Balance of Transcription Factors Downstream of Notch Signaling Determines the Fate of Myeloid Progenitors toward Differentiation to Mast Cells or Immortalization without Differentiation.

Notch signaling represents one of the fundamental communication channels in various types of cells. While Notch activation has been shown to inhibit myeloid differentiation in a subset of hematopoietic progenitors, the role of Notch signaling in mast cell differentiation is not clear. When common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) purified from mouse bone marrow cells were stimulated with Delta1-Fc, a soluble form of Notch ligand, in the presence of stem cell factor, IL-3, IL-6, and thrombopoietin, granulocyte and macrophage differentiation, which is observed at day 7 of culture in the absence of Delta1-Fc, was markedly inhibited. Instead, Lin-c-Kit+FcεR+ mast cells dominated in the culture. Delta1-Fc did not increase mast cell generation from either CMPs or GMPs of the bone marrow of pI:pC-treated Mx-Cre x Notch2 flox/flox (N2-MxcKO) mice, in contrast to littermate Notch2 flox/flox mice treated with pI:pC, which suggests that Notch2 is responsible for the Delta1-Fc-augmented mast cell generation from CMPs and GMPs in culture. Retroviral transfer of constitutive active form of Notch2 (aN2) into CMPs and GMPs resulted in the complete loss of granulocyte-macrophage colony-forming cells and the emergence of basophilic granules-containing blast like cells, indicating the cell fate instruction. Real-time PCR analysis revealed that Delta1-Fc stimulation and aN2 introduction up-regulated the expression of Hes1, a transcriptional suppressor that is known to be a direct target of Notch activation in several cell types, within 12 h. Moreover, among GATA genes, Delta1-Fc stimulation and aN2 introduction resulted in increase of GATA3 mRNA, while expression levels of GATA1 and GATA2, which have been suggested to play a role in regulating mast cell differentiation, were unchanged. Next, we retrovirally expressed Hes1 and/or a GATA gene into CMPs and GMPs to see if the same effects were observed. Mast cells were increased only when both genes were expressed. On the other hand, when Hes1 alone was transduced, we observed rapid growth and immortalization of these cells without differentiation. C/EBPa, which is known to be suppressed in mast cell differentiation and upregulated in myeloid cell differentiation, was down-regulated within 48 h from the initiation of Hes1 retroviral transduction, suggesting that C/EBPa is a downstream target of Hes1 in this myeloid cell fate determination. Theses results indicate that, at the downstream of Notch activation, there are a C/EBPa down-regulation pathway that is Hes1-dependent and a GATA3 up-regulation pathway. Balanced regulation of these pathways should play a physiological role in myeloid and mast cell differentiation, while imbalance between these two pathways might provide a new model of myeloid transformation.