Phenotypic Changes of Hematopoietic Stem Cells and Stromal Cells by Aberrant Expression of GATA−2; Possible Role for the Development of Aplastic Anemia.

(Introduction) Aplastic anemia (AA) is characterized by a fatty bone marrow and the reduced number of hematopoietic stem cell (HSC)s, resulting in peripheral pancytopenia. Several clinical studies have reported that the expression of transcription factor GATA−2 is significantly decreased in CD34 positive cells in AA, suggesting that downregulation of GATA−2 in HSCs may cause the reduction of number of HSCs in AA. Of note, GATA−2 has been shown to be essential for the maintenance of immaturity of preadipocytes as well as HSCs. If GATA−2 functions in the differentiation of stromal preadipocytes in bone marrow, GATA−2 may be involved in the formation of a fatty bone marrow in AA. In order to explore the role of GATA−2 for development of AA, phenotypic changes of HSCs and stromal preadipocytes by aberrant expression of GATA−2 were examined in this study.

(Method) First, to analyze phenotypic changes of HSCs by decreased expression of GATA-2, lineage negative, sca-1 positive, c-kit positive (LSK) cells were sorted both from wild-type and GATA-2 haplodeficient mice (kindly provided Dr SH Orkin), and the gene expression profile was compared by cDNA array. The difference of expression level on cDNA array was confirmed by RQ-PCR. Next, in order to examine the role of GATA-2 in the differentiation of stromal preadipocytes, the GATA-2-GFP expression vector was transfected into TBR343, a mouse stromal preadipocyte cell line, and GFP positive cells were sorted. The differentiation capacity of these GATA-2 overexpressing TBR343 cells was compared to that of control cells. In addition, GATA-2 expression was suppressed by using siRNA in TBR343, and the phenotypic change was also examined.

(Results) By comparing the gene expression profile, the expression levels of cyclin D1, D3, p21, E2F, c-Myc were found to be decreased in GATA-2 haplodeficient LSKs, suggesting GATA-2 down-regulation in HSCs may inhibit the cell cycle progression. When GATA-2 was overexpressed in TBR343, the oil drop formation and adipocyte-specific gene expression was significantly suppressed. Conversely, When GATA-2 was suppressed in TBR343 by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated, suggesting that decrease of GATA-2 expression accelerates the differentiation of stromal preadipocytes.

(Conclusion) These results suggest that suppression of GATA-2 in the bone marrow leads to an inactive cell cycle of HSCs and the accumulation of mature adipocytes, which may contribute to the expression of the representative features of AA, a fatty bone marrow and the reduced number of HSCs.