Induction of Senescence by Loss of Gata4 in Cardiac Fibroblasts.

Cardiac fibroblasts are a major source of cardiac fibrosis during heart repair processes in various heart diseases. Although it has been shown that cardiac fibroblasts become senescent in response to heart injury, it is unknown how the senescence of cardiac fibroblasts is regulated in vivo. Gata4, a cardiogenic transcription factor essential for heart development, is also expressed in cardiac fibroblasts. However, it remains elusive about the role of Gata4 in cardiac fibroblasts. To define the role of Gata4 in cardiac fibroblasts, we generated cardiac fibroblast-specific Gata4 knockout mice by cross-breeding Tcf21-MerCreMer mice with Gata4fl/fl mice. Using this mouse model, we could genetically ablate Gata4 in Tcf21 positive cardiac fibroblasts in an inducible manner upon tamoxifen administration. We found that cardiac fibroblast-specific deletion of Gata4 spontaneously induces senescence in cardiac fibroblasts in vivo and in vitro. We also found that Gata4 expression in both cardiomyocytes and non-myocytes significantly decreases in the aged heart. Interestingly, when αMHC-MerCreMer mice were bred with Gata4fl/fl mice to generate cardiomyocyte-specific Gata4 knockout mice, no senescent cells were detected in the hearts. Taken together, our results demonstrate that Gata4 deficiency in cardiac fibroblasts activates a program of cellular senescence, suggesting a novel molecular mechanism of cardiac fibroblast senescence.

Gata4-Dependent Differentiation of c-Kit+-Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

BACKGROUND: Although c-Kit+ adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation, we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells. METHODS: Kit allele-dependent lineage tracing and fusion analysis were performed in mice following simultaneous Gata4 and Gata6 cell type-specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit+ cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. RESULTS: Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart, and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion because of defective endothelial cell differentiation in the absence of Gata4. CONCLUSIONS: Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit+ cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit+ endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice.

BACKGROUND/AIMS: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. METHODS: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. RESULTS: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. CONCLUSION: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox ; Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1 and Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.

Role of GATA binding protein 4 (GATA4) in the regulation of tooth development via GNAI3.

Transcription factor GATA4 regulates cardiac and osteoblast differentiation. However, its role in tooth development is not clear. Therefore, we generated Wnt1-Cre;GATA4 fl/fl mice, with conditional inactivation of the GATA4 gene in the dental papilla mesenchymal cells. Phenotypic analysis showed short root deformity along with reduced expressions of odonto/osteogenic markers. Proliferation (but not apoptosis) of cells around the apical area of the root was attenuated. In vitro, we knocked down GATA4 expression in stem cells of dental apical papilla (SCAPs). Proliferation, migration and odonto/osteogenic differentiation of SCAPs were affected in the shGATA4 group. Overexpression of GATA4 in SCAPs increased mineralization. Based on our previous iTRAQ results, guanine nucleotide binding proteins 3 (GNAI3) is one of the distinct proteins after GATA4 deletion. G protein signaling is involved in bone development, remodeling, and disease. In this study, both GATA4 deletion in the mouse root and knock-down in human SCAPs decreased the expression of GNAI3. Dual-luciferase and ChIP assay confirmed the direct binding of GATA4 to the GNAI3 promoter, both in vitro and in vivo. GNAI3 knock-down significantly decreased the odonto/osteogenic differentiation ability of SCAPs. We thus establish the role of GATA4 as a novel regulator of root development and elucidate its downstream molecular events.

Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve.

Bicuspid aortic valve (BAV) is a heritable congenital heart defect and an important risk factor for valvulopathy and aortopathy. Here we report a genome-wide association scan of 466 BAV cases and 4,660 age, sex and ethnicity-matched controls with replication in up to 1,326 cases and 8,103 controls. We identify association with a noncoding variant 151 kb from the gene encoding the cardiac-specific transcription factor, GATA4, and near-significance for p.Ser377Gly in GATA4. GATA4 was interrupted by CRISPR-Cas9 in induced pluripotent stem cells from healthy donors. The disruption of GATA4 significantly impaired the transition from endothelial cells into mesenchymal cells, a critical step in heart valve development.

Identification of GATA-4 as a novel transcriptional regulatory component of regenerating islet-derived family members.

Intestinal epithelial cells are exposed to luminal bacterial threat and require adequate defense mechanisms to ensure host protection and epithelium regeneration against possible deleterious damage. Differentiated intestinal epithelial cells produce antimicrobial and regenerative components that protect against such challenges. Few intestinal specific transcription factors have been identified to control the switching from repression to activation of this class of gene. Herein, we show that gene transcription of some regenerating islet-derived (REG) family members is dependent on the transcription factor GATA-4. Silencing of GATA-4 expression in cultured intestinal epithelial cells identified Reg3ß as a target gene using an unbiased approach of gene expression profiling. Co-transfection and RNA interference assays identified complex GATA-4-interactive transcriptional components required for the activation or repression of Reg3ß gene activity. Conditional deletion of Gata4 in the mouse intestinal epithelium supported its regulatory role for Reg1, Reg3α, Reg3ß and Reg3γ genes. Reg1 dramatic down-modulation of expression in Gata4 conditional null mice was associated with a significant decrease in intestinal epithelial cell migration. Altogether, these results identify a novel and complex role for GATA-4 in the regulation of REG family members gene expression.

Disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation.

Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4(+/-);Tbx5(+/-) mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo, mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 (Gata4(MyoDel/wt);Tbx5(+/-) and Gata4(EndoDel/wt);Tbx5(+/-), respectively) were generated. Gata4(MyoDel/wt);Tbx5(+/-) mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4(EndoDel/wt);Tbx5(+/-) embryos were normal. Cdk4 and Cdk2, cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4(+/-);Tbx5(+/-) hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.

Regulation of a vascular plexus by gata4 is mediated in zebrafish through the chemokine sdf1a.

Using the zebrafish model we describe a previously unrecognized requirement for the transcription factor gata4 controlling embryonic angiogenesis. The development of a vascular plexus in the embryonic tail, the caudal hematopoietic tissue (CHT), fails in embryos depleted of gata4. Rather than forming a normal vascular plexus, the CHT of gata4 morphants remains fused, and cells in the CHT express high levels of osteogenic markers ssp1 and runx1. Definitive progenitors emerge from the hemogenic aortic endothelium, but fail to colonize the poorly vascularized CHT. We also found abnormal patterns and levels for the chemokine sdf1a in gata4 morphants, which was found to be functionally relevant, since the embryos also show defects in development of the lateral line, a mechano-sensory organ system highly dependent on a gradient of sdf1a levels. Reduction of sdf1a levels was sufficient to rescue lateral line development, circulation, and CHT morphology. The result was surprising since neither gata4 nor sdf1a is obviously expressed in the CHT. Therefore, we generated transgenic fish that conditionally express a dominant-negative gata4 isoform, and determined that gata4 function is required during gastrulation, when it is co-expressed with sdf1a in lateral mesoderm. Our study shows that the gata4 gene regulates sdf1a levels during early embryogenesis, which impacts embryonic patterning and subsequently the development of the caudal vascular plexus.

Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis.

Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis.

GATA4 and GATA6 control mouse pancreas organogenesis.

Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1+ cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis.

Intestinal GATA4 deficiency protects from diet-induced hepatic steatosis.

BACKGROUND & AIMS: GATA4, a zinc finger domain transcription factor, is critical for jejunal identity. Mice with an intestine-specific GATA4 deficiency (GATA4iKO) are resistant to diet-induced obesity and insulin resistance. Although they have decreased intestinal lipid absorption, hepatic de novo lipogenesis is inhibited. Here, we investigated dietary lipid-dependent and independent effects on the development of steatosis and fibrosis in GATA4iKO mice. METHODS: GATA4iKO and control mice were fed a Western-type diet (WTD) or a methionine and choline-deficient diet (MCDD) for 20 and 3 weeks, respectively. Functional effects of GATA4iKO on diet-induced liver steatosis were investigated. RESULTS: WTD-but not MCDD-fed GATA4iKO mice showed lower hepatic concentrations of triglycerides, free fatty acids, and thiobarbituric acid reactive species and had reduced expression of lipogenic as well as fibrotic genes compared with controls. Reduced nuclear sterol regulatory element-binding protein-1c protein levels were accompanied by lower lipogenic gene expression. Oil red O and Sirius Red staining of liver sections confirmed the observed reduction in hepatic lipid accumulation and fibrosis. Immunohistochemical staining revealed an increased number of jejunal glucagon-like peptide 1 (GLP-1) positive cells in GATA4iKO mice. Consequently, we found enhanced phosphorylation of hepatic AMP-activated protein kinase and acetyl-CoA carboxylase alpha. CONCLUSIONS: Our results provide strong indications for a protective effect of intestinal GATA4 deficiency on the development of hepatic steatosis and fibrosis via GLP-1, thereby blocking hepatic de novo lipogenesis.

GATA5 interacts with GATA4 and GATA6 in outflow tract development.

Members of the GATA family of transcription factors are critical regulators of heart development and mutations in 2 of them, GATA4 and GATA6 are associated with outflow tract and septal defects in human. The heart expresses 3 GATA factors, GATA4, 5 and 6 in a partially overlapping pattern. Here, we report that compound Gata4/Gata5 and Gata5/Gata6 mutants die embryonically or perinatally due to severe congenital heart defects. Almost all Gata4(+/-)Gata5(+/-) mutant embryos have double outlet right ventricles (DORV), large ventricular septal defects (VSD) as well as hypertrophied mitral and tricuspid valves. Only 25% of double compound Gata4/Gata5 heterozygotes survive to adulthood and these mice have aortic stenosis. Compound loss of a Gata5 and a Gata6 allele also leads to DORVs associated with subaortic VSDs. Expression of several transcription factors important for endocardial and myocardial cell differentiation, such as Tbx20, Mef2c, Hey1 and Hand2, was reduced in compound heterozygote embryos. These findings suggest the existence of important genetic interactions between Gata5 and the 2 other cardiac GATA factors in endocardial cushion formation and outflow tract morphogenesis. The data identify GATA5 as a potential genetic modifier of congenital heart disease and provide insight for elucidating the genetic basis of an important class of human birth defects.

Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation.

Assembly of functioning testis and ovary requires a GATA4-FOG2 transcriptional complex. To define the separate roles for GATA4 and FOG2 proteins in sexual development of the testis we have ablated the corresponding genes in somatic gonadal cells. We have established that GATA4 is required for testis differentiation, for the expression of Dmrt1 gene, and for testis cord morphogenesis. While Sf1Cre-mediated excision of Gata4 permitted normal expression of most genes associated with embryonic testis development, gonadal loss of Fog2 resulted in an early partial block in male pathway and sex reversal. We have also determined that testis sexual differentiation is sensitive to the timing of GATA4 loss during embryogenesis. Our results now demonstrate that these two genes also have non-overlapping essential functions in testis development.

Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death.

Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes.

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice.

Despite significant advances in identifying signaling molecules that induce cardiogenesis in mammals, the transcription factors that control the onset of cardiac myocyte gene expression have remained elusive. Candidates include the zinc finger transcription factors GATA binding proteins 4 and 6 (GATA4, GATA6). The individual loss of either protein in mice results in lethality prior to the onset of heart development due to defects in the extra-embryonic endoderm; however, when this extra-embryonic deficiency is circumvented using tetraploid embryo complementation, cardiac myocyte differentiation initiates normally. Here we show that these factors have redundant roles in controlling the onset of cardiac myocyte differentiation. As a consequence, Gata4(-/-)Gata6(-/-) embryos completely lack hearts, although second heart field progenitor cells are still generated. Our data support a model whereby GATA4 or GATA6 are essential for expression of the network of transcription factors that regulate the onset of cardiac myocyte gene expression during mammalian development.

Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects.

Congenital diaphragmatic hernia (CDH) is an often fatal birth defect that is commonly associated with pulmonary hypoplasia and cardiac malformations. Some investigators hypothesize that this constellation of defects results from genetic or environmental triggers that disrupt mesenchymal cell function in not only the primordial diaphragm but also the thoracic organs. The alternative hypothesis is that the displacement of the abdominal viscera in the chest secondarily perturbs the development of the heart and lungs. Recently, loss-of-function mutations in the gene encoding FOG-2, a transcriptional co-regulator, have been linked to CDH and pulmonary hypoplasia in humans and mice. Here we show that mutagenesis of the gene for GATA-4, a transcription factor known to functionally interact with FOG-2, predisposes inbred mice to a similar set of birth defects. Analysis of wild-type mouse embryos demonstrated co-expression of Gata4 and Fog2 in mesenchymal cells of the developing diaphragm, lungs, and heart. A significant fraction of C57Bl/6 mice heterozygous for a Gata4 deletion mutation died within 1 day of birth. Developmental defects in the heterozygotes included midline diaphragmatic hernias, dilated distal airways, and cardiac malformations. Heterozygotes had any combination of these defects or none. In chimeric mice, Gata4(-/-) cells retained the capacity to contribute to cells in the diaphragmatic central tendon and lung mesenchyme, indicating that GATA-4 is not required for differentiation of these lineages. We conclude that GATA-4, like its co-regulator FOG-2, is required for proper mesenchymal cell function in the developing diaphragm, lungs, and heart.