RESUMO
Huntington's disease (HD) is a genetic disorder marked by transcriptional alterations that result in neuronal impairment and death. MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation and fine-tuning of gene expression. Several studies identified altered miRNA expression in HD and other neurodegenerative diseases, however their roles in early stages of HD remain elusive. Here, we deep-sequenced miRNAs from the striatum of the HD mouse model, BACHD, at the age of 2 and 8 months, representing the pre-symptomatic and symptomatic stages of the disease. Our results show that 44 and 26 miRNAs were differentially expressed in 2- and 8-month-old BACHD mice, respectively, as compared to wild-type controls. Over-representation analysis suggested that miRNAs up-regulated in 2-month-old mice control the expression of genes crucial for PI3K-Akt and mTOR cell signaling pathways. Conversely, miRNAs regulating genes involved in neuronal disorders were down-regulated in 2-month-old BACHD mice. Interestingly, primary striatal neurons treated with anti-miRs targeting two up-regulated miRNAs, miR-449c-5p and miR-146b-5p, showed higher levels of cell death. Therefore, our results suggest that the miRNAs altered in 2-month-old BACHD mice regulate genes involved in the promotion of cell survival. Notably, over-representation suggested that targets of differentially expressed miRNAs at the age of 8 months were not significantly enriched for the same pathways. Together, our data shed light on the role of miRNAs in the initial stages of HD, suggesting a neuroprotective role as an attempt to maintain or reestablish cellular homeostasis.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doença de Huntington/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Neuroproteção/fisiologia , Sintomas Prodrômicos , Animais , Células Cultivadas , Feminino , Doença de Huntington/metabolismo , Doença de Huntington/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência de RNA/métodos , Regulação para Cima/fisiologiaRESUMO
GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor α (ERα)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ERα-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ERα-positive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca2+]i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca2+]i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca2+]i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ERα expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ERα/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen.
Assuntos
Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Receptores de Estrogênio/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Tamoxifeno/administração & dosagem , Neoplasias da Mama/patologia , Proliferação de Células/fisiologia , Feminino , Humanos , Células MCF-7 , Receptores Acoplados a Proteínas G/agonistas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H+-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.
Assuntos
Canais Iônicos Sensíveis a Ácido/biossíntese , Doença de Fabry/metabolismo , Regulação para Cima/fisiologia , Canais Iônicos Sensíveis a Ácido/genética , Animais , Células Cultivadas , Doença de Fabry/genética , Doença de Fabry/patologia , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/toxicidade , Venenos de Aranha/toxicidade , Regulação para Cima/efeitos dos fármacosRESUMO
Climatic change is pointed as one of the major challenges for global food security. Based on current models of climate change, reduction in precipitations and in turn, increase in the soil salinity will be a sharp constraint for crops productivity worldwide. In this context, root fungi appear as a new strategy to improve plant ecophysiological performance and crop yield under abiotic stress. In this study, we evaluated the impact of the two fungal endophytes Penicillium brevicompactum and P. chrysogenum isolated from Antarctic plants on nutrients and Na+ contents, net photosynthesis, water use efficiency, yield and survival in tomato and lettuce, facing salinity stress conditions. Inoculation of plant roots with fungal endophytes resulted in greater fresh and dry biomass production, and an enhanced survival rate under salt conditions. Inoculation of plants with the fungal endophytes was related with a higher up/down-regulation of ion homeostasis by enhanced expression of the NHX1 gene. The two endophytes diminished the effects of salt stress in tomato and lettuce, provoked a higher efficiency in photosynthetic energy production and an improved sequestration of Na+ in vacuoles is suggested by the upregulating of the expression of vacuolar NHX1 Na+/H+ antiporters. Promoting plant-beneficial interactions with root symbionts appears to be an environmentally friendly strategy to mitigate the impact of climate change variables on crop production.
Assuntos
Produtos Agrícolas/metabolismo , Produtos Agrícolas/fisiologia , Endófitos/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Estresse Salino/fisiologia , Sódio/metabolismo , Regiões Antárticas , Biomassa , Mudança Climática , Produtos Agrícolas/microbiologia , Regulação para Baixo/fisiologia , Homeostase/fisiologia , Íons/metabolismo , Lactuca/metabolismo , Lactuca/microbiologia , Lactuca/fisiologia , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Solanum lycopersicum/fisiologia , Penicillium chrysogenum/fisiologia , Fotossíntese/fisiologia , Raízes de Plantas/microbiologia , Salinidade , Trocadores de Sódio-Hidrogênio/metabolismo , Solo , Estresse Fisiológico/fisiologia , Taxa de Sobrevida , Regulação para Cima/fisiologia , Água/metabolismoRESUMO
Posttraumatic Stress Disorder (PTSD) is an anxiety disorder which occurs after a traumatic event. The NR3C1 gene codes for the Glucocorticoid Receptor, which participate in the Hypothalamic-Pituitary-Adrenal (HPA) axis and is altered in PTSD patients. To evaluate whether the NR3C1 gene expression in peripheral blood could be useful as a diagnosis biomarker, a total of 32 PTSD patients and 59 healthy controls were analyzed with quantitative RT-PCR. Also, to assess if NR3C1 dysregulation is associated with hypocortisolism in PTSD patients, serum cortisol was quantified by ELISA in a subset of these samples. Significant NR3C1 over-expression was found in PTSD patients compared with controls, and this was higher in patients with acute PTSD. The Area Under the Curve (AUC) of NR3C1 gene expression was 0.797. The sensibility and specificity of NRC1 gene expression to diagnose PTSD was 62.5% and 89.8%, respectively. We also found that an up-regulation of NR3C1 increased the risk for being diagnosed with PTSD (OR= 12.8, 95%, CI 4-41.4). Finally, the NR3C1 gene expression was inversely related with serum cortisol in PTSD patients. The present results suggest that NR3C1 gene expression could be a promising biomarker for PTSD diagnosis and estimate the risk for disease development.
Assuntos
Marcadores Genéticos/genética , Receptores de Glucocorticoides/genética , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Transtornos de Estresse Pós-Traumáticos/genética , Adulto , Feminino , Expressão Gênica , Humanos , Hidrocortisona/genética , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , México/epidemiologia , Sistema Hipófise-Suprarrenal/fisiologia , Receptores de Glucocorticoides/biossíntese , Fatores de Risco , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Regulação para Cima/fisiologiaRESUMO
AgRP neurons are important players in the control of energy homeostasis and are responsive to several hormones. In addition, STAT5 signalling in the brain, which is activated by metabolic hormones and growth factors, modulates food intake, body fat and glucose homeostasis. Given that, and the absence of studies that describe STAT5 function in AgRP cells, the present study investigated the metabolic effects of Stat5a/b gene ablation in these neurons. We observed that STAT5 signalling in AgRP neurons regulates body fat in female mice. However, male and female STAT5-knockout mice did not exhibit altered food intake, energy expenditure or glucose homeostasis compared to control mice. The counter-regulatory response or glucoprivic hyperphagia induced by 2-deoxy-d-glucose treatment were also not affected by AgRP-specific STAT5 ablation. However, under 60% food restriction, AgRP STAT5-knockout mice had a blunted upregulation of hypothalamic Agrp mRNA expression and corticosterone serum levels compared to control mice, suggesting a possible role for STAT5 in AgRP neurons for neuroendocrine adaptations to food restriction. Interestingly, ad libitum fed knockout male mice had reduced Pomc and Ucp-1 mRNA expression compared to control group. Taken together, these results suggest that STAT5 signalling in AgRP neurons regulates body adiposity in female mice, as well as some neuroendocrine adaptations to food restriction.
Assuntos
Adaptação Fisiológica/fisiologia , Adiposidade/fisiologia , Proteína Relacionada com Agouti/metabolismo , Metabolismo Energético/fisiologia , Neurônios/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Ingestão de Alimentos/fisiologia , Feminino , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Desacopladora 1/metabolismo , Regulação para Cima/fisiologiaRESUMO
The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.
Assuntos
Sistema Colinérgico não Neuronal/fisiologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologia , Acetilcolina/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Antagonistas Nicotínicos/farmacologia , Sistema Colinérgico não Neuronal/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Regulação para Cima/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Protease-activated receptors (PARs) have been described in a wide diversity of vertebrate cells, including human immune cells. Macrophages are pivotal cells in the host-pathogen interaction and their polarization in M1 or M2 cells has been described as a new central paradigm in the immune response to pathogens. In this context, we explored the involvement of PAR activation by serine proteases on M1/M2 macrophage differentiation and their impact on the Th1/Th2 cytokine profile in response to Mycobacterium tuberculosis antigen. Our results demonstrate that the serine proteases, thrombin and trypsin, induce interleukin (IL)-4 release from human monocytes, together with upregulation of the macrophage mannose receptor (CD206) in the same way that alternative M2a differentiated cells with M-CSF/IL-4. Protease stimulation of monocytes in the presence of PAR-1 (SCH-79797) or PAR-2 (FSLLRY-NH2) antagonists abolished IL-4 release from monocytes, whereas the use of the peptide agonist for PAR-1 (SFLLRNPNDKYEPF-NH2) or PAR-2 (SLIGKV-NH2) induced the secretion of IL-4 at a level comparable to thrombin or trypsin. When these protease-induced M2 macrophages from healthy human PPD + donors were co-cultured with autologous lymphocyte population in the presence of Mycobacterium tuberculosis antigen, we found a consistent inhibition of IFN-γ/IL-12 release together with persistent IL-4 expression, in contrast to the expected Th1 profile obtained with M2a macrophages. To our knowledge, this is the first observation that proteolytic activation of PAR1/2 receptors in monocytes induces M2-like macrophages with impaired plasticity and their implication in the driving of the Th1/Th2 cytokine profile.
Assuntos
Polaridade Celular/fisiologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Diferenciação Celular/fisiologia , Plasticidade Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Interleucina-4/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Ativação de Macrófagos/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Monócitos/metabolismo , Monócitos/fisiologia , Mycobacterium tuberculosis/patogenicidade , Tripsina/metabolismo , Tuberculose/metabolismo , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Long non-coding RNA H19 (H19) plays an important role by regulating protein expression in different tissues and organs of the body. However, whether H19 induces hypoxia/reoxygenation (h/R) injury via increase of autophagy in the hepatoma carcinoma cells is unknown. RESULTS: H19 was expressed in the hepatoma carcinoma cells (Hep G2 and HCCLM3 cells) and its expression was most in 8 h/24R. The knockdown of H19 and 3-MA (an autophagy inhibitor) protected against h/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The knockdown of H19 and 3-MA also decreased the autophagic vesicles (AVs) and the expression of Beclin-1 and the ration of LC3-II/LC3-I, and increased cell viability, the expression of Bcl-2 and P62 and the phosphorylation of PI3K, Akt and mTOR. In addition, chloroquine (CQ, an inhibitor of autophagy flux) markedly decreased formation of autophagy flux (the ration of LC3-II/LC3-I). The results of the knockdown of H19 group were similar to those of the 3-MA (or CQ) group. Rapamycin (a mTOR inhibitor, an autophagy activator) further down-regulated h/R-induced decrease of the phosphorylated PI3K, Akt and mTOR. The knockdown of H19 cancelled the effect of rapamycin. The overexpression of H19 further expanded h/R-induced increase of the ration of LC3-II/LC3-I and decrease of the phosphorylated PI3K, Akt and mTOR. CONCLUSIONS: Our results suggest that the long non-coding RNA H19 induces h/R injury by up-regulation of autophagy via activation of PI3K-Akt-mTOR pathway in the hepatoma carcinoma cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Oxigênio/metabolismo , Regulação para Cima/fisiologiaRESUMO
Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1ß and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1ß-induced GHS-R1a upregulation. IL-1ß and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1ß and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1ß and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1ß and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.