Specific Regulation of NFAT (Nuclear Factors of Activated T Cells) Expression in CD34⁺ Cells Differentiating into Diverse Hematopoietic Lineages.

NFAT (Nuclear Factor of Activated T cells) transcription factors are a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells. It is now clear that NFAT proteins are also expressed in various non-immune cell types, where they regulate the expression of genes involved in such diverse cellular processes as proliferation, apoptosis and differentiation. We have previously shown that NFATc2 is strongly expressed in human CD34+ cells and megakaryocytes, but not in purified peripheral blood neutrophil granulocytes and monocytes. Furthermore, granulocytic differentiation of CD34+ cells in vitro was paralleled by the rapid and profound suppression of NFATc2 mRNA and protein. The function of NFATc2 in CD34+ cells, however, is unknown, and no information exists on the expression or regulation of other NFAT family members in CD34+ cells or during heamtopoietic differentiation. To provide a systematic basis for further functional analysis, we established in the present study a comprehensive expression profile of all five NFAT family members in CD34+ cells and during their in vitro differentiation into neutrophil, eosinophil, erythroid and megakaryocytic lineages. CD34+ cells were purified from umbilical cord blood and cultured in the presence of cytokines or cytokine combinations inducing differentiation of the respective lineages. At several time-points during the culture, the efficacy and specificity of the differentiation was monitored by morphological examination of cytospin preparations as well as by analysis of lineage-specific cell surface markers. By quantitative RT-PCR, NFATc3 and NFAT5 were the NFAT family member found to be most prominently expressed in CD34+ cells of both peripheral blood and umbilical cord blood, as well as in the immature CD34+CD38− subpopulation of cells. NFAT expression during the differentiation of umbilical cord blood CD34+ cells into the diverse hematopoietic lineages followed a family member- and lineage-specific pattern. Neutrophil differentiation was accompanied by a rapid suppression of transcript level for all NFAT family members. In contrast, eosinophil, erythrocyte and megakaryocyte differentiation was paralleled by an upregulation of NFATc3, NFATc1/NFATc3 and NFATc1 mRNA, respectively. The most obvious lineage-specific pattern was observed for NFATc4, where transcript levels were low in CD34+ cells and either not or only transiently increased in neutrophil, eosinophil and erythrocyte differentiation; in contrast, they were specifically upregulated about 10-fold in the megakaryocytic lineage. The expression profile of NFAT family members in developing hematopoietic cells of diverse lineages presented here will allow predicting and directly assessing the role of individual NFAT family members in hematopoietic differentiation.