GATA-3 mRNA Levels Research Articles

Expression imbalance of transcription factors GATA-3 and T-bet in post-resuscitation myocardial immune dysfunction in a porcine model of cardiac arrest

BackgroundThis study investigated whether an imbalance in Th1/Th2 cells is involved in the post-resuscitation myocardial immune dysfunction. Methods26 Wuzhishan miniature pigs were randomly divided into return of spontaneous circulation (ROSC) group (n=20) and sham-operated group (n=6), 20 pigs were subjected to 8min of electrically induced cardiac arrest, After successful ROSC, the 16 surviving pigs were randomly assigned to be sacrificed (n=8 per group) at 12 and 24h after ROSC, respectively. CD4+ and CD8+ lymphocyte subsets were determined by flow cytometry, interleukin (IL)-4 and interferon (IFN)-γ in the myocardium were measured by ELISA, and protein and mRNA levels of GATA-3 and T-bet were detected in the myocardium by Western blotting and quantitative real-time PCR in the post-ROSC group (n=8 per group) at 12 and 24h after ROSC and sham-operated group (n=6) at 24h after ROSC, respectively. ResultsCD4+ lymphocyte subsets were significantly lower in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC. The levels of myocardium IFN-γ were markedly increased, while IL-4 was significantly decreased in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC. Protein expression and mRNA levels of T-bet were markedly increased in the myocardium of pigs in the post-ROSC group compared with the sham-operated group (P<0.05) at 12 and 24h after ROSC, while GATA-3 was significantly reduced (P<0.05). ConclusionThe myocardial immune dysfunction induced by the change in expression levels of the transcription factors GATA-3 and T-bet may be involved in the process of post-resuscitation myocardial injury in a porcine model of cardiac arrest.

Soluble LR11, a Novel Acute Leukemia Marker, Drastically Induces WT1 mRNA Expression Together with Synergic Activation of Gatas, and the Migration Activity

Abstract 4795 Introduction:We have recently reported that LR11 (also called SorLA or SORL1) is specifically expressed on the surface of acute leukemia cells, and the circulating levels of soluble form (sLR11) are highly associated with peripheral blast numbers in acute leukemia patients at diagnosis and decreased in remission (2012, Sakai et al). Although the functional role of sLR11 in the migration of undifferentiated vascular cells has been studied, the pathological significance of sLR11 has not been elucidated for the development of leukemia blast properties. The expressions of WT1, an oncogene for the various malignant tumors, are associated with poor prognosis of patients with acute leukemias, and the WT1 levels have been thought to indicate the blast properties. In this study, in order to know the significance of sLR11 for the oncogenic properties of leukemia cells, we first examined the correlations of circulating sLR11 levels with WT1 mRNA levels in the bone marrow of acute leukemia patients. Next, we analyzed the effects of sLR11 on WT1 mRNA expressions, particularly in association with GATAs, factors for the WT1 gene transcription, in leukemia cell lines. Materials and methods:Levels of serum sLR11 and bone marrow cell WT1 mRNA were measured by ELISA and real-time PCR, respectively, using samples of acute leukemia patients (AML n=6, ALL n=5). Statistical analyses were performed by Spearman rank correlation system. mRNA levels of GATA1, GATA2 and WT1 were analyzed using K562 cells with or without recombinant sLR11 and LR11-overexpressing K562 cells. Results:Figure 1 shows the relationship between sLR11 in serum and WT1 mRNA in bone marrow mononuclear cells in acute leukemia patients at diagnosis. The sLR11 levels were significantly positively correlated with the WT1 levels in the patients. In order to know the pathological link between sLR11 and WT1 in leukemia cells in detail, we then analyzed the effects of sLR11 on the WT1 mRNA expressions in K562 cells. WT1 mRNA levels were gradually increased after the incubation with 1 ng/ml sLR11, and the levels at 4 h were elevated to 2.5-fold of those before the incubation (Figure 2). The levels were then decreased to the levels equivalent to those before the incubation. The mRNA levels of GATA1 and GATA2 were also drastically elevated at 4 h and then sharply decreased after adding sLR11. GATA1 mRNA levels were particularly increased by 5-fold of those before the incubation. Thus, sLR11 were suggested to cause the activation of transcription factor network of GATAs, and induce the WT1 gene transcription in the cells. We finally investigated the effects of sLR11 overexpression on the elevation of WT1 mRNA in K562 cells. The LR11-overexpressing cells showed significantly increased expression levels of GATA1 and GATA2 in addition to WT1 mRNAs. Conclusions:Circulating sLR11 levels were significantly correlated with the WT1 mRNA expression levels in acute leukemia. sLR11 induces the WT1 expression, possibly through the activations of GATAs in leukemia cells. These results suggest that sLR11 may be a novel marker indicating the malignant properties of leukemia cells. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Regulatory T‐lymphocytes mediate amyotrophic lateral sclerosis progression and survival

In amyotrophic lateral sclerosis (ALS) mice, regulatory T-lymphocytes (Tregs) are neuroprotective, slowing disease progression. To address whether Tregs and FoxP3, a transcription factor required for Treg function, similarly influence progression rates of ALS patients, T-lymphocytes from patients were assessed by flow cytometry. Both numbers of Tregs and their FoxP3 protein expressions were reduced in rapidly progressing ALS patients and inversely correlated with progression rates. The mRNA levels of FoxP3, TGF-β, IL4 and Gata3, a Th2 transcription factor, were reduced in rapidly progressing patients and inversely correlated with progression rates. Both FoxP3 and Gata3 were accurate indicators of progression rates. No differences in IL10, Tbx21, a Th1 transcription factor or IFN-γ expression were found between slow and rapidly progressing patients. A 3.5-year prospective study with a second larger cohort revealed that early reduced FoxP3 levels were indicative of progression rates at collection and predictive of future rapid progression and attenuated survival. Collectively, these data suggest that Tregs and Th2 lymphocytes influence disease progression rates. Importantly, early reduced FoxP3 levels could be used to identify rapidly progressing patients.

Type I interferon inhibits acute IL-5 and IL-13 expression in human memory CD4+ T cells (125.12)

Abstract Type I Interferon (IFN-α/β) is an innate cytokine important for fighting infections and is used to treat hepatitis C infection and multiple sclerosis, among other diseases. Although the innate role of this cytokine has been studied extensively, our group has shown that IFN-α/β plays a role in shaping the adaptive immune response in humans. Recently, we demonstrated that IFN-α/β inhibits Th2 development by regulating mRNA and protein levels of GATA3. Regulation of GATA3 in turn suppresses Th2 cytokine production, without effecting IFN-γ production. In addition to blocking Th2 development, we now demonstrate an acute inhibition of human memory T cell function upon treatment with IFN-α/β. Human memory CD4+ T cells acutely activated with anti-CD3 in the presence of IFN-α/β resulted in reduced mRNA levels of Th2 cytokine genes, IL5 and IL13, but did not effect IL4 expression. GATA3 levels were not affected, suggesting a GATA3-independent mechanism. Further experiments have determined that this regulatory mechanism does not require new protein synthesis. This mechanism seems to be specific to IFN-α/β, as neither IFN-γ nor IFN-λ treatment were able to repress Th2 cytokine mRNA expression to the extent of IFN-α/β. This study has uncovered an additional level of regulation that blocks early TCR signaling events that affect Th2 function. This novel pathway suggests that treatment of atopic asthmatic individuals with IFN-α/β can regulate both Th2 development and memory Th2 function.

The Degree of Leukocytosis and Urine GATA-3 mRNA Levels Are Risk Factors for Severe Acute Kidney Injury in Puumala Virus Nephropathia Epidemica

Puumala hantavirus (PUUV) infection, also known as nephropathia epidemica, is the most common cause of hemorrhagic fever with renal syndrome (HFRS) in Europe. The pathogenesis of PUUV nephropathia epidemica is complex and multifactorial, and the risk factors for severe acute kidney injury (AKI) during acute PUUV infection are not well defined. We conducted a prospective study of hospitalized patients with PUUV infection in Tampere, Finland to identify acute illness risk factors for HFRS severity. Serial daily blood and urine samples were collected throughout acute illness and at 2 week and 6 month convalescent visits. By univariate analyses, the maximum white blood cell count during acute illness was a risk factor for severe AKI. There were no significant associations between PUUV-induced AKI severity and platelet counts, C-reactive protein, or alanine aminotransferase levels. Maximum plasma interleukin (IL)-6, urine IL-6, and urine IL-8 concentrations were positively associated with PUUV-induced AKI. Finally, the maximum urinary sediment GATA-3 mRNA level was positively correlated with the peak fold-change in serum creatinine, regardless of AKI severity classification. By multivariate analyses, we found that the maximum levels of leukocytes and urinary sediment GATA-3 mRNA during acute illness were independent risk factors for severe PUUV-induced AKI. We have identified novel acute illness risk factors for severe PUUV-induced AKI.

Monitoring of urinary messenger RNA levels for the prediction of flare in systemic lupus erythematosus

Background Systemic lupus erythematosus (SLE) is characterized by disease flares and remission. We hypothesize that in clinically quiescent SLE patients, the mRNA level of target genes in the urinary sediment is an early indicator of disease flare. Methods From a cohort of 134 adult SLE patients prospectively followed for 56 weeks, we identified 19 patients with a single disease flare. The mRNA level of eight pre-defined target genes in their urinary sediment before disease flare was compared to 19 matched controls with no disease flare during the same period. Results Urinary mRNA level remained static in the control group during the study period. Before disease flare, there was a significant increase in the mRNA level of monocyte chemotactic protein (MCP)-1and forkhead box P3 (FOXP3), and decrease in interleukin (IL)-17 and GATA-3, in the urinary sediment. The mRNA level of FOXP3 in urinary sediment increases 8 weeks prior to a flare, which precedes the corresponding change in serum complement and anti-DNA antibody titer, while that of MCP-1, IL-17, and GATA3 began to change 4 weeks prior to a flare. The same pattern of change in urinary mRNA level was observed in patients with mild-to-moderate or severe flare, and those with renal or non-renal flare. The SLE Disease Activity Index (SLEDAI) score at the time of flare significantly correlated with the change in urinary level of IL-17 (r = − 0.462, p = 0.046) and GATA-3 (r = − 0.455, p = 0.05), but not MCP-1 or FOXP3, prior to the flare. Conclusion Monitoring of MCP-1, IL-17, GATA-3 and FOXP3 mRNA level in urinary sediment may provide an early clue for detecting disease flare in SLE patients.

Stat6 activity-related Th2 cytokine profile and tumor growth advantage of human colorectal cancer cells in vitro and in vivo

Signal transducer and activator of transcription 6 (Stat6) is critical in Th2 polarization of immune cells and active Stat6 activity has been suggested in anti-tumor immunity in animal models. The present study aims at investigating the impact of natural Stat6 activity on tumor microenvironment in human colorectal cancer cells in vitro and in vivo. Using colorectal cancer cell lines HT-29 and Caco-2 whose IL-4/Stat6 activities were known and nude mice as a model, we examined correlative relationships between Stat6 activities and gene expression profiles together with cellular behaviors in vitro and in vivo. HT-29 cells carrying active Stat6 signaling displayed spontaneous expression profiles favoring Th2 cytokines, cell cycle promotion, anti-apoptosis and pro-metastasis with increased mRNA levels of IL-4, IL-13, GATA-3, CDK4, CD44v6 and S100A4 using RT-PCR. In contrast, Caco-2 cells carrying defective Stat6 signaling exhibited spontaneous expression profiles favoring Th1 and Th17 cytokines, cell cycle inhibition, pro-apoptosis and anti-metastasis with elevated mRNA expression of IFNγ, TNFα, IL-12A, IL-17, IL-23, T-bet, CDKN1A, CDKNIB, CDKN2A and NM23-H1. Xenograft tumors of Stat6-active HT-29 cells showed a growth advantage over those of Stat6-defective Caco-2 cells. Furthermore, mice bearing HT-29 tumors expressed increased levels of Th2 cytokines IL-4 and IL-5 in the blood and pro-growth and/or pro-metastasis proteins CDK4 and CD44v6 in the tumor. To the contrary, mice bearing Caco-2 tumors expressed heightened levels of Th1 cytokines IFNγ and TNF in the blood and pro-apoptosis and anti-metastatic proteins p53 and p27kip1 in the tumor. Colorectal cancer cells carrying active Stat6 signaling may create a microenvironment favoring Th2 cytokines and promoting expression of genes related to pro-growth, pro-metastasis and anti-apoptosis, which leads to a tumor growth advantage in vivo. These findings may imply why Stat6 pathway is constitutively activated in a number of human malignancies.

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.

Effects of retinoic acid and butyric acid on the expression of prestin and gata‐3 in organotypic cultures of the organ of corti of newborn rats

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high level of sensitivity of mammalian hearing. The factors that influence prestin expression are still largely unknown. We studied the effects of the application of retinoic acid, a ligand of a nuclear receptor, and of butyric acid, an inhibitor of histone deacetylase activity, on the expression of mRNA of prestin and Gata-3 in the organotypic culture of the organ of Corti of newborn rats using RT-PCR. Application of retinoic acid at concentrations of 1-50 μM results in a dose-dependent expression decrease after two days in culture. Treatment with sodium butyrate (0.5-2 mM) elevated the expression of prestin and Gata-3. Statistically significant correlations between Gata-3 and prestin mRNA levels were observed under all conditions. The data indicate that retinoid nuclear transcription factors, GATA-3 and histone acetylation/deacetylation processes may have a regulatory role to play in prestin expression.

Abstract 2281: GATA3 and progestin interaction in mammary cancer

Abstract GATA3 is a Master Transcription Factor crucial in mammary gland development and differentiation. Due to the role of progesterone in mammary gland development and in the etiology and progression of breast cancer, we explored the regulation of GATA3 by the synthetic progestin medroxyprogesterone acetate (MPA) in breast cancer cells. The experiments were performed in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Our results indicate that MPA produces GATA3 downregulation, as measured by Western Blot (WB) experiments, and this effect is abolished by the progestin antagonist RU486. In order to assess MPA regulation of GATA3 mRNA levels we performed Real Time PCR, and observed that MPA induced GATA3 transcriptional downregulation. By performing in silico analysis, we detected that GATA3 aminoacidic sequence contains a PEST sequence, recognized as a site for protein degradation. This finding lead us to explore whether MPA affects GATA3 post-translational regulation. For that purpose we treated T47D cells with cicloheximide, and a reduction of GATA3 half-life was observed with MPA treatment. To evaluate whether the B isoform of Progesterone Receptor (PR-B) was sufficient to maintain GATA3 downregulation, we used T47D – YB cell line, which expresses only PR-B. As assessed by WB analysis, PR-B proved to be sufficient for MPA-induced GATA3 downregulation. Remarkably, MPA treatment of T47D-Y- DBD cells, which express a mutant form of PR that is unable to bind to DNA or to tether with other transcription factors, was able to cause GATA3 downregulation. This result suggests that GATA3 downregulation occurs via PR citoplasmatic signaling. In order to dissect the signaling pathway involved in MPA regulation of GATA3 expression, cells were incubated with U0126 + MPA. We found that the addition of U0126 abrogated GATA3 dowregulation by MPA. This result suggests that ERK 1/2 phosphorylation is necessary to maintain this regulation. However, when Src phosphorylation was prevented, together with Src downstream target ERK 1/2 phosphorylation, GATA3 downregulation persisted. This result points to an off-target effect of U0126 inhibitor, probably preventing ERK 5 phosphorylation, as it was previously reported. Finally, we performed in silico analysis of cyclin D1 gene proximal promoter and found three potential GATA3 binding sites. To assess the effect of GATA3 on cyclin D1 expression, we overexpressed human GATA3 in T47D cells, and measured cyclin D1 levels by WB. Overexpression of GATA3 resulted in lower cyclin D1 levels. This study shows for the first time GATA3 downregulation by MPA in breast cancer cells, both at transcriptional and post-translational levels. It also sheds light over the PR mechanism involved in this process and the signaling pathway potentially implicated. Finally, we demonstrate that GATA3 overexpression affects cyclin D1 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2281. doi:10.1158/1538-7445.AM2011-2281

Distribution and expression analysis of transcription factors in tissues and progenitor cell populations of the goldfish (Carassius auratus L.) in response to growth factors and pathogens

We report on the mRNA levels of a panel of transcription factors in the kidney and spleen tissues, and in the cell populations from the blood, the spleen, and in the sorted kidney progenitor cells. The mRNA levels of cebpα, cjun, cmyb, egr1, gata1, gata2, gata3, lmo2, mafb, pax5, pu.1 and runx1 were assessed in healthy goldfish as well as in fish challenged with two different pathogens, Aeromonas salmonicida A449 or Trypanosoma carassii. Spleen tissue from healthy goldfish showed higher expression of myeloid (cjun), erythroid (gata1) and lymphoid (gata3, pax5) transcription factors, and lower expression of the myeloid transcription factor cebpα when compared to that of kidney. Splenocytes and PBLs had significantly higher mRNA levels of the transcription factors involved in myeloid (pu.1, mafb, cjun, egr1, cebpa), erythroid (gata1, lmo2), and lymphoid pathways (gata3 and pax5) compared to sorted kidney R1 progenitor cells, while R1 progenitor cells had higher mRNA levels of early progenitor transcription factors (runx1 and cmyb). Furthermore, the R1 progenitor cells had higher mRNA levels of the transcription factors involved in early progenitor cells (egr1, gata2) and the lymphoid lineage progenitors (gata3, pax5) compared to those in kidney. The mRNA levels of the transcription factors (gata2, mafb, cjun, gata1, lmo2, gata3, and pax5) in R1 progenitor cells changed during cultivation; they were elevated in day 2 R1 cells and down-regulated by day 6 of cultivation, when compared to those of day 0 R1 cells. Treatment of day 2 R1 progenitor cells with rgCSF-1 resulted in an up-regulation of transcription factors important for myeloid cell development (cjun and egr1). Similarly, rgkitla up-regulated the expressions of myeloid (mafb, egr1 and cebpa) transcription factors. Changes in the expression of transcription factors in the R1 progenitor cells were related to the observed developmental processes of myeloid progenitor cells during cultivation or treatment with recombinant growth factors in vitro. We also observed differential expressions of the transcription factors in R1 progenitor cells following exposure of the goldfish to either prokaryotic (heat-killed A. salmonicida A449) or eukaryotic (T. carassii) pathogens.

Type I interferon inhibits expression of the alternative GATA3 transcript in human Th2 cells (57.22)

Abstract The GATA3 transcription factor contributes to multiple immune processes, including the differentiation of T helper type 2 (Th2) cells. Th2 cells regulate many aspects of allergic disorders and express GATA3 in response to IL-4 signaling. Subsequently, GATA3 promotes transcription of the Th2 cytokines while also creating a feed-forward loop that stabilizes Th2 commitment. A previous study in mice has shown that Th2 commitment is accompanied by increased utilization of an alternate first exon (exon 1A) in the GATA3 transcript via an upstream promoter. The resulting transcript contains the same protein coding sequence but permits cell-specific control of expression levels. We previously demonstrated that the antiviral cytokine type I interferon (IFN-α/β) suppresses GATA3 mRNA and protein levels both in developing Th2 cells and in fully committed Th2 cells, leading to a decrease in Th2 cytokine expression. Here, we show that IL-4 significantly enhances utilization of exon 1A in the GATA3 transcripts in human Th2 cells. IFN-α/β preferentially inhibits expression of the exon 1A transcript, suggesting that IFN-α/β can regulate Th2 cells without affecting the basal levels of GATA3 required for other immune processes. Thus, IFN-α/β suppresses GATA3 in human Th2 cells and may be useful as a novel therapeutic for asthma and allergy.

Correlation between the ratio of T-bet/GATA-3 and the levels of IL-4 and IFN-γ in patients with allergic asthma

The aim of this study was to investigate the ratio of the transcription factors T-bet/GATA-3 in patients with allergic asthma. Forty-seven individuals with allergic asthma and 47 healthy control individuals provided 5ml of anticoagulated peripheral venous blood. Lymphocytes in peripheral blood were isolated by Ficoll and treated with phytohemagglutinin (PHA) at a final concentration of 100mg/l for 48h. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels were detected using an enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of both T-bet and GATA-3 were detected using reverse transcription polymerase chain reaction (RT-PCR). IFN-γ levels in the lymphocytes of asthmatic patients were lower than those of the control group (P<0.05) and were positively correlated with the ratio of T-bet/GATA-3. By contrast, IL-4 levels were significantly higher in asthmatic patients than in the control group (P<0.01) and were negatively correlated with the ratio of T-bet/GATA-3. In conclusion, the ratio of T-bet/GATA-3 can be used as an objective indicator of immune imbalance in patients with allergic asthma.

Intravenous immunoglobulin attenuates airway hyperresponsiveness in a murine model of allergic asthma

Intravenous immunoglobulin (IVIG) has potent anti-inflammatory and immune-modulating properties. IVIG has been utilized as a steroid-sparing agent in severe asthma, but the results of clinical trials have been conflicting. To determine whether IVIG is able to attenuate bronchial reactivity, pulmonary inflammation and T cell function using a murine model of allergic airways disease. BALB/c or C57BL/6 mice were sensitized to ovalbumin (OVA) or a phosphate-buffered saline control using local nasal sensitization, and then received five intranasal challenges on days 28-32 before sacrifice. Mice were treated intraperitoneally with either IVIG (1-2 g/kg) or equivalent human serum albumin 24 h before the first OVA challenge. Bronchial reactivity to methacholine was examined using the FlexiVent small animal ventilator. We evaluated pulmonary histology, mRNA from lung digests for T-helper type 2 (Th2)-related genes and bronchoalveolar lavage for cell counts and cytokines. Splenocytes were utilized to study OVA-induced cell proliferation, cytokine production and dendritic cell maturation. IVIG markedly attenuated the perivascular and peribronchial pulmonary inflammation, and decreased bronchial hyperresponsiveness to methacholine. IVIG treatment of splenocytes from sensitized animals diminished cellular proliferation to OVA, whereas IVIG treatment in vivo markedly attenuated OVA-driven splenocyte proliferation. This is accompanied by diminished IL-13 and TNF-α levels in splenocyte culture, decreased expression of Jagged-1, increased Delta-4 and decreased GATA-3 mRNA levels, signs that IVIG has suppressed the expected Th2 response that accompanies repeated allergen exposure. Increased regulatory T cells were found in draining pulmonary lymph nodes in IVIG-treated mice but not in controls. IVIG was effective in ameliorating allergic airway disease in our model. IVIG may be a promising adjunct therapy requiring further study for patients with severe asthma.

Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice

An inflammatory response is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a relentless and devastating degenerative disease of motoneurons. This response is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity; initially infiltrating lymphocytes and microglia slow disease progression, while later, they contribute to the acceleration of disease. Since motor weakness begins in the hindlimbs of ALS mice and only later involves the forelimbs, we determined whether differential protective versus injurious inflammatory responses in the cervical and lumbar spinal cords explained the temporally distinct clinical disease courses between the limbs of these mice. Densitometric evaluation of immunohistochemical sections and quantitative RT-PCR (qRT-PCR) demonstrated that CD68 and CD11c were differentially increased in their spinals cords. qRT-PCR revealed that protective and anti-inflammatory factors, including BDNF, GDNF, and IL-4, were increased in the cervical region compared with the lumbar region. In contrast, the toxic markers TNF-α, IL-1β and NOX2 were not different between ALS mice cervical and lumbar regions. T lymphocytes were observed infiltrating lumbar spinal cords of ALS mice prior to the cervical region; mRNA levels of the transcription factor gata-3 (Th2 response) were differentially elevated in the cervical cord of ALS mice whereas t-bet (Th1 response) was increased in the lumbar cord. These results reinforce the important balance between specific protective/injurious inflammatory immune responses in modulating clinical outcomes and suggest that the delayed forelimb motor weakness in ALS mice is partially explained by augmented protective responses in the cervical spinal cords.

Vegf regulates embryonic erythroid development through Gata1 modulation

AbstractTo determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

TCR-Dependent Translational Control of GATA-3 Enhances Th2 Differentiation

The differentiation of CD4(+) T cells into the Th2 subset is controlled by the transcription factor GATA-3. GATA-3 is both necessary and sufficient for Th2 differentiation and works through the induction of chromatin remodeling at the Th2 effector cytokine loci. We show in this study that IL-4 stimulation induces GATA-3 mRNA upregulation, but the level of GATA-3 protein induced is insufficient for Th2 differentiation. The levels of GATA-3 protein and Th2 differentiation are enhanced by concomitant TCR signaling through the PI3K/mammalian target of rapamycin pathway. The PI3K-mediated increase in GATA-3 protein occurs without increasing the GATA-3 mRNA level. Rather, TCR signaling through PI3K specifically enhances the translation rate of GATA-3 without affecting the protein stability. Importantly, this role of TCR signaling is independent of the effects of TCR signaling in T cell survival and expansion. Thus, TCR signaling through PI3K may play a critical role in Th2 differentiation by the specific enhancement of GATA-3 translation.

Abstract 4063: Estrogen receptor alpha functions as an RNA binding protein to regulate GATA3 protein levels: a novel role for ER

Abstract The role of estrogen receptor alpha (ER) as a transcription factor has been well established. ER transcriptionally activates genes by binding to DNA directly within the gene promoter region or by binding to DNA indirectly through protein-protein dependent mechanisms. Many ER-responsive genes that are activated through ER-mediated transcriptional activation have been identified, including GATA3. While the role of GATA3 in breast cancer still remains largely unknown, numerous reports have shown that ER and GATA3 are coordinately expressed in breast cancer and that ER may play a role in regulating GATA3 expression. A recent report by Eeckhoute et al (2007) has shown that ER transcriptionally activated GATA3 expression in T47D breast cancer cells. Using MCF7 cells, we have recently identified a novel role for ER as an RNA binding protein that binds to GATA3 mRNA. Interestingly, silencing of ER using siRNA probes slightly increased GATA3 mRNA levels, however GATA3 protein levels decreased significantly. This decrease in protein was not due to improper export of GATA3 mRNA from the nucleus to the cytoplasm where protein translation occurs, as GATA3 mRNA expression was equivalent in both the nuclear and cytoplasmic compartments. This suggested that the decrease in protein expression was not due to impaired GATA3 mRNA transport but could be due to impaired protein translation. Since silencing of ER had dramatically different effects on RNA and protein levels, this data suggested that ER protein may function as an RNA binding protein that binds to the GATA3 mRNA to regulate its protein translation. Using biotin pulldown techniques, we found that ER does function as an RNA binding protein by binding directly to a site within a 125 nucleotide region of the GATA3 3‘UTR. This role of ER as an RNA binding protein is consistent with the data of Lanz et al (1999, 2000) that demonstrated that ER protein can bind to an RNA transcript, the steroid receptor co-activator, SRA. When bound to SRA, however, ER did not alter the RNA levels or protein expression of SRA but did result in increased activation of ER. Therefore, our data is the first to identify a novel role for ER as a RNA binding protein that can regulate the protein expression of the RNA transcript to which it is bound. Altering such ER-RNA interactions could potentially provide a new therapeutic avenue for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4063.

Increased T-bet to GATA-3 Ratio During Acute Allograft Rejection in the Rat Lung

Acute allograft rejection (AR) remains a major problem in solid organ transplantation. The pivotal mechanism hinges on alloantigen recognition by recipient T helper (T h) cells that differentiate into T h1 and T h2. This study investigated the association of mRNA levels of the transcription factors T-box expressed in T cells and GATA-binding protein 3 with the development of T h1/T h2-directed immune responses. We investigated the expression of T-bet and GATA-3 mRNA levels and the protein levels of their marker cytokines interleukin (IL)-2 and IL-4 in orthotopically transplanted rat lungs during AR. We observed a nonsignificant increase in T-bet expression following allografting at days 3 and 5 but there was a significant reduction in GATA-3 expression on day 5 compared with controls. The ratio of T-bet to GATA-3 expression showed a trend to increase at day 3 following allografting reaching significance at 5 days. These changes were associated with a significant increase in the expression of IL-2 over IL-4 on days 3 and 5. This study suggests that T h1 responses play a major role during AR in the rat lung, and that this differentiation can be monitored by measuring mRNA levels of T-bet and GATA-3.

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.