C/EBP alpha 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/EBP alpha is expressed only in myeloid cells, and P U.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBP alpha-defici ent mice lack the neutrophil lineage and retain monocytes, whereas PU.1-def icient mice lack monocytes and have severely reduced neutrophils. We expres sed a C/EBP alpha-estrogen receptor ligand-binding domain fusion protein, C /EBP alpha WT-ER, in 32D cl3 myeloblasts. 32D cl3 cells proliferate in inte rleukin-3 (IL-3) and differentiate to neutrophils in granulocyte colony-sti mulating factor (G-CSF). In the presence of estradiol, C/EBP alpha WT-ER in duced morphologic differentiation and the expression of the myeloperoxidase , lactoferrin, and G-CSF receptor mRNAs. C/EBP alpha WT-ER also induced a G 1/S cell cycle block, with induction of p27 and Rb hypophosphorylation. bcr -abl(p210) prevented 32D cl3 cell differentiation. Activation of C/EBP alph a-ER in 32D-bcr-abl(p210) or Ba/F3 B-lymphoid cells induced cell cycle arre st independent of terminal differentiation. C/EBP alpha WT-ER induced endog enous PU.1 mRNA within 8 hours in both 32D cl3 and Ba/F3 cells, even in the presence of cycloheximide, indicating that C/EBP alpha directly activates the PU.1 gene. However, activation of a PU.1-ER fusion protein in 32D cl3 c ells induced myeloperoxidase (MPO) RNA but not terminal differentiation. Th us, C/EBP alpha acts downstream of G-CSF and upstream of PU.1, p27, and pot entially other factors to induce myeloblasts to undergo granulocytic differ entiation and cell cycle arrest. (C) 1999 by The American Society of Hemato logy.