C/EBPα bypasses granulocyte colony-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts

Within hematopoiesis, C/EBPα is expressed only in myeloid cells, and PU.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBPα-deficient mice lack the neutrophil lineage and retain monocytes, whereas PU.1-deficient mice lack monocytes and have severely reduced neutrophils. We expressed a C/EBPα-estrogen receptor ligand-binding domain fusion protein, C/EBPαWT- ER, in 32D cl3 myeloblasts. 32D cl3 cells proliferate in interleukin-3 (IL- 3) and differentiate to neutrophils in granulocyte colony-stimulating factor (G-CSF). In the presence of estradiol, C/EBPαWT-ER induced morphologic differentiation and the expression of the myeloperoxidase, lactoferrin, and G-CSF receptor mRNAs. C/EBPαWT-ER also induced a G1/S cell cycle block, with induction of p27 and Rb hypophosphorylation, bcr-abl(p210) prevented 32D cl3 cell differentiation. Activation of C/EBPα-ER in 32D-bcr-abl(p210) or Ba/F3 B-lymphoid cells induced cell cycle arrest independent of terminal differentiation. C/EBPαWT-ER induced endogenous PU.1 mRNA within 8 hours in both 32D cl3 and Ba/F3 cells, even in the presence of cycloheximide, indicating that C/EBPα directly activates the PU.1 gene. However, activation of a PU.1-ER fusion protein in 32D cl3 cells induced myeloperoxidase (MPO) RNA but not terminal differentiation. Thus, C/EBPα acts downstream of G-CSF and upstream of PU.1, p27, and potentially other factors to induce myeloblasts to undergo granulocytic differentiation and cell cycle arrest.