Overexpression of GATA-2 inhibits erythroid and promotes megakaryocyte differentiation

Objective. GATA-1 and GATA-2 transcription factors are required for effecti ve hematopoiesis. These regulatory proteins present overlapping yet distinc t patterns of expression in hematopoietic cells. Absence of GATA-2 leads to defective hematopoiesis and an embryonic lethal phenotype. Disruption of G ATA-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 differ entiation. 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 wer e used to examine the effects of increased levels of GATA-2 in altering cel l phenotype and activation of megakaryocytic markers. Human progenitor eryt hroid cells also were transfected with a GATA-2 expression vector. Growth c urve analysis, benzidine staining, and high-performance liquid chromatograp hic analysis were used to study the effects of GATA-2 on erythroid maturati on and proliferation. Results. K562/GATA-2 cell lines expressing high levels of GATA-2 mRNA showe d a marked decrease in proliferation and a shift in phenotype toward the me gakaryocyte lineage. Ploidy analyses showed that these cell lines developed a multinuclear phenotype, including tetraploids and octaploids. PCR analys is showed activation of megakaryocyte-specific genes including thrombopoiet in 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 GAT A-2 expression vector, production of total hemoglobin and cell proliferatio n decreased in a dose-dependent manner. Conclusions. GATA-2 plays an important role in deciding cell lineage throug hout hematopoiesis, and increased expression of GATA-2 determines megakaryo cytic differentiation. Downregulation of GATA-2 is required for erythroid d ifferentiation. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.