Abstract
C/EBPα (−/−) cells do not effectively generate the granulocyte-monocyte progenitor (GMP) from the common myeloid progenitor (CMP). However, the role of C/EBPα in the further commitment of the GMP remains uncertain. Transduction of murine marrow or fetal liver cells with KRAB-C/EBPα-ER, an estradiol-regulated, dominant inhibitor of C/EBPs, suppresses formation of CFU-M, CFU-G, and CFU-GM without affecting formation of BFU-E, suggesting a role for C/EBPα in both myeloid lineages. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells and several other cell lines, but induces monocytic development from transduced B cell progenitors. We have now assessed the effect of C/EBPα on the myeloid commitment and maturation of murine myeloid progenitors. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPα-ER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient and only then subjected to lineage-depletion, just prior to culture +/− estradiol. This protocol, combined with rapid assessment of lineage maturation aims to avoid biases due to effects on proliferation or differentiation during transduction. C/EBPα-ER was expressed approximately 4-fold above endogeous levels. By 48 hrs, 80% of the lin- cells had acquired myeloid surface markers. Mac1+/Gr1+ or Mac1+/MPO+ cells, assessed by FACS, were granulocytes and Mac1+/Gr1− or Mac1+/MPO− cells were monocytes, as confirmed by staining cytospins. Activation of C/EBPα-ER with estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold at 48 hrs in five assessments (P < 0.002). A similar effect was seen when the estradiol dose was reduced from 1.0 to 0.3, 0.1, 0.03, or 0.01 μM, suggesting that observed effects on myeloid maturation occur near a physiologic activity level. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68, a 2-fold increase in M-CSFR and PU.1, and a 4-fold decrease in NE by 48 hrs. Estradiol had little effect on the myeloid development of vector-transduced cells or those expressing a leucine zipper mutant. Interestingly, transduction with the BR3 basic region mutant favored granulopoiesis in 5 independent experiments (P < 0.005), potentially reflecting dominant inhibition of endogenous C/EBPα or the the inability of this variant to interact with NF-κB p50. To assess effects on more immature progenitors, transduced cells were plated in methylcellulose. If estradiol was included, a 3- to 5-fold reduction in colony number precluded assessment of lineage commitment. However, if transduced progenitors were cultured with reduced concentrations of estradiol for 24 hrs and then plated without estradiol, C/EBPα-ER only reduced CFU numbers 1.2 to 2-fold. In two independent experiments, culture in IL3/IL6/SCF or in GM-CSF after transient activation of C/EBPα-ER resulted in a 2.6- to 5-fold increase in the ratio of CFU-M to CFU-G, indicating an effect on monocyte commitment at an early progenitor stage. For example, in one experiment no estradiol gave an average of 111 CFU per dish in IL3/IL6/SCF, culture for 24 hrs with 0.1 μM estradiol prior to plating gave 93 CFU, and pretreatment with estradiol increased the ratio of CFU-M to CFU-G from 1.1 to 3.9. We suggest a model of myelopoiesis in which C/EBPα first directs commitment of stem cells to the GMP by inducing PU.1 transcription. C/EBPα then has the capacity to direct monocytic commitment and maturation, likely dependent on cytokine conditions and availability of cooperating factors such as AP-1 or NF-κB.
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