RESUMEN
TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPß activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.
Asunto(s)
Proteínas 14-3-3/inmunología , Macrófagos/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Proteínas 14-3-3/química , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Autoinmunidad , Humanos , Inflamación/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Complejos Multiproteicos/química , Complejos Multiproteicos/inmunología , Complejos Multiproteicos/metabolismo , Progranulinas/inmunología , Progranulinas/metabolismo , Células RAW 264.7 , Receptores Tipo II del Factor de Necrosis Tumoral/química , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
Asunto(s)
Proteínas 14-3-3/metabolismo , Biomarcadores de Tumor/metabolismo , Exorribonucleasas/metabolismo , Mitocondrias/enzimología , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mitocondrial/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Biomarcadores de Tumor/genética , Exorribonucleasas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mitocondrial/genética , Células Sf9 , SpodopteraRESUMEN
YWHAZ (14-3-3ζ) plays crucial roles in regulating proliferation, apoptosis, migration, and invasion of gastric cancer (GC) cells. However, its extensive roles and potential mechanisms in GC cells remain unknown, and need to be researched deeply. In this study, we focus on the role of miR-375/YWHAZ axis in migration, invasion and epithelial-to-mesenchymal transition (EMT) of GC cells. YWHAZ level was assessed by western blot and qPCR assays in GC cells. Scratch and transwell assays were used to determine the migration and invasion of GC cells. The protein levels of correlative molecules were detected by western blot. The regulation of miR-375 on the expression of its target gene YWHAZ was verified by dual-luciferase report system. According to the results, knockdown of YWHAZ inhibited the migration, invasion and EMT of GC cells. Moreover, silencing of YWHAZ restrained the activation of wnt/ß-catenin signalling pathway. YWHAZ was confirmed to be a target gene of miR-375, and its expression was regulated by miR-375 in GC cells. Transfection of miR-375 inhibitor promoted the migration, invasion, EMT and activation of wnt/ß-catenin pathway in GC cells, which was suppressed by inhibition of YWHAZ. Taken together, this study suggests that miR-375/YWHAZ axis may be served as a novel therapeutic target for GC patients.
Asunto(s)
Proteínas 14-3-3/metabolismo , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , Neoplasias Gástricas/patología , beta Catenina/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Invasividad Neoplásica , Vía de Señalización Wnt/genéticaRESUMEN
BACKGROUND: Ion homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes involved in high-affinity phosphate uptake (PHO genes) are strongly induced during both phosphate and potassium starvation, indicating a link between phosphate and potassium homeostasis. However, the signal transduction processes involved are not completely understood. As 14-3-3 proteins are key regulators of signal transduction processes, we investigated the effect of deletion of the 14-3-3 genes BMH1 or BMH2 on gene expression during potassium starvation and focused especially on the expression of genes involved in phosphate uptake. RESULTS: Genome-wide analysis of the effect of disruption of either BMH1 or BMH2 revealed that the mRNA levels of the PHO genes PHO84 and SPL2 are greatly reduced in the mutant strains compared to the levels in wild type strains. This was especially apparent at standard potassium and phosphate concentrations. Furthermore the promoter of these genes is less active after deletion of BMH1. Microscopic and flow cytometric analysis of cells with GFP-tagged SPL2 showed that disruption of BMH1 resulted in two populations of genetically identical cells, cells expressing the protein and the majority of cells with no detectible expression. Heterogeneity was also observed for the expression of GFP under control of the PHO84 promoter. Upon deletion of PHO80 encoding a regulator of the transcription factor Pho4, the effect of the BMH1 deletion on SPL2 and PHO84 promoter was lost, suggesting that the BMH1 deletion mainly influences processes upstream of the Pho4 transcription factor. CONCLUSION: Our data indicate that that yeast cells can be in either of two states, expressing or not expressing genes required for high-affinity phosphate uptake and that 14-3-3 proteins are involved in the process(es) that establish the activation state of the PHO regulon.
Asunto(s)
Proteínas 14-3-3/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Simportadores de Protón-Fosfato/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas 14-3-3/deficiencia , Transporte Biológico/genética , Fosfatos/metabolismo , Potasio/metabolismoRESUMEN
BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.
Asunto(s)
Proteínas 14-3-3/metabolismo , Aedes/inmunología , Inmunidad Celular , Proteínas de Insectos/metabolismo , Mosquitos Vectores/inmunología , Fagocitosis , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Actinas/metabolismo , Aedes/citología , Animales , Línea Celular , Citoplasma/química , Citoesqueleto/fisiología , Escherichia coli/inmunología , Silenciador del Gen , Proteínas de Insectos/deficiencia , Proteínas de Insectos/genética , Mosquitos Vectores/citología , Fagosomas/metabolismo , Fagosomas/microbiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Staphylococcus aureus/inmunologíaRESUMEN
Previous studies show that mice with Ywhae deficiency show abnormalities in brain development including defects in neuronal migration of post-mitotic pyramidal neurons as well as neuronal differentiation and proliferation in neuronal progenitor cells. Also, our previous research indicated that the Ywhae knockout mice show moderate defects in working memory and anxiety-like behavior. This previous work was performed using heterozygous mutant mice. Here we performed behavioral analyses using homozygous Ywhae knockout mice and found that the homozygous Ywhae knockout mice have increased locomotor activity, decreased working memory, and increased sociability. Taken together with the results obtained from the previous pathophysiological analyses in the Ywhae knockout mice, the Ywhae knockout mouse is useful for pathophysiological analyses of neuropsychiatric disorders caused by defects during neurodevelopment.
Asunto(s)
Proteínas 14-3-3/deficiencia , Ansiedad/genética , Trastornos de la Memoria/genética , Proteínas 14-3-3/genética , Análisis de Varianza , Animales , Peso Corporal/genética , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Femenino , Locomoción/genética , Masculino , Memoria a Corto Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de TiempoRESUMEN
BACKGROUND: The seven 14-3-3 protein isoforms bind to numerous proteins and are involved in a wide variety of cellular events, including the cell cycle, cell division, apoptosis and cancer. We previously found the importance of 14-3-3 proteins in neuronal migration of pyramidal neurons in the developing cortex. Here, we test the function of 14-3-3 proteins in the development of neural crest cells in vivo using mouse genetic approaches. RESULTS: We found that 14-3-3 proteins are important for the development of neural crest cells, in particular for the pigmentation of the fur on the ventral region of mice. CONCLUSIONS: Our data obtained from the 14-3-3ε/14-3-3ζ/Wnt1-Cre mice strongly indicate the importance of 14-3-3 proteins in the development of melanocyte lineages.
Asunto(s)
Proteínas 14-3-3/deficiencia , Integrasas/metabolismo , Pigmentación/genética , Regiones Promotoras Genéticas , Proteína Wnt1/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Peso Corporal , Anomalías Craneofaciales/patología , Cruzamientos Genéticos , Femenino , Masculino , Ratones , Cresta Neural/metabolismoRESUMEN
14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects.
Asunto(s)
Proteínas 14-3-3/deficiencia , Movimiento Celular/fisiología , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Neuronas/fisiología , Proteínas 14-3-3/genética , Factores de Edad , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Movimiento Celular/genética , Electroporación , Embrión de Mamíferos , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , ARN Mensajero , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND/OBJECTIVES: 14-3-3η protein, a dimeric phosphoserine-binding protein, provides protection against adverse cardiac remodeling during pressure-overload induced heart failure in mice. To identify its role in myocardial infarction (MI), we have used mice with cardio-specific expression of dominant-negative 14-3-3η protein mutant (DN14-3-3) and performed the surgical ligation of left anterior descending coronary artery. METHODS: We have performed echocardiography to assess cardiac function, protein expression analysis using Western blotting, mRNA expression by real time-reverse transcription polymerase chain reaction and histopathological analyses. RESULTS: DN14-3-3 mice with MI displayed reduced survival, left ventricular ejection fraction and fractional shortening. Interestingly, DN14-3-3 mice subjected to MI showed increased cardiac hypertrophy, inflammation, fibrosis and apoptosis as compared to their wild-type counterparts. Mechanistically, DN14-3-3 mice with MI exhibited activation of endoplasmic reticulum (ER) stress and markers of maladaptive cardiac remodeling. Cardiac regeneration marker expression also decreased drastically in the DN14-3-3 mice with MI. CONCLUSION: Depletion of the 14-3-3η protein causes cardiac dysfunction and reduces survival in mice with MI, probably via exacerbation of ER stress and death signaling pathways and suppression of cardiac regeneration. Thus, identification of drugs that can modulate cardiac 14-3-3η protein levels may probably provide a novel protective therapy for heart failure.
Asunto(s)
Proteínas 14-3-3/deficiencia , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Remodelación Ventricular/fisiología , Proteínas 14-3-3/biosíntesis , Animales , Vasos Coronarios/cirugía , Estrés del Retículo Endoplásmico/fisiología , Humanos , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ(-/-) mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ(-/-) phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ(-/-) BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ(-/-) BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory.
Asunto(s)
Proteínas 14-3-3/genética , Fibras Musgosas del Hipocampo/patología , Células Piramidales/patología , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Memoria Espacial , Proteínas 14-3-3/deficiencia , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Expresión Génica , Ventrículos Laterales/metabolismo , Ventrículos Laterales/patología , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fibras Musgosas del Hipocampo/metabolismo , Células Piramidales/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patología , Transducción de SeñalRESUMEN
BACKGROUND: TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive. RESULTS: We have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA. CONCLUSION: These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.
Asunto(s)
Proteínas 14-3-3/deficiencia , Membrana Celular/metabolismo , Glutamatos/toxicidad , Neuronas/citología , Canales Catiónicos TRPM/metabolismo , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Animales , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Hipocampo/citología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Serina/metabolismo , Relación Estructura-Actividad , Canales Catiónicos TRPM/químicaRESUMEN
14-3-3 is a family of regulatory proteins highly expressed in the brain. Previous invertebrate studies have demonstrated the importance of 14-3-3 in the regulation of synaptic functions and learning and memory. However, the in vivo role of 14-3-3 in these processes has not been determined using mammalian animal models. Here, we report the behavioral and electrophysiological characterization of a new animal model of 14-3-3 proteins. These transgenic mice, considered to be a 14-3-3 functional knock-out, express a known 14-3-3 inhibitor in various brain regions of different founder lines. We identify a founder-specific impairment in hippocampal-dependent learning and memory tasks, as well as a correlated suppression in long-term synaptic plasticity of the hippocampal synapses. Moreover, hippocampal synaptic NMDA receptor levels are selectively reduced in the transgenic founder line that exhibits both behavioral and synaptic plasticity deficits. Collectively, our findings provide evidence that 14-3-3 is a positive regulator of associative learning and memory at both the behavioral and cellular level.
Asunto(s)
Proteínas 14-3-3/fisiología , Aprendizaje por Asociación/fisiología , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Proteínas 14-3-3/deficiencia , Animales , Reacción de Prevención/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Condicionamiento Psicológico/fisiología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/genética , Miedo/fisiología , Hipocampo/metabolismo , Técnicas In Vitro , Potenciación a Largo Plazo/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-ClampRESUMEN
14-3-3 proteins have important roles in several cellular processes such as cell cycle progression, the DNA-damage checkpoint and apoptosis. We have shown previously that depleting 14-3-3η, a 14-3-3 isoform, enhances mitotic cell death, and that combining it with microtubule agents is more effective for anticancer therapeutics. In this study, we investigated whether depleting 14-3-3η can be combined with radiotherapy to enhance its therapeutic efficacy. We found that depleting 14-3-3η resulted in a synergistic radiosensitizing effect when combined with radiotherapy in several glioblastoma cell lines, where its specific expression and correlation of its expression level with malignancy have been reported. The radiosensitizing effect was associated with enhanced mitotic cell death by 14-3-3η depletion but not with mitotic catastrophe, which is one of the major cell death mechanisms observed in response to irradiation of most solid tumors. These results suggest that 14-3-3η may be a therapeutic target to overcome radioresistance in glioblastoma.
Asunto(s)
Proteínas 14-3-3/deficiencia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de la radiación , Muerte Celular/efectos de la radiación , Procesos de Crecimiento Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Glioblastoma/genética , Glioblastoma/patología , Células HeLa , Humanos , Mitosis/efectos de la radiación , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Tolerancia a Radiación/fisiología , TransfecciónRESUMEN
Neuron navigator 2 (NAV2) is required for all-trans retinoic acid (atRA) to induce neurite outgrowth in human neuroblastoma cells. Further, ectopic overexpression of full-length human NAV2 rescues an axonal elongation defect in the Caenorhabditis elegans unc-53 (NAV2 ortholog) mutant. Using a region of NAV2 that independently associates with the cytoskeleton as bait in a yeast-two-hybrid screen, 14-3-3ε was identified as a novel NAV2 interacting partner. Amino acids 761-960 of NAV2 are sufficient to confer a positive interaction with 14-3-3ε as evidenced by a two-hybrid screen and co-immunoprecipitation assay. Knockdown of 14-3-3ε leads to a decrease in atRA-mediated neurite outgrowth, similar to the elongation defects observed when NAV2 is depleted or mutated. Likewise, posterior lateral microtubule (PLM) defects in C. elegans fed unc-53 RNAi are similar to those fed ftt-2 (14-3-3 homolog) RNAi. The discovery of an interaction between NAV2 and 14-3-3ε could provide insight into the mechanism by which NAV2 participates in promoting cell migration and neuronal elongation.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Caenorhabditis elegans/citología , Línea Celular Tumoral , ADN Helicasas , Técnicas de Silenciamiento del Gen , Humanos , Neuritas/efectos de los fármacos , Unión Proteica , Tretinoina/farmacologíaRESUMEN
Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G(2)/M and the accumulation of cardiomyocytes in the G(0)/G(1) phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27(Kip1), possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27(Kip1). These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.
Asunto(s)
Proteínas 14-3-3/metabolismo , Cardiopatías Congénitas/embriología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Ciclina D1/metabolismo , Ciclina E/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Femenino , Corazón Fetal/anomalías , Corazón Fetal/embriología , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Proteínas Oncogénicas/metabolismoRESUMEN
OBJECTIVES: To determine the effect of 5-aza-2'-deoxycytidine (DAC) on human endometrial carcinoma cell (HECC) oncogenicity and demonstrate a molecular mechanism by which DAC modulates HECC oncogenicity. METHODS: The effect of DAC was tested on HECC RL95-2, AN3, Ishikawa and ECC1 cells. The role of 14-3-3σ on HECC oncogenicity in response to DAC treatment was evaluated in RL95-2 and AN3 cells after forced expression or silencing of 14-3-3σ gene expression. RESULTS: Treatment of HECC with DAC produced non-cytotoxic cell growth inhibition and G2/M cell cycle arrest. This effect was strongly correlated with increased expression of p21 and 14-3-3σ. Silencing of 14-3-3σ induced cellular proliferation and reduced the effect of DAC on cell cycle arrest in G2/M phases. Conversely, forced expression of 14-3-3σ showed the opposite effect. Furthermore, forced expression of 14-3-3σ in human endometrial cell lines reduced cell growth and colony formation. CONCLUSIONS: We suggest that 14-3-3σ in HECC suppresses cell proliferation and mediates DAC induced G2/M arrest and inhibition of cell proliferation in HECC.
Asunto(s)
Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Azacitidina/análogos & derivados , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Exonucleasas/genética , Exonucleasas/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas 14-3-3/biosíntesis , Proteínas 14-3-3/deficiencia , Azacitidina/farmacología , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/deficiencia , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Metilación de ADN/efectos de los fármacos , Decitabina , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/metabolismo , Exonucleasas/biosíntesis , Exonucleasas/deficiencia , Exorribonucleasas , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Genes Supresores de Tumor/efectos de los fármacos , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genética , TransfecciónRESUMEN
Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells.
Asunto(s)
Proteínas 14-3-3/metabolismo , Movimiento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Óvulo/citología , Espermatozoides/clasificación , Proteínas 14-3-3/química , Proteínas 14-3-3/deficiencia , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Femenino , Eliminación de Gen , Masculino , Datos de Secuencia Molecular , Óvulo/metabolismo , Estabilidad Proteica , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Espermatozoides/metabolismoRESUMEN
Ling and colleagues demonstrated that loss of the conditional 14-3-3σ allele results in accelerated HER2/ERBB2-driven mammary tumorigenesis and metastasis. This study underscores the role of 14-3-3σ as a potent tumor suppressor in ERBB2-driven tumor initiation and progression.
Asunto(s)
Proteínas 14-3-3/deficiencia , Neoplasias Mamarias Experimentales/metabolismo , Receptor ErbB-2/metabolismo , Animales , FemeninoRESUMEN
UNLABELLED: 14-3-3σ is a putative tumor suppressor involved in cell-cycle progression and epithelial polarity. We demonstrate that loss of one or both copies of the conditional 14-3-3σ allele results in accelerated mammary and salivary tumorigenesis in mice expressing an activated erbB2 oncogene under the endogenous erbB2 promoter. Significantly, the majority of tumors bearing a single conditional 14-3-3σ allele lose expression of the remaining 14-3-3σ allele, which is associated with epigenetic methylation of the 14-3-3σ locus. In addition to accelerated tumor onset, in a mouse mammary tumor virus-driven ErbB2 tumor model, loss of 14-3-3σ results in enhanced metastatic phenotype that is correlated with loss of cellular junctions. Taken together, these results provide compelling evidence that 14-3-3σ is a potent tumor suppressor involved in ErbB2-driven breast cancer initiation and metastasis. SIGNIFICANCE: 14-3-3σ has been identified as a normal mammary epithelial cell marker frequently downregulated during neoplastic development. Consistent with its potential role as a tumor suppressor, we demonstrate that targeted disruption of 14-3-3σ in a number of epithelial tissues can profoundly impact both the initiation and metastatic phases of ErbB2-mediated tumor progression through modulation of a number of distinct signaling networks.
Asunto(s)
Proteínas 14-3-3/deficiencia , Neoplasias Mamarias Experimentales/metabolismo , Receptor ErbB-2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Animales , Progresión de la Enfermedad , Femenino , Genes Supresores de Tumor , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Receptor ErbB-2/genéticaRESUMEN
Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.