C/EBPα directs monocytic commitment of primary myeloid progenitors

C/EBP alpha is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBP alpha in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBP(x-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradlol reduced activated C/EBP(x-ER to near the physiologic range. C/EBP alpha-ER increased Mac1(+)/Gr1(-)/MPO-/flow monocytes 1.9-fold while reducing Mac1(+/)Gr1(+/)MPO(hl) granulocytes 2.5-fold at 48 hours, even in 0.01 mu M estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transcluced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in lL-3/1L-6/SCF or GM-CSF, but not G-CSF, even in 0.01 mu M estradiol. C/EBP alpha-ER induced PU.1 mRNA, and PUA-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBP alpha contributes to monopoiesis. A C/EBPa variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-kappa B p50 stimulated granulopoiesis, suggesting their cooperation with C/EBP alpha during monocytic commitment.