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Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tissue-resident macrophages are highly heterogeneous in terms of their functions and phenotypes as a consequence of adaptation to different tissue environments. Local tissue-derived signals are thought to control functional polarization of resident macrophages; however, the identity of these signals remains largely unknown. It is also unknown whether functional heterogeneity is a result of irreversible lineage-specific differentiation or a consequence of continuous but reversible induction of diverse functional programs. Here, we identified retinoic acid as a signal that induces tissue-specific localization and functional polarization of peritoneal macrophages through the reversible induction of transcription factor GATA6. We further found that GATA6 in macrophages regulates gut IgA production through peritoneal B-1 cells. These results provide insight into the regulation of tissue-resident macrophage functional specialization by tissue-derived signals., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Piceatannol is found in grapes, passion fruit, and Japanese knotweed. Piceatannol pretreatment suppresses cardiac hypertrophy induced by isoproterenol as assessed by heart weight/body weight ratio, cross-sectional area, and expression of hypertrophic markers. The anti-hypertrophic effect of piceatannol in rat neonatal cardiomyocytes is the same as that in vivo. Piceatannol inhibits lentiviral-GATA6-induced cardiomyocyte hypertrophy. Furthermore, piceatannol reduces the interaction between GATA4 and GATA6 as well as the DNA-binding activity of endogenous GATA6 in the ANP promoter. Our results suggest that piceatannol may be a novel therapeutic agent for the prevention of cardiac hypertrophy., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate the expression of Dickkopf-1 and GATA-6 in laryngeal carcinoma and to discuss their relevance and the roles in carcinogenesis and development of laryngeal carcinoma., Method: Immunohistochemical technique was used to detect the expression of Dickkopf-1 and GATA-6 protein in 48 tissues of larynge al carcinoma, 48 para-carcinoma tissues and 20 normal laryngeal mucosal tissues., Result: (1) The expression of Dick kopf-1 protein in laryngeal cancer is significantly lower than in para-carcinoma tissues and normal laryngeal mucosa tissues (P < 0.05). (2) The expression of GATA-6 protein in laryngeal cancer is significantly higher than in para-carcinoma tissues and normal laryngeal mucosa tissues (P < 0.05). (3) The expression of Dickkopf-1 and GATA-6 protein in laryngeal cancer is correlated with lymph node metastasis, clinical stage, histological grade (P < 0.05). (4) The expression of Dickkopf-1 and GATA-6 are negatively correlated in laryngeal cancer., Conclusion: The expression of Dickkopf-1 and GATA-6 may contribute to the carcinogenesis and development of laryngeal carcinoma.

Background: The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2(-/-))., Results: We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2(-/-) mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression., Conclusions: Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.

Background: GATA transcription factors are essential for self-renewal of the small intestinal epithelium. Gata4 is expressed in the proximal 85% of small intestine while Gata6 is expressed throughout the length of small intestine. Deletion of intestinal Gata4 and Gata6 results in an altered proliferation/differentiation phenotype, and an up-regulation of SAM pointed domain containing ETS transcription factor (Spdef), a transcription factor recently shown to act as a tumor suppressor. The goal of this study is to determine to what extent SPDEF mediates the downstream functions of GATA4/GATA6 in the small intestine. The hypothesis to be tested is that intestinal GATA4/GATA6 functions through SPDEF by repressing Spdef gene expression. To test this hypothesis, we defined the functions most likely regulated by the overlapping GATA6/SPDEF target gene set in mouse intestine, delineated the relationship between GATA6 chromatin occupancy and Spdef gene regulation in Caco-2 cells, and determined the extent to which prevention of Spdef up-regulation by Spdef knockout rescues the GATA6 phenotype in conditional Gata6 knockout mouse ileum., Results: Using publicly available profiling data, we found that 83% of GATA6-regulated genes are also regulated by SPDEF, and that proliferation/cancer is the function most likely to be modulated by this overlapping gene set. In human Caco-2 cells, GATA6 knockdown results in an up-regulation of Spdef gene expression, modeling our mouse Gata6 knockout data. GATA6 occupies a genetic locus located 40 kb upstream of the Spdef transcription start site, consistent with direct regulation of Spdef gene expression by GATA6. Prevention of Spdef up-regulation in conditional Gata6 knockout mouse ileum by the additional deletion of Spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression., Conclusion: SPDEF is a key, immediate downstream effecter of the crypt cell proliferation function of GATA4/GATA6 in the small intestine.

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.

The majority of the cases diagnosed as primary aldosteronism (PA) are caused by aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). Histopathologically, both IHA and adjacent adrenal glands of APA demonstrate remodeled subcapsular zone (RSZ) but these zones in two disorders are markedly different in terms of steroidogenesis. 3β-Hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) expression has been known to be activated synergistically by GATA6 and SF1, and repressed by DAX1 through abolishing the activation. Nerve growth factor-induced clone B (NGFIB) is also known as one of the transcription factors to bind to and activate 3β-HSD promoter. The results of our immunohistochemical analysis demonstrated the expression levels of 3β-HSD in RSZ of IHA were higher than in RSZ of adjacent adrenals of APA, while those in the zona glomerulosa (ZG) of normal adrenal gland (NA) were in between these two RSZs. The expression levels of GATA6, SF1 and DAX1 did not prominently differ among these three types of adrenals, especially between in RSZs of IHA and APA cases, indicating the marked difference of 3β-HSD expression was unlikely to be explained by the levels of these three factors. However, the levels of NGFIB expression were significantly higher in RSZ of IHA than in RSZ of adjacent adrenals of APA and the ZG of NA (P<0.05), which may partly account for the expression levels of 3β-HSD among the three groups of adrenals. These results may imply NGFIB plays important roles in the marked differences in steroidogenic functions in the two distinct types of RSZ of PA cases.

Aberrant activation of Wnt signalling results in colorectal tumours. Lgr5 is specifically expressed in stem cells of the intestine and has an essential role in maintaining tissue homeostasis. Lgr5-positive stem cells are responsible for the intestinal adenoma initiated by mutations in adenomatous polyposis coli. Furthermore, Lgr5 interacts with R-spondins and thereby activates Wnt signalling. However, the function of Lgr5 in colorectal tumourigenesis is unclear. Here we show that LGR5 is required for the tumourigenicity of colorectal cancer cells. We show that the transcription factor GATA6 directly enhances the expression of LGR5. We further demonstrate that GATA6 is upregulated in colorectal cancer cells due to the downregulation of miR-363, which directly targets GATA6. Moreover, we show that overexpression of miR-363 suppresses the tumourigenicity of colorectal cancer cells. These results suggest that the miR-363-GATA6-LGR5 pathway is critical for colorectal tumourigenesis and would be a promising target for the treatment of colorectal cancer.

The transcriptional code that programs cardiac hypertrophy involves the zinc finger-containing DNA binding factors GATA-4 and GATA-6, both of which are required to mount a hypertrophic response of the adult heart. Here we performed conditional gene deletion of Gata4 or Gata6 in the mouse heart in conjunction with reciprocal gene replacement using a transgene encoding either GATA-4 or GATA-6 in the heart as a means of parsing dosage effects of GATA-4 and GATA-6 versus unique functional roles. We determined that GATA-4 and GATA-6 play a redundant and dosage-sensitive role in programming the hypertrophic growth response of the heart following pressure overload stimulation. However, non-redundant functions were identified in allowing the heart to compensate and resist heart failure after pressure overload stimulation, as neither Gata4 nor Gata6 deletion was fully rescued by expression of the reciprocal transgene. For example, only Gata4 heart-specific deletion blocked the neoangiogenic response to pressure overload stimulation. Gene expression profiling from hearts of these gene-deleted mice showed both overlapping and unique transcriptional codes, which is presented. These results indicate that GATA-4 and GATA-6 play a dosage-dependent and redundant role in programming cardiac hypertrophy, but that each has a more complex role in maintaining cardiac homeostasis and resistance to heart failure following injury that cannot be compensated by the other.

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. Despite sharing the common property of pluripotency, hESCs are notably distinct from epiblast cells of the preimplantation blastocyst. Here we use a combination of three small-molecule inhibitors to sustain hESCs in a LIF signaling-dependent hESC state (3iL hESCs) with elevated expression of NANOG and epiblast-enriched genes such as KLF4, DPPA3, and TBX3. Genome-wide transcriptome analysis confirms that the expression signature of 3iL hESCs shares similarities with native preimplantation epiblast cells. We also show that 3iL hESCs have a distinct epigenetic landscape, characterized by derepression of preimplantation epiblast genes. Using genome-wide binding profiles of NANOG and OCT4, we identify enhancers that contribute to rewiring of the regulatory circuitry. In summary, our study identifies a distinct hESC state with defined regulatory circuitry that will facilitate future analysis of human preimplantation embryogenesis and pluripotency., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hepcidin is an antimicrobial peptide hormone that plays a central role in the metabolism of iron and its expression in the liver can be induced through two major pathways: the inflammatory pathway, mainly via IL-6; and the iron-sensing pathway, mediated by BMP-6. GATA-proteins are group of evolutionary conserved transcriptional regulators that bind to the consensus motif-WGATAR-in the promoter region. In hepatoma cells, GATA-proteins 4 and 6 in conjunction with the co-factor friend of GATA (FOG) were shown to modulate the transcription of HAMP. However, it is unclear as to which of the GATA-proteins drive the expression of HAMP in vivo. In this study, using in vitro and in vivo approaches, we investigated the relevance of GATA and FOG proteins in the expression of hepcidin following treatment with IL-6 and BMP-6. We found that treatment of Huh7 cells with either IL-6 or BMP-6 increased the HAMP promoter activity. The HAMP promoter activity following treatment with IL-6 or BMP-6 was further increased by co-transfection of the promoter with GATA proteins 4 and 6. However, co-transfection of the HAMP promoter with FOG proteins 1 or 2 repressed the promoter response to treatments with either IL-6 or BMP-6. The effects of both GATA and FOG proteins on the promoter activity in response to IL-6 or BMP-6 treatment were abrogated by mutation of the GATA response element-TTATCT-in the HAMP promoter region -103/-98. In vivo, treatment of mice with lipopolysaccharide led to a transient increase of Gata-6 expression in the liver that was positively correlated with the expression of hepcidin. Our results indicate that during inflammation GATA-6 is up-regulated in concert with hepcidin while GATA-4 and FOG (1 and 2) are repressed.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis., (© 2013 Wiley Periodicals, Inc.)

Knockdown of the transcription factors GATA4 and GATA6 in granulosa cells (GCs) impairs folliculogenesis and induces infertility. To investigate the pathways and genes regulated by these factors, we performed microarray analyses on wild-type GCs or GCs lacking GATA4, GATA6, or GATA4/6 (G4(gcko), G6(gcko), and G4/6(gcko)) after in vivo treatment with equine chorionic gonadotropin. GATA4 deletion affected a greater number of genes than GATA6, which correlates with the subfertility observed in G4(gcko) mice and the normal reproductive function found in G6(gcko) animals. An even greater number of genes were affected by the deletion of both factors. Moreover, the expression of FSH receptor, LH receptor, inhibin α and β, versican, pregnancy-associated plasma protein A, and the regulatory unit 2b of protein kinase A, which are known to be crucial for ovarian function, was greatly affected in double GATA4 and GATA6 knockouts when compared with single GATA-deficient animals. This suggests that GATA4 and GATA6 functionally compensate for each other in the regulation of key ovarian genes. Functional enrichment revealed that ovulation, growth, intracellular signaling, extracellular structure organization, gonadotropin and growth factor actions, and steroidogenesis were significantly regulated in G4/6(gcko) mice. The results of this analysis were confirmed using quantitative polymerase chain reaction, immunohistochemical, and biological assays. Treatment of GCs with cAMP/IGF-I, to bypass FSH and IGF-I signaling defects, revealed that most of the affected genes are direct targets of GATA4/6. The diversity of pathways affected by the knockdown of GATA underscores the important role of these factors in the regulation of GC function.

Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.

Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.

In this study, we isolated and characterized spontaneously differentiated human embryonic stem cells (SD-hESCs) found in hESC colonies in comparison to the morphologically premature ESCs in the colonies to investigate the potential role of SD-hESCs in embryogenesis. SD-hESCs were distinguished from undifferentiated hESCs by their higher expression of GATA6, a marker for primitive endoderm and transthyretin, a marker visceral endoderm in embryoid bodies (EBs). SD-hESCs expressed OCT4 and NANOG, markers for pluripotent stem cells, at significantly lower levels than undifferentiated hESCs. EBs derived from isolated SD-hESCs were morphologically distinct from cells directly derived from the undifferentiated hESCs; they contained higher number of cysts compared to EBs from undifferentiated hESC-derived EBs (42% vs. 20%). Furthermore, the extracellular signal molecule, BMP2/4, induced a higher GATA4/6 expression and cystic EB formation than control and noggin-treated EBs. Since cystic formation in EBs play a role in primitive endoderm formation during embryogenesis, the SD-hESC may be a relevant cell type equipped to differentiate into primitive endoderm. Our results suggest that SD-ESCs generated during routine hESC culture are not just an artifact of in vitro culture and these cells could serve as a useful model to study the process of embryogenesis.

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real-time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α-MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL-2-antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA-6 or peroxisome proliferator-activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α-MHC, and GATA-4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation., (© 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.)

Background/aims: Pancreatic cancer has the worst prognosis of any gastrointestinal cancer with a mortality rate approaching its incidence. Previous studies have indicated that GATA6 plays a key role in organ development and function, and that abnormal expression of GATA6 may induce tumorigenesis. Meanwhile, it has been reported that generation of reactive oxygen species contributes to carcinogenesis. In this study, we set out to study the role of GATA6 expression on proliferation and apoptosis of pancreatic cancer cells and the role of reactive oxygen species., Methods: Four target miRNA sequences against GATA6 mRNA were synthesized and used to transfect SW1990 cells. Then, GATA6 expression in SW1990 cells was examined by western blot and quantative real-time polymerase chain reaction. Cell proliferation was examined by WST-8 and colony formation assay. Cell cycle progression and apoptosis were measured by flow cytometry. We also measured the generation of reactive oxygen species by immunofluorescence and flow cytometry., Results: RNA interference against GATA6 successfully inhibited mRNA and protein expression of GATA6 in the SW1990 pancreatic cancer cell line. Silencing of GATA6 by RNA interference inhibited cell proliferation and increased apoptosis of SW1990, and enhanced the expression of reactive oxygen species., Conclusions: These results suggest that the RNA interference approach against GATA6 may be an effective therapeutic approach for treatment of pancreatic cancer.

Liver metastasis is a major cause of mortality for colorectal cancer (CRC). However, the underlying mechanisms remain largely unknown. GATA binding protein 6 (GATA6), a zinc-finger transcription factor, is expressed in the colorectal epithelium. We investigated the clinical significance of GATA6 and its role in invasion and metastasis in CRC. Expression of GATA6 in 89 cancerous, 35 adjacent normal and 39 liver metastatic samples from 89 CRC patients undergoing surgical resection was detected by immunohistochemical (IHC) methods. The effect of GATA6 on invasion and metastasis was assessed in CRC cells by shRNA lentivirus or expressed-plasmid transfection. We found that GATA6 expression was significantly higher in liver metastatic tissues compared with adjacent normal tissues. Aberrant GATA6 expression in CRC was associated with liver metastasis. Kaplan-Meier analysis showed GATA6 expression correlated with poor overall survival (OS) in CRC. The cell invasion and migration of established CRC cell lines were decreased by GATA6 knockdown and enhanced by GATA6 overexpression in vitro. Thus, aberrant expression of GATA6 correlates with poor prognosis and liver metastasis in CRC.

Molecular programs that mediate normal cell differentiation are required for oncogenesis and tumor cell survival in certain cancers. How cell-lineage-restricted genes specifically influence metastasis is poorly defined. In lung cancers, we uncovered a transcriptional program that is preferentially associated with distal airway epithelial differentiation and lung adenocarcinoma (ADC) progression. This program is regulated in part by the lineage transcription factors GATA6 and HOPX. These factors can cooperatively limit the metastatic competence of ADC cells, by modulating overlapping alveolar differentiation and invasogenic target genes. Thus, GATA6 and HOPX are critical nodes in a lineage-selective pathway that directly links effectors of airway epithelial specification to the inhibition of metastasis in the lung ADC subtype., (Copyright © 2013 Elsevier Inc. All rights reserved.)

At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2-3 weeks.

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Transcription factor GATA6 is expressed in the fetal and adult adrenal cortex and has been implicated in steroidogenesis. To characterize the role of transcription factor GATA6 in adrenocortical development and function, we generated mice in which Gata6 was conditionally deleted using Cre-LoxP recombination with Sf1-cre. The adrenal glands of adult Gata6 conditional knockout (cKO) mice were small and had a thin cortex. Cytomegalic changes were evident in fetal and adult cKO adrenal glands, and chromaffin cells were ectopically located at the periphery of the glands. Corticosterone secretion in response to exogenous ACTH was blunted in cKO mice. Spindle-shaped cells expressing Gata4, a marker of gonadal stroma, accumulated in the adrenal subcapsule of Gata6 cKO mice. RNA analysis demonstrated the concomitant upregulation of other gonadal-like markers, including Amhr2, in the cKO adrenal glands, suggesting that GATA6 inhibits the spontaneous differentiation of adrenocortical stem/progenitor cells into gonadal-like cells. Lhcgr and Cyp17 were overexpressed in the adrenal glands of gonadectomized cKO vs control mice, implying that GATA6 also limits sex steroidogenic cell differentiation in response to the hormonal changes that accompany gonadectomy. Nulliparous female and orchiectomized male Gata6 cKO mice lacked an adrenal X-zone. Microarray hybridization identified Pik3c2g as a novel X-zone marker that is downregulated in the adrenal glands of these mice. Our findings offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells.

Objective: Barrett's oesophagus (BE) is a metaplastic condition of the distal oesophagus which predisposes to oesophageal adenocarcinoma (EAC). It has been suggested that microRNAs (miRNAs) are involved in the process of development of BE and EAC; however, few functional miRNA data are available. The aim of the study was to perform a tissue-specific miRNA profile and, based on this, to examine the function of miRNA-145 in the oesophagus., Design: miRNA expression profiling using microarray analysis in EAC, BE and normal squamous epithelium of the oesophagus (SQ) was performed and validated using real-time PCR in samples from 15 patients and in situ hybridisation in samples from 10 patients. The proliferative effect of miRNA-145 precursor transfection in the SQ (HET-1A) and BE cell line (BAR-T) was measured. Downstream targets of miRNA-145 were determined by analysing mRNA and protein expression from miRNA-145 transfected cells., Results: Three unique miRNA expression profiles were found in tissue from EAC, BE and SQ, which showed that miRNA-145 was upregulated in BE compared with EAC and SQ. Overexpression of miRNA-145 in HET-1A and BAR-T cells reduced cell proliferation and inhibited GATA6, BMP4 and SOX9 mRNA expression. Furthermore, altered BMP4 signalling was observed in vitro on miRNA-145 overexpression. These effects were blocked when cells were co-transfected with a miRNA-145 specific inhibitor. Additionally, BMP4 incubation of HET-1A cells altered miRNA-145 and GATA6 expression over time., Conclusion: These results imply that miRNA-145 indirectly targets BMP4 via GATA6 and is potentially involved in the development of BE.

Aim: GATA6, a zinc-finger transcription factor, functions as a tumour promoter or suppresser according to different tumour origins. We investigated the clinical significance of GATA6 and its role in invasion and metastasis in cholangiocarcinoma (CCA)., Methods: Expression of GATA6 in 87 cancerous, 24 paracancerous, 32 lymph-node metastatic and 8 liver metastatic samples from 87 CCA patients undergoing surgical resection was detected by immunohistochemistry. Impact of GATA6 on invasion, metastasis and 67kDa laminin receptor expression (67LR) was evaluated in CCA cells by shRNA lentivirus or expressed-plasmid transfection., Results: Aberrant expression of GATA6 in CCAs was significantly associated with lymph-node metastasis. GATA6 expression was higher in lymph-node and liver metastatic tissues compared with primary cancerous tissues. Kaplan-Meier analysis showed GATA6 expression correlated with poor overall survival and early recurrence in CCAs. Cox analysis suggested GATA6 was an independent prognostic marker for overall survival and recurrence-free survival. CCA cell invasion and migration were decreased by GATA6 knockdown and enhanced by GATA6 overexpression in vitro. Knockdown of GATA6 reduced CCA cell metastasis by xenotransplantation into nude mice. 67LR, which is overexpressed in CCAs and promotes invasion and metastasis through several pathways, positively correlated with GATA6 expression in 87 CCAs. Both mRNA and protein levels of 67LR were regulated by GATA6 in CCA cells. Moreover, ChIP analysis showed GATA6 bound to 67LR gene promoter in CCA cells., Conclusion: Aberrant expression of GATA6 correlates with poor prognosis and promotes invasion and metastasis in CCA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca(2+) sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

We recently reported de novo GATA6 mutations as the most common cause of pancreatic agenesis, accounting for 15 of 27 (56%) patients with insulin-treated neonatal diabetes and exocrine pancreatic insufficiency requiring enzyme replacement therapy. We investigated the role of GATA6 mutations in 171 subjects with neonatal diabetes of unknown genetic etiology from a cohort of 795 patients with neonatal diabetes. Mutations in known genes had been confirmed in 624 patients (including 15 GATA6 mutations). Sequencing of the remaining 171 patients identified nine new case subjects (24 of 795, 3%). Pancreatic agenesis was present in 21 case subjects (six new); two patients had permanent neonatal diabetes with no enzyme supplementation and one had transient neonatal diabetes. Four parents with heterozygous GATA6 mutations were diagnosed with diabetes outside the neonatal period (12-46 years). Subclinical exocrine insufficiency was demonstrated by low fecal elastase in three of four diabetic patients who did not receive enzyme supplementation. One parent with a mosaic mutation was not diabetic but had a heart malformation. Extrapancreatic features were observed in all 24 probands and three parents, with congenital heart defects most frequent (83%). Heterozygous GATA6 mutations cause a wide spectrum of diabetes manifestations, ranging from pancreatic agenesis to adult-onset diabetes with subclinical or no exocrine insufficiency.

Osteoblastic differentiation is controlled by complex interplay of several signaling pathways and associated key transcription factors, as well as by microRNAs (miRNAs). In our current study, we found miR-181a to be highly upregulated during BMP induced osteoblastic differentiation of C2C12 and MC3T3 cells. Overexpression of miR-181a led to upregulation of key markers of osteoblastic differentiation as well as enhanced ALP levels and Alizarin red staining, indicating the importance of this miRNA for osteoblastic differentiation. Further, we show that miR-181 isoforms (181a, 181b, 181c) are expressed during different stages of mouse calvarial and tibial development, implying their role in both endochondral and intramembranous ossification. We found several direct and indirect targets of miR-181a to be downregulated by global mRNA expression profiling. Our results demonstrate that miR-181a promotes osteoblastic differentiation via repression of TGF-β signaling molecules by targeting the negative regulator of osteoblastic differentiation Tgfbi (Tgf-beta induced) and TβR-I/Alk5 (TGF-β type I receptor). Furthermore, our findings suggest that Rgs4 and Gata6 are direct targets of miR-181a. Taken together, we provide evidence for a crucial functional link between a specific miRNA, miR-181a and osteoblastic differentiation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Early mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and differentiation. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription factors in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription factor OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in 128-256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription factor CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast growth factor receptor-1 (FGFR1), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription factor SOX17 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4-5dpf. SOX17 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription factor GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in differentiation mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human embryonic stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the outgrowth of TE-like cells, which impairs human embryonic stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Preimplantation development culminates with the emergence of three distinct populations: the inner cell mass, primitive endoderm and trophectoderm. Here, we define the mechanisms underlying the requirement of Suds3 in pre/peri-implantation development. Suds3 knockdown blastocysts exhibit a failure of both trophectoderm proliferation as well as a conspicuous lack of primitive endoderm. Expression of essential lineage factors Nanog, Sox2, Cdx2, Eomes, Elf5 and Sox17 are severely reduced in the absence of Suds3. Importantly, we document deficient FGF4/ERK signaling and show that exogenous FGF4 rescues primitive endoderm formation and trophectoderm proliferation in Suds3 knockdown blastocysts. We also show that Hdac1 knockdown reduces Sox2/FGF4/ERK signaling in blastocysts. Collectively, these data define a role for Suds3 in activation of FGF4/ERK signaling and determine an essential molecular role of Suds3/Sin3/HDAC complexes in lineage specification in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3β pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3β pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3β pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3β significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease associated with significant morbidity and mortality in humans. However, the molecular etiology underlying TOF in most patients remains largely unknown. In the present study, sequence analysis of the GATA6 gene was performed from fresh-frozen cardiac tissues and matched blood samples of 52 unrelated patients who underwent surgical repair of TOF. The cardiac tissues and matched blood specimens from 46 patients who underwent cardiac valve replacement due to rheumatic heart disease and blood samples from 200 healthy individuals as controls were genotyped. The functional characteristics of the mutations were assessed using a luciferase reporter assay system. Based on the results, two novel heterozygous GATA6 mutations, p.G367X and p.G394C, were identified in the cardiac tissues of 2 TOF patients, respectively. No mutations were found in the cardiac tissues from 46 patients with rheumatic heart disease and in the blood samples from the 298 participants. Functional analysis demonstrated that the GATA6 mutants were consistently associated with significantly reduced transcriptional activation compared with their wild-type counterpart. This is the first report on the link of somatic GATA6 mutation to TOF, providing novel insight into the molecular mechanism involved in TOF.

The goal of this study was to investigate the differential effect of tanshinone IIA on the induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by TNF-α and the possible molecular mechanisms by which it regulates ICAM-1 and VCAM-1 expression differentially. Stimulation of human umbilical vein endothelial cells (HUVEC) with TNF-α increased ICAM-1 and VCAM-1 expressions, and the pretreatment with tanshinone IIA concentration dependently inhibited VCAM-1 expression but not ICAM-1 expression. In previous study, PI3K/Akt, PKC and Jak/STAT-3 pathways were involved in the TNF-α-mediated induction of VCAM-1 but not ICAM-1. Thus, we examined the effect of tanshinone IIA on TNF-α-mediated activations of PI3K/Akt, PKC and Jak/STAT-3 pathways. Tanshinone IIA efficiently inhibited the phosphorylations of Akt, PKC and STAT-3 by TNF-α. Moreover, we determined the effect of tanshinone IIA on IRF-1 or GATAs induction and binding activity to VCAM-1 promoter since the upstream promoter region of VCAM-1 but not ICAM-1 contains IRF-1 and GATA binding motifs. Western blot analysis and ChIP assay showed that tanshinone IIA efficiently inhibited TNF-α-increased nuclear level of IRF-1 and GATA-6 and their binding affinity to VCAM-1 promoter region. Taken together, tanshinone IIA selectively inhibits TNF-α-mediated expression of VCAM-1 but not ICAM-1 through modulation of PI3/Akt, PKC and Jak/STAT-3 pathway as well as IRF-1 and GATA-6 binding activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis involves the differentiation of inner cell mass cells into primitive and then into parietal endoderm. These processes can be recapitulated in vitro using F9 teratocarcinoma cells, which differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm with subsequent treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on how primitive endoderm develops revealed that the Wnt6 gene is upregulated by RA, leading to the activation of the canonical WNT-β-catenin pathway. The mechanism by which Wnt6 is regulated was not determined, but in silico analysis of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 transcription factors might be involved [1]. Subsequent analysis determined that both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced molecular and morphological markers of primitive endoderm, which occurred concomitantly with the upregulation of the Wnt6 gene. Gata6- or Foxa2-overexpressing cells were also found to have increased levels in T-cell factor (TCF)-dependent transcription, and when these cells were treated with db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and overexpression studies showed that these transcription factors were sufficient to switch on the gene expression of a Wnt6 reporter construct. Together, these results provide evidence for the direct regulation of Wnt6 that leads to the activation of the canonical WNT-β-catenin pathway and subsequent induction of primitive extraembryonic endoderm.

Objective: Sulforaphane, a naturally occurring organosulfur compound in broccoli, has chemopreventive properties in cancer. However, the effects of sulforaphane in vascular diseases have not been examined. We therefore aimed to investigate the effects of sulforaphane on vascular smooth muscle cell (VSMC) proliferation and neointimal formation and the related mechanisms., Methods: The expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) was examined in VSMCs. The nuclear translocation of nuclear factor-κB (NF-κB) and GATA6 expression was examined in VSMCs and in a carotid artery injury model by Western blot and immunohistochemistry. We also investigated whether local delivery of sulforaphane affected neointimal formation., Results: Sulforaphane inhibited the mRNA and protein expression of VCAM-1 induced by tumor necrosis factor (TNF)-α in VSMCs. Treatment of VSMCs with sulforaphane blocked TNF-α-induced IκBα degradation and NF-κB p65 and GATA6 expression. Furthermore, NF-κB p65 and GATA6 expression were reduced in sulforaphane-treated carotid injury sections. Notably, binding of GATA6 to the VCAM-1 promoter was dramatically reduced by sulforaphane. The MTT, BrdU incorporation, and in vitro scratch assays revealed that the proliferation and migration of VSMCs were reduced by sulforaphane. Furthermore, local administration of sulforaphane significantly reduced neointima formation 14 days after vascular injury in rats., Conclusions: Our results indicate that sulforaphane inhibits neointima formation via targeting of adhesion molecules through the suppression of NF-κB/GATA6. Furthermore, sulforaphane regulates migration and proliferation in VSMCs. Sulforaphane may be a potential therapeutic agent for preventing restenosis after vascular injury., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Atrial fibrillation (AF) is the most common type of sustained cardiac arrhythmia and is associated with substantial morbidity and mortality. Increasing evidence demonstrates that hereditary defects are involved in the pathogenesis of AF. However, AF is of remarkable genetic heterogeneity, and the heritable components responsible for AF in the majority of patients remain unclear. In this study, the entire coding region of the GATA6 gene, which encodes a zinc-finger transcription factor crucial for cardiogenesis, was sequenced in 138 unrelated patients with lone AF, and a novel heterozygous GATA6 mutation, c.704A > C equivalent to p.Y235S, was identified in a patient. The detected substitution, which altered the amino acid highly conserved evolutionarily across species, was absent in 200 unrelated ethnically matched healthy individuals, and was predicted to be disease-causing by MutationTaster. Genetic analysis of the available relatives of the mutation carrier showed that in the family the variation co-segregated with the disease transmitted as an autosomal dominant trait, with complete penetrance. The functional analysis performed using a luciferase reporter assay system revealed that the mutant GATA6 protein resulted in significantly decreased transcriptional activity compared with its wild-type counterpart. These findings provide novel insight into the molecular pathophysiology implicated in AF, suggesting the potential implications in the prophylactic strategy and effective therapy for this common arrhythmia., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

GATA-6 is a zinc-finger transcription factor essential for early embryogenesis. Ablation of GATA-6 in mice impairs endoderm differentiation and causes apoptosis of epiblast cells. The endoderm defects have been attributed to the loss of HNF4, disabled-2, and GATA-4. However, the mechanisms underlying epiblast apoptosis are unclear. In this study we used mouse embryonic stem cell-derived embryoid bodies (EBs) as a model for peri-implantation development and found that ablation of GATA-6 causes massive apoptosis during EB differentiation. Endoderm grafting experiments and ectopic basement membrane (BM) assembly suggest that both BM and non-BM factors contribute to cell survival. Furthermore, the increased cell death in mutant EBs is accompanied by reduced expression of bone morphogenetic protein 2 (BMP-2). Chromatin immunoprecipitation reveals direct binding of GATA-6 to the Bmp2 promoter. Treatment of the mutant EBs with BMP-2 markedly suppresses apoptosis, whereas stable overexpression of the BMP antagonist noggin or a dominant-negative BMP receptor in normal EBs leads to increased apoptosis. Last, activation of SMAD1/5 by phosphorylation is significantly inhibited in the absence of GATA-6, and this is reversed by exogenous BMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitor increases apoptosis. Collectively these results suggest that GATA-6 promotes cell survival by regulating endoderm expression of BMP-2 and BM during embryonic epithelial morphogenesis.

Controlled renewal of the epithelium with precise cell distribution and gene expression patterns is essential for colonic function. GATA6 is expressed in the colonic epithelium, but its function in the colon is currently unknown. To define GATA6 function in the colon, we conditionally deleted Gata6 throughout the epithelium of small and large intestines of adult mice. In the colon, Gata6 deletion resulted in shorter, wider crypts, a decrease in proliferation, and a delayed crypt-to-surface epithelial migration rate. Staining techniques and electron microscopy indicated deficient maturation of goblet cells, and coimmunofluorescence demonstrated alterations in specific hormones produced by the endocrine L cells and serotonin-producing cells. Specific colonocyte genes were significantly downregulated. In LS174T, the colonic adenocarcinoma cell line, Gata6 knockdown resulted in a significant downregulation of a similar subset of goblet cell and colonocyte genes, and GATA6 was found to occupy active loci in enhancers and promoters of some of these genes, suggesting that they are direct targets of GATA6. These data demonstrate that GATA6 is necessary for proliferation, migration, lineage maturation, and gene expression in the mature colonic epithelium.

Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: This study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT)., Methods: Blastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4-8 cell and blastocyst stage embryos by real-time PCR., Results: Developmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4-8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos., Conclusion: MSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs.

During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

Single GATA-6 (G6(gcko)), GATA-4 (G4(gcko)), and double GATA-4/6 (G4/6(gcko)) granulosa cell-specific knockout mice were generated to further investigate the role of GATA transcription factors in ovarian function in vivo. No reproductive defects were found in G6(gcko) animals. G4(gcko) animals were subfertile as indicated by the reduced number of pups per litter and the release of significantly fewer oocytes at ovulation. In marked contrast, G4/6(gcko) females fail to ovulate and are infertile. Furthermore, G4/6(gcko) females had irregular estrous cycles, which correlate with the abnormal ovarian histology found in unstimulated adult G4/6(gcko) females showing lack of follicular development and increased follicular atresia. Moreover, treatment with exogenous gonadotropins did not rescue folliculogenesis or ovulation in double-knockout G4/6(gcko) mice. In addition, ovary weight and estradiol levels were significantly reduced in G4(gcko) and G4/6(gcko) animals when compared with control and G6(gcko) mice. Aromatase, P450scc, and LH receptor expression was significantly lower in G4(gcko) and G4/6(gcko) mice when compared with control animals. Most prominently, FSH receptor (FSHR) protein was undetectable in granulosa cells of G4(gcko) and G4/6(gcko). Accordingly, gel shift and reporter assays revealed that GATA-4 binds and stimulates the activity of the FSHR promoter. These results demonstrate that GATA-4 and GATA-6 are needed for normal ovarian function. Our data are consistent with a role for GATA-4 in the regulation of the FSHR gene and provide a possible molecular mechanism to explain the fertility defects observed in animals with deficient GATA expression in the ovary.

Cell adhesion molecules (CAMs) are involved in a variety of pathologies including cancer, inflammation, pathogenic infections and autoimmune disease. In particular, VCAM-1, rather than ICAM-1, plays a major role in the initiation of atherosclerosis and tumor progression. Therefore, we attempted to elucidate differential mechanisms that regulate VCAM-1 and ICAM-1 expressions. Down-regulation of JNK by a specific inhibitor (SP600125) or dominant negative (DN) JNK1 plasmid enhanced TNF-α-induced VCAM-1 but not ICAM-1 expression. Moreover, transfection with a JNK1-overexpressing vector resulted in the inhibition of VCAM-1 expression stimulated by TNF-α in HUVECs, suggesting that JNK negatively regulates TNF-α-induced VCAM-1 expression in endothelial cells (ECs). Next, we investigated whether JNK signaling affects IRF-1 and/or GATA6, which are transcription factors that mediate TNF-α induction of VCAM-1 but not ICAM-1. The DN-JNK1 plasmid-transfected cells enhanced TNF-α up-regulation of IRF-1 whereas JNK1-overexpressing cells displayed down-regulation; however, neither DN-JNK1 transfection nor JNK1 overexpression affected GATA6 protein levels in the nuclear fraction. Chromatin immunoprecipitation (ChIP) assay confirmed that the inhibition of JNK by DN-JNK1 transfection increases the binding of IRF-1 to the VCAM-1 promoter whereas the overexpression of JNK1 inhibits IRF-1 binding to the VCAM-1 promoter. However, neither DN-JNK1 nor JNK1 overexpression altered GATA6 affinity for the VCAM-1 promoter region. We also examined whether MKP-7 affects ICAM-1 or VCAM-1 by regulating JNK. TNF-α-induced phosphor-JNK levels increased after 5min, peaked at 10 min, and decreased after 30 min. Interestingly, MKP-7 protein levels increased after 30 min, when phosphor-JNK induction by TNF-α was decreased. In addition, silencing MKP-7 with specific siRNA resulted in an increase in phosphor-JNK and inhibited the expression of VCAM-1 but not ICAM-1. Moreover, silencing MKP-7 caused the down-regulation of IRF-1 protein levels and binding to the VCAM-1 promoter. Thus, we suggest that MKP-7, a negative regulator of JNK, regulates VCAM-1 expression in activated endothelial cells through IRF-1 but not GATA6., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Gene amplification is a tumor-specific event during malignant transformation. Recent studies have proposed a lineage-dependency (addiction) model of human cancer whereby amplification of certain lineage transcription factors predisposes a survival mechanism in tumor cells. These tumor cells are derived from tissues where the lineage factors play essential developmental and maintenance roles. Here, we show that recurrent amplification at 18q11.2 occurs in 21% of esophageal adenocarcinomas (EAC). Utilization of an integrative genomic strategy reveals a single gene, the embryonic endoderm transcription factor GATA6, as the selected target of the amplification. Overexpression of GATA6 is found in EACs that contain gene amplification. We find that EAC patients whose tumors carry GATA6 amplification have a poorer survival. We show that ectopic expression of GATA6, together with FGFR2 isoform IIIb, increases anchorage-independent growth in immortalized Barrett's esophageal cells. Conversely, siRNA-mediated silencing of GATA6 significantly reduces both cell proliferation and anchorage-independent growth in EAC cells. We further demonstrate that induction of apoptotic/anoikis pathways is triggered upon silencing of GATA6 in EAC cells but not in esophageal squamous cells. We show that activation of p38α signaling and up-regulation of TNF-related apoptosis-inducing ligand are detected in apoptotic EAC cells upon GATA6 deprivation. We conclude that selective gene amplification of GATA6 during EAC development sustains oncogenic lineage-survival of esophageal adenocarcinoma.

Although luteinizing hormone (LH) affects androstenedione (A4) and progesterone (P4) production in theca cells, it is still unknown how LH influences molecular mechanism of A4 and P4 production. To examine the relationship between LH and transcription factors involved in A4 and P4 production, ovarian theca cells were cultured in the presence or absence of high concentrations of LH for 24 h (pre-treatment with high concentration of LH) and then cultured in the presence or absence of low concentration of LH for 48 h. Low LH enhanced production of A4 and P4, and expressions of CYP17 and StAR mRNA in theca cells without pre-treatment with high LH. In addition, low LH stimulated the expression of SF-1 protein in nuclear fractions from theca cells with or without pre-treatment with high LH. The binding of SF-1 to the CYP17 and StAR promoter regions increased in theca cells treated with low LH. Although GATA-4 and GATA-6 are both found in the nuclear fraction but not in the cytosol of theca cells, low LH enhanced the binding of GATA-6, but not of GATA-4, to the CYP17 promoter region without pre-treatment with high LH. Acetylation histone H3 in StAR and CYP17 promoter regions were changed by different LH-dosage. Overall, we showed that LH regulates the production of A4 and P4 by affecting the nuclear localization and switching of transcription factors in theca cells and that target transcription factors involved in steroid production in theca cells are changed by different LH concentration., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)