RESUMO
The process by which fibroblasts are directly reprogrammed into cardiomyocytes involves two stages; initiation and maturation. Initiation represents the initial expression of factors that induce fibroblasts to transdifferentiate into cardiomyocytes. Following initiation, the cell undergoes a period of maturation before becoming a mature cardiomyocyte. We wanted to understand the role of cardiac development transcription factors in the maturation process. We directly reprogram fibroblasts into cardiomyocytes by a combination of miRNAs (miR combo). The ability of miR combo to induce cardiomyocyte-specific genes in fibroblasts was lost following the knockdown of the cardiac transcription factors Gata4, Mef2C, Tbx5 and Hand2 (GMTH). To further clarify the role of GMTH in miR combo reprogramming we utilized a modified CRISPR-Cas9 approach to activate endogenous GMTH genes. Importantly, both miR combo and the modified CRISPR-Cas9 approach induced comparable levels of GMTH expression. While miR combo was able to reprogram fibroblasts into cardiomyocyte-like cells, the modified CRISPR-Cas9 approach could not. Indeed, we found that cardiomyocyte maturation only occurred with very high levels of GMT expression. Taken together, our data indicates that while endogenous cardiac transcription factors are insufficient to reprogram fibroblasts into mature cardiomyocytes, endogenous cardiac transcription factors are necessary for expression of maturation genes.
Assuntos
Fator de Transcrição GATA4/genética , Proteínas com Domínio T/genética , Animais , Sistemas CRISPR-Cas/genética , Transdiferenciação Celular , Células Cultivadas , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/metabolismo , Edição de Genes , Fatores de Transcrição MEF2/antagonistas & inibidores , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas com Domínio T/antagonistas & inibidores , Proteínas com Domínio T/metabolismoRESUMO
Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.
Assuntos
Fator de Transcrição GATA4/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Isoxazóis/farmacologia , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Fator de Transcrição GATA4/química , Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5/química , Proteína Homeobox Nkx-2.5/metabolismo , Isoxazóis/síntese química , Isoxazóis/química , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A combination of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3ß (GSK3ß) inhibitors, called 2i, is widely used for maintaining the pluripotency of mouse embryonic stem cells (ESCs) in vitro. Without 2i, a few mouse ESCs spontaneously gives rise to primitive endoderm (PrE) cells, whereas 2i completely blocks this PrE cell differentiation. However, the molecular mechanisms underlying the inhibitory action of 2i on PrE cell differentiation remain unclear. Robust PrE cell induction is achieved by enforced expression of the transcription factor Gata4. Here, we analyzed how 2i inhibits the PrE cell differentiation using mouse ESCs carrying an inducible Gata4 expression cassette. We found that 2i effectively inhibited the Gata4-induced PrE cell differentiation and the ERK1/2 inhibitor was responsible for this effect. We further revealed that the transcriptional activation ability of Gata4 was necessary for PrE cell induction and its disruption by the ERK1/2 inhibitor. The phosphorylation of Ser105, Ser266, and Ser411 of the Gata4 protein was not involved in the PrE cell induction. Overexpression of Klf4, an ERK1/2 substrate, inhibited the Gata4-mediated transcriptional activation. Our data indicated that ERK1/2 supported the PrE cell induction via the indirect transcriptional activation of Gata4.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Doxiciclina/farmacologia , Endoderma/citologia , Endoderma/efeitos dos fármacos , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Fator 4 Semelhante a Kruppel , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Células-Tronco Embrionárias Murinas/citologia , Fosforilação , Piridinas/farmacologia , Pirimidinas/farmacologiaRESUMO
GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells.
Assuntos
Movimento Celular , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Mesoderma/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células , Criança , Pré-Escolar , Feminino , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Hepatoblastoma/genética , Humanos , Lactente , Neoplasias Hepáticas/genética , Masculino , Mesoderma/metabolismo , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/genética , Adulto JovemRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by abnormal inflammation, angiogenesis, and cartilage destruction. In RA, neoangiogenesis is an early and crucial event to promote the formation of pannus, causing further inflammatory cell infiltration. The transcription factor GATA4 is a critical regulator of cardiac differentiation-specific gene expression. We find that a higher level of GATA4 exists in synovium of rheumatoid arthritis (RA) patients, but the function of GATA4 in RA remains unclear. In the present study, IL-1ß induces inflammation in fibroblast-like synoviocytes (FLS) MH7A, which is accompanied with the increased expression of GATA4 and VEGF production. Through application of GATA4 loss-of-function assays, we confirm the requirement of GATA4 expression for inflammation induced by IL-1ß in FLS. In addition, we demonstrate for the first time that GATA4 plays key roles in regulating VEGF secretion from RA FLS to promote cellular proliferation, induce cell migration, and angiogenic tube formation of endothelial cells. GATA4 induces the angiogenic factors VEGFA and VEGFC, by directly binding to the promoter and enhancing transcription. The knockdown of GATA4 attenuates the development of collagen-induced arthritis (CIA) and prevents RA-augmented angiogenesis in vivo, which are accompanied with decreased VEGF level. These results reveal a previously unrecognized function for GATA4 as a regulator of RA angiogenesis and we provide experimental data validating the therapeutic target of GATA4 in RA mice.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Fator de Transcrição GATA4/genética , Neovascularização Patológica/genética , Sinoviócitos/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Masculino , Camundongos Endogâmicos DBA , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.
Assuntos
Fator de Transcrição GATA4/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Hipertensão/prevenção & controle , Isoxazóis/farmacologia , Infarto do Miocárdio/prevenção & controle , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Bibliotecas de Moléculas Pequenas/farmacologia , Angiotensina II/toxicidade , Animais , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nkx-2.5/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Fosforilação , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND/AIMS: GATA4, a protein related to osteoblast differentiation and mineralization, whose acetylation is essential for cardiac defects. Here, we aimed to explore the functional impacts of GATA4 acetylation on osteoporosis (OS). METHODS: GATA4 acetylation in hFOB1.19 and 293T cells was detected after exposure of HDAC inhibitors (TSA and SAHA). Co-immunoprecipitation was conducted to determine which HATs and HDACs was involved in the modulation of GATA4 acetylation/deacetylation, and to identify the acetylation site. The transcriptional activity of GATA4 was measured in the presence or absence of cycloheximide. Furthermore, hFOB1.19 cells viability and apoptosis were evaluated after transfection with acetylation-defective mutant of GATA4. RESULTS: As a result, GATA4 acetylation was identified as a pivotal event in hFOB1.19 cells. GATA4 can be acetylated by P300/CBP, and the acetylation site was on lysine residue K313. Besides, the acetylation of GATA4 can be impaired by HDAC1, rather than by HDAC2-5. GATA4 acetylation contributed to the stability and transcription of GATA4. Moreover, GATA4 acetylation activated CCND2 transcription, and mutation of GATA4 on K-313 reduced cell viability and increased a mitochondria-dependent apoptosis in hFOB1.19 cells. CONCLUSION: Our data suggest that GATA4 exists as an acetylated protein in hFOB1.19 cells. Acetylation regulates the stability and transcription of GATA4, and activates CCND2 transcription, which may explain the growth-promoting functions of GATA4 in hFOB1.19 cells.
Assuntos
Fator de Transcrição GATA4/metabolismo , Lisina/química , Acetilação , Apoptose , Linhagem Celular , Proliferação de Células , Ciclina D2/genética , Ciclina D2/metabolismo , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Células HEK293 , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The role of GATA-binding protein 4 (GATA4) in neural crest cells (NCCs) is poorly defined. Here we showed that mouse NCCs lacking GATA4 exhibited developmental defects in craniofacial bone, teeth, and heart. The defects likely occurred due to decreased cell proliferation at the developmental stage. The in vitro results were consistent with the mouse model. The isobaric tags for relative and absolute quantitation assay revealed that BARX1 is one of the differentially expressed proteins after GATA4 knockdown in NCCs. On the basis of the results of dual-luciferase, electro-mobility shift, and chromatin immunoprecipitation assays, Barx1 expression is directly regulated by GATA4 in NCCs. In zebrafish, gata4 knockdown affects the development of NCCs derivatives. However, the phenotype in zebrafish could be partly rescued by co-injection of gata4 morpholino oligomers and barx1 mRNA. This study identified new downstream targets of GATA4 in NCCs and uncovered additional evidence of the complex regulatory functions of GATA4 in NCC development.
Assuntos
Ossos Faciais/crescimento & desenvolvimento , Fator de Transcrição GATA4/metabolismo , Proteínas de Homeodomínio/metabolismo , Crista Neural/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células , Ossos Faciais/diagnóstico por imagem , Ossos Faciais/metabolismo , Feminino , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/citologia , Crista Neural/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Microtomografia por Raio-X , Peixe-Zebra/metabolismoRESUMO
Neurons in the central nervous system (CNS) lose their intrinsic ability and fail to regenerate, but the underlying mechanisms are largely unknown. Polycomb group (PcG) proteins, which include PRC1 and PRC2 complexes function as gene repressors and are involved in many biological processes. Here we report that PRC1 components (polycomb chromobox (CBX) 2, 7, and 8) are novel regulators of axon growth and regeneration. Especially, knockdown of CBX7 in either embryonic cortical neurons or adult dorsal root ganglion (DRG) neurons enhances their axon growth ability. Two important transcription factors GATA4 and SOX11 are functional downstream targets of CBX7 in controlling axon regeneration. Moreover, knockdown of GATA4 or SOX11 in cultured DRG neurons inhibits axon regeneration response from CBX7 downregulation in DRG neurons. These findings suggest that targeting CBX signaling pathway may be a novel approach for promoting the intrinsic regenerative capacity of damaged CNS neurons.
Assuntos
Axônios/fisiologia , Proteínas do Grupo Polycomb/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Gânglios Espinais/citologia , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regeneração , Fatores de Transcrição SOXC/antagonistas & inibidores , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Nervo Isquiático/lesõesRESUMO
RATIONALE: Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3+ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction. OBJECTIVE: The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair. METHODS AND RESULTS: In vivo dynamics of Nano-lantern-labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]-specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]-introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)-S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4-gene-silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. CONCLUSIONS: Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.
Assuntos
Lisofosfolipídeos/fisiologia , Infarto do Miocárdio/cirurgia , Células-Tronco Pluripotentes/transplante , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/análogos & derivados , Aloenxertos , Animais , Autoenxertos , Diferenciação Celular , Movimento Celular/fisiologia , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/fisiologia , Sobrevivência de Enxerto , Proteínas de Fluorescência Verde/análise , Xenoenxertos , Humanos , Luciferases/análise , Proteínas Luminescentes/análise , Masculino , Infarto do Miocárdio/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Coelhos , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Proteínas Recombinantes de Fusão/análise , Especificidade da Espécie , Esfingosina/fisiologia , Receptores de Esfingosina-1-FosfatoRESUMO
Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC50 = 3 µM), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.
Assuntos
Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Isoxazóis/química , Isoxazóis/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Linhagem Celular , Fator de Transcrição GATA4/agonistas , Fator de Transcrição GATA4/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/agonistas , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Humanos , Camundongos , Modelos Moleculares , Mapas de Interação de Proteínas/efeitos dos fármacosRESUMO
BACKGROUND Myocardial infarction affects the health of many people. Post-infarction myocardial fibrosis has attracted much attention, but details of the mechanism remain elusive. In this study, the role of microRNA-208b (miR-208b) in modulating post-infarction myocardial fibrosis and the related mechanism were investigated. MATERIAL AND METHODS A rat model of myocardial infarction induced by ligating the left anterior descending artery was used to analyze the expression and roles of miR-208b by overexpression with the lentivirus vector of pre-miR-208b. Myocardial function was assessed and the expression of fibrosis-related factors type I collagen (COL1) and ACTA2 (alias αSMA) was detected. Myocardial fibroblasts isolated from newborn rats were transfected with luciferase reporter vectors containing wild-type or mutant Gata4 3' UTR to verify the relationship between Gata4 and miR-208b. We then transfected the specific small interference RNA of Gata4 to detect changes in COL1 and ACTA2. RESULTS miR-208b was down-regulated in hearts of model rats (P<0.01). Overexpressing miR-208b improved myocardial functions, such as reducing the infarction area (P<0.05) and promoting LVEF and LVFS (P<0.01), and inhibited COL1 and ACTA2 (P<0.01). Luciferase reporter assay proved Gata4 to be the direct target of miR-208b, with the target sequence in the 3'UTR. Inhibiting GATA4 resulted in the down-regulation of COL1 and ACTA2, suggesting that the role of miR-208b was achieved via regulating GATA4. CONCLUSIONS This study demonstrates the protective function of miR-208b via GATA4 in post-infarction myocardial fibrosis, providing a potential therapeutic target for treating myocardial fibrosis.
Assuntos
Fibrose Endomiocárdica/genética , Fator de Transcrição GATA4/antagonistas & inibidores , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Regiões 3' não Traduzidas , Actinas/biossíntese , Actinas/genética , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Modelos Animais de Doenças , Regulação para Baixo , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , MicroRNAs/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
The transforming growth factor (TGF-ß)-inducible early gene (TIEG1), a member of the Sp1/Krüppel-like family of transcription factors, plays an important role in regulating cell growth, differentiation, and apoptosis in many human diseases, including breast cancer, osteoporosis, and atherosclerosis. However, little is known about the role of TIEG1 in the heart. In this study, we investigated the role of TIEG1 in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and its underlying mechanism. Our results showed that TIEG1 expression was downregulated in Ang II-induced hypertrophic cardiomyocytes. Gene silencing of TIEG1 by RNA interference upregulated cellular surface area and ANF and BNP messenger RNA levels, whereas TIEG1 overexpression inhibited the expression of those genes. Mechanistically, TIEG1 could inhibit the expression and transcriptional activity of transcription factor GATA4 in cardiomyocytes, which was recognized as an important factor mediating cardiac gene transcription. In summary, our data disclose a novel role of TIEG1 as an inhibitor in Ang II-induced hypertrophic cardiomyocytes through GATA4 signal pathway.
Assuntos
Angiotensina II/toxicidade , Proteínas de Ligação a DNA/biossíntese , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/biossíntese , Animais , Animais Recém-Nascidos , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA4/metabolismo , Osteoblastos/citologia , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação para Baixo , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Pressure overloadinduced myocardial hypertrophy is associated with a poor prognosis in humans and contributes to the development of cardiac arrhythmias, diastolic dysfunction and ultimate congestive heart failure. 3Hydroxy3methylglutarylCoA (HMGCoA) reductase inhibitors, also known as statins, have been previously shown to induce regression of myocardial hypertrophy in aortic banding models. However, there is limited knowledge regarding the underlying molecular mechanisms. Therefore, we hypothesized that the myocardial hypertrophyrelated signaling pathways protein kinase B (Akt), extracellular signalregulated kinases 1 or 2 (ERK1/2) and GATA binding protein 4 (GATA4) activation pathways constitute targets of rosuvastatin (RSV). Therefore, the abovementioned activation pathways were hypothesized to be involved in the regression of pressure overloadinduced myocardial hypertrophy treated by RSV. Twentyeight Wistar rats were randomly allocated into 4 groups: the sham operationvehicle (SHV), abdominal aortic constrictionvehicle (AACV), abdominal aortic constrictionRSV 10 mg/kg/day (AACLO) and the abdominal aortic constrictionRSV 20 mg/kg/day (AACHI) group. Following the establishment of the abdominal aorta constriction model, we investigated the effect of RSV, a new hydrophilic statin, on abdominal aortic constrictioninduced myocardial hypertrophy as well as the underlying intercellular signaling pathways after 5 days and 4 weeks of drug intervention. Moreover, echocardiographic features and the left ventricular weight to final body weight ratio (LVW/BW) were determined. Crosssectional areas (CSAs) of cardiomyocytes were assessed by hematoxylin and eosin (H&E) staining. Atrial natriuretic factor (ANF), ßmyosin heavy chain (ßMHC) and peroxisome proliferatoractivated receptor α (PPARα) messenger RNA (mRNA) expression was assessed using RTPCR. The phosphorylation of Akt, ERK1/2 and GATA4 were also examined using western blot analysis. Our results showed that RSV significantly attenuates pressure overloadinduced myocardial hypertrophy by preventing myocardial hypertrophyrelated activation of Akt, ERK1/2 and GATA4 signaling pathways.
Assuntos
Cardiomegalia/metabolismo , Fluorbenzenos/farmacologia , Fator de Transcrição GATA4/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Eletrocardiografia , Fator de Transcrição GATA4/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Masculino , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Rosuvastatina CálcicaRESUMO
Histone acetylase (HAT) p300 plays an important role in the regulation of cardiac gene expression. During cardiac development, bone morphogenetic protein (BMP)-2 induces the expression of cardiac transcription factors. However, the underlying molecular mechanism(s) is not clear. In the present study, we tested the hypothesis that p300-mediated histone acetylation was essential for the regulation of cardiac transcription factors by BMP2. Cultured rat H9c2 embryonic cardiac myocytes (H9c2 cells) were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without curcumin, a specific p300-HAT inhibitor. Quantitative real-time RT-PCR analysis showed that curcumin substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and MEF2C, but not Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that curcumin inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and MEF2C, but not of Tbx5. In addition, curcumin selectively suppressed AdBMP2-induced expression of HAT p300, but not HAT GCN5 in H9c2 cells. The data indicated that inhibition of histone H3 acetylation with curcumin diminished BMP2-induced expression of GATA4 and MEF2C, suggesting that p300-mediated histone acetylation was essential for the regulation of GATA4 and MEF2C by BMP2 in H9c2 cells.
Assuntos
Proteína Morfogenética Óssea 2/antagonistas & inibidores , Proteína Morfogenética Óssea 2/biossíntese , Proteína p300 Associada a E1A/fisiologia , Fator de Transcrição GATA4/fisiologia , Histonas/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Animais , Células Cultivadas , Curcumina/farmacologia , Fator de Transcrição GATA4/antagonistas & inibidores , Histonas/metabolismo , Humanos , Fatores de Transcrição MEF2/antagonistas & inibidores , Fatores de Transcrição MEF2/fisiologia , Ratos , Transfecção/métodosRESUMO
Curcumin is a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric, Curcuma longa. It is commonly used as a dietary spice and coloring agent in cooking, and is used anecdotally as an herb in traditional Indian and Chinese medicine. It has been reported that curcumin has the potential to protect against cardiac inflammation through suppression of GATA-4 and nuclear factor-κB (NF-κB); however, no study to date has addressed the effect of curcumin on experimental autoimmune myocarditis (EAM) in rats. In this study, 8-week-old male Lewis rats were immunized with cardiac myosin to induce EAM. They were then divided randomly into a treatment or vehicle group and orally administrated curcumin (50 mg/kg/d) or 1% gum arabic, respectively, for 3 weeks after myosin injection. We performed hemodynamic, echocardiographic, hematoxylin and eosin staining, mast cell staining and Western blotting studies to evaluate the protective effect of curcumin in the acute phase of EAM. Cardiac functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by curcumin treatment. Furthermore, curcumin reduced the heart weight-to-body weight ratio, area of inflammatory lesions and the myocardial protein level of NF-κB, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and GATA-4. Our results indicate that curcumin has the potential to protect against cardiac inflammation through suppression of IL-1ß, TNF-α, GATA-4 and NF-κB expresses, and may provide a novel therapeutic strategy for autoimmune myocarditis.
Assuntos
Doenças Autoimunes/prevenção & controle , Curcumina/farmacologia , Miocardite/prevenção & controle , Animais , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/metabolismo , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Members of the GATA family of transcription factors are zinc finger proteins that were shown to play evolutionary conserved roles in cell differentiation and proliferation in different organisms. We hypothesized that by finding new molecules that inhibit their function to be crucial in future therapeutical interventions for various diseases. By virtual high throughput screening using a version of glide (Schrodinger®) program with both crystal and NMR structure of GATA C-terminal zinc finger, we identified new small molecular weight chemicals with lead-like properties. We used in vitro cell-based assays to show that these molecules selectively and efficiently inhibit GATA4 activity by inhibiting its interaction with the DNA. In addition we showed that these molecules can block the activation of downstream target genes by GATA4. Moreover these compounds can moderately enhanced a mouse model of myoblast differentiation into myotubes. This might be partially due to decreased GATA4/DNA interaction as shown by gel retardation assays. Further investigation is needed to reach selectivity and efficacy. Our study however do show that in silico screening combined with in vitro studies are efficient tools to unravel new molecules that interact with zinc finger proteins such as GATA4.
Assuntos
Desenho de Fármacos , Fator de Transcrição GATA4/antagonistas & inibidores , Ribavirina/síntese química , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Química Farmacêutica , Técnicas de Química Combinatória , Simulação por Computador , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Ratos , Ribavirina/química , Ribavirina/farmacologia , Alinhamento de Sequência , Relação Estrutura-AtividadeAssuntos
Fatores de Transcrição GATA/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Xenopus/embriologia , Animais , Fatores de Transcrição GATA/genética , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/antagonistas & inibidores , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Cardiogenesis is inhibited by canonical Wnt/beta-catenin signalling and stimulated by non-canonical Wnt11/JNK signalling, but how these two signalling pathways crosstalk is currently unknown. Here, we show that Wnt/beta-catenin signalling restricts cardiogenesis via inhibition of GATA gene expression, as experimentally reinstating GATA function overrides beta-catenin-mediated inhibition and restores cardiogenesis. Furthermore, we show that GATA transcription factors in turn directly regulate Wnt11 gene expression, and that Wnt11 is required to a significant degree for mediating the cardiogenesis-promoting function of GATA transcription factors. These results demonstrate that GATA factors occupy a central position between canonical and non-canonical Wnt signalling in regulating heart muscle formation.