Overexpression of GATA-2 inhibits erythroid and promotes megakaryocyte differentiation
Overexpression of GATA-2 inhibits erythroid and promotes megakaryocyte differentiation
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DOI：
10.1016/s0301-472x(00)00553-1
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发表时间：
2000-12-01
影响因子：
2.6
通讯作者：
Noguchi, CT
中科院分区：
文献类型：
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作者：
Ikonomi, P;Rivera, CE;Noguchi, CT
Objective.加塔-1和加塔-2转录因子是有效造血所必需的。这些调节蛋白在造血细胞中呈现重叠但不同的表达模式。加塔-2的缺失导致造血缺陷和胚胎致死表型。加塔-1的破坏导致早期红系细胞中加塔-2的补偿性增加和不完全的红细胞生成，胚胎在11.5天时死亡。我们研究了加塔-2在红系分化过程中造血中的特异性作用。利用加塔-2表达质粒构建了稳定表达不同水平加塔-2的K562细胞系，通过定量聚合酶链反应（PCR）检测加塔-2转录本的过表达。细胞离心涂片、生长曲线分析、PCR和流式细胞术用于检查增加的加塔-2水平在改变细胞表型和巨核细胞标志物活化中的作用。人祖红系细胞也用加塔-2表达载体转染。通过生长曲线分析、联苯胺染色、高效液相色谱分析等方法研究加塔-2对红系成熟和增殖的影响。表达高水平加塔-2 mRNA的K562/加塔-2细胞系显示增殖显著降低，表型向巨核细胞谱系转变。倍性分析表明，这些细胞系开发了多核表型，包括四倍体和八倍体。PCR分析显示巨核细胞特异性基因的激活，包括血小板生成素受体（c-mpl）。血小板糖蛋白受体Ib/IX（CD 42 b/CD 42 a）和IIb/IIIa（CD 41/CD 61）的表面表达也通过流式细胞术证实。在用加塔-2表达载体转染的原代成人红系培养物中，总血红蛋白的产生和细胞增殖以剂量依赖性方式降低。加塔-2在决定整个造血过程中的细胞谱系中起重要作用，并且增加的加塔-2表达决定巨核细胞分化。加塔-2的下调是红系分化所必需的。(C)2000年国际实验血液学学会。出版社：Elsevier Science Inc.
Objective. GATA-1 and GATA-2 transcription factors are required for effective hematopoiesis. These regulatory proteins present overlapping yet distinct patterns of expression in hematopoietic cells. Absence of GATA-2 leads to defective hematopoiesis and an embryonic lethal phenotype. Disruption of GATA-1 results in a compensatory increase in GATA-2 in early erythroid cells and incomplete erythropoiesis with embryos dying at 11.5 days. We examine the specific role of GATA-2 later in hematopoiesis, during erythroid differentiation.Materials and Methods. Stable K562 cell lines expressing various levels of GATA-2 were generated using a GATA-2 expression plasmid, Overexpression of GATA-2 transcripts was determined by quantitative polymerase chain reaction (PCR). Cytospin smears, growth curve analysis, PCR, and flow cytometry were used to examine the effects of increased levels of GATA-2 in altering cell phenotype and activation of megakaryocytic markers. Human progenitor erythroid cells also were transfected with a GATA-2 expression vector. Growth curve analysis, benzidine staining, and high-performance liquid chromatographic analysis were used to study the effects of GATA-2 on erythroid maturation and proliferation.Results. K562/GATA-2 cell lines expressing high levels of GATA-2 mRNA showed a marked decrease in proliferation and a shift in phenotype toward the megakaryocyte lineage. Ploidy analyses showed that these cell lines developed a multinuclear phenotype, including tetraploids and octaploids. PCR analysis showed activation of megakaryocyte-specific genes including thrombopoietin receptor (c-mpl). Surface expression of platelet glycoprotein receptors Ib/IX (CD42b/CD42a) and IIb/IIIa (CD41/CD61) also was demonstrated by flow cytometry. In primary human adult erythroid cultures transfected with a GATA-2 expression vector, production of total hemoglobin and cell proliferation decreased in a dose-dependent manner.Conclusions. GATA-2 plays an important role in deciding cell lineage throughout hematopoiesis, and increased expression of GATA-2 determines megakaryocytic differentiation. Downregulation of GATA-2 is required for erythroid differentiation. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.