Impairment of the Hypothalamus-Pituitary-Thyroid Axis Caused by Naturally Occurring GATA2 Mutations In Vitro.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus-pituitary-thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone ß (TSHß). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHß promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHß promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

The in vitro and in vivo effects of yerba mate (Ilex paraguariensis) extract on adipogenesis.

The aim of this study was to evaluate the effects of yerba mate extract and its principal bioactive compounds on adipogenesis. The anti-adipogenic effects of yerba mate, chlorogenic acid, quercetin and rutin were evaluated in 3T3-L1 cells using a PCR array. The results obtained in vitro were validated in vivo in a high-fat diet-induced model of obesity. The in vitro and in vivo results demonstrated that yerba mate extract down-regulated the expression of genes that regulate adipogenesis, such as Creb-1and C/EBPα, and the extract up-regulated the expression of genes related to the inhibition of adipogenesis, including Dlk1, Gata2, Gata3, Klf2, Lrp5, Pparγ2, Sfrp1, Tcf7l2, Wnt10b, and Wnt3a. In summary, it was demonstrated that yerba mate and its bioactive compounds regulate the expression of genes related to in vitro adipogenesis. Furthermore, yerba mate might regulate adipogenesis through the Wnt pathway.

Omega-3 fatty acids suppress Th2-associated cytokine gene expressions and GATA transcription factors in mast cells.

Because the interaction between omega-3 fatty acids and mast cells has remained largely unknown in allergies, we investigated whether omega-3 fatty acids affect the activation of mast cells by examining Th2-associated cytokine production and possible molecular mechanisms. Alpha-linolenic acid and its metabolites including eicosapentaenoic acid and decosahexaenoic acid induced a dramatic decrease in the production of interleukin (IL)-4, IL-5 and IL-13 in a dose-dependent manner, as well as mRNA expression of their genes, in activated MC/9 mast cells and bone marrow-derived mast cells. The effects were comparable to those of cyclosporin A (1 µM), a well-known immunosuppressive agent. Nuclear expression of GATA binding protein-1 (GATA-1) and GATA binding protein-2 (GATA-2), essential transcription factors for mast cell activation, was also greatly suppressed. However, their mRNA expressions were not affected. In P815 mast cells, which do not express GATA-1, the suppressive effects on cytokines were abolished. On the contrary, omega-3 fatty acids had less significant effects on IL-4 and IL-5 and resulted in a slight decrease in IL-13 production in EL-4 T cells. Finally, oral administration of fish oil containing high level of omega-3 fatty acids significantly reduced the severity of dermatitis and the thickening of epidermis/dermis in a NC/Nga murine atopic model. The number of cells expressing CD117(+) and FcεRIα(+) was greatly decreased and GATA-1 expression in the cells was also diminished. Taken together, omega-3 fatty acids might target mast cells to a greater extent than T cells to suppress Th2 cytokine expression by inhibiting GATAs for alleviation of allergic disease.

Transcriptional regulatory mechanisms underlying the GABAergic neuron fate in different diencephalic prosomeres.

Diverse mechanisms regulate development of GABAergic neurons in different regions of the central nervous system. We have addressed the roles of a proneural gene, Ascl1, and a postmitotic selector gene, Gata2, in the differentiation of GABAergic neuron subpopulations in three diencephalic prosomeres: prethalamus (P3), thalamus (P2) and pretectum (P1). Although the different proliferative progenitor populations of GABAergic neurons commonly express Ascl1, they have distinct requirements for it in promotion of cell-cycle exit and GABAergic neuron identity. Subsequently, Gata2 is activated as postmitotic GABAergic precursors are born. In P1, Gata2 regulates the neurotransmitter identity by promoting GABAergic and inhibiting glutamatergic neuron differentiation. Interestingly, Gata2 defines instead the subtype of GABAergic neurons in the rostral thalamus (pTh-R), which is a subpopulation of P2. Without Gata2, the GABAergic precursors born in the pTh-R fail to activate subtype-specific markers, but start to express genes typical of GABAergic precursors in the neighbouring P3 domain. Thus, our results demonstrate diverse mechanisms regulating differentiation of GABAergic neuron subpopulations and suggest a role for Gata2 as a selector gene of both GABAergic neuron neurotransmitter and prosomere subtype identities in the developing diencephalon. Our results demonstrate for the first time that neuronal identities between distinct prosomeres can still be transformed in postmitotic neuronal precursors.

Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia.

We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

MicroRNA-24 regulates vascularity after myocardial infarction.

BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.

Inhibitory effect and transcriptional impact of berberine and evodiamine on human white preadipocyte differentiation.

It has been reported that the botanical alkaloids, berberine and evodiamine inhibit mouse preadipocyte 3T3-L1 differentiation. The aim of this study was to investigate the effect and transcriptional impact of berberine and evodiamine individually and in combination on human white preadipocyte (HWP) differentiation. We have shown that treatment with 8 microM berberine or 4 microM evodiamine resulted in a major inhibition of HWP differentiation accompanied by up-regulation of both GATA binding protein 2 and 3 (GATA-2 and GATA-3) mRNA and protein expression, suggesting that both compounds may have excellent potential as agents to prevent obesity.

Berberine increases expression of GATA-2 and GATA-3 during inhibition of adipocyte differentiation.

It is known that a number of transcription factors are key regulators in the complex process of adipocyte differentiation including peroxisome proliferator activated receptor gamma (PPARgamma) and the CCAAT enhancer binding protein alpha (C/EBPalpha). Studies have demonstrated that in pre-adipocyte 3T3-L1 cells constitutive expression of the DNA binding proteins GATA-2 and GATA-3 results in protein/protein interactions with C/EBPalpha resulting in down regulation of PPARgamma and subsequent suppressed adipocyte differentiation with cells trapped at the pre-adipocyte stage. Thus it appears that GATA-2 and GATA-3 are of critical importance in regulating adipocyte differentiation through molecular interactions with PPARgamma and C/EBPalpha. Recent reports suggest that berberine, an isoquinoline derivative alkaloid isolated from many medicinal herbs prevents differentiation of 3T3-L1 cells via a down regulation of PPARgamma and C/EBPalpha expression. The aim of this study was to determine the effect of berberine on GATA-2 and 3 gene and protein expression levels during differentiation of 3T3-L1 cells. MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was used to detect the cytotoxic effects of berberine on the viability of 3T3-L1 cells during proliferation and differentiation. Differentiation of 3T3-L1 cells was monitored by Oil Red O staining and RT-PCR of PPARgamma and C/EBPalpha and the expression of GATA-2 and 3 was determined by RT-PCR and Western Blot. Results show that following treatment with 8microM berberine the mRNA and protein expression levels of GATA-2 and 3 were elevated and accompanied by inhibited adipocyte differentiation. These results may lead to the use of berberine to target the induction of specific genes such as GATA-2 and GATA-3 which affect adipocyte differentiation.

Molecular pharmacological basis of the YiSui ShenXu Granule in beta-thalassemia therapy.

OBJECTIVES: To study the molecular pharmacological basis of the YiSui ShenXu Granule, a complex prescription of the Chinese traditional medicine used to treat beta-thalassemia. METHODS: Real-time quantitative PCR method had been applied to analyze the genes expression: gamma-globin, Ckit, EpoR, Spi, FKLF, GATA1 and GATA2 in K562 cell treated and untreated with this complex prescription and its each single herbal medicine. RESULTS: The results showed that this complex prescription increased the gamma-globin, EpoR, Spi and FKLF expression and decreased the Ckit, GATA1 and GATA2 expression. And all single herbal medicines of this complex prescription could change some of those gene expressions, but not the same as the complex prescription. Even that, this study results indicated that the YiSui ShenXu Granule has its molecular pharmacological basis in treating beta-thalassemia.

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.

Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.