Dissertationen zum Thema „Jak2-Stat3“

Background: Ovarian cancer is the most lethal gynaecological malignancy, accounting for an estimated 140,000 deaths per year worldwide. Five year survival rates have not increased significantly in the last 10 years and the acquisition of resistance to chemotherapy remains a significant barrier to improving patient survival. Isogenic cell line models of in vivo acquired resistance to chemotherapy were used to examine cellular responses to cisplatin and identify differences between sensitive and resistant pairs that might be exploited to sensitise cells to treatment. Results: Microarray analysis of the isogenic paired sensitive/resistant high grade serous ovarian cell lines PEO1 and PEO4 revealed IL6 expression is induced by cisplatin exposure. This result was replicated by QRT-PCR and validated in the additional isogenic pair PEA1/PEA2. Western blotting demonstrated the lack of a correlation between IL6 expression and phosphorylation of either Y1007/1008 JAK2 or Y705 STAT3 levels, suggesting IL6 is not driving the constitutive activation of these proteins. Cells did however display dose dependant changes in STAT3, JAK2 and ERBB2 activation in response to cisplatin that differed between sensitive and resistant isogenic pairs. Both sensitive clones increased their activation of JAK2 and ERBB2 when exposed to low dose, 2μM, cisplatin but reversed these increases at higher concentrations. Resistant clones, PEO4 and PEA2, experienced no low dose increases in ERBB2 JAK2 or STAT3 activation instead reducing the activation of these proteins with greater sensitivity to cisplatin dose. Common to all cell lines was a high degree of correlation in the levels of activated JAK2 and ERBB2. Interfering with cisplatin dependent STAT3 deactivation using IL6 treatment was able to both sensitise cells and reduce cisplatin IC50, suggesting a functional role for STAT3 in both response, and acquired resistance, to cisplatin. Overexpression and knockdown of STAT3 demonstrated it promotes proliferation and the expression of cyclin D1 and BCL xL/S. STAT3 knockdown increased cisplatin resistance as quantified by IC50 whereas STAT3 overexpression was able to potentiate cisplatin induced apoptosis and decrease cisplatin IC50. Similarly the overexpression and knockdown of JAK2 demonstrated it also promotes proliferation, in part by regulating the activity of STAT3. The inhibition of JAK2 activity also increased resistance to cisplatin; small molecule inhibition of JAK2 both lowered background levels of apoptosis as well as attenuating cisplatin induced apoptosis. In common with STAT3 ablation JAK2 knockdown also increased cisplatin IC50. Surprisingly knockdown, overexpression and inhibition of JAK2 were all associated with changes in the activation of ERBB2. Knockdown and inhibition were associated with decreases in Y1248 phosphorylated ERBB2 whereas overexpression was associated with an increase, changes in activation appear to be driven by changes at the level of total protein. GP130 was investigated for due to its role in IL6 signalling and STAT3 activation, mRNA overexpressed was detected in the resistant pair of 2/3 isogenic cell lines. GP130 overexpression was associated with growth promotion and cisplatin resistance as revealed by siRNA knockdowns, which had no effect in cisplatin sensitive non overexpressing cells lines. Knockdown also revealed different pathways to constitutive STAT3 activation, in each high grade serous line assessed there was no effect on pSTAT3 levels, which were almost completely abolished in SKOV3. RNAi of GP130 was also associated with an increase in ERK1/2 activation which was recapitulated by JAK2 knockdown, inhibition as well as cisplatin and doxorubicin exposure. Treatment with a MEK1/2 inhibitor was able to reverse cisplatin and doxorubicin dependent activation and attenuate cytotoxic induced apoptosis. MEK1/2 inhibition was associated with an increase in JAK2, pSTAT3 and pERBB2 which was capable of reversing cisplatin dependent down regulation of these protein, highlighting a mutual feedback mechanism between the GP130/JAK2 and MAPK pathways. Central Conclusion Transcriptional regulation of JAK2 in response to cisplatin exposure drives differential behaviour of paired isogenic cell lines. Greater sensitivity of cisplatin resistant cells lines, in their deactivation of STAT3 and ERBB2 is regulated by their greater extent of JAK2 downregulation upon cisplatin exposure. Downregulation of JAK2 and its commensurate reduction in STAT3 activation were associated with reduced proliferation rates, rendering cells in a state refractory to cisplatin toxicity. This may be due to either or both of reducing the accumulation of DNA double stranded breaks associated with cell division and allowing more time for the repair of single stranded DNA adducts before cell division. While STAT3 has been suggested as a target for adjuvant chemotherapy, data presented here suggests that in combination with cisplatin STAT3 abrogation might actually reduce the effectiveness of treatment.

La maladie d'Alzheimer (MA) est une maladie neurodégénérative et la première cause de démence à travers le monde. Au cours de la MA, les astrocytes développent un phénotype réactif et présentent des changements moléculaires, morphologiques et fonctionnels, pouvant induire des effets délétères et/ou bénéfiques sur le fonctionnement des neurones. Récemment, des données de séquençage ARN (RNAseq) de cellules/noyaux uniques ont révélé l'existence de sous-populations distinctes d'astrocytes dans des cerveaux de patients atteints de la MA et dans les modèles murins. Cependant, les cascades de signalisation contrôlant ces sous-populations, leurs caractéristiques fonctionnelles et leurs impacts sur le fonctionnement du cortex préfrontal (PFC) restent inconnus. Les voies de signalisation JAK2-STAT3 et NF-kB sont connues pour être activées au cours de la MA et contrôler l'état réactif des astrocytes. Notre équipe a développé des lentivirus astrocyte-spécifiques rapporteurs de ces deux voies de signalisation, dans lesquels l'expression de CFP ou de GFP est contrôlée par des séquences promotrices NF-kB et STAT3-dépendantes, respectivement. Ces rapporteurs nous ont permis de mettre en évidence trois sous-populations astrocytaires selon leurs cascades activées, NF-kB+, STAT3+ et NF-κB+/STAT3+ dans le PFC de souris modèle de la MA, les souris APP/PS1dE9, mais aussi les souris APPNL-F/NL-F. Tout d'abord, nous avons observé que les populations ne sont topographiquement pas déterminées par leur proximité aux plaques amyloïdes. Ces observations nous ont ensuite permis de constater que celles-ci sont aussi présentes dans les souris WT, nous guidant vers l'hypothèse que les sous-populations sont probablement innées. Nous observons ensuite que la surface du territoire astrocytaire en 2 dimensions des astrocytes NF-κB+ est plus grande que celle des astrocytes STAT3+ et STAT3+/NF-κB+. Après analyse RNAseq des trois sous-populations, nous avons pu identifier des gènes différentiellement exprimés entre les trois sous-populations, associés à des fonctions spécifiques d'intérêt dans le contexte de la MA (protéostasie, régulation synaptique, métabolisme des lipides). Nous avons confirmé que les sous-populations STAT3+ et NF-κB+ présentent des activités du protéasome et du lysosome significativement différentes dans le PFC de souris APP/PS1dE9. Ayant observé une expression plus élevée des transcrits de Cx30 et 43 dans les astrocytes STAT3+, nous avons aussi étudié le couplage astrocytaire par les jonctions gap et l'activité des hémicanaux. Nous avons montré par redistribution de fluorescence après photoblanchiment (FRAP), que les sous-populations sont couplées de façon équivalente via les jonctions gap, mais que la population STAT3+ présente une augmentation de l'activité des hémicanaux par rapport aux deux autres. Enfin, à l'aide de nouveaux vecteurs lentiviraux, nous avons inhibé spécifiquement les sous-populations STAT3+ ou NF-κB+ en inhibant les voies de signalisation correspondantes chez les souris APP/PS1dE9. Nous observons que ces populations ont un impact sur l'anxiété et la préférence et mémoire sociale des souris APP/PS1dE9. Les autres caractéristiques de la MA (plaques amyloïdes, neurites dystrophiques, inflammation) sont en cours d'étude. Ce projet a permis de mettre en évidence que l'hétérogénéité astrocytaire est contrôlée par différentes voies de signalisation, et que ces sous-populations présentent différents profils morphologiques, moléculaires et fonctionnels, avec potentiellement des impacts différentiels au cours de la MA qui restent à caractériser
Alzheimer's disease (AD) is a neurodegenerative disease and the leading cause of dementia worldwide. During AD, astrocytes develop a reactive phenotype and exhibit molecular, morphological, and functional changes, which can induce deleterious and/or beneficial effects on neuronal function. Recently, single-cell/nucleus RNA sequencing (RNAseq) data have revealed the existence of distinct subpopulations of astrocytes in the brains of AD patients and in mouse models. However, the signaling cascades controlling these subpopulations, their functional characteristics, and their impacts on the functioning of the prefrontal cortex (PFC) remain unknown. The JAK2-STAT3 and NF-kB signaling pathways are known to be activated during AD and control the reactive state of astrocytes. Our team has developed astrocyte-specific lentivirus reporters for these two signaling pathways, in which the expression of CFP or GFP is controlled by NF-κB- and STAT3-dependent promoter sequences, respectively. These reporters allowed us to identify three astrocytic subpopulations based on their activated cascades, NF-kB+, STAT3+, and NF-kB+/STAT3+, in the PFC of AD model mice, APP/PS1dE9 mice, as well as APPNL-F/NL-F mice. First, we observed that the subpopulations are not topographically determined by their proximity to amyloid plaques. These observations subsequently led us to note that these subpopulations are also present in WT mice, suggesting that the subpopulations are probably innate. We then observed that the 2D territory area of NF-kB+ astrocytes is larger than that of STAT3+ and STAT3+/NF-kB+ astrocytes. After RNAseq analysis of the three subpopulations, we identified differentially expressed genes between the three subpopulations associated with specific functions of interest in the context of AD (proteostasis, synaptic regulation, lipid metabolism). We confirmed that STAT3+ and NF-κB+ subpopulations exhibit significantly different proteasome and lysosome activities in the PFC of APP/PS1dE9 mice. Observing higher expression of Cx30 and 43 transcripts in STAT3+ astrocytes, we also studied astrocytic coupling via gap junctions and hemichannel activity. We demonstrated through fluorescence recovery after photobleaching (FRAP) that the subpopulations are equivalently coupled via gap junctions, but the STAT3+ population shows increased hemichannel activity compared to the other two. Finally, using new lentiviral vectors, we specifically inhibited the STAT3+ or NF-kB+ subpopulations by inhibiting the corresponding signaling pathways in APP/PS1dE9 mice. We observed that these populations impact anxiety and social preference and memory in APP/PS1dE9 mice. Other characteristics of AD (amyloid plaques, dystrophic neurites, inflammation) are under study. This project has highlighted that astrocytic heterogeneity is controlled by different signaling pathways, and that these subpopulations exhibit different morphological, molecular, and functional profiles, potentially with differential impacts during AD that remain to be characterized

[EN] Idiopathic pulmonary fibrosis (IPF) is the pulmonary disease with higher incidence and worse prognosis. Recent evidence suggests that cucurbitaceae, selective inhibitors of the JAK2/STAT3 pathway, may improve the pathogenesis of IPF, as anti-inflammatory and antioxidative properties have been confirmed in other diseases. However the role in IPF is unknown. Two pharmacological models were investigated in the present study. In the preventive model, Wistar rats were instilled intratracheally with a single dose of bleomycin (BLM)(3.75 U/kg; n=12) to induce lung injury. CuI (20mg/kg/day; n=6) or CuI vehicle (control and IPF group) was administered intraperitoneally daily for 21 days. The therapeutic model was exactly the same starting CuI administration on day 7 until day 21. Animal evolution together with the ventilation-perfusion ratio was controlled through CT/SPECT imaging. Cellular count and characterization was performed in broncoalveolar lavage (BAL) together with the total protein content and the IL-6 and IL-13 concentration in BAL and lung tissue. Hematoxilin-eosin and masson trichrome stains allowed the study of the lung's tissue histology. TGF-ß1, CTGF, COL1A and ET-1 gene and protein expression were both measured by real time PCR and WB as pulmonary vascular remodeling markers. P-STAT3, P-SMAD3 and P-JAK2 proteins were determined by protein and immunohistochemistry quantification. Finally, JAK2 and STAT3 expression and distribution was studied in lung tissue of fibrotic patients with pulmonary hypertension. CT/SPECT quantification showed reduction of the fibrotic areas in the CuI treated group by reestablishing the air space in the lungs back to day 0 levels for the preventive and the therapeutic model. Furthermore, ventilation/perfusion correlation was restored in the therapeutic model after administrating CuI during 14 days. Hematoxilin-eosine stains demonstrated how the group treated pharmacologically presented an improvement in the lung tissue architecture, reversing vascular remodeling and right heart hypertrophy. Masson trichrome stain revealed a reduction of the collagen deposits. Ashcroft score, used to determine the severity of pulmonary fibrosis, was measured and diminished significantly in the CuI treated group. The results showed a gene and protein overexpression of TGF-ß1, CTGF, COL1A and ET-1 in BLM relative to Control rats. This was counteracted with CuI treatment which reduced the expression back to control. In terms of immunohistochemistry, the results demonstrated a decrease in the COL1A deposits in the treated group versus the absence of treatment group. Protein and immunohistochemistry analysis of JAK2, STAT3 and SMAD3 demonstrated an overexpression in bleomycin rats while the protein expression was inhibited in the CuI treated group. In accordance to the results obtained, the immunohistochemistry analysis of the lung parenchyma in patients with pulmonary hypertension related to pulmonary fibrosis showed and overexpression of the phosphorylated forms of JAK2 and STAT3, lacking its expression in healthy lung tissue. The preventive and therapeutic administration of JAK2/STAT3 inhibitor can be a potential treatment for pulmonary fibrosis, as it improves the parameters related to the disease.
[ES] La fibrosis pulmonar idiopática (FPI) es la enfermedad pulmonar con mayor incidencia y peor pronóstico. Estudios recientes sugieren que la familia de las cucurbitaceae, inhibidores selectivos de la ruta JAK2/STAT3, pueden mejorar la patogénesis de la enfermedad, al haberse confirmado sus propiedades antinflamatorias y antioxidantes en otras enfermedades. Sin embargo se desconoce su papel en FPI. En el presente trabajo se estudiaron dos modelos farmacológicos. En el modelo preventivo las ratas Wistar fueron instiladas con una dosis única de bleomicina intratraqueal (BLM)(3.75 U/kg; n=12) para inducir las lesión pulmonar. Durante 21 días se administró CuI (20mg/kg/día; n=6) o vehículo de CuI (control y grupo FPI) por vía intraperitoneal. El modelo curativo se diferencia del preventivo en el comienzo de la administración farmacológica a los 7 días de inducir la enfermedad. El seguimiento de la evolución animal y el ratio de ventilación-perfusión se realizó mediante las técnicas de imagen TC/SPECT. Se realizó el recuento y caracterización de las células totales extravasadas en lavado broncoalveolar (LBA) así como el contenido de proteína total y la concentración de IL- 6 e IL-13 en LBA y tejido pulmonar. Las tinciones de hematoxilina-eosina y masson tricrómico permitieron el estudio de la histología del tejido pulmonar. Se determinó la expresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 mediante las técnicas de real time PCR y WB como marcadores de remodelado vascular. Las proteínas P-STAT3, P-SMAD3 y P-JAK2, fueron determinadas mediante cuantificación proteica e inmunohistoquimia. Por último se estudió la expresión y distribución de JAK2 y STAT3 en tejido de pacientes con fibrosis pulmonar e hipertensión pulmonar. La cuantificación de las imágenes TC/SPECT mostraron una reducción de las áreas fibróticas en el grupo tratado con CuI. El tratamiento farmacológico permitió el restablecimiento del espacio aéreo pulmonar hasta valores control en ambos modelos estudiados. El grupo con tratamiento farmacológico restauró el ratio de ventilación/perfusión tras administrar CuI durante 14 días. Las tinciones de hematoxilina eosina revelaron como el grupo animal tratado farmacológicamente presenta una mejora de la histología pulmonar, revirtiendo el remodelado vascular y la hipertrofia del ventrículo derecho. La tinción de masson tricrómico mostró una disminución de los depósitos de colágeno. Se determinó el valor de Ashcroft, evaluador del grado de fibrosis pulmonar, que descendió significativamente en el grupo tratado con CuI. Los resultados presentan una sobrexpresión génica y proteica de TGF-ß1, CTGF, COL1A y ET-1 en los grupos de bleomicina frente a las ratas control. Dicha condición fue revertida mediante el tratamiento con CuI que restableció los valores a niveles control. Los análisis proteicos e inmunohistoquíimicos de JAK2, STAT3 y SMAD3 revelaron una sobreexpresión en las ratas con bleomicina mientras que la expresión proteica fue inhibida en el grupo tratado con CuI. En consonancia con los resultados obtenidos, el análisis inmunohistoquímico del parénquima pulmonar de pacientes con FPI e HP asociada muestran una sobreeexpresión de las formas fosforiladas de JAK2 y STAT3 frente a la ausencia de expresión en tejido pulmonar sano. La administración curativa y preventiva de un inhibidor de la ruta JAK2/STAT3 puede ser un potencial tratamiento para la fibrosis pulmonar, ya que mejora parámetros indicativos de la patología.
[CAT] La fibrosi pulmonar idiopàtica (FPI) és la enfermetat pulmonar amb major incidència i pitjor pronostic. Estudis recents suggereixen que la família de les cucurbitàcies, Inhibidors selectius de la ruta JAK2 / STAT3, poden millorar la patogènesi de la malaltia, en haver-se confirmat les seues propietats antiinflamatòries i antioxidants En altres patologies. No obstant això es desconeix el seu paper en FPI. En el present treball es van estudiar 2 models farmacològics. En el model preventiu les rates Wistar foren instilades amb una dosi única de bleomicina intratraqueal (BLM) (3,75 U / kg; n = 12) per a induir les lesions pulmonars. Durant 21 dies es va administrar CuI (20 mg / kg / dia, n = 6) o Vehicle de CuI (control i grup FPI) per vía intraperitoneal. El model curatiu es diferència del preventiu en el començament de l'administració farmacológica als 7 dies d'induir l'enfermetat. El seguiment de l'evoluciò dels animals i el ratio de Ventilació-perfusió es va realitzar mitjançant tècniques d'imatge TC/SPECT. Es realitzà el recompte i caracterització de les cèl·lules totals extravasades en el llavat broncoalveolar (LBA). Així com el contingut de proteina total i la concentració d'IL-6 i IL-13 en LBA i teixit pulmonar. Les tincions d'hematoxilina-eosina i tricròmic de Masson van permetre l'estudi de la histologia del teixit pulmonar. Es va determinar l'expressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 mitjançant tècniques de PCR en temps real i WB com marcadors de remodelat vascular. Les proteïnes P-STAT3, P-SMAD3 i P-JAK2, varen ser determinades mitjançant quantificació proteica i inmunohistoquimia. Per últim se estudià l'expressió i distribució de JAK2 i STAT3 en teixit de pacients amb fibrosi pulmonar e hipertensió pulmonar. La quantificació de les imatges TC/SPECT mostraren una reducció de les àrees fibrótiques en el grup tractat amb CuI. El tractament farmacològic permet el restabliment de l'espai aeri pulmonar fins valors de control en els dos models estudiats. El grup amb tractament farmacològic va restaurar el ratio de ventilació/perfusió tras administrar Cul durant 14 dies. Les tincions d'hematoxilina eosina van revelar com el grup animal tractat farmacològicament presenta una Millora de la histologia pulmonar, revertint el remodelat vascular i la hipertròfia del ventricle dret. La tinció de tricròmic de Masson va mostrar una disminució dels dipòsits de col·lagen. Es va determinar el valor d'Ashcroft, avaluador del grau de fibrosi pulmonar, que va baixar significativament a el grup tractat amb CuI. Els resultats presenten una sobreexpressió gènica i proteica de TGF-ß1, CTGF, COL1A i Et-1 en els grups de bleomicina en comparacióa les rates control. Aquesta condició va ser revertida mitjançant el tractament amb CuI que va restablir els valors fins a nivel dels control. A nivell immunohistoquímic els resultats mostren una disminució dels dipòsits de COL1A en el grup tractat comparativament al grup sense tractament. Els anàlisi proteics i inmunohistoquíimics de JAK2, STAT3 i SMAD3 van revelar una sobreexpressió en les rates amb bleomicina mentre que l'expressió proteica va ser inhibida en el grup tractat amb CuI. D'acord amb els resultats obtinguts, l'anàlisi immunohistoquímic del parènquima pulmonar de pacients amb FPI i HP associada mostren sobreeexpresió de les formes fosforilades de JAK2 i STAT3 davant l'absència d'expressió en teixit pulmonar sa. La administració curativa i preventiva d'un inhibidor de la ruta JAK2 / STAT3 pot ser un potencial tractament per la fibrosi pulmonar, ja que millora paràmetres indicatius de la patologia.
Hernández Ribes, G. (2016). ESTUDIO DE LA RUTA CELULAR JAK2/STAT3 COMO POTENCIAL INHIBIDOR EN EL MODELO DE FIBROSIS PULMONAR [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/64087
TESIS

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A via Janus Kinase (JAK) e do transdutor de sinal e ativador de transcrição (STAT) desempenham papéis importantes na patogênese de neoplasias hematopoiéticas. A ativação da via JAK2/STAT3 promove o crescimento e sobrevivência celular em uma variedade de linfomas humanos. Há uma necessidade de compreender a participação da via JAK2/STAT3 em linfomas caninos difusos de grandes células B e do potencial terapêutico dos inibidores de JAK no tratamento dessa doença. O objetivo do presente estudo foi avaliar a expressão de JAK2-STAT3 em linfomas difusos de grandes células B e o impacto do uso de inibidores de JAK2 como AZD1480 e CYT387 no crescimento in vitro dessa linhagem tumoral. Foi realizada técnica de imuno-histoquímica com os anticorpos anti-STAT3 e anti-STAT3 fosforilado (p-STAT3) em linfonodos acometidos por linfoma difuso de grandes células B e comparado à linfonodos normais e reativos. Para avaliação do efeito terapêutico dos inibidores de JAK2 (AZD1480 e CYT387) foi realizado ensaio de viabilidade celular pelo método de azul de tripan utilizando linhagens celulares de linfoma difuso de grandes células B (CLBL-1) e análise de apoptose por citometria de fluxo utilizando o sistema Annexin V. Houve aumento significativo na expressão de STAT3 e p-STAT3 em linfomas difusos de grandes células B em comparação com linfonodos normais. Ambos os fármacos inibiram o crescimento celular em proporções dependentes da dose administrada e houve um aumento significativo nas taxas de apoptose das células tratadas com inibidores de JAK-2 em comparação ao grupo controle tratado com DMSO. Este é o primeiro estudo a avaliar a via JAK2/STAT3 em linfomas difusos de grandes céluslas B canino e esses dados permitem compreender e explorar o potencial terapêutico dos inibidores de JAK permitindo estudos futuros da eficácia clínica desses fármacos na oncologia veterinária
The Janus Kinase (JAK) and signal transducer and activator of transcription (STAT) pathway play important roles in the pathogenesis of hematologic malignancies. Activated JAK2-STAT3 signaling pathway promotes the growth and survival of a variety of lymphomas in human. There is a great demand for understanding JAK-STAT pathway in canine diffuse large B cell lymphoma (DLBCLs) and evaluating the therapeutic potential of JAK inhibitors. Our study aims to evaluate the expression of JAK2-STAT3 pathway in canine DLBCLs and to assess the impact of AZD1480 and CYT387, two novel JAK inhibitors, on canine DLBCL cell growth. Immunohistochemistry was performed in canine DLBCLs, normal and reactive lymph nodes with primary antibodies against STAT3 and phosphorylated STAT3 (p-STAT3). To evaluate the therapeutic effect of novel JAK inhibitors, canine DLBCL cell line CLBL-1 was treated with either AZD1480 or CYT387 and trypan blue viability assay was performed post treatment. There was a significant increase in expression of STAT3 and pSTAT3 in canine DLBCLs compared with the normal lymph node. Both AZD1480 and CYT387 inhibited canine DLBCL cells in a dose dependent manner. This is the first study to evaluate the JAK2/STAT3 pathway in canine DLBCLs. The knowledge of JAK2-STAT3 activity in canine DLBCLs enables us to understand and explore the therapeutic potential of JAK inhibitors. The dose dependent cell growth inhibition by novel JAK inhibitors in this study will lead into the future studies of the underlying mechanism

Orientador: Mirela Tinucci Costa
Banca: Felipe Augusto Ruiz Sueiro
Banca: Lucas Campos de Sá Rodrigues
Banca: Andrigo Barboza De Nardi
Banca: Letícia Abrahão Anai
Resumo: A via Janus Kinase (JAK) e do transdutor de sinal e ativador de transcrição (STAT) desempenham papéis importantes na patogênese de neoplasias hematopoiéticas. A ativação da via JAK2/STAT3 promove o crescimento e sobrevivência celular em uma variedade de linfomas humanos. Há uma necessidade de compreender a participação da via JAK2/STAT3 em linfomas caninos difusos de grandes células B e do potencial terapêutico dos inibidores de JAK no tratamento dessa doença. O objetivo do presente estudo foi avaliar a expressão de JAK2-STAT3 em linfomas difusos de grandes células B e o impacto do uso de inibidores de JAK2 como AZD1480 e CYT387 no crescimento in vitro dessa linhagem tumoral. Foi realizada técnica de imuno-histoquímica com os anticorpos anti-STAT3 e anti-STAT3 fosforilado (p-STAT3) em linfonodos acometidos por linfoma difuso de grandes células B e comparado à linfonodos normais e reativos. Para avaliação do efeito terapêutico dos inibidores de JAK2 (AZD1480 e CYT387) foi realizado ensaio de viabilidade celular pelo método de azul de tripan utilizando linhagens celulares de linfoma difuso de grandes células B (CLBL-1) e análise de apoptose por citometria de fluxo utilizando o sistema Annexin V. Houve aumento significativo na expressão de STAT3 e p-STAT3 em linfomas difusos de grandes células B em comparação com linfonodos normais. Ambos os fármacos inibiram o crescimento celular em proporções dependentes da dose administrada e houve um aumento significativo nas taxas de apopto... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Janus Kinase (JAK) and signal transducer and activator of transcription (STAT) pathway play important roles in the pathogenesis of hematologic malignancies. Activated JAK2-STAT3 signaling pathway promotes the growth and survival of a variety of lymphomas in human. There is a great demand for understanding JAK-STAT pathway in canine diffuse large B cell lymphoma (DLBCLs) and evaluating the therapeutic potential of JAK inhibitors. Our study aims to evaluate the expression of JAK2-STAT3 pathway in canine DLBCLs and to assess the impact of AZD1480 and CYT387, two novel JAK inhibitors, on canine DLBCL cell growth. Immunohistochemistry was performed in canine DLBCLs, normal and reactive lymph nodes with primary antibodies against STAT3 and phosphorylated STAT3 (p-STAT3). To evaluate the therapeutic effect of novel JAK inhibitors, canine DLBCL cell line CLBL-1 was treated with either AZD1480 or CYT387 and trypan blue viability assay was performed post treatment. There was a significant increase in expression of STAT3 and pSTAT3 in canine DLBCLs compared with the normal lymph node. Both AZD1480 and CYT387 inhibited canine DLBCL cells in a dose dependent manner. This is the first study to evaluate the JAK2/STAT3 pathway in canine DLBCLs. The knowledge of JAK2-STAT3 activity in canine DLBCLs enables us to understand and explore the therapeutic potential of JAK inhibitors. The dose dependent cell growth inhibition by novel JAK inhibitors in this study will lead into the future studies of the underlying mechanism
Doutor

Les astrocytes deviennent réactifs dans les maladies neurodégénératives (MND) comme la maladie d’Alzheimer (MA) et de Huntington (MH) mais les conséquences fonctionnelles de cette réactivité sont peu connues. Dans cette étude, nous avons évalué 1) les voies de signalisation impliquées dans la réactivité astrocytaire, 2) la contribution des astrocyte réactifs (AR) à la dysfonction neuronale dans des modèles de MND et 3) les caractéristiques fonctionnelles des AR.Nous avons montré que la voie JAK2/STAT3 est responsable de la réactivité astrocytaire dans des modèles murins de la MA et la MH. Nous avons développé de nouveaux vecteurs viraux ciblant cette voie dans les astrocytes, in vivo. Grâce à ces outils, nous avons étudié la contribution des AR à la dysfonction neuronale dans deux modèles murins de la MH. Nos résultats suggèrent que les AR ne jouent pas un rôle central dans ces modèles de pathologie. En ciblant la voie JAK2/STAT3, nous avons induit la réactivité astrocytaire chez la souris sauvage et avons montré que cette voie régule la transcription de gènes impliqués dans des fonctions cellulaires importantes. De plus, nous avons observé que l’activation des astrocytes conduit à une diminution de la plasticité synaptique dans le cerveau de souris.En conclusion, nous avons montré que la voie JAK2/STAT3 est une voie centrale dans les AR. Nous avons développé des vecteurs viraux innovants pour évaluer 1) la contribution des AR à la dysfonction neuronale dans des modèles de MND et 2) les propriétés fonctionnelles des AR in vivo. L’étude des AR permettra d’identifier de nouvelles cibles moléculaires pour manipuler ces cellules pléiotropes à des fins thérapeutiques
Astrocyte reactivity is a hallmark of pathological conditions in the CNS including neurodegenerative diseases (ND) such as Alzheimer’s (AD) and Huntington’s (HD) diseases. Reactive astrocytes (RA) are identified by morphological changes but their functional features and influence on neurons are poorly understood, especially in ND. Therefore, we aimed at 1) identifying the signaling cascades involved in astrocyte reactivity in ND, 2) evaluating RA contribution to disease phenotype in ND models and 3) deciphering RA functional features. The JAK2/STAT3 pathway is a known trigger of astrocyte reactivity in CNS injuries. Here, we show that this pathway is a common inducer of astrocyte reactivity in AD and HD models. We developed new viral vectors to target this cascade in astrocytes and manipulate astrocyte reactivity in vivo. We used these vectors to determine the contribution of RA to neuronal dysfunction in HD mouse models. We found that RA do not primarily influence disease phenotype in HD. Last, we targeted the JAK2/STAT3 pathway in WT mice to characterize RA functional features in vivo. We show RA undergo transcriptional changes of numerous genes involved in metabolism, protein degradation pathways and immune response. Moreover, we show that astrocyte reactivity alters synaptic plasticity in the mouse hippocampus. Our results identify the JAK2/STAT3 pathway as a central cascade for astrocyte reactivity. The viral vectors developed in this project represent powerful tools to decipher the roles of RA in various ND models and to characterize RA functional features in vivo. Better understanding RA functions may lead to the identification of new therapeutic targets for ND

Les astrocytes sont des éléments clés de la physiologie cérébrale. Dans les maladies neurodégénératives comme la maladie d’Alzheimer (MA), les astrocytes deviennent réactifs. Cette réactivité astrocytaire (RA) est essentiellement caractérisée par des changements morphologiques. En revanche, les effets de la réactivité sur les fonctions de support des astrocytes sont mal connus. De plus, les cascades de signalisation qui conduisent à la RA restent à déterminer. Les objectifs de ce projet étaient de : 1/ démontrer que la voie JAK2-STAT3 (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) joue un rôle central dans le contrôle de la RA au cours des maladies neurodégénératives ; 2/ comprendre quelle est l’implication de la RA dans les altérations moléculaires, cellulaires et fonctionnelles observées dans la MA. Nous avons montré que la voie JAK2-STAT3 est une cascade de signalisation centrale dans la RA (Ben Haim et al., 2015). Dans ce projet, nous démontrons en utilisant de nouveaux outils moléculaires basés sur des vecteurs viraux, que cette voie est nécessaire et suffisante à la RA. Nos résultats montrent également que la modulation de la RA dans deux modèles murins de la MA (souris APP/PS1dE9 et 3xTg-AD) influence certains index pathologiques, mais de façon contexte-dépendante. L’ensemble de ce travail a permis de valider de nouveaux outils pour étudier les astrocytes réactifs in situ et souligne l’importance et la complexité de leur fonctions au cours des maladies neurodégénératives
Astrocytes are emerging as key players in brain physiology. In Alzheimer’s disease (AD), astrocytes become reactive. Astrocyte reactivity (AR) is essentially characterized by morphological changes. But how the normal supportive functions of astrocytes are changed by their reactive state is unclear. Moreover, signaling cascades leading to AR are not yet determined. In this study, we aim to: 1/ demonstrate the JAK2-STAT3 pathway (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) is responsible for AR in neurodegenerative diseases ; 2/ understand the contribution of reactive astrocytes to molecular, cellular and functional alterations in AD. We already reported that the JAK2- STAT3 pathway is a central cascade for AR (Ben Haim et al., 2015). Here, we demonstrate, with new molecular tools based on viral vectors, that this pathway is necessary and sufficient to AR. Our results also show that the modulation of AR in two AD mouse models (APP/PS1dE9 and 3xTg-AD mice) influence several pathological hallmarks, but in a context-dependent manner. Overall, this work has generated new original tools to study reactive astrocytes in situ and it underlines the importance and complexity of their functions in neurodegenerative diseases

La maladie de Huntington (MH) est une maladie neurodégénérative causée par une extension de répétitions du codon CAG dans le gène de la Huntingtine (Htt). Cette maladie est caractérisée par la mort des neurones striataux et la présence d’agrégats de Htt mutée (mHtt). De plus, au cours de la MH, les astrocytes, qui sont essentiels au bon fonctionnement neuronal, changent d’état et deviennent réactifs. La réactivité astrocytaire est caractérisée par des changements morphologiques et transcriptomiques mais l’impact fonctionnel de cette réactivité reste peu compris.Afin d’étudier le rôle des astrocytes réactifs dans la MH, nous avons utilisé des vecteurs viraux récemment développés par notre équipe, qui induisent ou bloquent la réactivité astrocytaire in vivo en ciblant la voie JAK2-STAT3. Nous avons montré que les astrocytes réactifs diminuent le nombre et la taille des agrégats de mHtt majoritairement présents dans les neurones. Ceci est associé à l’amélioration de plusieurs altérations neuronales observées dans ces modèles. Une analyse transcriptomique réalisée sur des astrocytes réactifs révèle des changements majeurs d’expression de gènes liés aux systèmes de protéostasie. De plus, l’activité du lysosome et du protéasome est augmentée dans les astrocytes réactifs de souris modèles de la MH. Nous montrons également que les astrocytes réactifs éliminent plus efficacement leurs propres agrégats de mHtt, suggérant qu’au cours de la MH, ces cellules pourraient dégrader plus efficacement la mHtt provenant des neurones. De plus, certaines protéines chaperonnes sont induites dans les astrocytes réactifs. En particulier, la co-chaperonne DNAJB1/Hsp40 est surexprimée dans les astrocytes réactifs et est retrouvée dans les exosomes isolés à partir de striata de souris MH. Des expériences de gain et perte de fonction suggèrent que cette chaperonne est impliquée dans les effets bénéfiques des astrocytes réactifs sur l’agrégation de la mHtt et l’état des neurones. Les astrocytes réactifs pourraient donc libérer des protéines anti-agrégantes qui favorise l’élimination de la mHtt dans les neurones.Notre étude montre que les astrocytes peuvent, en devenant réactifs au cours de la MH, acquérir des propriétés bénéfiques pour les neurones et favoriser, via un dialogue complexe avec les neurones, l’élimination des agrégats de mHtt
Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by an expansion of CAG codons in the Huntingtin gene. It is characterized by the death of striatal neurons and the presence of mutant Huntingtin (mHtt) aggregates. In pathological conditions, as in HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological and significant transcriptomic changes. Astrocytes are essential for the proper functioning of neurons but the functional changes associated with reactivity are still unclear.To better understand the roles played by reactive astrocytes in HD, we took advantage of our recently developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors in two complementary mouse models of HD and found that reactive astrocytes decrease the number and the size of mHtt aggregates that mainly form in neurons. Reduced mHtt aggregation was associated with improvement of neuronal alterations observed in our mouse models of HD. A genome-wide transcriptomic analysis was performed on acutely sorted reactive astrocytes and revealed an enrichment in genes linked to proteolysis. Lysosomal and proteosomal activities were also increased in reactive astrocytes in HD mice. Moreover, we show that reactive astrocytes degrade more efficiently their own mHtt aggregates, suggesting that these cells could siphon mHtt away from neurons. Alternatively, several chaperones were induced in reactive astrocytes. In particular, the co-chaperone DNAJB1/Hsp40 was upregulated in reactive astrocytes and was present in exosomal fraction from HD mouse striatum. Loss and gain of function experiments suggest that this chaperone is involved in the beneficial effects of reactive astrocytes on mHtt aggregation and neuronal status. Therefore, reactive astrocytes could release anti-aggregation proteins that could promote mHtt clearance in neurons.Overall, our data show that astrocytes, by becoming reactive in HD, develop a protective response that involves complex bidirectional signaling with neurons to reduce mHtt aggregation

碩士
中國醫藥大學
基礎醫學研究所碩士班
99
Abstract The first induced pluripotent stem (iPS) cells were generated in 2006 from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient, and maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required leukemia induced factor (LIF) as an expensive reagent. Our goal is to find a pure compound that not only maintains ES and iPS cell pluripotency, but also increases iPS cell generation efficiency. From 15 candidate compounds we determined that 10 μg/ml n-Butylidenephthalide (BP), an Angelica sinensis extract, triggers up-regulation of Oct4 and Sox2 gene expression levels in MEF cells. We used ES and iPS cells treated with different concentrations of BP to test its usefulness for maintaining stem cell pluripotency. Results indicate higher expression levels of several stem cell markers in BP-treated ES and iPS cells compared to controls that did not contain LIF including alkaline phosphatase, SSEA1, and Nanog. Embryoid body formation and differentiation results confirm that BP containing medium culture is capable of maintaining ES cell pluripotency. Microarray analysis data identified PPAR, ECM and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phospholation-Jak2 and phospholation-Stat3 protein levels increased following BP treatment, and that cytokines associated with the Jak2-Stat3 pathway were up-regulated. Last, we used pou5f1-GFP MEF cells to test iPS generation efficiency following BP treatment. Our data demonstrate the ability of BP to maintain stem cell pluripotency via the Jak2-Stat3 pathway by inducing cytokine expression levels, at the same time improving iPS generation efficiency.

碩士
臺北醫學大學
醫學研究所
95
In this study, we investigated the signaling pathway by thrombin- induced CTGF expression. Thrombin induced increase in CTGF protein expression and CTGF-luciferase activity in WI-38 lung fibroblasts. AG490 (JAK inhibitor) and dominant negative of JAK2 (JAK2 DN) inhibited thrombin-CTGF expression and CTGF-luciferase activity in a dose-dependent manner. Thrombin also caused a time-dependent increase JAK2 phosphorylation at Tyr1007/1008. Transfection of cells with STAT3 DN and STAT3 ODN (oligodeoxynucleotides) inhibited CTGF protein expression and CTGF-luciferase activity. Thrombin induced STAT3 at Tyr705 phosphorylation in a time-dependent manner, which was inhibited by AG490 and JAK2 DN. Thrombin induced STAT binding to CTGF promoter by DNA-binding affinity pull down assay. We also found that thrombin-induced CTGF protein expression was inhibited by STAT3-1 oligodeoxynucleotides(ODN) and STAT3-2 (ODN). Furthermore, thrombin-indused CTGF protein expression and CTGF-luciferase activity was inhibited by cell transfected with c-Src DN. Thrombin induced STAT3 phosphorylation and JAK2 phosphorylation also inhibited by c-Src DN. Taken together, thrombin though Src, JAK2, and STAT3 signaling pathway to induced CTGF protein expression and activity in lung fibroblasts.

碩士
中國醫藥大學
免疫學研究所碩士班
100
Stem cells provide hope for many diseases that currently lack effective therapeutic methods. In all type of stem cells, embryonic stem (ES) and induced pluripotent stem (iPS) cells are the most powerful cells that could differentiate into all three-layer cells. However, maintaining ES and iPS cells self-renewal in vitro must be need leukemia induced factor (LIF) -- an expensive reagent. Our goal is to find a cheaper pure compound that can maintain ES cells pluripotency. After testing Oct4 and Sox2 gene expression levels in 15 candidate compounds, 500μg/ml of Salvia miltiorrhizae triggers the up-regulation of Oct4 gene expression levels in MEF cells. Salvianolic acid B (Sal B), Salvia miltiorrhizae extract, also could up-regulate Oct4 and Sox2 gene expression levels in MEF cells. Viability analysis and BrdU analysis showed that Sal B not only did not cause cell death, but induced cell proliferation, especially in 0.01nM. We used ES cells treated with different concentrations of Sal B to test whether it is useful for maintaining stem cell pluripotency. Results indicated that compared to controls (did not contain LIF), Sal B -treated ES cells have higher expression levels of several stem cell markers in 0.01nM, including alkaline phosphatase, SSEA1, and Nanog. According to previous study, Sal B direct targeted to EGFR, and activated EGFR, then EGFR turn on Stat3 and ERK1/2. We demonstrated that Sal B could bind to LIF receptor by Docking analysis and EGFR to turn-on the Jak2-Stat3 signaling pathway and EGFR-ERK1/2 signaling pathways. We subsequently determined that phospholation-EGFR, phospholation-Jak2, phospholation-Stat3 and phospholation-ERK protein levels were increased after treated with Sal B by western blotting analysis . Cytokines associated with Jak2-Stat3 signaling pathway were also up-regulated.We add Jak2 antagonist AG490 and EGFR antagonist ZD1839 to comfirmed Jak2-Stat3 signaling pathway and EGFR-ERK1/2 signaling pathways. We subsequently determined that phospholation-EGFR, phospholation-Jak2, phospholation-Stat3 and phospholation-ERK protein levels were decreased after treated by Sal B and antagonist. In addition, after antagonist treatment, the stem cell marker expression were also down regulated in Sal B treated ES cells. Through viability assay, we determined that Sal B could increase cell proliferation. In conclusion, we determined that Sal B not only could replace LIF to maintain the pluripotency of ES cells but increase the proliferation rate. It may become a power reagent in stem cell research.