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Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

Purpose: GATA4 has emerged as a novel regulator that plays a critical role in mediating senescence. However, the role of GATA4 in age-related cataract (ARC), the leading cause of visual impairment, requires further elucidation., Methods: GATA4 expression was measured by quantitative RT-PCR and capillary Western immunoassay (WES). The MTT assay, EdU assay, and rhodamine-123/Hoechst and calcein-AM/propidium iodide double staining were used to investigate the role of GATA4 in the viability, proliferation, and apoptosis of cultured human lens epithelial cells (HLECs)., Results: HLECs were subjected to 3 different treatment models, including prolonged exposure to low-dose H2O2, UVB irradiation, and mild heating, to simulate senescence and apoptosis. GATA4 expression was significantly increased in these models in a time- and dose-dependent manner. Overexpression of GATA4 reduced cell viability, accelerated apoptosis development, and reduced the proliferation of HLECs. Furthermore, the expression of GATA4 from ARC was up-regulated at both mRNA and at protein level compared with clear lenses., Conclusion: GATA4 is up-regulated in all 3 models of HLECs in vitro and the cells from ARC lenses in vivo. Up-regulation of GATA4 mediates HLEC dysfunction. GATA4-mediated effects in HLECs would provide a novel insight into the pathogenesis of ARC., (© 2020 S. Karger AG, Basel.)

GATA-binding protein 4 (GATA-4)，a member of the GATA family of transcription factors, is expressed in the normal brain and participates in the neural inflammatory response and senescence. However, few studies have investigated whether GATA-4 is involved in the brain damage induced by intracerebral hemorrhage (ICH). The aim of this study was to investigate in vivo and in vitro the role of GATA-4 in ICH-induced secondary brain injury (SBI) and its potential underlying mechanisms. A rat model of ICH was established by autologous blood injection in vivo. In vitro, oxidized hemoglobin was applied to mimic the effects of ICH in neuronal culture. The function of GATA-4 and its mechanism of action after ICH were investigated using siRNA-mediated knockdown and plasmid-mediated overexpression techniques combined with immunofluorescence, western blot, and other molecular methods. It was found that the expression of GATA-4 was increased in the brain of rats after ICH, and its phosphorylation also increased correspondingly. Furthermore, knocking down the expression of GATA-4 led to a significant decrease in neurobehavioral scores and neuronal apoptosis, indicating that secondary brain damage was improved. Conversely, the overexpression of GATA-4 aggravated brain damage. Blockade of a critical phosphorylation site on the GATA-4 overexpression plasmid alleviated the exacerbated damage in vitro and in vivo. Moreover, GATA-4 promoted the activation of NF-κB, and increased the expression of Bax, and cysteine aspartate-specific protease 3 (caspase-3) in its cleaved form, causing neuronal apoptosis. In conclusion, the expression of GATA-4 was increased in the brain of rats after ICH. GATA-4 phosphorylation mediates the function of the protein in ICH-induced SBI. Neuronal apoptosis after ICH was mainly induced by NF-κB activation, which was promoted by GATA-4., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pancreatic ductal carcinoma (PDAC) is a common malignant tumor of the digestive system. GATA4 is one of the transcriptional regulatory factors, which regulates the development of endoderm-derived organs, including heart and gut. GATA4 may act as a putative tumor suppressor gene. However, the role of GATA4 in pancreatic carcinogenesis is not yet clarified. This study showed that GATA4 was highly expressed in pancreatic cancer tissues, and its expression level was positively related to the grade of pathological differentiation, suggesting that it may contribute to the progression of pancreatic neoplasia. Ectopic expression of GATA4 gene reduced cell viability and interference of GATA4 expression significantly increased the colony formation ability of pancreatic cancer cells. Furthermore, GATA4 inhibited tumor growth in xenograft mice. Agilent expression microarray profiling analysis indicated that the genes with significant levels of differential expression in GATA4 over-expressing cells were enriched in the cell differentiation process. Analysis of KEGG signaling pathway demonstrated that the regulated genes were partially enriched in MAPK and JAK-STAT signaling pathways. Re-expression of GATA4 up-regulated P53 gene expression. Our data indicate that GATA4 gene might play a role in cell proliferation and differentiation during the progression of pancreatic cancer., Competing Interests: The authors have declared that no competing interests exist.

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Bone marrow mesenchymal stem cells (BMMSCs) can differentiate into cardiomyocytes and be used in cardiac tissue engineering for heart regeneration. However, the effective clinical application of cardiomyocytes derived from BMMSCs is limited because of their immature phenotype. The aim of this study was to investigate the potential of triiodo-L-thyronine (T3) to drive cardiomyocytes derived from BMMSCs to a more mature state. BMMSCs were divided into 3 groups: untreated controls, differentiated, and T3 treated. The differentiation potential was evaluated by immunofluorescence microscopy and flow cytometry. Data were represented as the numbers of cells positive for the troponin I (cTnI), α-actinin, GATA4, and the connexin-43 (Cx-43). The mRNA levels of these specific markers of cardiomyocytes were determined by quantitative real-time polymerase chain reaction. The levels of cardiomyocytes markers protein and octamer-binding transcription factor 4 (Oct-4) were determined by Western blot analyses. Our data demonstrate that T3 treatment leads to a significant increase in cells positive for cTnI, GATA4, Cx-43, and α-actinin. The mRNA and protein expression levels of these specific markers of cardiomyocytes were also increased after T3 treatment. At the same time, the protein expression level of Oct-4 was substantially downregulated in T3-treated cells. These results demonstrate that T3 treatment increases the differentiation of BMMSCs induced to cardiomyocytes and promotes their maturation.

During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/β-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation., (© 2016 AlphaMed Press.)

3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

Background: RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a "noncoding RNAi". However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen)., Results: Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response., Conclusions: Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and "non coding" siRNA is a hindrance in the development of siRNA based therapeutics.

Background: Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear., Methods: Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records., Results: MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06 ± 70.94; P = 0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76 ± 105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P = 0.035) and minimal residual disease MRD (P = 0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P = 0.014)., Conclusions: Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required.

It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications., Design: KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice., Results: KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4α, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs., Conclusions: KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells., Methods and Results: Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways., Conclusions: Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Nodal modulator 1 (NOMO1), a highly conserved transmembrane protein, has been identified as a part of a protein complex that participates in the Nodal signaling pathway, a critical determinant of heart and visceral organ formation. We previously found that the NOMO1 gene was substantially downregulated in human ventricular septal defect (VSD) myocardium and, thus, may be an important molecular pathway in human heart development. In this study, we aimed to investigate the effects of NOMO1 gene silencing by RNA interference (RNAi) during early mouse cardiac differentiation using P19 cells as a model system. Our results revealed that the differentiated P19 cell population exhibited downregulated NOMO1 levels and expressed lower levels of Nodal signaling mediators, such as Nodal, Cripto and Smad2, than the negative control. Similarly, cardiomyocyte-specific sarcomeric markers, such as cardiac troponin T, as well as expression of cardiogenesis-related transcriptional factors, such as Nkx2.5, Gata4 and Tbx5 were found to be downregulated in P19 differentiated cardiomyocytes in NOMO1-silenced cells when compared to controls. In conclusion, our results indicate that NOMO1 gene knockdown inhibits the differentiation of P19 cells into cardiomyocytes, which highlights a potential role for NOMO1 in early cardiogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rationale: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach., Objective: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming., Methods and Results: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers., Conclusions: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies., (© 2014 American Heart Association, Inc.)

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increasingly recognized as important regulators of embryo development in mammals. This study investigated the importance of FGF signaling during in vitro development of ovine embryo. The mRNAs of four FGFR subtypes were detected throughout preimplantation development of in vitro fertilized (IVF) embryos, peaked in abundance at the morula stage, and decreased significantly at the blastocyst stage. To gain insight into the role of these mRNAs in embryo development, IVF embryos were cultured in the presence of FGF2 (100 or 500 ng/ml: beginning from days 1 or 4 to 7) or PD173074 (1 µM: beginning from days 1 to 7) as usual treatments for activation or inhibition of FGFRs, respectively. FGF2-supplementation did not affect the percentage of embryos that developed to the blastocyst, blastocyst cell count and the proportion of cells allocated in inner cell mass (ICM) and trophectoderm (TE) compared to control (p > 0.05). Also, increasing the dosage or duration of FGF2 treatment did not significantly alter blastocyst yield or differential cell count (p > 0.05). PD173074-mediated inhibition of FGFRs did not significantly affect blastocyst yield (p > 0.05). Assessment of expression profiles of lineage-associated markers revealed that FGF2 (500 ng/ml) supplementation: (i) significantly increased expression of putative hypoblast marker (GATA4), (ii) significantly decreased expression of putative epiblast (EPI) marker (NANOG) and (iii) did not change TE markers (CDX2 and IFNT) and pluripotency makers (OCT4, SOX2 and REX1). In summary, FGF2-mediated activation of FGFRs may promote a switch in transcriptional profile of ovine ICM from EPI- to hypoblast-associated gene expression.

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.

Granulosa cell tumors (GCTs) carry a risk of recurrence also at an early stage, but reliable prognostic factors are lacking. We assessed clinicopathological prognostic factors and the prognostic roles of the human epidermal growth factor receptors (HER 2-4) and the transcription factor GATA4 in GCTs. We conducted a long-term follow-up study of 80 GCT patients with a mean follow-up time of 16.8 years. A tumor-tissue microarray was immunohistochemically stained for HER2-4 and GATA4. Expression of HER2-4 mRNA was studied by means of real time polymerase chain reaction and HER2 gene amplification was analyzed by means of silver in situ hybridization. The results were correlated to clinical data on recurrences and survival. We found that GCTs have an indolent prognosis, with 5-year disease-specific survival (DSS) being 97.5%. Tumor recurrence was detected in 24% of the patients at a median of 7.0 years (range 2.6-18 years) after diagnosis. Tumor stage was not prognostic of disease-free survival (DFS). Of the molecular prognostic factors, high-level expression of HER2, and GATA4, and high nuclear atypia were prognostic of shorter DFS. In multivariate analyses, high-level coexpression of HER2 and GATA4 independently predicted DFS (hazard ratio [HR] 8.75, 95% CI 2.20-39.48, P = 0.002). High-level expression of GATA4 also predicted shorter DSS (HR 3.96, 95% CI 1.45-12.57, P = 0.006). In multivariate analyses, however, tumor stage (II-III) and nuclear atypia were independent prognostic factors of DSS. In conclusion HER2 and GATA4 are new molecular prognostic markers of GCT recurrence, which could be utilized to optimize the management and follow-up of patients with early-stage GCTs., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Cardiomyocyte hypertrophy is an adaptive response of the heart to various types of stress. During the period of stress accumulation, the transition from physiological hypertrophy to pathological hypertrophy results in the promotion of heart failure. Gelsolin (GSN) is a member of the actin-binding proteins, which regulate dynamic actin filament organization by severing and capping. Moreover, GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we used H9c2 and H9c2-GSN stable clones in an attempt to understand the mechanisms of GSN overexpression in cardiomyocytes. These data showed that the overexpression of GSN in H9c2-induced cardiac hypertrophy and increased the pathological hypertrophy markers atrial natriuretic peptide brain natriuretic peptide. Furthermore, we found that E-cadherin expression decreased with the overexpression of GSN in H9c2, but β-catenin expression increased. These data presume that the cytoskeleton is loose. Further, previous studies show that the mitogen-activated protein kinase pathway can induce cardiac hypertrophy. Our data showed that p-p38 expression increased with the overexpression of GSN in H9c2, and the transcription factor p-GATA4 expression also increased, suggesting that the overexpression of GSN in H9c2-induced cardiac hypertrophy seemed to be regulated by the p38/GATA4 pathway. Moreover, we used both the p38 inhibitor (SB203580) and GSN siRNA to confirm our conjecture. We found that both of these factors significantly suppressed gelsolin-induced cardiac hypertrophy through p38/GATA4 signaling pathway. Therefore, we predict that the gene silencing of GSN and/or the downstream blocking of GSN along the p38 pathway could be applied to ameliorate pathological cardiac hypertrophy in the future.

The transcription factors GATA-4 and GATA-6, members of the GATA family, play an important role in ovarian cell proliferation, differentiation and apoptosis. In this study, the full-length coding sequences of goose GATA-4 and GATA-6 were cloned and characterized. GATA-4 and GATA-6 consist of 1236 and 1104 nucleotides encoding proteins with 411 and 367 amino acids, respectively. The deduced amino acid sequences of both proteins include two adjacent zinc finger domains with the distinctive form (CVNC-X17-CNAC)-X29-(CANC-X17-CNAC) and share 84.76% identity within this domain. In silico prediction together with matching of the high affinity RRXS(T)Y motif revealed that the GATA-4 protein might be phosphorylated predominantly at S(233), but no phosphorylation site was found in the GATA-6 protein. Real-time quantitative PCR analysis showed that GATA-4 and GATA-6 mRNAs were co-expressed in goose follicles, moderately expressed in granulosa cells and weakly expressed in theca cells. The expression level of GATA-4 mRNA in healthy follicles was significantly higher than in atretic follicles or postovulatory follicles (P<0.01), and the expression level of GATA-6 mRNA in healthy follicles was significantly lower than in atretic follicles or postovulatory follicles (P<0.01). The expression level of GATA-4 mRNA in granulosa cells was downregulated during follicle development; the peak of expression occurred in the 8-10 mm follicles, and the lowest expression was in the F1 follicles. GATA-6 was upregulated and reached its peak expression in the F1 follicles. These results indicate that the molecular structural differences in goose GATA-4 and GATA-6 may be related to their different roles during follicle development.

In the limb bud, patterning along the anterior-posterior (A-P) axis is controlled by Sonic Hedgehog (Shh), a signaling molecule secreted by the "Zone of Polarizing Activity", an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient. Thus, to specifically examine the role of GATA6 in limb patterning we generated Prx1-Cre; GATA6(fl/fl) mice, which conditionally delete GATA6 from their developing limb buds. We found that these animals display ectopic expression of both Shh and its transcriptional targets specifically in the anterior mesenchyme of the hindlimb buds. Loss of GATA6 in the developing limbs results in the formation of preaxial polydactyly in the hindlimbs. Conversely, forced expression of GATA6 throughout the limb bud represses expression of Shh and results in hypomorphic limbs. We have found that GATA6 can bind to chromatin (isolated from limb buds) encoding either Shh or Gli1 regulatory elements that drive expression of these genes in this tissue, and demonstrated that GATA6 works synergistically with FOG co-factors to repress expression of luciferase reporters driven by these sequences. Most significantly, we have found that conditional loss of Shh in limb buds lacking GATA6 prevents development of hindlimb polydactyly in these compound mutant embryos, indicating that GATA6 expression in the anterior region of the limb bud blocks hindlimb polydactyly by repressing ectopic expression of Shh.

Background/purpose: MicroRNA-208a (miR208a) and mechanical stress play a key role in cardiac hypertrophy. The relationship between miR208a and mechanical stress in cultured cardiomyocytes has not been investigated. The molecular mechanisms underlying miR208a-induced hypertrophy of cardiomyocytes by mechanical stress is poorly understood. This study investigated whether miR208a is a critical regulator in cardiomyocyte hypertrophy under mechanical stretch., Methods: Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched at 60 cycles/minute. MiR real-time quantitative assays were used to quantify miRs. A quantitative sandwich enzyme immunoassay technique was used to measure transforming growth factor-β1 (TGF-β1). A (3)H-proline incorporation assay was used to measure protein synthesis., Results: Mechanical stretch significantly enhanced miR208a expression. Stretch significantly induced cardiomyocyte hypertrophic protein expression such as β-myosin heavy chain (MHCβ), thyroid hormone receptor-associated protein 100, myostatin, connexin 40, GATA4, and brain natriuretic peptide. MHCα was not induced by stretch. Overexpression of miR208a significantly increased MHCβ protein expression while pretreatment with antagomir208a significantly attenuated MHCβ protein expression induced by stretch and overexpression of miR208a. Mechanical stretch significantly increased the secretion of TGF-β1 from cultured cardiomyocytes. Exogenous addition of TGF-β1 recombinant protein significantly increased miR208a expression and pretreatment with TGF-β1 antibody attenuated miR208a expression induced by stretch. Mechanical stretch and overexpression of miR208a increased protein synthesis while antagomir208a attenuated protein synthesis induced by stretch and overexpression of miR208a., Conclusion: Cyclic stretch enhances miR208a expression in cultured rat cardiomyocytes. MiR208a plays a role in stretch-induced cardiac hypertrophy. The stretch-induced miR208a is mediated by TGF-β1., (Copyright © 2013. Published by Elsevier B.V.)

In present study, we report on bone marrow (BM) mesenchymal stem cells (MSCs) that are isolated from giant pandas. Cells were collected from the BM of two stillborn giant pandas. The cells were cultured and expanded in 10% fetal bovine serum medium. Cell morphology was observed under an inverted microscopy, and the proliferation potential of the cells was evaluated by counting cell numbers for eight consecutive days. Differentiation potentials of the cells were determined by using a variety of differentiation protocols for osteocytes, adipocytes, neuron cells, and cardiomyocytes. Meanwhile, the specific gene expressions for MSCs or differentiated cells were analyzed by RT-PCR. The isolated cells exhibited a fibroblast-like morphology; expressed mesenchymal specific markers such as cluster of differentiation 73 (CD73), SRY (sex determining region Y)-box 2 (SOX-2), guanine nucleotide-binding protein-like 3 (GNL3), and stem cell factor receptor (SCFR); and could be differentiated into osteocytes and adipocytes that were characterized by Alizarin Red and Oil Red O staining. Under appropriate induction conditions, these cells were also able to differentiate into neuroglial-like or myocardial-like cells that expressed specific myocardial markers such as GATA transcription factors 4 (GATA-4), cardiac troponin T (cTnT), and myosin heavy chain 7B (MYH7B), or neural specific markers such as Nestin and glial fibrillary acidic protein (GFAP). This study demonstrated stem cells recovery and growth from giant pandas. The findings suggest that cells isolated from the BM of giant pandas have a high proliferative capacity and multiple differentiation potential in vitro which might aid conservation efforts.

Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo.

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3'UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3'UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3'UTR of GATA4. ΔMFE ≥4 kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3-0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3'UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.

Human lung cancer is a disease with high incidence and accounts for most cancer-related deaths in both men and women. Metastasis is a common event in non-small cell lung carcinoma (NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified. Here, we focus on GATA4 as a MYC target in the development of metastasis with origin in lung adenocarcinoma, the most common type of NSCLC. Epigenetic alterations at the GATA4 promoter level were observed after MYC expression in lung adenocarcinoma in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. Our results thus identify a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in lung adenocarcinoma, suggesting novel potential targets for the development of antimetastatic therapy.

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and α-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK)1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.

Erythropoietin (EPO), the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and delay myogenic differentiation. However, the underlying mechanism is not yet fully understood. Here, we report that EPO changes the skeletal myogenic regulatory factor expression program and delays differentiation via induction of GATA-4 and the basic helix-loop-helix TAL1 and that knockdown of both factors promotes differentiation. EPO increases the Sirt1 level, a NAD(+)-dependent deacetylase, and also induces the NAD(+)/NADH ratio that further increases Sirt1 activity. Sirt1 knockdown reduced GATA-4 and TAL1 expression, impaired EPO effect on delayed myogenic differentiation, and the Sirt1 knockdown effect was abrogated when combined with overexpression of GATA-4 or TAL1. GATA-4 interacts with Sirt1 and targets Sirt1 to the myogenin promoter and represses myogenin expression, whereas TAL1 inhibits myogenin expression by decreasing MyoD binding to and activation of the myogenin promoter. Sirt1 was found to bind to the GATA-4 promoter to directly regulate GATA-4 expression and GATA-4 binds to the TAL1 promoter to regulate TAL1 expression positively. These data suggest that GATA-4, TAL1, and Sirt1 cross-talk each other to regulate myogenic differentiation and mediate EPO activity during myogenic differentiation with Sirt1 playing a role upstream of GATA-4 and TAL1. Taken together, our findings reveal a novel role for GATA-4 and TAL1 to affect skeletal myogenic differentiation and EPO response via cross-talk with Sirt1.

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3' untranslated region (3'-UTR) of the flavivirus genome, in particular the terminal 3' stem-loop (3'SL) fulfils multiple functions in virus replication and virus-host interactions. Using the Kunjin strain of WNV (WNV(KUN)), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3'SL. Transcription of WNV(KUN) pre-miRNA (3'SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNV(KUN) virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.

Background: The high death rate of the transplanted stem cells in the infarcted heart and low efficiency of differentiation toward cardiomyocytes show that mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) is not effective. Csx/Nkx2.5 and GATA-4 are considered to be key regulators of cardiogenesis. The aim of the present study was to investigate the effect of transplanting MSC overexpressing Csx/Nkx2.5 and GATA-4 (MSCs-CG) after MI., Methods and Results: According to acridine orange/ethidium bromide staining, MSCs-CG were more resistant to anoxia as compared with MSCs in vitro. In a mouse MI model, ejection fraction and fractional shortening were higher in the MSC-CG group than in the MSC or phosphate-buffered saline group. Wall thickness of the infarct area was increased and collagen deposition was clearly reduced in the MSC-CG group as compared with the other groups. There were more surviving MSCs in the MSC-CG group than in the MSC group. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). Cx-43 was localized between Y chromosome-positive cells and recipient cardiomyocytes. Microvessel density in the peri-infarct regions and infarct regions increased significantly in the MSC-CG group., Conclusions: Transplantation of MSCs overexpressing Csx/Nkx2.5 and GATA-4 represents a new treatment strategy with the potential to improve cardiac function after MI.

Doxorubicin (DOX) is one of the most effective anticancer drugs. However, its cardiotoxicity remains a clinical concern that severely restricts its therapeutic usage. We designed this study to investigate whether tadalafil, a long-acting phosphodiesterase-5 (PDE-5) inhibitor, protects against DOX-induced cardiotoxicity. We also sought to delineate the cellular and molecular mechanisms underlying tadalafil-induced cardioprotection. Male CF-1 outbred mice were randomized into three groups (n = 15-24/group) to receive either saline (0.2 ml i.p.), DOX (15 mg/kg, given by a single intraperitoneal injection), or tadalafil (4 mg/kg p.o. daily for 9 days) plus DOX. Left ventricular function was subsequently assessed by transthoracic echocardiography and Millar conductance catheter. Cardiac contractile function was impaired by DOX, and it was significantly improved by cotreatment with tadalafil. Tadalafil attenuated DOX-induced apoptosis and depletion of prosurvival proteins, including Bcl-2 and GATA-4, in myocardium. Cardiac oxidative stress was attenuated and antioxidant capacity was enhanced by tadalafil possibly via up-regulation of mitochondrial superoxide dismutase (MnSOD). Moreover, the tadalafil-treated group demonstrated increased cardiac cGMP level and protein kinase G (PKG) activity. Tadalafil did not interfere with the efficacy of DOX in killing human osteosarcoma cells in vitro or its antitumor effect in vivo in tumor xenograft model. We conclude that tadalafil improved left ventricular function and prevented cardiomyocyte apoptosis in DOX-induced cardiomyopathy through mechanisms involving up-regulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of DOX.

The corpus luteum (CL) is the major site of progesterone (P4) production during the luteal phase of the estrous cycle in cattle. To better understand the molecular mechanisms underlying P4 production, we compared the mRNA and protein expression profiles of key components of the steroidogenic pathway (StAR, CYP11A, and 3beta-HSD) during the bovine CL luteal phase with that of several transcription factors (NR5A1, NR5A2, GATA4, GATA6) known for their roles in the control of steroidogenic gene expression. In the bovine CL, StAR, CYP11A, and 3beta-HSD mRNA and protein levels remained constant at the mid and late luteal phases but markedly declined at the regressed luteal stage. NR5A1 and NR5A2 exhibited a similar pattern with a significant decrease in expression at the regressed luteal stage. Both GATA4 and GATA6 mRNA and proteins could be detected in bovine CL; GATA6 levels, however, were generally higher. Although GATA4 expression did not change during the luteal phase, GATA6 showed a marked decrease at the regressed luteal stage, like NR5A1, NR5A2, and the other steroidogenic markers. Thus, we suggest that NR5A1, NR5A2, and GATA6, but not GATA4, contribute to the transcriptional regulation of steroidogenic gene expression, and hence P4 production, in the bovine CL. Furthermore, we have demonstrated the association of NR5A1 and NR5A2 with the bovine StAR promoter in the mid-luteal CL using chromatin immunoprecipitation, suggesting that these factors have definitive roles in the regulation of StAR gene transcription in vivo.

Background: Transforming growth factor beta 1 (TGF-beta1) promotes epithelial healing in inflammatory bowel disease. We hypothesized that GATA-4, a transcription factor cooperating with TGF-beta signaling pathway, is upregulated by TGF-beta1 in the inflamed intestinal epithelium., Methods: Normal and inflamed intestinal samples were subjected to immunohistochemistry for GATA-4/6 and the TGF-beta signaling pathway components Smad2/3/4. Proliferation and apoptosis were analyzed using Ki-67 and in situ DNA 3'-end labeling assays and Bax and Bcl-2 immunohistochemistry. Furthermore, GATA-4 was assessed in intestinal Caco-2 cells stimulated with TGF-beta1, or interleukin-6 and tumor necrosis factor alpha., Results: GATA-4 was detected in only 20% of normal intestinal samples, but was upregulated in corresponding inflamed regions. GATA-6 expression remained unchanged during inflammation. TGF-beta1 and Smad3/4, but not Smad2, were expressed concomitantly with GATA-4 in inflamed bowel mucosa. In intestinal Caco-2 cells, TGF-beta1 upregulated GATA-4 and Smad2/3/4, whereas treatment with control cytokines had no effect. Inflammation was associated with increased epithelial cell apoptosis and the enhancement of Bcl-2, but not Bax., Conclusions: We surmise GATA-4 expression is upregulated in inflamed intestine correlating with the activation of TGF-beta signaling pathway. We speculate that TGF-beta1 drives GATA-4 expression during intestinal inflammation, these two components cooperating to promote epithelial healing.

Overexpression of the ERBB2 gene, linked to genomic and transcriptional amplifications, is a poor prognosis indicator in 25% to 30% of breast cancers. In contrast to some well-documented genomic amplifications, molecular mechanisms leading to ERBB2 transcriptional overexpression remain poorly characterized. Gene expression analyses of breast cancer have characterized distinct transcriptional signatures allowing a molecular classification of breast carcinoma. Coexpression of the ERBB2 and GATA4 genes was originally observed in tumors. Both genes are essential for cardiovascular development and GATA4 has been proposed to control the transcription of critical genes for the differentiation and the function of myocardium. We determined that ERBB2-targeted small interfering RNA repressed both ERBB2 and GATA4 genes, whereas GATA4-targeted small interfering RNA repressed GATA4 and activated ERBB2 transcription. Transfected GATA4-expressing construct repressed ERBB2 promoter. Phylogenetic foot printing revealed multiple putative GATA4 binding sites conserved in mammals within the ERBB2 promoter region. Chromatin immunoprecipitation showed that GATA4 binds specifically to several ERBB2 gene noncoding regions. Electrophoretic mobility shift assay revealed GATA4 binding to a well-conserved consensus motif. Site-directed mutagenesis confirmed the role of this new regulatory element for the activity of the ERBB2 gene enhancer. In agreement with a repressor role of GATA4 on ERBB2 gene expression balanced by ERBB2 activation of the GATA4 gene, a negative correlation between the relative levels of ERBB2 and GATA4 mRNA was observed in breast cancer cell lines and breast tumor samples. We propose that the negative feedback loop linking ERBB2 and GATA4 plays a role in the transcriptional dysregulation of ERBB2 gene expression in breast cancer.

Background & Aims: Although the zinc-finger transcription factor GATA4 has been implicated in regulating jejunal gene expression, the contribution of GATA4 in controlling jejunal physiology has not been addressed., Methods: We generated mice in which the Gata4 gene was specifically deleted in the small intestinal epithelium. Measurements of plasma cholesterol and phospholipids, intestinal absorption of dietary fat and cholesterol, and gene expression were performed on these animals., Results: Mice lacking GATA4 in the intestine displayed a dramatic block in their ability to absorb cholesterol and dietary fat. Comparison of the global gene expression profiles of control jejunum, control ileum, and GATA4 null jejunum by gene array analysis revealed that GATA4 null jejunum lost expression of 53% of the jejunal-specific gene set and gained expression of 47% of the set of genes unique to the ileum. These alterations in gene expression included a decrease in messenger RNAs (mRNAs) encoding lipid and cholesterol transporters as well as an increase in mRNAs encoding proteins involved in bile acid absorption., Conclusions: Our data demonstrate that GATA4 is essential for jejunal function including fat and cholesterol absorption and confirm that GATA4 plays a pivotal role in determining jejunal vs ileal identity.

Background: In the mouse, the parenchyma of both the liver and ventral pancreas is specified from adjacent domains of the ventral foregut endoderm. GATA4, a zinc finger transcription factor, is strongly expressed in these endodermal domains and molecular analyses have implicated GATA4 in potentiating liver gene expression during the onset of hepatogenesis. We therefore hypothesized that GATA4 has an integral role in controlling the early stages of pancreatic and liver development., Results: To determine whether GATA4 contributes to development of either the pancreas or liver we characterized the formation of pancreatic and hepatic tissues in embryos derived from Gata4-/- ES cells by tetraploid embryo complementation. In the absence of GATA4, development of the liver and ventral pancreas was disrupted. At embryonic day (E) 9.5, the liver bud failed to expand although, contrary to expectations, the hepatic endoderm was able to form a pseudo-stratified epithelial liver bud that expressed hepatic genes. Moreover, as we had shown previously, the embryos lacked septum transversum mesenchyme suggesting that liver defects may be cell non-autonomous. Analyses of pancreatic development revealed a complete absence of the ventral but not the dorsal pancreas in Gata4-/- embryos. Moreover, Gata6-/- embryos displayed a similar, although less dramatic phenotype, suggesting a critical role for multiple GATA factors at the earliest stages of ventral pancreas development., Conclusion: This study defines integral roles for GATA factors in controlling early development of the mammalian liver and pancreas.

GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5'-untranslated region (5'-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (-65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5'-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5'-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca(2+) levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca(2+) stores and PKC.

Cardiomyocyte regeneration is limited in adult life and is not sufficient to compensate for cell loss with myocardial infarction. Hence, the identification of a useful source of cardiomyocyte progenitors is of great interest for possible use in regenerative therapy. In this study, we isolated stem cells derived from human subcutaneous adipose tissue. The expression of Nkx2.5 and GATA-4 can be observed by PCR directly after extraction and during cultivation in some of these cells. Cardiac Troponin T and myosin light chain-2v become positive after 12 days of cultivation. To define respective factors responsible for spontaneous differentiation, we measured VEGF level in ADSC conditioned medium. Our data showed that ADSC secrete significant amount of VEGF (283.5pg per microgram DNA) and that anti-VEGF receptor antibodies blocked the cardiac differentiation. In conclusion, we demonstrated the spontaneous differentiation of human subcutaneous adipose-derived stem cells into a cardiomyocyte phenotype under standard culturing conditions.

Objective: To investigate the effect of cardiotrophin-1 (CT-1) on the GATA4 expression and related signaling pathways (JAK-STAT3, ERK1/2 and PI3-K) in rat cardiomyocytes., Methods: Using semi-quantitative RT-PCR and EMSA, we measured the dose and time dependent effects of CT-1 on GATA4 mRNA and binding activity in cultured rat cardiomyocytes. Parthenolide (a STAT inhibitor), U-0126 (an ERK inhibitor) and LY-294002 (a PI3-K inhibitor) alone or in combination were added to the culture medium to assess the role of above signaling pathways in CT-1 mediated effects., Results: GATA4 mRNA expression significantly increased at 3 h post 0.1 nmol/L CT-1 exposure, peaked at 6 h and remained high till 24 h post exposure. The GATA4 binding activity began to increase at 10 min and peaked at 60 min and returned to baseline level 180 min. Six hours post CT-1 (0.01 nmol/L, 0.1 nmol/L, 1 nmol/L) exposure, the GATA4 mRNA expression increased in a dose-dependent manner. The GATA4 binding activity peaked with 0.1 nmol/L CT-1 and higher dose did not further increase the binding activity. U-0126 increased the GATA4 mRNA expression and enhanced the GATA4 binding activity and these effects could be partially attenuated with addition of Parthenolide. Parthenolide also prevented the increase of GATA4 mRNA and binding activity induced by CT-1. LY-294002 had no effects GATA4 mRNA and binding activity., Conclusion: CT-1 increases the GATA4 mRNA expression and binding activity in rat cardiomyocytes via STAT3/ERK1/2 pathways and these effects are independent of PI3-K pathway.

Objective: Adult stem cells have been detected in several human tissues. The object of this study was to investigate whether they also occur in the human thyroid gland., Design: The expression of the stem cell marker Oct- 4 and the early endodermal markers GATA-4 and HNF4alpha was analyzed in histologic slides and cultured cells derived from goiters, in the FRTL5 cell line, and the HTh74, HTC, C643, and XTC133 thyroid carcinoma cell lines., Main Outcome: Stem cell markers were detectable in all primary cultures whereas in the differentiated FRTL5 cell line no expression was observed. Expression of stem cell marker mRNA was not affected by thyrotropin (TSH) stimulation and did not decrease when cells underwent several passages. Immunostaining of cultured cells and of histologic slides of goitrous tissues showed only single cells that were immunoreactive for Oct-4, GATA-4, and HNF4a. Expression of Oct-4 but not of endodermal marker GATA-4 was also detectable in some thyroid carcinoma cell lines. Fluorescence-activated cell sorter (FACS) analysis demonstrated cell populations that were positive for either Oct-4, GATA-4, or HNF4alpha but negative for thyroglobulin. When these putative, FACS-sorted stem cell populations were further analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), expression of all stem cell markers and of Pax8 but not of thyroglobulin mRNA was detectable., Conclusions: These data provide evidence for the presence of adult stem and precursor cells of endodermal origin in the human thyroid gland.

Aim: To investigate effects of icariin on cardiac gene expression and the modulation of nitric oxide (NO) signal transduction during the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro., Methods: The expression levels of cardiac developmental-dependent genes were measured using reverse transcription-polymerase chain reaction (RT-PCR). The chronotropic responses of cardiomyocytes to b-adrenoceptor stimulation were determined. The levels of cAMP and cGMP in ES cells were measured using radioimmunoassay. Endogenous NO levels were measured by using the Griess reaction. Aminoguanidine (AG) was used to confirm the influence of icariin on the endogenous NO signal pathway., Results: Icariin significantly elevated mRNA levels of cardiac transcription factors GATA4 and Nkx2.5, and cardiac-specific alpha-MHC, MLC-2v and beta-AR genes in a concentration- and time-dependent manner (P<0.05). Cardiomyocytes derived from embryoid body (EB) treated with icariin were more sensitive to isoprenaline (P<0.01). Treatment of ES cells with icariin resulted in a continued elevation in the cAMP/cGMP ratio before a shift to the cardiomyocyte phenotype (P<0.05). AG decreased the NO level, and delayed and decreased the incidence of contracting EB to only approximately 35% on d 5+11, an effect that could be rescued by icariin. When cells were cocultured with icariin and AG, the percentage of beating EB reached a peak level of 73% on d 5+11 (P<0.05)., Conclusion: The inducible effects of icariin were partly related to increase in the expression of cardiac developmental-dependent genes, and elevation of the cAMP/cGMP ratio in ES cells, as well as upregulation of endogenous NO generation during the early stages of cardiac development.

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.

Proteins of the LIM family play important roles in a variety of fundamental biological processes including cell lineage specification and organ development. Here we examined the function in cardiogenesis of a new member of the LIM family, hhLIM, by molecular analysis of early stages of cardiomyocyte differentiation in hhLIM-deficient P19 cell line and P19 cells stably overexpressing hhLIM. The results indicate that hhLIM is a potent transcriptional activator of several cardiac muscle-specific genes. Inhibition of hhLIM expression by antisense transcripts can interfere with expression of cardiac muscle genes and block development of beating cardiomyocytes in P19 embryonic stem cells. Overexpression of hhLIM in P19 cells can enhance expression of cardiac marker genes Nkx2.5 and GATA-4 and potentiate development of cardiomyocyte-like morphology. These findings suggest that, in addition to its role in activation of the cardiac genetic program, hhLIM may be the nuclear target of inductive factor for precardiac cells.

Disaccharidases are important digestive enzymes whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression, either by impairment to enterocyte differentiation or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron-deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested as well as the activity of intestinal alkaline phosphatase (IAP)-II because it is unrelated to carbohydrate digestion. mRNA levels of beta-actin, sucrase, lactase, and the associated transcription factors pancreatic duodenal homeobox (PDX)-1, caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridization, respectively. It was found that iron-deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower) but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9%, respectively, by iron deficiency, suggesting that enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5-fold higher in iron deficiency, whereas CDX-2, GATA-4, and HNF-1 levels were not significantly different. These data suggest that decreases in sucrase and lactase activities result from a reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.

Objective: To explore the effect of the microenvironment induced by damaged mouse hepatic cells on the conversion of human umbilical cord blood-derived cells into hepatocyte-like cells., Methods: A hepatic injury-like microenvironment was mimicked using carbon tetrachloride damaged mouse hepatic cells, where mononuclear cells (MNC) from human umbilical cord blood were cultured in a compartment separated by trans-well membrane. Histochemical staining, reversed transcription-polymerase chain reaction (RT-PCR) and gene sequencing were performed for the information on the conversion of human umbilical cord blood MNC., Results: A number of PAS positive stained cells in MNC co-cultured with damaged mouse hepatic cells were observed after 72 h. Cells expressing mature hepatocyte markers, human albumin (hALB) and human GATA-4 (hGATA-4) mRNA were detected by RT-PCR, which was further confirmed with sequencing. Relevant control groups, MNC co-cultured with normal mouse hepatic cells and MNC cultured alone remained negative., Conclusion: The culture system using damaged mouse hepatic cells as stimulator could be a potential in vitro system for the conversion of human umbilical cord blood-derived cells into hepatocyte-like cells.