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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.
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Unmethylated CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to protect against myocardial ischemia/reperfusion injury. However, the potential effects of CpG-ODN on myocardial infarction (MI) induced by persistent ischemia remains unclear. Here, we investigated whether and how CpG-ODN preconditioning protects against MI in mice. C57BL/6 mice were treated with CpG-ODN by i.p. injection 2 hr prior to MI induction, and cardiac function, and histology were analyzed 2 weeks after MI. Both 1826-CpG and KSK-CpG preconditioning significantly improved the left ventricular (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS) when compared with non-CpG controls. Histological analysis further confirmed the cardioprotection of CpG-ODN preconditioning. In vitro studies further demonstrated that CpG-ODN preconditioning increases cardiomyocyte survival under hypoxic/ischemic conditions by enhancing stress tolerance through TLR9-mediated inhibition of the SERCA2/ATP and activation of AMPK pathways. Moreover, CpG-ODN preconditioning significantly increased angiogenesis in the infarcted myocardium compared with non-CpG. However, persistent TLR9 activation mediated by lentiviral infection failed to improve cardiac function after MI. Although CpG-ODN preconditioning increased angiogenesis in vitro, both the persistent stimulation of CpG-ODN and stable overexpression of TLR9 suppressed the tube formation of cardiac microvascular endothelial cells. CpG-ODN preconditioning significantly protects cardiac function against MI by suppressing the energy metabolism of cardiomyocytes and promoting angiogenesis. Our data also indicate that CpG-ODN preconditioning may be useful in MI therapy.
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Infarto del Miocardio/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Oligodesoxirribonucleótidos/administración & dosificación , Función Ventricular Izquierda/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Humanos , Precondicionamiento Isquémico Miocárdico/métodos , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Receptor Toll-Like 9/genéticaRESUMEN
CXCL10, the chemokine with potent chemotactic activity on immune cells and other non-immune cells expressing its receptor CXCR3, has been demonstrated to involve in myocardial infarction, which was resulted from hypoxia/ischemia. The cardiac microvascular endothelial cells (CMECs) are the first cell type which is implicated by hypoxia/ischemia. However, the potential molecular mechanism by which hypoxia/ischemia regulates the expression of CXCL10 in CMECs remains unclear. In the present study, the expression of CXCL10 was firstly examined by real-time PCR and ELISA analysis. Several potential binding sites (BS) for transcription factors including NF-kappaB (NFkB), HIF1 alpha (HIF1α) and FoxO3a were identified in the promoter region of CXCL10 gene from -2000 bp to -1 bp using bioinformatics software. Luciferase reporter gene vectors for CXCL10 promoter and for activation of above transcription factors were constructed. The activation of NFkB, hypoxia-inducible transcription factor-1 alpha (HIF-1α) and FoxO3a was also analyzed by Western blotting. It was shown that the production of CXCL10 in CMECs was significantly increased by hypoxia/ischemia treatment, in parallel with the activation of CXCL10 promoter examined by reporter gene vector system. Furthermore, transcription factors including NFkB, HIF1α and FoxO3a were activated by hypoxia/ischemia in CMECs. However, over-expression of NFkB, but not that of HIF1α or FoxO3a, significantly promoted the activation of CXCL10 promoter reporter gene. These findings indicated that CXCL10 production in CMECs was significantly increased by hypoxia/ischemia, at least in part, through activation of NFkB pathway and subsequently binding to CXCL10 promoter, finally promoted the transcription of CXCL10 gene.
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Quimiocina CXCL10/metabolismo , Vasos Coronarios/citología , Células Endoteliales/metabolismo , FN-kappa B/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Hipoxia de la Célula , Células Cultivadas , Quimiocina CXCL10/genética , Ensayo de Inmunoadsorción Enzimática , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.
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Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL10/farmacología , Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Receptores CXCR3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Hipoxia de la Célula , Células Cultivadas , Vasos Coronarios/citología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Expresión Génica/efectos de los fármacos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Ratas Sprague-Dawley , Receptores CXCR3/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
FoxO3a plays an important role in the aging process and decreases with age. However, the potential regulatory roles of FoxO3a in processes involved in cardiac microvascular endothelial cell (CMEC) senescence, and its underlying molecular mechanisms have not been elucidated. This study demonstrates that FoxO3a is deactivated in senescent CMECs together with the inhibition of proliferation and tube formation. Furthermore, the activation of the antioxidant enzymes catalase and SOD, downstream FoxO3a targets, was significantly decreased, thereby leading to cell cycle arrest in G1-phase by increased ROS generation and subsequently the activation of the p27(Kip1) pathway. However, FoxO3a overexpression in primary low-passage CMECs not only significantly suppressed the senescence process by increasing the activation of catalase and SOD but also markedly inhibited ROS generation and p27(Kip1) activation, although it failed to reverse cellular senescence. Moreover, both cell viability and tube formation were greatly increased by FoxO3a overexpression in primary CMECs during continuous passage. In addition, FoxO3a, deficiency in low-passage CMECs, accelerated the senescence process. Collectively, our data suggest that FoxO3a suppresses the senescence process in CMECs by regulating the antioxidant/ROS/p27(Kip1) pathways, although it fails to reverse the cellular senescent phenotype.
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Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/genética , Miocardio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Secuencia de Bases , Catalasa/genética , Catalasa/metabolismo , Supervivencia Celular , Senescencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Endoteliales/patología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación de la Expresión Génica , Genes Reporteros , Lentivirus/genética , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Miocardio/patología , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. However, the relationship between apoptosis and autophagy in lymphoma cells exposed to statins remains unclear. The objective of this study was to elucidate the potential involvement of autophagy in fluvastatin-induced cell death of lymphoma cells. We found that fluvastatin treatment enhanced the activation of pro-apoptotic members such as caspase-3 and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells. The process was accompanied by increases in numbers of annexin V alone or annexin V/PI double positive cells. Furthermore, both autophagosomes and increases in levels of LC3-II were also observed in fluvastatin-treated lymphoma cells. However, apoptosis in fluvastatin-treated lymphoma cells could be blocked by the addition of 3-methyladenine (3-MA), the specific inhibitor of autophagy. Fluvastatin-induced activation of caspase-3, DNA fragmentation, and activation of LC3-II were blocked by metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggest that autophagy contributes to fluvastatin-induced apoptosis in lymphoma cells, and that these regulating processes require inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.
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FoxO3a, a member of the forkhead transcription factors, has been demonstrated to be involved in myocardial ischemia/reperfusion (I/R) injury. Cardiac microvascular endothelial cells (CMECs) are some of the predominant cells damaged immediately after myocardial I/R injury. Despite the importance of injured CMECs in an ischemic heart, little is known about the involvement of FoxO3a in regulating CMECs injury. Thus, we used rat CMECs following simulated I/R to examine FoxO3a activation and signaling in relation to survival, the cell cycle and apoptosis in CMECs. We found that Akt negatively regulates activation of the FoxO3a pathway by phosphorylating FoxO3a in CMECs as demonstrated with an Akt inhibitor and activator. Upon I/R injury, the FoxO3a pathway was significantly activated in CMECs, which was accompanied by Akt deactivation. In parallel, the I/R of CMECs induced G1-phase arrest through p27(Kip1) up-regulation and significant activation of caspase-3. Accordingly, inhibition of the FoxO3a pathway by IGF-1, an Akt activator, could significantly block the I/R-enhanced activation of p27(Kip1) and caspase-3 in CMECs. Collectively, our results indicate that the FoxO3a pathway is involved in the I/R injury of CMECs at least in part through the regulation of cell cycle arrest and apoptosis, suggesting that the FoxO3a pathway may be a novel therapeutic target that protects against microvascular endothelial damage in ischemic hearts.
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Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Transducción de Señal , Animales , Apoptosis/fisiología , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Proteína Forkhead Box O3 , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Circular RNAs (circRNAs) are generally formed by the back-splicing of precursor mRNA. Increasing evidence implicates the important role of circRNAs in cardiovascular diseases. However, the role of circ-insulin-like growth factor 1 receptor (circIGF1R) in cardiomyocyte (CM) proliferation remains unclear. Here, we investigated the potential role of the circIGF1R in the proliferation of CMs. We found that circIGF1R expression in heart tissues and primary CMs from adult mice was significantly lower than that in neonatal mice at postnatal 1 day (p1). Increased circIGF1R expression was detected in the injured neonatal heart at 0.5 and 1 days post-resection. circIGF1R knockdown significantly decreased the proliferation of primary CMs. Combined prediction software, luciferase reporter gene analysis, and quantitative real time-PCR (qPCR) revealed that circIGF1R interacted with miR-362-5p. A significant increase in miR-362-5p expression was detected in the adult heart compared with that in the neonatal heart. Further, heart injury significantly decreased the expression of miR-362-5p in neonatal mice. Treatment with miR-362-5p mimics significantly suppressed the proliferation of primary CMs, whereas knockdown of miR-362-5p promoted the CMs proliferation. Meanwhile, miR-362-5p silencing can rescue the proliferation inhibition of CMs induced by circIGF1R knockdown. Target prediction and qPCR validation revealed that miR-362-5p significantly inhibited the expression of Phf3 in primary CMs. In addition, decreased Phf3 expression was detected in adult hearts compared with neonatal hearts. Consistently, increased Phf3 expression was detected in injured neonatal hearts compared with that in sham hearts. Knockdown of Phf3 markedly repressed CMs proliferation. Taken together, these findings suggest that circIGF1R might contribute to cardiomyocyte proliferation by promoting Pfh3 expression by sponging miR-362-5p and provide an important experimental basis for the regulation of heart regeneration.
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Enfermedades Cardiovasculares , MicroARNs , Animales , Ratones , Miocitos Cardíacos , ARN Circular/genética , Proliferación Celular/genética , MicroARNs/genética , Línea Celular TumoralRESUMEN
Bambusae caulis in Liquamen (BCL), traditional herbal medicine used in East Asia, is known to have antioxidative and immune-regulating properties. We hypothesized that the potential antioxidant effects of BCL might suppress the production of thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in human keratinocytes (HaCaT cell). The immune-regulating effect of BCL was demonstrated by antioxidant capacity using DPPH analysis and DCFH-DA analysis. We found that BCL had strong ROS scavenge effect in HaCaT cell. BCL also showed suppression of IFN-γ-induced expression of TARC and MDC, activation of NF-κB, and, moreover, significant block of IFN-γ-induced degradation and phosphorylation of IκB. However, it had no effects on phosphorylation of p38 MAPK. Collectively, these results suggest that BCL may have a therapeutic potential on skin disease such as atopic dermatitis by inhibiting Th2 chemokines which is due, at least in part, to its antioxidant capacities.
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It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which can be employed to elucidate the mechanisms of adult heart regeneration.
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Insuficiencia Cardíaca , Lesiones Cardíacas , Animales , Xenopus , Ventrículos Cardíacos , Pericardio , MamíferosRESUMEN
Upon injury, the liver is capable of substantial regeneration from the original tissue until an appropriate functional size. The underlying mechanisms controlling the liver regeneration processes are not well elucidated. Previous studies have proposed that the transcription factor FoxO3 is involved in various liver diseases, but its exact role in the regulation of liver regeneration remains largely unclear. To directly test the detailed role of FoxO3 in liver regeneration, both a constitutive Albumin-Cre driver line and adeno-associated virus serotype 8 (AAV8)-Tbg-Cre (AAV-Cre)-injected adult FoxO3fl/fl mice were subjected to 70% partial hepatectomy (PH). Our data demonstrate that FoxO3 deletion accelerates liver regeneration primarily by limiting polyploidization and promoting the proliferation of hepatocytes during liver regeneration. RNA-seq analysis indicates that FoxO3 deficiency greatly alters the expression of gene sets associated with cell proliferation and apoptosis during liver regeneration. Chromatin immunoprecipitation-PCR (ChIP-PCR) and luciferase reporter assays reveal that FoxO3 promotes the expression of Nox4 but suppresses the expression of Nr4a1 in hepatocytes. AAV8 virus-mediated overexpression of Nox4 and knockdown of Nr4a1 significantly suppressed hepatocyte proliferation and liver regeneration in FoxO3-deficient mice. We demonstrate that FoxO3 negatively controls hepatocyte proliferation through Nox4 upregulation and Nr4a1 downregulation, thereby ensuring appropriate functional regeneration of the liver. Our findings provide novel mechanistic insight into the therapeutic mechanisms of FoxO3 in liver damage and repair.
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The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon-gamma (IFN-γ)-treated HaCaT keratinocytes. We found that IFN-γ-induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N-acetyl cysteine (NAC), significantly inhibited the IFN-γ-induced production of chemokines as well as the activation of I kappa-B (IκB)-nuclear factor-kappa B (NF-κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen-activated kinase (MAPK), and NF-κB suppressed IFN-γ-induced production of TARC and MDC. NF-κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN-γ-stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L-buthionine sulfoximine (L-BSO). However, IFN-γ-stimulated activation of IκB and NF-κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN-γ-stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN-γ-stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF-κB activation.
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Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Queratinocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Quimiocina CCL17/genética , Quimiocina CCL22/genética , Depuradores de Radicales Libres , Humanos , Queratinocitos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismoRESUMEN
PTEN has been considered as one of the important anti-oncogenes, which possesses very wide biological activities. Endogenous PTEN genes begin to express in epiblast during chick embryo gastrulation, and then the expression extends to neural plate and mesoderm. This suggests that PTEN might be involved in cell migration, proliferation, and differentiation during early embryo development. In this study, we employed in vivo approach to explore if endogenous PTEN participates in EMT (Epithelial-mesenchymal transition) in early chick embryo. PTEN was initially detected to highly express in primitive streak during chick gastrulation, in which EMT occurs, and subsequently mesoderm structure such as somites etc. Then, overexpression of both Wt PTEN-GFP and Wt PTEN-GFP positive transplantation of primitive streak resulted in cell accumulation in primitive streak in the development hereafter, indicating that EMT was blocked in both of our assays, either whole embryo transfection of Wt PTEN-GFP or transplantation of Wt PTEN-GFP primary streak tissue. Finally down-regulation of PTEN gene in one side using PTEN siRNA led to reduce the more number of mesoderm cells in PTNE siRNA side than normal side, which suggests that PTEN gene is probably involved in regulating EMT process in development of early embryonic gastrulation.
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Embrión de Pollo/embriología , Transición Epitelial-Mesenquimal , Gastrulación , Fosfohidrolasa PTEN/metabolismo , Animales , Embrión de Pollo/metabolismo , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/genética , Gastrulación/genética , Fosfohidrolasa PTEN/genéticaRESUMEN
Cardiovascular disease is the leading cause of death in the world due to losing regenerative capacity in the adult heart. Frogs possess remarkable capacities to regenerate multiple organs, including spinal cord, tail, and limb, but the response to heart injury and the underlying molecular mechanism remains largely unclear. Here we demonstrated that cardiomyocyte proliferation greatly contributes to heart regeneration in adult X. tropicalis upon apex resection. Using RNA-seq and qPCR, we found that the expression of Fos-like antigen 1 (Fosl1) was dramatically upregulated in early stage of heart injury. To study Fosl1 function in heart regeneration, its expression was modulated in vitro and in vivo. Overexpression of X. tropicalis Fosl1 significantly promoted the proliferation of cardiomyocyte cell line H9c2. Consistently, endogenous Fosl1 knockdown suppressed the proliferation of H9c2 cells and primary cardiomyocytes isolated from neonatal mice. Taking use of a cardiomyocyte-specific dominant-negative approach, we show that blocking Fosl1 function leads to defects in cardiomyocyte proliferation during X. tropicalis heart regeneration. We further show that knockdown of Fosl1 can suppress the capacity of heart regeneration in neonatal mice, but overexpression of Fosl1 can improve the cardiac function in adult mouse upon myocardium infarction. Co-immunoprecipitation, luciferase reporter, and ChIP analysis reveal that Fosl1 interacts with JunB and promotes the expression of Cyclin-T1 (Ccnt1) during heart regeneration. In conclusion, we demonstrated that Fosl1 plays an essential role in cardiomyocyte proliferation and heart regeneration in vertebrates, at least in part, through interaction with JunB, thereby promoting expression of cell cycle regulators including Ccnt1.
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Stress-responsive genes play critical roles in many biological functions that includes apoptosis, survival, differentiation and regeneration. We have identified a novel stress-responsive gene called BRE which interacts with TNF-receptor-1 and blocks the apoptotic effect of TNF-alpha. BRE enhances tumor growth in vivo and is up-regulated in hepatocellular and esophageal carcinomas. BRE also regulates the ubiquitination of the DNA repair complex BRCC, and the synthesis of steroid hormones. Here, we examined BRE-mRNA in cells after treatments with UV and ionizing radiation (IR). UV and IR treatment alone suppressed BRE-mRNA levels by more than 90% at 24 h, while hydroxyurea, fluorodeoxyuridine, aphidicolin, known inhibitors of S-phase DNA synthesis, had no significant effect. BRE protein expression was unaltered in cells treated with TNF-alpha, Interleukin-1 and Dexamethasone, while a threefold increase was observed following chorionic gonadotropin exposure. Although BRE plays a regulatory role in many different pathways, yet its expression is apparently under very stringent control.
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Factores Biológicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Afidicolina/farmacología , Cadaverina/análogos & derivados , Cadaverina/farmacología , Línea Celular , Gonadotropina Coriónica/farmacología , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Dexametasona/farmacología , Dimetilsulfóxido/farmacología , Fluorodesoxiuridilato/farmacología , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Hidroxiurea/farmacología , Interleucina-1/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Estrés Fisiológico/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Rayos UltravioletaRESUMEN
The forkhead-box transcription factors of O subfamily (FOXO) play important roles in regulation of various biological functions. We cloned foxo1, foxo3, foxo4, and foxo6 from Xenopus tropicalis (hereafter X. tropicalis), and examined their expression in embryos and adult tissues. Maternal transcripts of foxo1 and foxo3 genes are detected within the animal half of the early embryo, their zygotic transcripts show distinct patterns. At late tailbud stages, foxo1 expression is observed mainly in eye, brain, branchial arches, and pronephros. In addition to eye, brain, branchial arches and pronephros, foxo3 expression is also evident in heart and somites. Foxo4 expression was not detected in oocytes. At late tailbud stages, foxo4 is mainly expressed in eye, brain, branchial arches and otic vesicle. Foxo6 expression was not detectable until stage 36, with a specific expression in nasal pits. Obvious expression of foxo1, foxo3 and foxo4, but not foxo6, is detected by RT-PCR both in oocytes and in embryos at examined stages. The expression of foxo1, foxo3 and foxo4 is observed in all tested adult tissues including heart, muscle, liver, lung, stomach and small intestine, while foxo6 is only detectable in stomach and small intestine. The differential expression pattern of foxo genes suggests that they exert distinct functions during embryonic development and in various organs of X. tropicalis.
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Proteínas Anfibias/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Anfibias/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Bronquios/embriología , Bronquios/metabolismo , Ojo/embriología , Ojo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Corazón/embriología , Riñón/embriología , Riñón/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Miocardio/metabolismo , XenopusRESUMEN
Protein arginine methyltransferases (PRMTs) are involved in many cellular processes via the arginine methylation of histone or non-histone proteins. We examined the expression patterns of prmt4, prmt7, and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR). Xenopus prmt4 and prmt7 were expressed in the neural crest, brain, and spinal cord, and also detected in the eye, branchial arches, and heart at the tailbud stage. Specific prmt9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches. Quantitative RT-PCR indicated that the expressions of prmt4 and prmt7 were up-regulated during the neurula stage, whereas prmt9 maintained its low expression until the late tailbud stage, consistent with the whole-mount in situ hybridization results. Thus, the developmental expression patterns of these three prmt genes in Xenopus embryos provide a basis for further functional study of such genes.
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Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína-Arginina N-Metiltransferasas/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Animales , Regulación Enzimológica de la Expresión Génica/fisiología , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
Fos-like antigens (Fosl) including Fosl1 and Fosl2 exclusively heterodimerize with Jun members to form AP-1 complex, thereby participating in various cellular progresses including cell cycle regulation. However, expression patterns of these two genes during embryonic development remains largely unknown. In the present study, both temporal and spatial expression patterns of fosl1 and fosl2 were examined during embryonic development of Xenopus tropicalis. Real-time quantitative PCR results showed that the expression of the two genes was increased from stage 2 to stage 42. However, expression level of fosl1 is much higher than that of fosl2 at stage 42. Whole-mount in situ hybridization showed that fosl1 was expressed in eyes, branchial arch, notochord, otic vesicle, and liver. However, fosl2 was expressed in lung primordium from stage 34 to stage 38, in addition to the moderate expression in eyes and branchial arch at stage 42. Thus, the developmental expression patterns of these two fosl genes is different in Xenopus embryos. These results provide a basis for further functional study of these two genes.
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Desarrollo Embrionario/genética , Proteínas Proto-Oncogénicas c-fos/genética , Xenopus/embriología , Secuencia de Aminoácidos/genética , Animales , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Proteínas Proto-Oncogénicas c-fos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMEN
Paraquat (PQ) promotes cell senescence in brain tissue, which contributes to Parkinson's disease. Furthermore, PQ induces heart failure and oxidative damage, but it remains unknown whether and how PQ induces cardiac aging. Here, we demonstrate that PQ induces phenotypes associated with senescence of cardiomyocyte cell lines and results in cardiac aging-associated phenotypes including cardiac remodeling and dysfunction in vivo. Moreover, PQ inhibits the activation of Forkhead box O3 (FoxO3), an important longevity factor, both in vitro and in vivo. We found that PQ-induced senescence phenotypes, including proliferation inhibition, apoptosis, senescence-associated ß-galactosidase activity, and p16INK4a expression, were significantly enhanced by FoxO3 deficiency in cardiomyocytes. Notably, PQ-induced cardiac remolding, apoptosis, oxidative damage, and p16INK4a expression in hearts were exacerbated by FoxO3 deficiency. In addition, both in vitro deficiency and in vivo deficiency of FoxO3 greatly suppressed the activation of antioxidant enzymes including catalase (CAT) and superoxide dismutase 2 (SOD2) in the presence of PQ, which was accompanied by attenuation in cardiac function. The direct in vivo binding of FoxO3 to the promoters of the Cat and Sod2 genes in the heart was verified by chromatin immunoprecipitation (ChIP). Functionally, overexpression of Cat or Sod2 alleviated the PQ-induced senescence phenotypes in FoxO3-deficient cardiomyocyte cell lines. Overexpression of FoxO3 and CAT in hearts greatly suppressed the PQ-induced heart injury and phenotypes associated with aging. Collectively, these results suggest that FoxO3 protects the heart against an aging-associated decline in cardiac function in mice exposed to PQ, at least in part by upregulating the expression of antioxidant enzymes and suppressing oxidative stress.
Asunto(s)
Envejecimiento/metabolismo , Antioxidantes/metabolismo , Proteína Forkhead Box O3/metabolismo , Paraquat/antagonistas & inhibidores , Sustancias Protectoras/metabolismo , Regulación hacia Arriba , Envejecimiento/efectos de los fármacos , Animales , Catalasa/genética , Catalasa/metabolismo , Corazón/efectos de los fármacos , Ratones , Ratones Noqueados , Paraquat/farmacología , Fenotipo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19-39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.