RESUMO
We previously described the protective role of the nuclear factor of activated T cells 5 (NFAT5) during hypoxia. Alternatively, inducible nitric oxide synthase (iNOS) is also induced by hypoxia. Some evidence indicates that NFAT5 is essential for the expression of iNOS in Toll-like receptor-stimulated macrophages and that iNOS inhibition increases NFAT5 expression in renal ischemia-reperfusion. Here we studied potential NFAT5 target genes stimulated by hypoxia in mouse embryonic fibroblast (MEF) cells. We used three types of MEF cells associated with NFAT5 gene: NFAT5 wild type (MEF-NFAT5+/+), NFAT5 knockout (MEF-NFAT5-/-), and NFAT5 dominant-negative (MEF-NFAT5Δ/Δ) cells. MEF cells were exposed to 21% or 1% O2 in a time course curve of 48 h. We found that, in MEF-NFAT5+/+ cells exposed to 1% O2, NFAT5 was upregulated and translocated into the nuclei, and its transactivation domain activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5-/- or MEF-NFAT5Δ/Δ cells, the basal levels of iNOS and AQP-1 expression were strongly downregulated, but not for UTA-1. The upregulation of AQP-1, UTA-1, and iNOS by hypoxia was blocked in both NFAT5-mutated cells. The iNOS induction by hypoxia was recovered in MEF-NFAT5-/- MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5Δ/Δ cells, confirming the dominant-negative effect of MEF-NFAT5Δ/Δ cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information about the signaling pathway of NFAT5 during responses to oxygen depletion in mammalian cells and suggests that the expression of iNOS induced by hypoxia is dependent on NFAT5.
Assuntos
Fibroblastos/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aquaporina 1/genética , Aquaporina 1/metabolismo , Hipóxia Celular , Células Cultivadas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Transdução de Sinais , Fatores de Transcrição/genética , Transportadores de UreiaRESUMO
RESUMEN Objetivo: Relacionar la pertenencia a la etnia Mapuche y los síntomas climatéricos de indicación de Terapia Hormonal de la Menopausia (THM), en una población del sector rural de Boyeco, región de La Araucanía. Materiales y métodos: Estudio observacional realizado en mujeres rurales en control de salud en CESFAM Boyeco entre octubre de 2016 y enero de 2017. Ninguna de las participantes evaluadas recibía THM. Para el estudio, se consideró el número de apellidos mapuches. Se utilizó el instrumento validado para población chilena, "Escala MRS" (Menopause Rating Scale), el cual permite discriminar los diferentes dominios sintomáticos del climaterio. Resultados: El grupo en estudio lo componen 36 mujeres de 41 a 78 años de edad, promedio (DE) 52,8(8,6) años, un 52,8% tiene dos apellidos mapuches y 25% uno. Un 92,8% de las mujeres mapuche tiene indicación de terapia, versus todas las no mapuche. En las menores de 50 años, todas tiene indicación de terapia, frente a un 71,4% en las mayores de 50 años. Conclusiones: Las pacientes mapuches tienen mayor sintomatología en los dominios somáticos y psicológicos, especialmente aquellas bajo 50 años. Todas las mujeres estudiadas bajo 50 años requieren terapia, sin variación estadísticamente significativa x etnicidad.
SUMMARY Objective: To stablish the relationship between belonging to Mapuche ethnic group on the climacteric symptoms for indication of menopause hormone therapy (HTM), in the rural population of Boyeco, inside of Araucania's region, Chile. Materials and methods: An observational and descriptive study, in a sample in time of 36 women belonging to the sector who attended CESFAM Boyeco, between October 2016 and January 2017. None of the evaluated participants received THM. As exposure variable, it was considered the number the mapuche surnames. We used the Menopause Rating Scale (MRS), an international instrument validated for Chilean population, to discriminate the different symptomatic domains of the climacteric period. Results: 94.7% of mapuche women and all non-mapuche population had prescribed hormonal therapy. Independent of ethnicity, those under 50 years of age, 100% have an indication for therapy compared to 71.4% in those over 50 years of age. Conclusions: Mapuche patients have greater symptomatology in the somatic-psychological domains, especially in those under 50 years of age. The totality of women under 50 requires therapy, however, variation according to ethnicity.
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Climatério , Menopausa/etnologia , Indígenas Sul-Americanos , Terapia de Reposição Hormonal , Povos Indígenas , Atenção Primária à Saúde/estatística & dados numéricos , Psicometria/métodos , Chile/epidemiologiaRESUMO
Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies.
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The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders.
RESUMO
Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSConset) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSCpeak). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSClate). Surprisingly, EAE atypical signs were detected in EAE+MSCpeak and EAE+MSClate mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, RORγT, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis.
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Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.
Assuntos
Embrião de Mamíferos/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Wnt/metabolismo , Envelhecimento/fisiologia , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/citologia , Humanos , Microdissecção , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Gânglios Espinais/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , PPAR gama/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular , Gânglios Espinais/patologia , Hipocampo/patologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Hipoglicemiantes/farmacologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/patologia , Oxidantes/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Células PC12 , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
BACKGROUND: The generation of the amyloid-beta peptide (Abeta) through the proteolytic processing of the amyloid precursor protein (APP) is a central event in the pathogenesis of Alzheimer's disease (AD). Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R) family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing. RESULTS: Here, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Abeta production and reduced levels of APP-CTFs. The increased Abeta production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased gamma-secretase activity, both of which might contribute to increased Abeta production. CONCLUSION: These findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Abeta production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting beta-secretase and gamma-secretase mediated amyloidogenic processing and also by incrementing the activity of gamma-secretase.
RESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder with progressive dementia accompanied by two main structural changes in the brain: intracellular protein deposits termed neurofibrillary tangles (NFT) and extracellular amyloid protein deposits surrounded by dystrophic neurites that constitutes the senile plaques. Currently, it is widely accepted that amyloid beta-peptide (A beta) metabolism disbalance is crucial for AD progression. A beta deposition may be enhanced by molecular chaperones, including metals like copper and proteins like acetylcholinesterase (AChE). At the neuronal level, several AD-related proteins interact with transducers of the Wnt/beta-catenin signaling pathway, including beta-catenin and glycogen synthase kinase 3 beta (GSK-3 beta) and both in vitro and in vivo studies suggest that Wnt/beta-catenin signaling is a target for A beta toxicity. Accordingly, activation of this signaling by lithium or Wnt ligands in AD-experimental animal models or in primary hippocampal neurons attenuate A beta neurotoxicity by recovering beta-catenin levels and Wnt-target gene expression of survival genes such as bcl-2. On the other hand, peroxisomal proliferator-activated receptor gamma (PPAR gamma) and muscarinic acetylcholine receptor (mAChR) agonists also activate Wnt/beta-catenin signaling and they have neuroprotective effects on hippocampal neurons. Our studies are consistent with the idea that a sustained loss of function of Wnt signaling components would trigger a series of events, determining the onset and development of AD and that modulation of this pathway through the activation of cross-talking signaling cascades should be considered as a possible therapeutic strategy for AD treatment.