CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors

According to the current view of hematopoiesis, all blood cell types derive from a common pluripotent stem cell (65). In the adult, the stem cells are found in bone marrow, where they divide to produce more stem cells (self-renewal) and various precursor cells committed to a single lineage which terminally differentiate to morphologically and functionally distinct erythroid, myeloid, or lymphoid cells. Within the myeloid compartment, the same committed precursor can give rise to monocytic or granulocytic cells. This raises a question: what are the molecular mechanisms that dictate the fate of the common precursor to one or the other of these two diverse myeloid lineages? Genetic manipulations such as gene knockout and gene transfer experiments provide increasing evidence that transcription factors are involved in execution of the differentiation program of a cell. Determination of the developmental role of a number of transcription factors in blood development has been achieved by gene disruption experiments. These studies indicated that the GATA-1 and GATA-2, SCL, PU.1, Ikaros, c-myb, and AML1 genes, among others, are key regulators of hematopoiesis (reviewed in references 63 and 68). Inactivation of these genes had multilineage effects. Complementary to these knockout experiments are expression studies which identified certain transcription factors as master regulators of development, defined as genes which once activated would establish a specific cell type. For example, ectopic expression of MyoD in diverse cell types converts them to muscle cells (17). Similarly, expression of the B-cell transcription factor Oct-2 or the helix-loop-helix protein E47 in non-B cells induces B cell-like phenotypes (55, 59). In addition, thanks to advances in cell and molecular biological techniques, isolation and analysis of single primary cells, including functional stem cells, is feasible, and their developmental patterns can be studied as well (12). CCAAT/enhancer binding protein α (C/EBPα) was initially identified in liver and adipose tissue, where it was found to be important for terminal differentiation (8, 16, 22, 39, 44, 75). More recently it was shown to be also expressed in early myeloid cells (12, 61, 68). In addition, a number of granulocyte-specific genes, including granulocyte colony-stimulating factor (G-CSF) receptor (64), neutrophil elastase (52), and myeloperoxidase (23, 52) genes, have been shown to be regulated by C/EBPα. Gene targeting experiments revealed a specific defect in the hematopoietic system of C/EBPα knockout mice. The C/EBPα null phenotype was characterized by lack of mature granulocytes, with all the other blood cell types present, including monocytes and peritoneal macrophages (81). These results strongly point to a critical role of C/EBPα in granulocytic differentiation (reviewed in reference 68). In the present study, we have investigated the expression pattern of C/EBPα in the hematopoietic system during monocytic and granulocytic differentiation. We also examined the effect of induced overexpression of C/EBPα on the differentiation program of early bipotential myeloid cells. Our results demonstrate that the C/EBPα gene is activated at the stage of myeloid commitment and is specifically expressed in granulocytic cells. Increased levels of C/EBPα expressed from an inducible promoter construct directed differentiation along the granulocytic pathway, as determined by morphological criteria. Furthermore, ectopic expression of C/EBPα resulted in upregulation of mRNA of the granulocyte-restricted genes encoding the G-CSF receptor and C/EBPɛ, as well as secondary granule protein genes lactoferrin and human neutrophil collagenase. Our findings identify C/EBPα as the molecular switch during early hematopoietic developmental events that directs cells to the granulocytic pathway.