verschiedener Tumorentitäten kann häufig mit einer vorliegenden Virusinfektion korreliert werden. So lässt sich beispielsweise ein Zusammenhang zwischen der Infektion mit den zur Gruppe der Herpesviren gehörenden Epstein-Barr-Viren (EBV) und der Entwicklung von Burkitt-Lymphomen, Hodgkin-Lymphomen sowie Nasopharynx-Karzinomen herstellen. Für die Transformation einer mit EBV infizierten humanen B-Zelle ist die Expression des Latenten Membranproteins 1 (LMP1) des Epstein-Barr-Virus essentiell. LMP1 ist ein Membranprotein mit einem funktionellen C-terminus, das Homologien zu den Tumornekrosefaktoren (TNF)- und Toll-like-Rezeptoren aufweist. Im Zusammenspiel mit anderen viralen Faktoren trägt LMP1 durch die von ihm aktivierten Signalwege (z.B. NF-κB, JNK, p38) zur Immortalisierung und malignen Entartung von EBV-infizierten Zellen bei. Einer dieser Signalwege ist der Jak/STAT-Signalweg. Da dessen EBV-abhängige Aktivierung bislang nur unzureichend geklärt ist, ist es Zielsetzung dieser Arbeit, die durch das Epstein-Barr-Virus vermittelte Aktivierung des Jak/STAT-Signalweges in Burkitt-Lymphomzellen näher zu untersuchen. Als Negativregulatoren des Jak/STAT-Signalweges sind hierbei die SOCS-Moleküle (Suppressor of Cytokine Signaling) von Bedeutung. Von besonderem Interesse sind dabei die Mechanismen der Aktivierung von SOCS3 durch LMP1 und dem damit verbundenen möglichen Einfluss auf den transformierten Zustand einer Zelle. In dieser Arbeit konnte gezeigt werden, dass in den untersuchten Burkitt-Lymphomzellen das exprimierte virale Onkoprotein LMP1 des Epstein-Barr-Virus ausreichend ist, den Jak/STAT-Signalweg zu aktivieren und SOCS3 zu induzieren. Die Aktivierung des Jak/STAT-Signalweges wird indirekt, das heißt über EBV-abhängig aktivierte Zytokinsignalwege, reguliert. Eine zusätzliche direkte Aktivierung durch eine Interaktion von Komponenten des Jak/STAT-Signalweges mit LMP1 kann derzeit jedoch nicht völlig ausgeschlossen werden.

Janus Kinase (JAK) activity specifies the cell fates of the follicular epithelium during Drosophila oogenesis by establishing a gradient of JAK activity with highest levels at the A/P poles. Unpaired (Upd), a ligand for the pathway, is expressed and secreted exclusively from the polar cells potentially establishing the JAK activity gradient. This project proposed that Upd acts as a morphogen to directly establish the JAK activity gradient, specifying the fates of the follicular epithelium. The aims of this work were to investigate the extracellular distribution of Upd and, in addition, factors that may be involved. Furthermore, upd3, a gene encoding a protein with sequence similarity to Upd, is also co-expressed with upd in the polar cells. An additional aim of this project was to determine what role, if any, Upd3 plays in follicular development. Immunostaining was used to reveal Upd distribution during oogenesis. The data revealed an Upd gradient on the apical membrane of the follicular epithelium. By virtue of the extracellular gradient, Upd fulfills the requirements necessary to be classified as a morphogen. Some morphogens are dependent on heparan sulphate proteoglycans (HSPGs) for distribution. Using mitotic recombination to make mosaics, this work reveals that Dally, a glypican, is essential for the distribution of Upd and establishment of the JAK gradient during oogenesis. The data suggests Dally is involved with stability of extracellular Upd. Mosaic analysis of an additional HSPGs revealed that they are not essential for the Upd gradient or JAK activity during oogenesis. upd3 mutant flies have small eyes and outstretched wings, a phenotype consistent reduced JAK activity. In upd3 mutant ovaries it is shown that there is a higher frequency of deteriorating egg chambers, a higher frequency of egg chamber fusions, and a decrease in border cells per egg chamber compared to wildtype controls; all of which support a reduction of JAK activity. Furthermore, ovarian phenotypes of upd3 get worse as the fly ages suggesting that upd3 is required over time. The data presented suggests that Upd3 does act to maintain JAK activity in the ovary as the fly ages.

Le polyomavirus humain BK (virus BK) établit une infection persistante asymptomatique dans les voies rénales de 80% de la population humaine. Chez les patients transplantés, la réactivation du virus BK est à l'origine de néphropathies et de cystites hémorragiques. L'augmentation des pathologies associées au virus BK en même temps que l'utilisation de traitements immunosuppresseurs de plus en plus puissants souligne un lien étroit entre la réponse immunitaire de l'hôte et la réactivation virale. Cependant la réponse immune à l'infection par le virus BK, en particulier le rôle des cytokines antivirales dans le contrôle de l'infection est peu documentée. Ici, nous avons étudié l'efficacité antivirale des interférons (IFN) sur la multiplication du virus BK. Nous avons testé les IFN-alpha, lambda et gamma sur la souche Dunlop du virus BK dans les cellules Véro et MRC 5. L'IFN-gamma inhibe de façon dose-dépendante la transcription virale de la région précoce et de la région tardive ainsi que l'expression de la protéine virale VP1. Un moindre effet antiviral a été observé avec l'IFN-alpha et l'IFN lambda. Ces résultats sont associés à une phosphorylation prolongée de STAT 1 avec l'IFN-gamma, non retrouvée avec l'IFN-alpha et lambda. La différence d'efficacité entre ces trois types d'IFN suggère que certaines protéines induites seulement par l'IFN-gamma ont un effet antiviral dans l'infection par le virus BK. L'analyse transcriptionnelle révèle neuf protéines qui pourraient être impliquées dans cet effet antiviral spécifique. Parmi elles, nous avons étudié l'effet antiviral de l'indoleamine 2,3-dioxygénase (IDO) et les protéines de liaison au guanylate (GBP ou guanylate binding protéines), GBP1 et GBP2, sur le virus BK. Nos résultats montrent que GBP1 et GBP2 mais pas IDO contribuent à l'activité antivirale de l'IFN-gamma sur le virus BK. Trouver le mécanisme d'action de ces protéines antivirales induites par l'IFN pourrait nous aider à développer une stratégie thérapeutique / The human polyomavirus BK (BK virus) establishes an asymptomatic persistent infection in the urinary tract of 80% of the human population. In transplant recipients, reactivation of the BK virus infection is the cause of nephropathy and hemorrhagic cystitis. Diseases associated with BK virus infections are increasing at the same time as potent immunosuppressive therapies are developing. This highlights the importance of components of the immune system in controlling viral reactivation. However, the immune response to the BK virus, particularly the role of antiviral cytokines in infection control, is poorly documented. Here, we investigated the antiviral efficacy of interferons (IFN) on the BK virus multiplication. We tested IFN-alpha, lambda and gamma on the Dunlop strain of BK virus in Vero cells and MRC 5 cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose dependent manner and decreased the expression of the early and late viral transcripts. A weaker antiviral effect was observed with IFN-alpha and IFN-lambda. These results are associated with a prolonged STAT1 phosphorylation with IFN-gamma but not with IFN-alpha and lambda. The difference of efficacy between these three types of interferon suggests that some interferon induced proteins only produced by IFN-gamma had an antiviral effect on BK virus infection. Transcriptomic analysis reveals that nine proteins could be involved in this specific antiviral effect. Among them, we selected and investigated the antiviral effect of indoleamine 2,3-dioxygenase (IDO) and guanylate binding protein 1 and 2 (GBP1 and GBP2) on the BK virus. Our results suggest that GBP1 and GBP2 but not IDO contribute to the antiviral activity of IFN-gamma on the BK virus. Finding the action mechanism of these IFN gamma induced antiviral proteins could help to develop a therapeutic strategy

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

Cell signaling is central to integration of internal and external cues that regulate development and homeostasis. Most development is thought of as pre-adult, but limited developmental processes occur in adults. Gametogenesis incorporates elements of both these facets, with a distinct developmental plan for gamete synthesis which is regulated by integration of homeostatic inputs such as nutrient status, and environmental cues. Signaling pathways integrate and transduce information from these cues to evoke a response. A decline in homeostasis and subsequent cues occurs over time, in the case of reproductive tissues leading to a progressive loss of fertility. The Janus Kinase and Signal Transducer and Activator of Transcription or Jak/Stat signaling pathway is conserved between vertebrates and invertebrates and is necessary for numerous functions needed to maintain organism and reproductive homeostasis, as well as contributing to various developmental events. The pathway in the fruit fly Drosophila melanogaster, is composed of a single receptor, Domeless, one Janus kinase, Hopscotch, one known effector, Stat92E, and the Unpaired family of ligands consisting of Upd, Upd2, and Upd3. Jak/Stat signaling is highly pleiotropic in both sexes with involvement in homeostasis and reproduction, making it an ideal model for studying the role of signaling in reproductive aging. Reduction of pathway activity in females results in a higher proportion of unfertilized eggs, which increases with age, and in males leads to a premature onset of infertility. Central to both is integration through cell signaling to evoke an appropriate response. This dissertation explores two of the requirements for Jak/Stat signaling: the pleiotropic requirement for Jak/Stat activity during oogenesis and male reproductive maintenance. Jak/Stat functions from the beginning of oogenesis, in the stem cell niche. From there it participates in multiple functions including specification of a subset of somatic cells called the border cells through the polar cells, a pair of cells at either pole of the egg. Pathway stimulation in the border cells drives their migration with the polar cells to the oocyte boundary, where the polar cells each form an extension in a coordinated manner into the micropyle, the means for sperm entrance during fertilization. Loss of Jak/Stat activity in the border cells prevents border cell migration. While border cell migration has been well studied, polar cell involvement after completion of border cell migration is less well known. To investigate the requirements for polar cell activity and Jak/Stat activity after the completion of border cell migration, we reduced Jak/Stat signaling in the polar cells which, while having no effect on border cell migration, results in blocked micropyles due to loss of coordination of extensions during their outgrowth. Reduced function in the polar cells did not significantly affect expression of adhesion molecules. But, the loss of Stat92E is phenocopied by loss of DE-cadherin. Hence, these results indicate a previously unknown autocrine requirement for Jak/Stat activity in the polar cells. The testes also have a continuous requirement for Jak/Stat activity for stem cell maintenance and differentiation of the germline into mature sperm. Reproductive maintenance not only requires sustained production of gametes, but reproductive tissues are also subject to deterioration of homeostatic functions that contribute to organismal aging. Males from thirty-nine lines of the Drosophila Genetic Reference Panel (DGRP), a panel of inbred, fully sequenced lines, were screened for age at infertility. Data were used to perform a genome-wide association study (GWAS) to identify the genetic architecture of reproductive aging. Candidate variants associated with cell signaling regulators, genes with functions in maintaining cell homeostasis, and organism behavior were uncovered. Notably, several SNPs fell in and near Ptp61F, a negative regulator of Jak/Stat activity. While variants in the primary components of the Jak/Stat pathway were not identified, the general classes of candidate loci functions reflect the requirements for homeostasis, metabolism, and development that have been shown by other studies examining the genetics of aging and fecundity. Thus, we show that Jak/Stat has an amazing amount of pleiotropy that encompasses both the real-time functions of fertility and the time related process of aging.