Asunto(s)
Factor de Transcripción GATA4/biosíntesis , Infarto del Miocardio/genética , Miocitos Cardíacos/metabolismo , Proteínas de Dominio T Box/biosíntesis , Animales , Fusión Celular , Reprogramación Celular/fisiología , Femenino , Factores de Transcripción MEF2/biosíntesis , Masculino , Ratones , Miocitos Cardíacos/citologíaRESUMEN
Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.
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Escherichia coli , Factor de Transcripción GATA4 , Línea Celular , Dicroismo Circular , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Factor de Transcripción GATA4/biosíntesis , Factor de Transcripción GATA4/química , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/aislamiento & purificación , Humanos , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
PURPOSE: GATA4 has emerged as a novel regulator that plays a critical role in mediating senescence. However, the role of GATA4 in age-related cataract (ARC), the leading cause of visual impairment, requires further elucidation. METHODS: GATA4 expression was measured by quantitative RT-PCR and capillary Western immunoassay (WES). The MTT assay, EdU assay, and rhodamine-123/Hoechst and calcein-AM/propidium iodide double staining were used to investigate the role of GATA4 in the viability, proliferation, and apoptosis of cultured human lens epithelial cells (HLECs). RESULTS: HLECs were subjected to 3 different treatment models, including prolonged exposure to low-dose H2O2, UVB irradiation, and mild heating, to simulate senescence and apoptosis. GATA4 expression was significantly increased in these models in a time- and dose-dependent manner. Overexpression of GATA4 reduced cell viability, accelerated apoptosis development, and reduced the proliferation of HLECs. Furthermore, the expression of GATA4 from ARC was up-regulated at both mRNA and at protein level compared with clear lenses. CONCLUSION: GATA4 is up-regulated in all 3 models of HLECs in vitro and the cells from ARC lenses in vivo. Up-regulation of GATA4 mediates HLEC dysfunction. GATA4-mediated effects in HLECs would provide a novel insight into the pathogenesis of ARC.
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Catarata/genética , Células Epiteliales/metabolismo , Factor de Transcripción GATA4/genética , Regulación de la Expresión Génica , Estrés Oxidativo , ARN/genética , Regulación hacia Arriba , Anciano , Apoptosis , Catarata/metabolismo , Catarata/patología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Células Epiteliales/patología , Factor de Transcripción GATA4/biosíntesis , Humanos , Persona de Mediana Edad , Transducción de Señal , Dedos de ZincRESUMEN
GATA-binding protein 4 (GATA-4)ï¼a member of the GATA family of transcription factors, is expressed in the normal brain and participates in the neural inflammatory response and senescence. However, few studies have investigated whether GATA-4 is involved in the brain damage induced by intracerebral hemorrhage (ICH). The aim of this study was to investigate in vivo and in vitro the role of GATA-4 in ICH-induced secondary brain injury (SBI) and its potential underlying mechanisms. A rat model of ICH was established by autologous blood injection in vivo. In vitro, oxidized hemoglobin was applied to mimic the effects of ICH in neuronal culture. The function of GATA-4 and its mechanism of action after ICH were investigated using siRNA-mediated knockdown and plasmid-mediated overexpression techniques combined with immunofluorescence, western blot, and other molecular methods. It was found that the expression of GATA-4 was increased in the brain of rats after ICH, and its phosphorylation also increased correspondingly. Furthermore, knocking down the expression of GATA-4 led to a significant decrease in neurobehavioral scores and neuronal apoptosis, indicating that secondary brain damage was improved. Conversely, the overexpression of GATA-4 aggravated brain damage. Blockade of a critical phosphorylation site on the GATA-4 overexpression plasmid alleviated the exacerbated damage in vitro and in vivo. Moreover, GATA-4 promoted the activation of NF-κB, and increased the expression of Bax, and cysteine aspartate-specific protease 3 (caspase-3) in its cleaved form, causing neuronal apoptosis. In conclusion, the expression of GATA-4 was increased in the brain of rats after ICH. GATA-4 phosphorylation mediates the function of the protein in ICH-induced SBI. Neuronal apoptosis after ICH was mainly induced by NF-κB activation, which was promoted by GATA-4.
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Apoptosis/genética , Caspasa 3/genética , Hemorragia Cerebral/patología , Factor de Transcripción GATA4/genética , FN-kappa B/genética , Neuronas/patología , Transducción de Señal/genética , Proteína X Asociada a bcl-2/genética , Animales , Conducta Animal , Química Encefálica , Hemorragia Cerebral/metabolismo , Factor de Transcripción GATA4/biosíntesis , Técnicas de Silenciamiento del Gen , Masculino , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Pancreatic ductal carcinoma (PDAC) is a common malignant tumor of the digestive system. GATA4 is one of the transcriptional regulatory factors, which regulates the development of endoderm-derived organs, including heart and gut. GATA4 may act as a putative tumor suppressor gene. However, the role of GATA4 in pancreatic carcinogenesis is not yet clarified. This study showed that GATA4 was highly expressed in pancreatic cancer tissues, and its expression level was positively related to the grade of pathological differentiation, suggesting that it may contribute to the progression of pancreatic neoplasia. Ectopic expression of GATA4 gene reduced cell viability and interference of GATA4 expression significantly increased the colony formation ability of pancreatic cancer cells. Furthermore, GATA4 inhibited tumor growth in xenograft mice. Agilent expression microarray profiling analysis indicated that the genes with significant levels of differential expression in GATA4 over-expressing cells were enriched in the cell differentiation process. Analysis of KEGG signaling pathway demonstrated that the regulated genes were partially enriched in MAPK and JAK-STAT signaling pathways. Re-expression of GATA4 up-regulated P53 gene expression. Our data indicate that GATA4 gene might play a role in cell proliferation and differentiation during the progression of pancreatic cancer.
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Diferenciación Celular , Proliferación Celular , Factor de Transcripción GATA4/biosíntesis , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/biosíntesis , Neoplasias Pancreáticas/metabolismo , Línea Celular Tumoral , Factor de Transcripción GATA4/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologíaRESUMEN
Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.
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Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Factor de Transcripción GATA4/biosíntesis , Hepatoblastoma/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina , Factor de Transcripción GATA4/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hepatoblastoma/genética , Hepatoblastoma/patología , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Interferente PequeñoRESUMEN
The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.
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Factor de Transcripción GATA4/genética , Factor de Transcripción GATA5/genética , Factor de Transcripción GATA6/genética , Intestinos/citología , Planarias/embriología , Regeneración/genética , Regeneración/fisiología , Células Madre/citología , Animales , Proliferación Celular/genética , Factor de Transcripción GATA4/biosíntesis , Factor de Transcripción GATA5/biosíntesis , Factor de Transcripción GATA6/biosíntesis , Mucosa Intestinal/metabolismo , Planarias/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/ß-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation.
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Diferenciación Celular/genética , Factor de Transcripción GATA4/biosíntesis , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Represoras/genética , Animales , Linaje de la Célula/genética , Factor de Transcripción GATA4/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Vía de Señalización WntRESUMEN
BACKGROUND: RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a "noncoding RNAi". However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). RESULTS: Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response. CONCLUSIONS: Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and "non coding" siRNA is a hindrance in the development of siRNA based therapeutics.
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Aorta/efectos de los fármacos , Biología Computacional , Regulación de la Expresión Génica/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Aorta/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Factor de Transcripción GATA4/biosíntesis , Regulación de la Expresión Génica/genética , Silenciador del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interleucina-1/biosíntesis , Músculo Liso Vascular/metabolismo , Complejo Represivo Polycomb 2/biosíntesis , Polietileneimina/administración & dosificación , ARN Interferente Pequeño/genética , Transfección/métodosRESUMEN
Bone marrow mesenchymal stem cells (BMMSCs) can differentiate into cardiomyocytes and be used in cardiac tissue engineering for heart regeneration. However, the effective clinical application of cardiomyocytes derived from BMMSCs is limited because of their immature phenotype. The aim of this study was to investigate the potential of triiodo-L-thyronine (T3) to drive cardiomyocytes derived from BMMSCs to a more mature state. BMMSCs were divided into 3 groups: untreated controls, differentiated, and T3 treated. The differentiation potential was evaluated by immunofluorescence microscopy and flow cytometry. Data were represented as the numbers of cells positive for the troponin I (cTnI), α-actinin, GATA4, and the connexin-43 (Cx-43). The mRNA levels of these specific markers of cardiomyocytes were determined by quantitative real-time polymerase chain reaction. The levels of cardiomyocytes markers protein and octamer-binding transcription factor 4 (Oct-4) were determined by Western blot analyses. Our data demonstrate that T3 treatment leads to a significant increase in cells positive for cTnI, GATA4, Cx-43, and α-actinin. The mRNA and protein expression levels of these specific markers of cardiomyocytes were also increased after T3 treatment. At the same time, the protein expression level of Oct-4 was substantially downregulated in T3-treated cells. These results demonstrate that T3 treatment increases the differentiation of BMMSCs induced to cardiomyocytes and promotes their maturation.
Asunto(s)
Médula Ósea , Células Madre Mesenquimatosas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Tironinas/farmacología , Actinina/biosíntesis , Animales , Diferenciación Celular , Células Cultivadas , Conexina 43/biosíntesis , Factor de Transcripción GATA4/biosíntesis , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Troponina I/biosíntesisRESUMEN
3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.
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Adipogénesis/genética , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Osteogénesis/genética , Actinas/biosíntesis , Animales , Proliferación Celular/genética , Factor de Transcripción GATA4/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas/citología , Ratones , Miocitos Cardíacos/metabolismo , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear. METHODS: Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. RESULTS: MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06 ± 70.94; P = 0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76 ± 105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P = 0.035) and minimal residual disease MRD (P = 0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P = 0.014). CONCLUSIONS: Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required.
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Metilación de ADN/genética , Factor de Transcripción GATA4/genética , Leucemia Mieloide Aguda/genética , Pronóstico , Adolescente , Niño , Preescolar , Islas de CpG/genética , Femenino , Factor de Transcripción GATA4/biosíntesis , Regulación Leucémica de la Expresión Génica , Células HL-60 , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/patología , Masculino , Regiones Promotoras GenéticasRESUMEN
It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification.
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Proteína Morfogenética Ósea 2/metabolismo , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Gástrula/embriología , Corazón/embriología , MicroARNs/genética , Animales , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 2/genética , Embrión de Pollo , Electroporación , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Factor 8 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 8 de Crecimiento de Fibroblastos/genética , Factor de Transcripción GATA4/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Reacción en Cadena de la Polimerasa , Transducción de Señal/genética , Factores de Transcripción/biosíntesisRESUMEN
Nodal modulator 1 (NOMO1), a highly conserved transmembrane protein, has been identified as a part of a protein complex that participates in the Nodal signaling pathway, a critical determinant of heart and visceral organ formation. We previously found that the NOMO1 gene was substantially downregulated in human ventricular septal defect (VSD) myocardium and, thus, may be an important molecular pathway in human heart development. In this study, we aimed to investigate the effects of NOMO1 gene silencing by RNA interference (RNAi) during early mouse cardiac differentiation using P19 cells as a model system. Our results revealed that the differentiated P19 cell population exhibited downregulated NOMO1 levels and expressed lower levels of Nodal signaling mediators, such as Nodal, Cripto and Smad2, than the negative control. Similarly, cardiomyocyte-specific sarcomeric markers, such as cardiac troponin T, as well as expression of cardiogenesis-related transcriptional factors, such as Nkx2.5, Gata4 and Tbx5 were found to be downregulated in P19 differentiated cardiomyocytes in NOMO1-silenced cells when compared to controls. In conclusion, our results indicate that NOMO1 gene knockdown inhibits the differentiation of P19 cells into cardiomyocytes, which highlights a potential role for NOMO1 in early cardiogenesis.
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Diferenciación Celular/genética , Corazón/embriología , Miocitos Cardíacos/citología , Proteína Nodal/genética , Animales , Línea Celular , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/genética , Factor de Transcripción GATA4/biosíntesis , Factor de Transcripción GATA4/genética , Defectos del Tabique Interventricular/genética , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Miocitos Cardíacos/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Transducción de Señal/genética , Proteína Smad2/biosíntesis , Proteína Smad2/genética , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Troponina T/biosíntesis , Troponina T/genéticaRESUMEN
BACKGROUND: Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells. METHODS AND RESULTS: Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways. CONCLUSIONS: Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.
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Apoptosis , Factor de Transcripción GATA4/genética , Regulación de la Expresión Génica , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Infarto del Miocardio/genética , Animales , Supervivencia Celular , Células Cultivadas , Exosomas , Factor de Transcripción GATA4/biosíntesis , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/terapia , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN/genética , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
RATIONALE: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. OBJECTIVE: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. METHODS AND RESULTS: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. CONCLUSIONS: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.
Asunto(s)
Reprogramación Celular/fisiología , Factor de Transcripción GATA4/biosíntesis , Miocitos Cardíacos/fisiología , Proteínas de Dominio T Box/biosíntesis , Animales , Células Cultivadas , Factor de Transcripción GATA4/genética , Factores de Transcripción MEF2/biosíntesis , Factores de Transcripción MEF2/genética , Ratones , Ratones Transgénicos , Proteínas de Dominio T Box/genéticaRESUMEN
Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increasingly recognized as important regulators of embryo development in mammals. This study investigated the importance of FGF signaling during in vitro development of ovine embryo. The mRNAs of four FGFR subtypes were detected throughout preimplantation development of in vitro fertilized (IVF) embryos, peaked in abundance at the morula stage, and decreased significantly at the blastocyst stage. To gain insight into the role of these mRNAs in embryo development, IVF embryos were cultured in the presence of FGF2 (100 or 500 ng/ml: beginning from days 1 or 4 to 7) or PD173074 (1 µM: beginning from days 1 to 7) as usual treatments for activation or inhibition of FGFRs, respectively. FGF2-supplementation did not affect the percentage of embryos that developed to the blastocyst, blastocyst cell count and the proportion of cells allocated in inner cell mass (ICM) and trophectoderm (TE) compared to control (p > 0.05). Also, increasing the dosage or duration of FGF2 treatment did not significantly alter blastocyst yield or differential cell count (p > 0.05). PD173074-mediated inhibition of FGFRs did not significantly affect blastocyst yield (p > 0.05). Assessment of expression profiles of lineage-associated markers revealed that FGF2 (500 ng/ml) supplementation: (i) significantly increased expression of putative hypoblast marker (GATA4), (ii) significantly decreased expression of putative epiblast (EPI) marker (NANOG) and (iii) did not change TE markers (CDX2 and IFNT) and pluripotency makers (OCT4, SOX2 and REX1). In summary, FGF2-mediated activation of FGFRs may promote a switch in transcriptional profile of ovine ICM from EPI- to hypoblast-associated gene expression.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Pirimidinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/biosíntesis , Animales , Blastocisto/metabolismo , Femenino , Factor de Transcripción GATA4/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Masculino , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , OvinosRESUMEN
OBJECTIVE: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications. DESIGN: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice. RESULTS: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs. CONCLUSIONS: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells.
Asunto(s)
Factor de Transcripción GATA4/genética , Factor de Transcripción GATA6/genética , Regulación Neoplásica de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Gástricas/genética , Animales , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Factor de Transcripción GATA4/biosíntesis , Factor de Transcripción GATA6/biosíntesis , Perfilación de la Expresión Génica/métodos , Silenciador del Gen , Predisposición Genética a la Enfermedad , Xenoinjertos , Humanos , Factores de Transcripción de Tipo Kruppel/biosíntesis , Ratones Desnudos , Trasplante de Neoplasias , Oncogenes/genética , Regiones Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Células Tumorales CultivadasRESUMEN
Granulosa cell tumors (GCTs) carry a risk of recurrence also at an early stage, but reliable prognostic factors are lacking. We assessed clinicopathological prognostic factors and the prognostic roles of the human epidermal growth factor receptors (HER 2-4) and the transcription factor GATA4 in GCTs. We conducted a long-term follow-up study of 80 GCT patients with a mean follow-up time of 16.8 years. A tumor-tissue microarray was immunohistochemically stained for HER2-4 and GATA4. Expression of HER2-4 mRNA was studied by means of real time polymerase chain reaction and HER2 gene amplification was analyzed by means of silver in situ hybridization. The results were correlated to clinical data on recurrences and survival. We found that GCTs have an indolent prognosis, with 5-year disease-specific survival (DSS) being 97.5%. Tumor recurrence was detected in 24% of the patients at a median of 7.0 years (range 2.6-18 years) after diagnosis. Tumor stage was not prognostic of disease-free survival (DFS). Of the molecular prognostic factors, high-level expression of HER2, and GATA4, and high nuclear atypia were prognostic of shorter DFS. In multivariate analyses, high-level coexpression of HER2 and GATA4 independently predicted DFS (hazard ratio [HR] 8.75, 95% CI 2.20-39.48, P = 0.002). High-level expression of GATA4 also predicted shorter DSS (HR 3.96, 95% CI 1.45-12.57, P = 0.006). In multivariate analyses, however, tumor stage (II-III) and nuclear atypia were independent prognostic factors of DSS. In conclusion HER2 and GATA4 are new molecular prognostic markers of GCT recurrence, which could be utilized to optimize the management and follow-up of patients with early-stage GCTs.