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1.
Int J Mol Sci ; 20(17)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480672

RESUMO

Cardiovascular diseases have a high prevalence worldwide and constitute the leading causes of mortality. Recently, malfunctioning of ß-catenin signaling has been addressed in hypertensive heart condition. Ang-II is an important mediator of cardiovascular remodeling processes which not only regulates blood pressure but also leads to pathological cardiac changes. However, the contribution of Ang-II/ß-catenin axis in hypertrophied hearts is ill-defined. Employing in vitro H9c2 cells and in vivo spontaneously hypertensive rats (SHR) cardiac tissue samples, western blot analysis, luciferase assays, nuclear-cytosolic protein extracts, and immunoprecipitation assays, we found that under hypertensive condition ß-catenin gets abnormally induced that co-activated LEF1 and lead to cardiac hypertrophy changes by up-regulating the IGF-IIR signaling pathway. We identified putative LEF1 consensus binding site on IGF-IIR promoter that could be regulated by ß-catenin/LEF1 which in turn modulate the expression of cardiac hypertrophy agents. This study suggested that suppression of ß-catenin expression under hypertensive condition could be exploited as a clinical strategy for cardiac pathological remodeling processes.


Assuntos
Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Angiotensina II , Animais , Biomarcadores/metabolismo , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Fator de Transcrição GATA4/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Quinase C-alfa/metabolismo , Ratos Endogâmicos SHR
2.
Genet Test Mol Biomarkers ; 23(9): 601-609, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31386585

RESUMO

Background: Tetralogy of Fallot (TOF) accounts for ∼10% of congenital heart disease cases. The blood vessel epicardial substance (BVES) gene has been reported to play a role in the function of adult hearts. However, whether allelic variants in BVES contribute to the risk of TOF and its possible mechanism remains unknown. Methods: The open reading frame of the BVES gene was sequenced using samples from 146 TOF patients and 100 unrelated healthy controls. qRT-PCR and western blot assays were used to confirm the expression of mutated BVES variants in the TOF samples. The online software Polyphen2 and SIFT were used to predict the deleterious effects of the observed allelic variants. The effects of these allelic variants on the transcriptional activities of genes were examined using dual-fluorescence reporter assays. Results: We genotyped four single nucleotide polymorphisms (SNPs) in the BVES gene from each of the 146 TOF patients. Among them, the minor allelic frequencies of c.385C>T (p.R129W) were 0.035% in TOF, but ∼0.025% in 100 controls and the Chinese Millionome Database. This allelic variant was predicted to be a potentially harmful alteration by the Polyphen2 and SIFT softwares. qRT-PCR and western blot analyses indicated that the expression of BVES in the six right ventricular outflow tract samples with the c.385C>T allelic variant was significantly downregulated. A dual-fluorescence reporter system showed that the c.385C>T allelic variant significantly decreased the transcriptional activity of the BVES gene and also decreased transcription from the GATA4 and NKX2.5 promoters. Conclusions: c.385C>T (p.R129W) is a functional SNP of the BVES gene that reduces the transcriptional activity of BVES in vitro and in vivo in TOF tissues. This subsequently affects the transcriptional activities of GATA4 and NKX2.5 related to TOF. These findings suggest that c.385C>T may be associated with the risk of TOF in the Han Chinese population.


Assuntos
Moléculas de Adesão Celular/genética , Proteínas Musculares/genética , Tetralogia de Fallot/genética , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Moléculas de Adesão Celular/metabolismo , China/etnologia , Fator de Transcrição GATA4/metabolismo , Genótipo , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Proteínas Musculares/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Análise de Sequência de DNA/métodos
3.
Mol Med Rep ; 20(3): 2325-2331, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322241

RESUMO

Congenital heart disease (CHD) is the most common type of developmental defect, with high rates of morbidity in infants. The transcription factor GATA­binding factor 4 (GATA4) has been reported to serve a critical role in embryogenesis and cardiac development. Our previous study reported a heterozygous GATA4 c.1306C>T (p.H436Y) mutation in four Chinese infants with congenital heart defects. In the present study, functional analysis of the GATA4 H436Y mutation was performed in vitro. The functional effect of GATA4 mutation was compared with GATA4 wild­type using a dual­luciferase reporter assay system and immunofluorescence. Electrophoretic mobility­shift assays were performed to explore the binding affinity of the mutated GATA4 to the heart and neural crest derivatives expressed 2 (HAND2) gene. The results revealed that the mutation had no effect on normal nuclear localization, but resulted in diminished GATA­binding affinity to HAND2 and significantly decreased gene transcriptional activation. These results indicated that this GATA4 mutation may not influence cellular localization in transfected cells, but may affect the affinity of the GATA­binding site on HAND2 and decrease transcriptional activity, thus suggesting that the GATA4 mutation may be associated with the pathogenesis of CHD.


Assuntos
Fator de Transcrição GATA4/genética , Cardiopatias Congênitas/genética , Grupo com Ancestrais do Continente Asiático/genética , Feminino , Fator de Transcrição GATA4/análise , Fator de Transcrição GATA4/metabolismo , Células HeLa , Cardiopatias Congênitas/metabolismo , Heterozigoto , Humanos , Lactente , Masculino , Mutação Puntual , Ativação Transcricional
4.
Nucleic Acids Res ; 47(17): 9069-9086, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350899

RESUMO

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.


Assuntos
Cromatina/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Endoderma/citologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 3-beta Nuclear de Hepatócito/genética , Código das Histonas , Histonas/metabolismo , Camundongos , Camundongos Knockout , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Transdução de Sinais
5.
Oxid Med Cell Longev ; 2019: 5432792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178965

RESUMO

LPS is a major endotoxin produced by gram-negative bacteria, and exposure to it commonly occurs in animal husbandry. Previous studies have shown that LPS infection disturbs steroidogenesis, including progesterone production, and subsequently decreases animal reproductive performance. However, little information about the underlying mechanisms is available thus far. In the present study, an in vitro-luteinized porcine granulosa cell model was used to study the underlying molecular mechanisms of LPS treatment. We found that LPS significantly inhibits progesterone production and downregulates the expressions of progesterone synthesis-associated genes (StAR, CYP11A1, and 3ß-HSD). Furthermore, the levels of ROS were significantly increased in an LPS dose-dependent manner. Moreover, transcriptional factors GATA4 and GATA6, but not NR5A1, were significantly downregulated. Elimination of LPS-stimulated ROS by melatonin or vitamin C could restore the expressions of GATA4, GATA6, and StAR. In parallel, StAR expression was also inhibited by the knockdown of GATA4 and GATA6. Based on these data, we conclude that LPS impairs StAR expression via the ROS-induced downregulation of GATA4 and GATA6. Collectively, these findings provide new insights into the understanding of reproductive losses in animals suffering from bacterial infection and LPS exposure.


Assuntos
Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/metabolismo , Células da Granulosa/metabolismo , Lipopolissacarídeos/metabolismo , Células Lúteas/metabolismo , Animais , Regulação para Baixo , Feminino , Humanos , Espécies Reativas de Oxigênio , Suínos , Transfecção
6.
PLoS One ; 14(5): e0214873, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086358

RESUMO

BACKGROUNDS: Reducing toxicants transplacental rates could contribute to the prevention of congenital heart defects (CHDs). Placental P-glycoprotein (P-gp) plays a vital role in fetal toxicants exposure and subsequently affects the risk of toxicants-induced birth defects. However, data on the role of placental P-gp in decreasing toxicants-induced cardiac anomalies is extremely limited. This study aimed to explore the protective role of placental P-gp in reducing the risk of Di-(2-ethylhexyl)-phthalate (DEHP) induced cardiac anomalies in mice. METHODS: The C57BL mice were randomly divided into four groups: the vehicle group (corn oil, n = 10), 500mg/Kg DEHP group (n = 15), 3mg/Kg verapamil group (n = 10) and 500mg/Kg DEHP & 3mg/Kg verapamil group (n = 20). Pregnant dams in different group received respective intervention by gavage once daily from E6.5-14.5. Maternal weights were monitored every day and samples were collected at E15.5. HE staining was used to examine fetal cardiac malformations. Real-time quantitative PCR (RT-qPCR) and Western-Blot were applied to detect Nkx2.5/Gata4/Tbx5/Mef2c/Chf1 mRNA and protein expression, respectively. The mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) was also determined using RT-qPCR. RESULTS: Co-administration of verapamil and DEHP significantly elevated fetal cardiac malformation rates, in comparison with the DEHP group, the verapamil group and the vehicle group. Different phenotypes of cardiac anomalies, including septal defects and ventricular myocardium noncompaction, were noted both in the DEHP group and the DEHP & verapamil group. The ventricular myocardium noncompaction appeared to be more severe in the DEHP & verapamil group. Fetal cardiac PPARγ mRNA expression was notably increased and Gata4/Mef2c/Chf1 expression was markedly decreased in the DEHP & verapamil group. CONCLUSION: Placental P-gp inhibition enhances susceptibility to DEHP induced cardiac malformations in mice.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Dietilexilftalato/toxicidade , Coração Fetal/efeitos dos fármacos , Cardiopatias Congênitas/patologia , Placenta/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Feminino , Coração Fetal/metabolismo , Coração Fetal/patologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Exposição Materna , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Gravidez , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Verapamil/farmacologia
7.
PLoS Genet ; 15(5): e1007711, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120883

RESUMO

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.


Assuntos
Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/genética , Proteínas Hedgehog/genética , Receptor Smoothened/genética , Obstrução do Fluxo Ventricular Externo/genética , Septo Interventricular/metabolismo , Animais , Movimento Celular , Proliferação de Células , Embrião de Mamíferos , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica , Haploinsuficiência , Proteínas Hedgehog/metabolismo , Heterozigoto , Humanos , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Receptor Smoothened/metabolismo , Tronco Arterial/anormalidades , Tronco Arterial/metabolismo , Obstrução do Fluxo Ventricular Externo/metabolismo , Obstrução do Fluxo Ventricular Externo/patologia , Septo Interventricular/patologia
8.
Dis Model Mech ; 12(6)2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31138536

RESUMO

Congenital heart defects affect ∼2% of live births and often involve malformations of the semilunar (aortic and pulmonic) valves. We previously reported a highly penetrant GATA4 p.Gly296Ser mutation in familial, congenital atrial septal defects and pulmonic valve stenosis and showed that mice harboring the orthologous G295S disease-causing mutation display not only atrial septal defects, but also semilunar valve stenosis. Here, we aimed to characterize the role of Gata4 in semilunar valve development and stenosis using the Gata4G295Ski/wt mouse model. GATA4 is highly expressed in developing valve endothelial and interstitial cells. Echocardiographic examination of Gata4G295Ski/wt mice at 2 months and 1 year of age identified functional semilunar valve stenosis predominantly affecting the aortic valve with distal leaflet thickening and severe extracellular matrix (ECM) disorganization. Examination of the aortic valve at earlier postnatal timepoints demonstrated similar ECM abnormalities consistent with congenital disease. Analysis at embryonic timepoints showed a reduction in aortic valve cushion volume at embryonic day (E)13.5, predominantly affecting the non-coronary cusp (NCC). Although total cusp volume recovered by E15.5, the NCC cusp remained statistically smaller. As endothelial to mesenchymal transition (EMT)-derived cells contribute significantly to the NCC, we performed proximal outflow tract cushion explant assays and found EMT deficits in Gata4G295Ski/wt embryos along with deficits in cell proliferation. RNA-seq analysis of E15.5 outflow tracts of mutant embryos suggested a disease state and identified changes in genes involved in ECM and cell migration as well as dysregulation of Wnt signaling. By utilizing a mouse model harboring a human disease-causing mutation, we demonstrate a novel role for GATA4 in congenital semilunar valve stenosis.This article has an associated First Person interview with the joint first authors of the paper.


Assuntos
Estenose da Valva Aórtica/genética , Valva Aórtica/patologia , Fator de Transcrição GATA4/genética , Cardiopatias Congênitas/genética , Mutação/genética , Animais , Animais Recém-Nascidos , Valva Aórtica/embriologia , Valva Aórtica/metabolismo , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Transição Epitelial-Mesenquimal , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/patologia , Heterozigoto , Camundongos Endogâmicos C57BL , Via de Sinalização Wnt
9.
Nat Commun ; 10(1): 1665, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971692

RESUMO

Lung cancer is the leading cause of cancer-related deaths worldwide. Tumor suppressor genes remain to be systemically identified for lung cancer. Through the genome-wide screening of tumor-suppressive transcription factors, we demonstrate here that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vivo. Ectopic GATA4 expression results in lung cancer cell senescence. Mechanistically, GATA4 upregulates multiple miRNAs targeting TGFB2 mRNA and causes ensuing WNT7B downregulation and eventually triggers cell senescence. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGF-ß2 level and is significantly associated with poor prognosis. TGFBR1 inhibitors show synergy with existing therapeutics in treating GATA4-deficient lung cancers in genetically engineered mouse model as well as patient-derived xenograft (PDX) mouse models. Collectively, our work demonstrates that GATA4 functions as a tumor suppressor in lung cancer and targeting the TGF-ß signaling provides a potential way for the treatment of GATA4-deficient lung cancer.


Assuntos
Fator de Transcrição GATA4/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Proteínas Wnt/metabolismo , Células A549 , Animais , Senescência Celular/genética , Regulação para Baixo , Feminino , Fator de Transcrição GATA4/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Biol Chem ; 294(23): 9134-9146, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31023824

RESUMO

Reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a potentially promising strategy for regenerating a damaged heart. However, low fibroblast-cardiomyocyte conversion rates remain a major challenge in this reprogramming. To this end, here we conducted a chemical screen and identified four agents, insulin-like growth factor-1, Mll1 inhibitor MM589, transforming growth factor-ß inhibitor A83-01, and Bmi1 inhibitor PTC-209, termed IMAP, which coordinately enhanced reprogramming efficiency. Using α-muscle heavy chain-GFP-tagged mouse embryo fibroblasts as a starting cell type, we observed that the IMAP treatment increases iCM formation 6-fold. IMAP stimulated higher cardiac troponin T and α-actinin expression and increased sarcomere formation, coinciding with up-regulated expression of many cardiac genes and down-regulated fibroblast gene expression. Furthermore, IMAP promoted higher spontaneous beating and calcium transient activities of iCMs derived from neonatal cardiac fibroblasts. Intriguingly, we also observed that the IMAP treatment repressed many genes involved in immune responses, particularly those in specific C-C chemokine signaling pathways. We therefore investigated the roles of C-C motif chemokine ligand 3 (CCL3), CCL6, and CCL17 in cardiac reprogramming and observed that they inhibited iCM formation, whereas inhibitors of C-C motif chemokine receptor 1 (CCR1), CCR4, and CCR5 had the opposite effect. These results indicated that the IMAP treatment directly suppresses specific C-C chemokine signaling pathways and thereby enhances cardiac reprogramming. In conclusion, a combination of four chemicals, named here IMAP, suppresses specific C-C chemokine signaling pathways and facilitates Mef2c/Gata4/Tbx5 (MGT)-induced cardiac reprogramming, providing a potential means for iCM formation in clinical applications.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Quimiocina CCL3/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Actinina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA4/metabolismo , Fatores de Transcrição MEF2/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Receptores CCR1/metabolismo , Proteínas com Domínio T/metabolismo , Troponina T/metabolismo
11.
Appl Biochem Biotechnol ; 189(2): 396-410, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31025171

RESUMO

Although embryonic stem (ES) cells (ESCs) may be a promising donor source for the repair of infarcted or ischemic heart tissues, their successful application in regenerative medicine has been hampered by difficulties in enriching, identifying, and selecting cardiomyocytes from the differentiating cells. We established transgenic human ES cell lines by transcriptional control of the α-cardiac myosin heavy chain (α-MHC) promoter driving green fluorescent protein (GFP) expression. Differentiated GFP-expressing cells display the characteristics of cardiomyocytes (CMs). Apela, a recently identified short peptide, up-regulated the expression of the cardiac-restricted transcription factors Tbx5 and GATA4 as well as differentiated the cardiomyocyte markers α-MHC and ß-MHC. Flow cytometric analysis showed that apela increased the percentage of GFP-expressing cells in the beating foci of the embryoid bodies. The percentage of cardiac troponin T (TNT)-positive cells and the protein expression of TNT were increased in the ES cell-derived CMs with apela treatment. Functionally, the contractile frequency of the ES-derived CMs responded appropriately to the vasoactive drugs isoprenaline and carbachol. Our work presented a protocol for specially labelling and enriching CMs by combining transgenic human ES cell lines and exogenous growth factor treatment.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Hormônios Peptídicos/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Hormônios Peptídicos/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Troponina T/metabolismo , Regulação para Cima , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
12.
Biochem Biophys Res Commun ; 512(2): 399-404, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30902394

RESUMO

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 , 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/farmacologia
13.
Int J Mol Med ; 43(4): 1839-1846, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720060

RESUMO

Congenital bicuspid aortic valve (BAV) represents the most common type of cardiac birth defect affecting 0.4­2% of the general population, and accounts for a markedly increased incidence of life­threatening complications, including valvulopathy and aortopathy. Accumulating evidence has demonstrated the genetic basis of BAV. However, the genetic basis for BAV in the majority of cases remains to be elucidated. In the present study, the coding regions and splicing donors/acceptors of the nuclear receptor subfamily 2 group F member 2 (NR2F2) gene, which encodes a transcription factor essential for proper cardiovascular development, were sequenced in 176 unrelated cases of congenital BAV. The available family members of the proband carrying an identified NR2F2 mutation and 280 unrelated, sex­ and ethnicity­matched healthy individuals as controls were additionally genotyped for NR2F2. The functional effect of the mutation was characterized using a dual­luciferase reporter assay system. As a result, a novel heterozygous NR2F2 mutation, NM_021005.3: c.288C>A; p.(Cys96*), was identified in a family with BAV, which was transmitted in an autosomal dominant mode with complete penetrance. The nonsense mutation was absent from the 560 control chromosomes. Functional analysis identified that the mutant NR2F2 protein had no transcriptional activity. Furthermore, the mutation disrupted the synergistic transcriptional activation between NR2F2 and transcription factor GATA­4, another transcription factor that is associated with BAV. These findings suggested NR2F2 as a novel susceptibility gene of human BAV, which reveals a novel molecular pathogenesis underpinning BAV.


Assuntos
Valva Aórtica/anormalidades , Fator II de Transcrição COUP/genética , Cardiopatias Congênitas/genética , Mutação com Perda de Função/genética , Valva Aórtica/patologia , Sequência de Bases , Linhagem Celular , Feminino , Fator de Transcrição GATA4/metabolismo , Doenças das Valvas Cardíacas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/metabolismo , Fenótipo , Ativação Transcricional/genética
14.
Circ Res ; 124(8): 1184-1197, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30744497

RESUMO

RATIONALE: Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. OBJECTIVE: We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. METHODS AND RESULTS: Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. CONCLUSIONS: Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.


Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Genes do Retinoblastoma/fisiologia , Genes p16/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Linhagem Celular , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Inativação Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteína Meis1/genética , Proteína Meis1/metabolismo , Células-Tronco Pluripotentes/transplante , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Suínos , Regulação para Cima
15.
Endocrinology ; 160(4): 817-826, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30759208

RESUMO

GATA4 is an essential transcriptional regulator required for gonadal development, differentiation, and function. In the developing testis, proposed GATA4-regulated genes include steroidogenic factor 1 (Nr5a1), SRY-related HMG box 9 (Sox9), and anti-Müllerian hormone (Amh). Although some of these genes have been validated as genuine GATA4 targets, it remains unclear whether GATA4 is a direct regulator of endogenous Amh transcription. We used a CRISPR/Cas9-based approach to specifically inactivate or delete the sole GATA-binding motif of the proximal mouse Amh promoter. AMH mRNA and protein levels were assessed at developmental time points corresponding to elevated AMH levels: fetal and neonate testes in males and adult ovaries in females. In males, loss of GATA binding to the Amh promoter significantly reduced Amh expression. Although the loss of GATA binding did not block the initiation of Amh transcription, AMH mRNA and protein levels failed to upregulate in the developing fetal and neonate testis. Interestingly, adult male mice presented no anatomical anomalies and had no evidence of retained Müllerian duct structures, suggesting that AMH levels, although markedly reduced, were sufficient to masculinize the male embryo. In contrast to males, GATA binding to the Amh promoter was dispensable for Amh expression in the adult ovary. These results provide conclusive evidence that in males, GATA4 is a positive modulator of Amh expression that works in concert with other key transcription factors to ensure that the Amh gene is sufficiently expressed in a correct spatiotemporal manner during fetal and prepubertal testis development.


Assuntos
Hormônio Antimülleriano/genética , Fator de Transcrição GATA4/genética , Ovário/metabolismo , Diferenciação Sexual/genética , Testículo/metabolismo , Animais , Hormônio Antimülleriano/metabolismo , Feminino , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Regiões Promotoras Genéticas
16.
Cardiovasc Drugs Ther ; 33(2): 231-237, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30809744

RESUMO

BACKGROUND: Endothelial cell (EC) dysfunction (enhanced inflammation, proliferation and permeability) is the initial trigger for atherosclerosis. Atherosclerosis shows preferential development near branches and bends exposed to disturbed blood flow. By contrast, sites that are exposed to non-disturbed blood flow are atheroprotected. Disturbed flow promotes atherosclerosis by promoting EC dysfunction. Blood flow controls EC function through transcriptional and post-transcriptional mechanisms that are incompletely understood. METHODS AND RESULTS: We identified the developmental transcription factors Twist1 and GATA4 as being enriched in EC at disturbed flow, atheroprone regions of the porcine aorta in a microarray study. Further work using the porcine and murine aortae demonstrated that Twist1 and GATA4 expression was enhanced at the atheroprone, disturbed flow sites in vivo. Using controlled in vitro flow systems, the expression of Twist1 and GATA4 was enhanced under disturbed compared to non-disturbed flow in cultured cells. Disturbed flow promoted Twist1 expression through a GATA4-mediated transcriptional mechanism as revealed by a series of in vivo and in vitro studies. GATA4-Twist1 signalling promoted EC proliferation, inflammation, permeability and endothelial-to-mesenchymal transition (EndoMT) under disturbed flow, leading to atherosclerosis development, as shown in a combination of in vitro and in vivo studies using GATA4 and Twist1-specific siRNA and EC-specific GATA4 and Twist1 Knock out (KO) mice. CONCLUSIONS: We revealed that GATA4-Twist1-Snail signalling triggers EC dysfunction and atherosclerosis; this work could lead to the development of novel anti-atherosclerosis therapeutics.


Assuntos
Artérias/metabolismo , Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Fator de Transcrição GATA4/metabolismo , Mecanotransdução Celular , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Artérias/patologia , Artérias/fisiopatologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Transição Epitelial-Mesenquimal , Humanos , Placa Aterosclerótica , Fluxo Sanguíneo Regional
17.
Mol Med Rep ; 19(4): 2861-2868, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720078

RESUMO

Coronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease; however, the genetic causes remain largely unknown. Recent epidemiological investigations indicated that the incidence of CAD in patients with congenital heart diseases is markedly higher than that observed in healthy controls. It was therefore hypothesized that the dysregulated expression of cardiac developmental genes may be involved in CAD development. GATA binding protein 4 (GATA4) serves essential roles in heart development and coronary vessel formation. In the present study, the GATA4 gene promoter was analyzed in patients with AMI (n=395) and in ethnically­matched healthy controls (n=397). A total of 14 DNA variants were identified, including two single­nucleotide polymorphisms. Three novel heterozygous DNA variants (g.31806C>T, g.31900G>C and g.32241C>T) were reported in three patients with AMI. These DNA variants significantly increased the activity of the GATA4 gene promoter. The electrophoretic mobility shift assay revealed that the DNA variant g.32241C>T influenced the binding ability of transcription factors. Taken together, the DNA variants may alter GATA4 gene promoter activity and affect GATA4 levels, thus contributing to AMI development.


Assuntos
Fator de Transcrição GATA4/genética , Predisposição Genética para Doença , Variação Genética , Infarto do Miocárdio/genética , Regiões Promotoras Genéticas , Adulto , Idoso , Sítios de Ligação , Feminino , Fator de Transcrição GATA4/metabolismo , Expressão Gênica , Genes Reporter , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Análise de Sequência de DNA , Ativação Transcricional
18.
Nat Commun ; 10(1): 674, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787297

RESUMO

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1ß signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/efeitos dos fármacos , Diclofenaco/farmacologia , Dinoprostona , Fibroblastos , Fator de Transcrição GATA4/metabolismo , Humanos , Inflamação , Interleucina-1beta , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas com Domínio T/metabolismo
19.
Life Sci ; 221: 319-326, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30802510

RESUMO

AIM: Myocardial infarction (MI), an important cause of morbidity and mortality, can be followed by left ventricular dysfunction and cardiomyocyte loss. Cardiac repair mechanisms may subsequently improve left ventricular function. Exercise training has been suggested to have cardioprotective effects against MI damage, but detailed knowledge is lacking on the effects of different types and intensities of exercise training on molecular targets of cardiomyocyte regeneration. MAIN METHODS: MI was induced in male Wistar rats by ligating the left anterior descending coronary artery. After MI induction, the rats were randomly assigned to one of five groups: sham operated, and experimental MI followed by no exercise, or low, moderate or high intensity exercise Cardiac function and infarct size were assessed by echocardiography and Evans blue/TTC staining, respectively. The expression of mRNA markers and proteins associated with myocardial regeneration was measured with RT-PCR and western blotting. KEY FINDINGS: Exercise training at different intensities improved cardiac function and levels of stem cell and cardiomyocyte markers, and reduced infarct size. mRNA levels of GATA4, Nkx2.5 and c-Kit and protein expression of Nkx2.5 and c-Kit were significantly increased in all MI-exercise groups. The high-intensity exercise group had greater increases than the low and moderate intensity exercise groups. In the high-intensity exercise group, Sca-1 and CITED4 increased more than in the low-intensity exercise group. C/EBPß mRNA and protein levels decreased after exercise training, with greater reductions in the high-intensity exercise group than the low- or moderate-intensity groups. SIGNIFICANCE: The findings suggest that by targeting cardiogenesis, high-intensity training can exert cardioprotective effects against cardiac dysfunction in an experimental model of MI.


Assuntos
Infarto do Miocárdio/metabolismo , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/fisiologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT , Vasos Coronários , Modelos Animais de Doenças , Ecocardiografia , Fator de Transcrição GATA4/metabolismo , Treinamento Intervalado de Alta Intensidade/métodos , Proteína Homeobox Nkx-2.5/metabolismo , Masculino , Miocárdio , Miócitos Cardíacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ratos , Ratos Wistar , Função Ventricular Esquerda
20.
Cardiovasc Toxicol ; 19(3): 264-275, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30535663

RESUMO

The wide use of anthracyclines represented by doxorubicin (DOX) has benefited cancer patients, yet the clinical application is limited due to its cardiotoxicity. Although numerous evidences have supported a role of microRNAs (miRNAs) in DOX-induced myocardial damage, the exact etiology and pathogenesis remain largely obscure. In this study, we focused on the role of miR-15b-5p in DOX-induced cardiotoxicity. We employed a public miRNA and gene microarray to screen differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) in rat cardiomyocytes, and 33 DEMs including miR-15b-5p and 237 DEGs including Bmpr1a and Gata4 were identified. The Gene ontology (GO) and pathway enrichment analysis of 237 DEGs indicated that the DEGs were mainly enriched in heart development and ALK pathway in cardiomyocyte which included the main receptor Bmpr1a and transcription factor Gata4. The up-regulated miR-15b-5p and down-regulated Bmpr1a and Gata4 mRNA expressions were further validated in H9c2 cardiomyocytes exposed to DOX. Moreover, the results showed overexpression of miR-15b-5p or inhibition of Bmpr1a may enhance the DOX-induced apoptosis, oxidative stress and mitochondria damage in H9c2 cardiomyocytes. The Bmpr1a was suggested as a potential target of miR-15b-5p by bioinformatics prediction. We further verified the negatively regulatory effect of miR-15b-5p on Bmpr1a signaling. Moreover, we also confirmed that overexpression of miR-15b-5p may exacerbate the DOX-induced apoptosis of H9c2 cardiomyocytes by affecting the protein expression ratio of Bcl-2/Bax and Akt activation, while this pro-apoptotic effect was able to be suppressed by Bmpr1a agonist. Collectively, the results suggest that miR-15b-5p is likely involved in doxorubicin-induced cardiotoxicity via inhibiting Bmpr1a signaling in H9c2 cardiomyocytes. Our study provides a novel insight for investigating DOX-induced cardiotoxicity.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Doxorrubicina/toxicidade , MicroRNAs/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Cardiotoxicidade , Linhagem Celular , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial/efeitos dos fármacos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo
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