Towards Understanding the Gene-Specific Roles of GATA Factors in Heart Development: Does GATA4 Lead the Way?

Transcription factors play crucial roles in the regulation of heart induction, formation, growth and morphogenesis. Zinc finger GATA transcription factors are among the critical regulators of these processes. GATA4, 5 and 6 genes are expressed in a partially overlapping manner in developing hearts, and GATA4 and 6 continue their expression in adult cardiac myocytes. Using different experimental models, GATA4, 5 and 6 were shown to work together not only to ensure specification of cardiac cells but also during subsequent heart development. The complex involvement of these related gene family members in those processes is demonstrated through the redundancy among them and crossregulation of each other. Our recent identification at the genome-wide level of genes specifically regulated by each of the three family members and our earlier discovery that gata4 and gata6 function upstream, while gata5 functions downstream of noncanonical Wnt signalling during cardiac differentiation, clearly demonstrate the functional differences among the cardiogenic GATA factors. Such suspected functional differences are worth exploring more widely. It appears that in the past few years, significant advances have indeed been made in providing a deeper understanding of the mechanisms by which each of these molecules function during heart development. In this review, I will therefore discuss current evidence of the role of individual cardiogenic GATA factors in the process of heart development and emphasize the emerging central role of GATA4.

Endothelial GATA5 positively regulates angiogenesis via cathepsin S-mediated Angpt2/Flk1 and MMP2/9 signaling pathways.

Although GATA5 is vital in maintaining the function of endothelial cells, the relationship between GATA5 and angiogenesis, however, remains unclear. Our study aims to determine how endothelial GATA5 mediates angiogenesis. Using the ischemic hindlimb of mice with GATA5 overexpression in the endothelia (EC-Ad mice), we showed that GATA5 overexpression could improve blood perfusion and increase capillary density. Furthermore, we showed that overexpression of GATA5 can increase the protein and mRNA levels of angiopoietin-2 (Angpt2) and fetal liver kinase 1 (Flk1) in the endothelia of EC-Ad mice, while GATA5 knockdown can inhibit the VEGF-165-induced proliferation, tube formation, and migration of human umbilical vein endothelial cells (HUVECs). In addition, we observed a decrease in the Angpt2 and Flk1, and the matrix metalloproteinase (MMP) family proteins: MMP2 and MMP9 while GATA5 was decreased. Meanwhile, our study also demonstrated that the expression of cathepsin S (Cat S) decreases when GATA5 is downregulated. Immunoprecipitation assay indicated that GATA5 could bind to Cat S directly. Furthermore, GATA5 or Cat S overexpression can promote tube formation and migration of HUVECs, restore the Angpt2 and Flk1 expression levels in the GATA5 knockdown HUVECs, and upregulate MMP2 and MMP9 protein levels. In summary, our study demonstrated that endothelial GATA5 could mediate angiogenesis by inducing the expression of Cat S, which mediates the Angpt2/Flk1 and MMP2/9 signaling pathways.

GATA binding protein 5-mediated transcriptional activation of transmembrane protein 100 suppresses cell proliferation, migration and epithelial-to-mesenchymal transition in prostate cancer DU145 cells.

It has been reported that transmembrane protein 100 (TMEM100) acts as a tumor regulator in several types of cancers. However, whether the expression of TMEM100 is associated with the development and prognosis of prostate cancer (PCa) remains elusive. Therefore, the present study aimed to uncover the role of GATA binding protein 5 (GATA5)-mediated activation of TMEM100 in the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of PCa cells. The expressions of TMEM100 and GATA5 in PCa patients were analyzed by the GEPIA database. The binding site of GATA5 and TMEM100 promoter was predicted by the JASPAR database. Expressions of TMEM100 and GATA5 in PCa cells were detected by qRT-PCR and Western blot analysis. Cell Counting Kit 8 and colony formation assays were performed to measure cell proliferation. In addition, cell migration, invasion and the expression of EMT-associated proteins were evaluated using wound healing, transwell assay and Western blotting assays, respectively. The bioinformatics analysis revealed that TMEM100 was downregulated in PCa and was associated with overall survival of PCa. In addition, TMEM10 overexpression attenuated cell proliferation, migration, invasion and EMT in PCa cells. The interaction between TMEM100 and GATA5 was verified using dual luciferase reporter and chromatin immunoprecipitation assays. Furthermore, the results showed that GATA5 was downregulated and GATA5 silencing reversed the inhibitory effects of TMEM10 on PCa cells. Overall, the current study suggested that the GATA5-mediated transcriptional activation of TMEM100 could affect the behavior of PCa cells and was associated with poor prognosis in PCa.

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Potential roles of GATA binding protein 5 in cardiovascular diseases.

It is known that functional defects of GATA binding protein 5 (GATA5), an important member of GATA transcription factor family, could cause multiple congenital defects. However, the mechanisms of this transcription factor in cardiovascular diseases are still little known. Finding a genetic approach should help with understanding the possible roles of GATA5 in different cardiovascular diseases and purpose it as a possible therapeutic agent. Hence, this review is divided into three chapters to summarize the roles and main regulatory mechanisms of GATA5 in hypertension, arrhythmia and congenital heart disease, respectively. In each chapter, this review firstly introduces the roles of GATA5 mutations, and then discusses the main regulatory mechanisms of GATA5 in the corresponding diseases (Such as the endothelial dysfunction signaling pathway in the chapter of hypertension, GATA5-NaV1.5 signaling pathway in the chapter of arrhythmia, GATA5-HEY2 and GATA5-Nodal signaling pathway in the chapter of congenital heart disease). Additionally, based on these regulatory networks, it is also speculated that abnormal methylation of the GATA5 gene promoter may lead to cardiovascular diseases such as congenital heart disease. This conjecture is proposed to enrich the regulatory networks of GATA5 and provide a theoretical basis for diagnosis and treatment of cardiovascular diseases.

Specificity, redundancy and dosage thresholds among gata4/5/6 genes during zebrafish cardiogenesis.

The Gata4/5/6 sub-family of zinc finger transcription factors regulate many aspects of cardiogenesis. However, critical roles in extra-embryonic endoderm also challenge comprehensive analysis during early mouse cardiogenesis, while zebrafish models have previously relied on knockdown assays. We generated targeted deletions to disrupt each gata4/5/6 gene in zebrafish and analyzed cardiac phenotypes in single, double and triple mutants. The analysis confirmed that loss of gata5 causes cardia bifida and validated functional redundancies for gata5/6 in cardiac precursor specification. Surprisingly, we discovered that gata4 is dispensable for early zebrafish development, while loss of one gata4 allele can suppress the bifid phenotype of the gata5 mutant. The gata4 mutants eventually develop an age-dependent cardiomyopathy. By combining combinations of mutant alleles, we show that cardiac specification depends primarily on an overall dosage of gata4/5/6 alleles rather than a specific gene. We also identify a specific role for gata6 in controlling ventricle morphogenesis through regulation of both the first and second heart field, while loss of both gata4/6 eliminates the ventricle. Thus, different developmental programs are dependent on total dosage, certain pairs, or specific gata4/5/6 genes during embryonic cardiogenesis.This article has an associated First Person interview with the first author of the paper.

The role of miR-92 in regulating early development and metamorphosis of Japanese flounder Paralichthys olivaceus.

MicroRNAs are a class of short non-coding RNAs that contain approximately 22 nucleotides and play a regulatory role in RNA silencing and translational repression. miR-92 belongs to the miR-17-92 family and has a regulatory effect on cell proliferation, apoptosis, and expression of proto-oncogenes and tumor suppressor genes. However, its function in flatfish is unclear. In this study, we used farmed Japanese flounder, Paralichthys olivaceus, and showed that gata5 is a target gene of miR-92. Experiments on miR-92 overexpression indicated that gata5 and sox17 were downregulated, while the transcription level of ntl increased. By contrast, depletion of miR-92 resulted in increased gata5 and sox17 levels and reduced ntl level. Moreover, thiourea treatment indicated that miR-92 may inhibit the metamorphic development of Japanese flounder. Our study suggests that miR-92 regulates the fate of endoderm and mesoderm by controlling gata5.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.

Endothelial SIRT6 Is Vital to Prevent Hypertension and Associated Cardiorenal Injury Through Targeting Nkx3.2-GATA5 Signaling.

RATIONALE: Endothelial dysfunction is an important determinant risk factor for the development of hypertension and its complications. Thus, identification of potential therapeutic targets for preventing endothelial dysfunction has major clinical importance. Emerging evidence indicates that epigenetic modifications are closely associated with the regulation of endothelial function. Among them, HDAC (histone deacetylase)-mediated epigenetic processes in vascular homeostasis and cardiovascular disease have attracted much attention. SIRT6 (sirtuin 6) is one member of SIRTs (class III HDAC) that are highly conserved NAD+-dependent deacetylases. OBJECTIVE: This study was designed to elucidate the role of SIRT6 in the pathogenesis of hypertension, discover the new targets of SIRT6, and explore related mechanisms on the regulation of endothelial function. METHODS AND RESULTS: The levels of endothelial SIRT6 were significantly reduced in 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Utilizing genetically engineered endothelial-specific SIRT6 knockout (Cre+/SIRT6fl/fl) mice, we found that endothelial-specific deletion of SIRT6 significantly enhanced blood pressure, exacerbated endothelial dysfunction and cardiorenal injury in experimental hypertension. Functionally, SIRT6 has pleiotropic protective actions in endothelial cells, which include promoting endothelium-dependent vasodilatation and vascular NO bioavailability, reducing cellular permeability, ameliorating endothelial senescence and apoptosis, and facilitating autophagy. Mechanistically, SIRT6 induced the expression of GATA5 (GATA-binding protein 5), a novel regulator of blood pressure, through inhibiting Nkx3.2 (NK3 homeobox 2) transcription by deacetylating histone H3K9 (histone H3 lysine 9), thereby regulating GATA5-mediated signaling pathways to prevent endothelial injury. Finally, we provide direct evidence for the therapeutic potential of SIRT6 in desoxycorticosterone acetate/salt-induced hypertensive mice by overexpression of SIRT6 in vivo. CONCLUSIONS: This study for the first time demonstrates that SIRT6 prevents hypertension and its complications by maintaining endothelial function. Pharmacological targeting of SIRT6 may be an innovative therapeutic strategy for treating patients with hypertension.

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes.

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with ß-catenin in the cytoplasm, preventing ß-catenin from entering the nucleus. Treatment with the specific Wnt/ß-catenin pathway inhibitor salinomycin was able to reduce the expression of ß-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored ß-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of ß-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/ß-catenin pathway and the reduction of reprogramming gene expression.

Zebrafish microRNA miR-210-5p inhibits primitive myelopoiesis by silencing foxj1b and slc3a2a mRNAs downstream of gata4/5/6 transcription factor genes.

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6-regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6-knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6-knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a, encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis.

Effect of Helicobacter pylori Infection on GATA-5 and TFF1 Regulation, Comparison Between Pediatric and Adult Patients.

BACKGROUND: GATA factors, which constitute a family of transcription regulatory proteins, participate in gastrointestinal development. Trefoil factor 1 (TFF1) plays a crucial role in mucosal defense and healing, and evidence suggests that GATA-5 mediated its regulation. Gastric cancer is a multiple-step process triggered by Helicobacter pylori and is characterized by accumulation of molecular and epigenetic alteration. The aim of this study was to evaluate the effect of H. pylori infection on the regulation of GATA-5 and TFF1 in vitro and in vivo. RESULTS: Infected cells exhibited upregulation of GATA-5 and TFF1 after 48 h. An increase in GATA-5 and TFF1 mRNA levels was also found in mice samples after 6 and 12 months of infection, respectively. In human samples, we found an association between H. pylori infection and GATA-5 upregulation. In fact, among H. pylori-infected patients, hypermethylation was observed in 45.5% of pediatric samples, in 62.6% of chronic gastritis samples, and in 63% of gastric cancer samples. Regarding TFF1, the expression levels were similar in pediatrics and adults patients, and were independent of H. pylori infection, and the expression of these factors was downregulated in gastric cancer samples. GATA-5 promoter methylation was associated with a decrease in TFF1 mRNA levels. CONCLUSIONS: Our results suggest that the upregulation of GATA-5 and TFF1 observed in vitro and in vivo may be correlated with a protective effect of the mucosa in response to infection. The epigenetic inactivation of GATA-5 observed in human biopsies from infected patients may suggest that this alteration is an early event occurring in association with H. pylori infection.

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Oxidative stress-induced apoptotic insults to rat osteoblasts are attenuated by nitric oxide pretreatment via GATA-5-involved regulation of Bcl-X L gene expression and protein translocation.

Nitric oxide (NO) has biphasic effects on regulating osteoblast survival and death. This study was aimed to evaluate the effects of NO pretreatment on hydrogen peroxide (HP)-induced insults of rat osteoblasts and the possible mechanisms. Exposure of osteoblasts prepared from rat calvarias to HP significantly increased intracellular reactive oxygen species levels, decreased alkaline phosphatase activity and cell survival, and ultimately induced cell apoptosis. However, NO pretreatment lowered HP-induced oxidative stress and apoptotic insults. In parallel, HP increased Bax levels and its translocation from the cytoplasm to mitochondria. NO pretreatment caused significant attenuations in HP-induced modulations in Bax synthesis and translocation. In contrast, pretreatment with NO enhanced levels and translocation of antiapoptotic Bcl-XL protein in rat osteoblasts. RNA analyses further revealed that HP inhibited Bcl-XL mRNA expression without affecting Bax mRNA levels. In comparison, NO induced Bcl-XL mRNA production and alleviated HP-caused inhibition of this mRNA expression. As to the mechanism, HP suppressed RNA and protein levels of transcription factor GATA-5 in rat osteoblasts. Pretreatment with NO induced GATA-5 mRNA and protein expressions and simultaneously attenuated HP-induced inhibition of this gene's expression. Consequently, GATA-5 knockdown using RNA interference inhibited Bcl-XL mRNA expression and concurrently lowered NO's protection against HP-induced apoptotic insults. Therefore, this study showed that NO can protect rat osteoblasts from HP-induced apoptotic insults. The protective mechanisms are mediated by GATA-5-mediated transcriptional induction of Bcl-X L gene, and translocational modulation of Bcl-XL and Bax proteins.

Endothelial Gata5 transcription factor regulates blood pressure.

Despite its high prevalence and economic burden, the aetiology of human hypertension remains incompletely understood. Here we identify the transcription factor GATA5, as a new regulator of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells and its genetic inactivation in mice (Gata5-null) leads to vascular endothelial dysfunction and hypertension. Endothelial-specific inactivation of Gata5 mimics the hypertensive phenotype of the Gata5-null mice, suggestive of an important role for GATA5 in endothelial homeostasis. Transcriptomic analysis of human microvascular endothelial cells with GATA5 knockdown reveals that GATA5 affects several genes and pathways critical for proper endothelial function, such as PKA and nitric oxide pathways. Consistent with a role in human hypertension, we report genetic association of variants at the GATA5 locus with hypertension traits in two large independent cohorts. Our results unveil an unsuspected link between GATA5 and a prominent human condition, and provide a new animal model for hypertension.

PTEN regulates lung endodermal morphogenesis through MEK/ERK pathway.

Pten is a multifunctional tumor suppressor. Deletions and mutations in the Pten gene have been associated with multiple forms of human cancers. Pten is a central regulator of several signaling pathways that influences multiple cellular functions. One such function is in cell motility and migration, although the precise mechanism remains unknown. In this study, we deleted Pten in the embryonic lung epithelium using Gata5-cre mice. Absence of Pten blocked branching morphogenesis and ERK and AKT phosphorylation at E12.5. In an explant model, Pten(Δ/Δ) mesenchyme-free embryonic lung endoderm failed to branch. Inhibition of budding in Pten(Δ/Δ) explants was associated with major changes in cell migration, while cell proliferation was not affected. We further examined the role of ERK and AKT in branching morphogenesis by conditional, endodermal-specific mutants which blocked ERK or AKT phosphorylation. MEK(DM/+); Gata5-cre (blocking of ERK phosphorylation) lung showed more severe phenotype in branching morphogenesis. The inhibition of budding was also associated with disruption of cell migration. Thus, the mechanisms by which Pten is required for early endodermal morphogenesis may involve ERK, but not AKT, mediated cell migration.

BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

BACKGROUND: MicroRNA-200 (miR-200) suppresses the epithelial-mesenchymal transition of various cancer cells, including lung adenocarcinoma cells. We found that bone morphogenetic protein 4 (BMP4) was decreased in miR-200-overexpressing cells and epithelial-like lung cancer cells. In this study, we investigated the mechanism and role of BMP4 depletion by miR-200 in murine lung adenocarcinoma cells. METHODS: BMP4 expression levels in murine lung cancer cells were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Promoter and 3'-untranslated region (UTR) luciferase reporter assays were performed to discover the mechanism of regulation of BMP4 by miR-200. Murine lung cancer cells were transfected with Bmp4 shRNAs, which were then injected into syngeneic mice to measure their tumorigenic and metastatic potential and cultured on Matrigel to study the influence of BMP4 on 3-D acinus formation. RESULTS: miR-200 down-regulated BMP4 via direct targeting of the GATA4 and GATA6 transcription factors that stimulate Bmp4 transcription. BMP4 up-regulated JAG2, an upstream factor of miR-200; therefore, JAG2, miR-200, and BMP4 form a regulatory loop. Bmp4 knockdown suppressed cancer cell growth, migration, and invasion and inhibited tumorigenesis and metastasis of lung cancer cells when injected into syngeneic mice. In addition, BMP4 was required for normal acinus formation in Matrigel 3-D culture of murine lung cancer cells, which may be mediated by MYH10, a downstream target of BMP4. CONCLUSION: BMP4 functions as a pro-tumorigenic factor in a murine lung cancer model, and its transcription is regulated by miR-200 and GATA4/6. Thus, we propose that BMP4 and its antagonists may be suitable therapeutic targets for the treatment of lung cancer.

ASK1 mediates the teratogenicity of diabetes in the developing heart by inducing ER stress and inhibiting critical factors essential for cardiac development.

Maternal diabetes in mice induces heart defects similar to those observed in human diabetic pregnancies. Diabetes enhances apoptosis and suppresses cell proliferation in the developing heart, yet the underlying mechanism remains elusive. Apoptosis signal-regulating kinase 1 (ASK1) activates the proapoptotic c-Jun NH2-terminal kinase 1/2 (JNK1/2) leading to apoptosis, suggesting a possible role of ASK1 in diabetes-induced heart defects. We aimed to investigate whether ASK1 is activated in the heart and whether deleting the Ask1 gene blocks diabetes-induced adverse events and heart defect formation. The ASK1-JNK1/2 pathway was activated by diabetes. Deleting Ask1 gene significantly reduced the rate of heart defects, including ventricular septal defects (VSDs) and persistent truncus arteriosus (PTA). Additionally, Ask1 deletion diminished diabetes-induced JNK1/2 phosphorylation and its downstream transcription factors and endoplasmic reticulum (ER) stress markers. Consistent with this, caspase activation and apoptosis were blunted. Ask1 deletion blocked the increase in cell cycle inhibitors (p21 and p27) and the decrease in cyclin D1 and D3 and reversed diabetes-repressed cell proliferation. Ask1 deletion also restored the expression of BMP4, NKX2.5, and GATA5, Smad1/5/8 phosphorylation, whose mutations or deletion result in reduced cell proliferation, VSD, and PTA formation. We conclude that ASK1 may mediate the teratogenicity of diabetes through activating the JNK1/2-ER stress pathway and inhibiting cell cycle progression, thereby impeding the cardiogenesis pathways essential for ventricular septation and outflow tract development.

GATA5 loss-of-function mutation in familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), the most common form of primary myocardial disease, is an important cause of sudden cardiac death and heart failure and is the leading indication for heart transplantation in children and adults worldwide. Recent studies have revealed a strong genetic basis for idiopathic DCM, with many distinct genes causally implicated. Nevertheless, DCM is a genetically heterogeneous disorder and the genetic determinants underlying DCM in a substantial proportion of patients remain unclear. In this study, the whole coding exons and flanking introns of the GATA binding protein 5 (GATA5) gene, which codes for a zinc-finger transcription factor essential for cardiovascular development and structural remodeling, were sequenced in 130 unrelated patients with idiopathic DCM. The available relatives of the index patient carrying an identified mutation and 200 unrelated ethnically matched healthy individuals used as the controls were genotyped for GATA5. The functional characteristics of the mutant GATA5 were analyzed in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. As a result, a novel heterozygous GATA5 mutation, p.G240D, was identified in a family with DCM inherited in an autosomal dominant pattern, which co-segregated with DCM in the family with complete penetrance. The missense mutation was absent in 400 reference chromosomes and the altered amino acid was completely conserved evolutionarily across species. Functional analyses revealed that the GATA5 mutant was associated with significantly diminished transcriptional activity. This study firstly links GATA5 mutation to DCM, which provides novel insight into the molecular mechanisms of DCM, suggesting a potential molecular target for the prenatal prophylaxis and allele-specific treatment of DCM.