RESUMEN
The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.
Asunto(s)
Ventrículos Cardíacos , Miocitos Cardíacos , Femenino , Embarazo , Animales , Ratones , Desarrollo Embrionario , Miocardio , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción , Proteínas Represoras , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
Given the pivotal role of neuronal populations in various biological processes, assessing their collective output is crucial for understanding the nervous system's complex functions. Building on our prior development of a spiral scanning mechanism for the rapid acquisition of Raman spectra from single cells and incorporating machine learning for label-free evaluation of cell states, we investigated whether the Paint Raman Express Spectroscopy System (PRESS) can assess neuronal activities. We tested this hypothesis by examining the chemical responses of glutamatergic neurons as individual neurons and autonomic neuron ganglia as neuronal populations derived from human-induced pluripotent stem cells. The PRESS successfully acquired Raman spectra from both individual neurons and ganglia within a few seconds, achieving a signal-to-noise ratio sufficient for detailed analysis. To evaluate the ligand responsiveness of the induced neurons and ganglia, the Raman spectra were subjected to principal component and partial least squares discriminant analyses. The PRESS detected neuronal activity in response to glutamate and nicotine, which were absent in the absence of calcium. Additionally, the PRESS induced dose-dependent neuronal activity changes. These findings underscore the capability of the PRESS to assess individual neuronal activity and elucidate neuronal population dynamics and pharmacological responses, heralding new opportunities for drug discovery and regenerative medicine advancement.
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Ácido Glutámico , Células Madre Pluripotentes Inducidas , Neuronas , Espectrometría Raman , Espectrometría Raman/métodos , Neuronas/metabolismo , Neuronas/fisiología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Nicotina/farmacología , Análisis de Componente PrincipalRESUMEN
Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.
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Endotelio Vascular/metabolismo , Elementos de Facilitación Genéticos , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Cromatina/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/embriología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Ratones , Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Sitio de Iniciación de la Transcripción , Regulador Transcripcional ERG/metabolismoRESUMEN
Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.
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Arterias/embriología , Proteínas de la Mielina/metabolismo , Neovascularización Fisiológica/genética , Animales , Arterias/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de la Mielina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Development of multi-chambered heart is associated with spatio-temporal regulation of gene expression. A basic helix-loop-helix transcription factor Hey2 is specifically expressed in the embryonic mouse ventricles and is indispensable for ventricular myocyte differentiation, compartment identity and morphogenesis of the heart. However, how Hey2 transcription is precisely regulated in the heart remains unclear. In this study, we identified a distal Hey2 enhancer conserved in the mouse and human to possess specific transcriptional activity in ventricular free wall myocytes at the looping stage of cardiac development. Deletion of the enhancer significantly decreased endogenous Hey2 expression in the ventricular myocardium but not in other tissues of mouse embryos. Mutation/deletion of the conserved binding sites for T-box and Gata proteins, but not NK-2 proteins, abolished the enhancer activity, and Tbx20 null mice completely lost the enhancer activity in the embryonic ventricles. Luciferase reporter analysis suggested that the ventricular enhancer activity was controlled by Tbx20 through its DNA binding and cooperative function with cardiac Gata proteins. These results delineate a regulatory mechanism of ventricular Hey2 expression and help fully understand molecular cascades in myocardial cell differentiation and cardiac morphogenesis during embryonic development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Elementos de Facilitación Genéticos , Factor de Transcripción GATA4/fisiología , Regulación del Desarrollo de la Expresión Génica , Ventrículos Cardíacos/embriología , Proteínas Represoras/biosíntesis , Proteínas de Dominio T Box/fisiología , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Secuencia Conservada , Genes Reporteros , Ventrículos Cardíacos/metabolismo , Humanos , Mamíferos/genética , Ratones , Ratones Transgénicos , Proteínas Represoras/genética , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.
Asunto(s)
Proteína ADAM17/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Membrana Celular/metabolismo , Receptor ErbB-4/metabolismo , Animales , Ratones , Dominios Proteicos , Células RAW 264.7RESUMEN
Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Endotelio/metabolismo , Receptores Notch/metabolismo , Animales , Arterias/crecimiento & desarrollo , Arterias/metabolismo , Región Branquial/irrigación sanguínea , Región Branquial/crecimiento & desarrollo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Embrión de Mamíferos/metabolismo , Endotelio/citología , Femenino , Humanos , Ratones , Ratones Noqueados , Morfogénesis , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Activación TranscripcionalRESUMEN
A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ventrículos Cardíacos/crecimiento & desarrollo , Corazón/crecimiento & desarrollo , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Femenino , Ventrículos Cardíacos/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Morfogénesis , Proteína Proto-Oncogénica N-Myc/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/deficiencia , Proteínas de Dominio T Box/genética , Función Ventricular DerechaRESUMEN
Heart disease is the most common cause of death in developed countries, but the medical treatments for heart failure remain limited. In this context, the development of cardiac regeneration therapy for severe heart failure is important. Owing to their unique characteristics, including multiple differentiation and infinitive self-renewal, pluripotent stem cells can be considered as a novel source for regenerative medicine. Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling plays critical roles in the induction, maintenance, and differentiation of pluripotent stem cells. In the heart, JAK/STAT3 signaling has diverse cellular functions, including myocardial differentiation, cell cycle re-entry of matured myocyte after injury, and anti-apoptosis in pathological conditions. Therefore, regulating STAT3 activity has great potential as a strategy of cardiac regeneration therapy. In this review, we summarize the current understanding of STAT3, focusing on stem cell biology and pathophysiology, as they contribute to cardiac regeneration therapy. We also introduce a recently reported therapeutic strategy for myocardial regeneration that uses engineered artificial receptors that trigger endogenous STAT3 signal activation.
Asunto(s)
Corazón/fisiopatología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Regeneración/fisiología , Medicina Regenerativa/métodos , Factor de Transcripción STAT3/metabolismo , Animales , Bioingeniería/métodos , Diferenciación Celular/fisiología , Humanos , Miocardio/metabolismoRESUMEN
Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Neovascularización Fisiológica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transcripción Genética , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Factor 2 de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/genética , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Proteínas Inmediatas-Precoces/genética , Ratones , Mutación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.
Asunto(s)
Reprogramación Celular/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratinocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFbeta, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.
Asunto(s)
Tejido Adiposo/citología , Células Madre Pluripotentes Inducidas/fisiología , Animales , Quimera , Técnicas de Cocultivo , Factores de Crecimiento de Fibroblastos , Fibroblastos/citología , Fibroblastos/fisiología , Fibronectinas/biosíntesis , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador alfa/biosíntesis , Vitronectina/biosíntesisRESUMEN
Induced pluripotent stem cells (iPSCs) are promising cell sources for regenerative medicine and disease modeling. iPSCs are commonly established by introducing the defined reprogramming factors Oct4, Sox2, Klf4, and c-Myc. However, iPSC reprogramming efficiency remains low. Although recent studies have identified microRNAs that contribute to efficient reprogramming, the underlying molecular mechanisms are not completely understood. miR-17-92 is highly expressed in embryonic stem cells and may play an important role in regulating stem cell properties. Therefore, we examined the role of miR-17-92 in the induction of mouse iPSC production. c-Myc-mediated miR-17-92 upregulation increased reprogramming efficiency, whereas CRISPR/Cas9-based deletion of the miR-17-92 cluster decreased reprogramming efficiency. A combination of in silico and microarray analyses revealed that Pten and cyclin-dependent kinase inhibitor 1 (known as p21) are common target genes of miR-17 and miR-20a, which are transcribed from the miR-17-92 cluster. Moreover, miR-17-92 downregulated p21 in the early phase and PTEN in the mid-to-late phase of reprogramming. These downregulations were perturbed by introducing the 3' UTR of PTEN and p21, respectively, suggesting that PTEN and p21 mRNAs are competing endogenous RNAs (ceRNA) against miR-17-92. Collectively, we propose that the c-Myc-mediated expression of miR-17-92 is involved in iPSC reprogramming through the phase-dependent inhibition of PTEN and p21 in a ceRNA manner, thus elucidating an underlying mechanism of iPSC reprogramming.
RESUMEN
BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.
RESUMEN
Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.
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To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.
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A zinc finger protein GATA4 is one of the hypertrophy-responsive transcription factors and forms a complex with an intrinsic histone acetyltransferase, p300. Disruption of this complex results in the inhibition of cardiomyocyte hypertrophy and heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9) as a novel GATA4-binding partner. Cdk9 also formed a complex with p300 as well as GATA4 and cyclin T1. We showed that p300 was required for the interaction of GATA4 with Cdk9 and for the kinase activity of Cdk9. Conversely, Cdk9 kinase activity was required for the p300-induced transcriptional activities, DNA binding, and acetylation of GATA4. Furthermore, the kinase activity of Cdk9 was required for the phosphorylation of p300 as well as for cardiomyocyte hypertrophy. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4- transcriptional pathway during cardiomyocyte hypertrophy.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Factor de Transcripción GATA4/metabolismo , Hipertrofia , Miocitos Cardíacos/metabolismo , Fenilefrina/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Histonas/metabolismo , Humanos , Ratas , Transcripción GenéticaRESUMEN
The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin-dependent kinase 9 (Cdk9), a core component of positive elongation factor-b, is a novel GATA4-binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac-specific Nkx2.5 promoter, were induced to differentiate on feeder-free gelatin-coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP-positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant-negative form of Cdk9 (DN-Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6-dichloro-1-ß-ribofuranosyl-benzimidazole (DRB) on TSA-induced myocardial cell differentiation. The introduction of the DN-Cdk9 inhibited TSA-induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA-induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Factor de Transcripción GATA4/metabolismo , Miocitos Cardíacos/citología , Animales , Diferenciación Celular , Línea Celular , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Diclororribofuranosil Benzoimidazol/farmacología , Factor de Transcripción GATA4/genética , Inhibidores de Histona Desacetilasas/farmacología , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ácidos Hidroxámicos/farmacología , Ratones , Miocitos Cardíacos/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure-induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans.
Asunto(s)
Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Insuficiencia Cardíaca/prevención & control , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Animales , Cardiomegalia/prevención & control , Curcumina/uso terapéutico , ADN/metabolismo , Factor de Transcripción GATA4/metabolismo , Hipertensión/complicaciones , Masculino , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Sístole/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
BACKGROUND: A natural p300-specific histone acetyltransferase (HAT) inhibitor, curcumin, may have therapeutic potential for heart failure. However, it is unclear whether curcumin exhibits beneficial additive or synergistic effects on conventional therapy with angiotensin-converting enzyme inhibitors (ACEIs). METHODS AND RESULTS: Rats were subjected to a sham operation or left coronary artery ligation. One week later, 34 rats with a moderate sized myocardial infarction (MI) were randomly assigned to 4 groups: solvents as control (n = 8), enalapril (an ACEI, 10 mg·kg⻹·day⻹) alone (n=8), curcumin (50 mg·kg⻹·day⻹) alone (n = 9) and enalapril plus curcumin (n = 9). Daily oral treatment was repeated and continued for 6 weeks. Echocardiographic data were similar among the 4 groups before treatment. After treatment, left ventricular (LV) fractional shortening (FS) was significantly higher in the enalapril (29.0 ± 1.9%) and curcumin (30.8 ± 1.7%) groups than in the vehicle group (19.7 ± 1.6%). Notably, LVFS further increased in the enalapril/curcumin combination group (34.4 ± 1.8%). Histologically, cardiomyocyte diameter in the non-infarct area was smaller in the enalapril/curcumin combination group than in the enalapril group. Perivascular fibrosis was significantly reduced in the enalapril/curcumin group compared with the curcumin group. CONCLUSIONS: A natural non-toxic dietary compound, curcumin, combined with an ACEI exerts beneficial effects on post-MI LV systolic function in rats.