GATA3 Protein Expression Research Articles

Apocynum Tablet Protects against Cardiac Hypertrophy via Inhibiting AKT and ERK1/2 Phosphorylation after Pressure Overload.

Background. Cardiac hypertrophy occurs in many cardiovascular diseases. Apocynum tablet (AT), a traditional Chinese medicine, has been widely used in China to treat patients with hypertension. However, the underlying molecular mechanisms of AT on the hypertension-induced cardiac hypertrophy remain elusive. The current study evaluated the effect and mechanisms of AT on cardiac hypertrophy. Methods. We created a mouse model of cardiac hypertrophy by inducing pressure overload with surgery of transverse aortic constriction (TAC) and then explored the effect of AT on the development of cardiac hypertrophy using 46 mice in 4 study groups (combinations of AT and TAC). In addition, we evaluated the signaling pathway of phosphorylation of ERK1/2, AKT, and protein expression of GATA4 in the cardioprotective effects of AT using Western blot. Results. AT inhibited the phosphorylation of Thr202/Tyr204 sites of ERK1/2, Ser473 site of AKT, and protein expression of GATA4 and significantly inhibited cardiac hypertrophy and cardiac fibrosis at 2 weeks after TAC surgery (P < 0.05). Conclusions. We experimentally demonstrated that AT inhibits cardiac hypertrophy via suppressing phosphorylation of ERK1/2 and AKT.

Effects of Psoraleae fructus and its major component psoralen on Th2 response in allergic asthma.

This study is aimed to evaluate the effects of Psoraleae fructus (PF) on Th2 responses in a rat model of asthma in vivo and psoralen, a major constituent in PF, on Th2 responses in vitro. A rat model of asthma was established by sensitization and challenged with ovalbumin (OVA). Airway hyperresponsiveness was detected by direct airway resistance analysis. Lung tissues were examined for cell infiltration and mucus hypersecretion. Bronchoalveolar lavage fluid (BALF) was assessed for cytokine levels. In vitro study, Th2 cytokine production was evaluated in the culture supernatant of D10.G4.1 (D10 cells) followed by the determination of cell viability, meanwhile Th2 transcription factor GATA-3 expression in D10 cells was also determined. The oral administration of PF significantly reduced airway hyperresponsiveness (AHR) to aerosolized methacholine and decreased IL-4 and IL-13 levels in the BALF. Histological studies showed that PF markedly inhibited inflammatory infiltration and mucus secretion in the lung tissues. In vitro study, psoralen significantly suppressed Th2 cytokines of IL-4, IL-5 and IL-13 by ConA-stimulated D10 cells without inhibitory effect on cell viability. Furthermore, GATA-3 protein expression was also markedly reduced by psoralen. This study demonstrated that PF exhibited inhibitory effects on hyperresponsiveness and airway inflammation in a rat model of asthma, which was associated with the suppression of Th2 response. Psoralen, a major constituent of PF, has immunomodulatory properties on Th2 response in vitro, which indicated that psoralen might be a critical component of PF for its therapeutic effects.

Identification of the E3 Deubiquitinase Ubiquitin-specific Peptidase 21 (USP21) as a Positive Regulator of the Transcription Factor GATA3

The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

Erythropoietin‐activated ERK/MAP kinase enhances GATA‐4 acetylation via phosphorylation of serine 261 of GATA‐4

GATA-4, a zinc finger transcription factor, plays a critical role in heart development. Previous studies have shown that p300-targeted GATA-4 acetylation increases GATA-4 stability and transcriptional activity, which then stimulates hypertrophy of cardiomyocyte. Erythropoietin (EPO), an essential hypoxia-induced hormone for normal erythropoiesis, is known to exert cardioprotective effects against heart disease of either ischemic or non-ischemic origins. Although, various action mechanisms of EPO have been proposed in the diseased heart, its action mechanism in normal condition has not been investigated. In this study, we aimed to investigate the influence of EPO-induced ERK signaling on the regulation of GATA-4 protein action. EPO treatment increased the protein level of endogenous GATA-4 via ERK signaling pathway. Inhibition of ERK activity by U0126, suppressed EPO-induced expression of GATA-4 protein in rat cardiac myocytes. In addition, ERK activation by over-expression of constitutively active MEK1 strongly increased GATA-4 phosphorylation and subsequently enhanced its acetylation in P19 cells. EPO-induced ERK activation further increased the association of GATA-4 with p300. On the other hand, knock-down of p300 using siRNA diminished ERK-induced GATA-4 acetylation. As EPO-induced GATA-4 phosphorylation via ERK signaling pathway directly correlated with GATA-4 acetylation, we investigated to identify the ERK-dependent phosphorylation sites in GATA-4. Site-directed mutagenesis implicated that Ser-261 in GATA-4 played an important role for ERK-mediated GATA-4 acetylation. Taken together, these results indicated that EPO-induced ERK signaling activation increased GATA-4 phosphorylation and acetylation, partly via increase in the association between GATA-4 and p300, and these processes required the phosphorylation of GATA-4 at Ser-261 residue.

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and β-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of β-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes.

Defective nuclear localization of Hsp70 is associated with dyserythropoiesis and GATA-1 cleavage in myelodysplastic syndromes

AbstractNormal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3–mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.

An Ebox Element in the Proximal Gata4 Promoter Is Required for Gata4 Expression In Vivo

GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5′ origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5′ flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult) revealed that the Ebox mutation leads to a robust and specific decrease (up to 89%) of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.

The Xla (X-linked anemia) Mouse: A Transient Neonatal Anemia Caused by a Gata1 Splicing Mutation,

Abstract 3162The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice. Disclosures:No relevant conflicts of interest to declare.

Expression of GATA1 in the ovine conceptus and endometrium during the peri‐attachment period

The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes.

Novel Hematopoietic Progenitor Populations Revealed by Direct Assessment of GATA1 Protein Expression and cMPL Signaling Events

Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.

Coordinate Regulation of GATA-3 and Th2 Cytokine Gene Expression by the RNA-Binding Protein HuR

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate-rich elements (AREs) present in the 3' untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3'UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

GATA3 Expression Is Decreased in Psoriasis and during Epidermal Regeneration; Induction by Narrow-Band UVB and IL-4

Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions.Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction.

Identification of the E3 deubiquitinase USP21 as a positive regulator of GATA3 (152.25)

Abstract TCR signaling induces GATA3 expression in FOXP3+ Treg, but the underlying mechanism for GATA3 accumulation and its role in Treg function remains unclear. Here we identify the E3 deubiquitinase USP21 as a positive regulator of GATA3 in FOXP3+ T cells. By screening for regulators which may affect the function of key transcription factors essential for T cell lineage differentiation and function, we found that USP21 dramatically increases the cellular level of GATA3. Reciprocal co-immunoprecipitation studies indicated that USP21 can interact with GATA3 in vivo. Overexpression of wild type USP21, but not the enzymatically inactive mutant C221A, specifically promotes the up-regulation of GATA3 protein expression, suggesting that the deubiquitinase activity of USP21 is essential for the upregulation of GATA3. Moreover, we found that T cell activation via TCR and CD28 costimulation may upregulate the level of USP21 and GATA3 in FOXP3 expressing T cells. shRNA mediated knockdown of USP21 can prevent GATA3 accumulation after T cell activation, suggesting that the mechanism by which USP21 stablizes GATA3 may occur through preventing GATA3 degradation. Functionally, overexpression of wild-type USP21 promotes GATA3 mediated upregulation of IL-4. Our studies have identified a unique pathway to upregulate GATA3 activity at posttranslational level by reversing ubiqutination-mediated protein degradation.

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

Higher levels of GATA3 predict better survival in women with breast cancer

The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. GATA3 in particular is necessary for mammary gland maturation, and its loss has been implicated in breast cancer development. Our goal was to validate the ability of GATA3 expression to predict survival in breast cancer patients. Protein expression of GATA3 was analyzed on a high-density tissue microarray consisting of 242 cases of breast cancer. We associated GATA3 expression with patient outcomes and clinicopathologic variables. Expression of GATA3 was significantly increased in breast cancer, in situ lesions, and hyperplastic tissue compared with normal breast tissue. GATA3 expression decreased with increasing tumor grade. Low GATA3 expression was a significant predictor of disease-related death in all patients, as well as in subgroups of estrogen receptor-positive or low-grade patients. In addition, low GATA3 expression correlated with increased tumor size and estrogen and progesterone receptor negativity. GATA3 is an important predictor of disease outcome in breast cancer patients. This finding has been validated in a diverse set of populations. Thus, GATA3 expression has utility as a prognostic indicator in breast cancer.

T helper 1, 2 and 17 cell subsets in renal transplant patients with delayed graft function

Ischemia-reperfusion injury (IRI) in kidney transplantation is the major cause of delayed graft function (DGF), an event associated with an increased risk of acute rejection. The aim of this study was to evaluate T helper (Th) cell phenotype in renal transplants with DGF. T-bet (Th1), GATA-3 (Th2) and IL-17 (Th17) protein expression was investigated in pretransplant biopsies, DGF and acute tubular damage (ATD) caused by calcineurin-inhibitor toxicity. Intracytofluorimetric analysis of IFN-γ, IL-4 and IL-17 was performed to analyze Th1, Th2 and Th17 responses in peripheral blood mononuclear cells of recipients with early graft function (EGF) and DGF, before (T0) and 24 h after transplantation (T24). In pretransplant biopsies, T-bet(+) , GATA-3(+) and IL-17(+) cells were barely detectable. In DGF, T-bet(+) and IL-17(+) cells were significantly increased compared with pretransplant and ATD. More than 90% of T-bet(+) and less then 5% of IL-17(+) cells were CD4(+) . GATA-3(+) cells were increased to a lower extent. T-bet(+) /GATA-3(+) cell ratio was significantly higher in DGF. Peripheral CD4(+) IFN-γ/IL-4 ratio was significantly decreased in DGF, while CD4(+) /IL-17(+) cells did not differ between T0 and T24 in DGF. Our data suggest that DGF is characterized by a prevalent Th1 phenotype within the graft. This event might represent a link between DGF and acute rejection.

PU.1 Positively Regulates GATA-1 Expression in Mast Cells

Coexpression of PU.1 and GATA-1 is required for proper specification of the mast cell lineage; however, in the myeloid and erythroid lineages, PU.1 and GATA-1 are functionally antagonistic. In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. We isolated a variant mRNA isoform of GATA-1 in murine mast cells that is significantly upregulated during mast cell differentiation. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. In contrast to erythroid and megakaryocyte cells, in mast cells we show that PU.1 and GATA-2 predominantly occupy potential cis-regulatory elements in the IB exon region in vivo. Using reporter assays, we identify an enhancer flanking the IB exon that is activated by PU.1. Furthermore, we observe that in PU.1(-/-) fetal liver cells, low levels of the IE GATA-1 isoform is expressed, but the variant IB isoform is absent. Reintroduction of PU.1 restores variant IB isoform and upregulates total GATA-1 protein expression, which is concurrent with mast cell differentiation. Our results are consistent with a transcriptional hierarchy in which PU.1, possibly in concert with GATA-2, activates GATA-1 expression in mast cells in a pathway distinct from that seen in the erythroid and megakaryocytic lineages.

Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

The RNA-binding protein HuR regulates GATA3 mRNA stability in human breast cancer cell lines

Meta-analyses of microarray data indicate that GATA3 is co-expressed with estrogen receptor alpha (ER) in breast cancer cells. While the significance of this remains unclear, it is thought that GATA3 may serve as a prognostic indicator in breast tumors and may play a role in ER signaling. Recently, reciprocal regulation of GATA3 and ER transcription was demonstrated, suggesting that control of their expression is intertwined. We sought to determine whether GATA3 and ER expression was also coordinately regulated at other levels. Unlike ER, GATA3 was not under epigenetic control and was not re-expressed in the presence of DNMT or HDAC inhibitors in ER/GATA3-negative cells. However, like ER, these inhibitors decreased GATA3 expression in ER/GATA3-positive cell lines. We have previously reported that ER mRNA stability is increased through binding of the RNA-binding protein HuR/ELAV1 to the 3'untranslated region (UTR) and that DNMT and HDAC inhibitors reduce ER expression by altering this interaction. Biotin pull-down assays using a biotinylated GATA3 RNA probe confirmed that HuR also binds to the GATA3 3'UTR. Inhibition of HuR using siRNA probes decreased GATA3 mRNA, mRNA stability and protein expression, indicating that HuR plays a role in regulating GATA3 expression. Inhibition of either HuR or GATA3 reduced cell growth of MCF7 cells. Based on our findings, it is clear that coordinate regulation of ER and GATA3 occurs, however differences do exist. These findings may aid in identification of new targets that control cell growth of breast cancer cells.

Ontogenetic development of erythropoiesis can be studied non-invasively in GATA-1:DsRed transgenic zebrafish

For the erythroid cell lineage development in vertebrates, GATA-1 transcription factor is essential. In our report, we have demonstrated that the approximate developmental status of erythrocytes and the progression of blood formation can be studied non-invasively in GATA-1:DsRed transgenic zebrafish ( Danio rerio) embryo and larva by characterization of fluorescence luminance spectra. The study was carried out for animals maintained under normoxic and hypoxic (152 and 20 torr PO 2 respectively) conditions up to 10 days post-fertilization (dpf) and total blood cell concentrations and fluorescent cells' percentage were determined for this purpose. The erythroids were classified into five intensity stages (IS) on the basis of their fluorescence intensity. The luminescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf to 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The total blood cell concentration dropped after 8 dpf in contrast to hypoxic group which showed further increasing trend. The fluorescent cells' percentage in normoxic group was generally higher as compared to the hypoxic ones. Our method successfully defined various stages of erythroid development. An effort was also made to correlate our luminance data (GATA-1 expression) and total blood cell concentrations with Epo mRNA production. Quantitative RT-PCR of 2–15 dpf old zebrafish was carried out for this purpose. Normoxic animals showed 1–3 Epo mRNA copies per ng RNA in contrast to the hypoxic larvae that showed remarkable fluctuation of 1 to 12 Epo mRNA copies per ng RNA during development. The blood volume (aortic diameter) and production time scale proved to be important factors to define the relationship of Epo mRNA with total blood cell concentration and GATA-1 protein expression respectively.