C/EBPalpha 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/EBPalpha is expressed only in myeloid cells, and PU.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBPalpha-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/EBPalpha-estrogen receptor ligand-binding domain fusion protein, C/EBPalphaWT-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/EBPalphaWT-ER induced morphologic differentiation and the expression of the myeloperoxidase, lactoferrin, and G-CSF receptor mRNAs. C/EBPalphaWT-ER also induced a G1/S cell cycle block, with induction of p27 and Rb hypophosphorylation. bcr-ablp210 prevented 32D cl3 cell differentiation. Activation of C/EBPalpha-ER in 32D-bcr-ablp210 or Ba/F3 B-lymphoid cells induced cell cycle arrest independent of terminal differentiation. C/EBPalphaWT-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/EBPalpha 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/EBPalpha 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.