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Granulopoiesis requires increased C/EBP? compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells.

ABSTRACT: C/EBP? is required for the formation of granulocyte-monocyte progenitors; however, its role in subsequent myeloid lineage specification remains uncertain. Transduction of murine marrow with either of two Cebpa shRNAs markedly increases monocyte and reduces granulocyte colonies in methylcellulose or the monocyte to neutrophil ratio in liquid culture. Similar findings were found after marrow shRNA transduction and transplantation and with CEBPA knockdown in human marrow CD34+ cells. These results apparently reflect altered myeloid lineage specification, as similar knockdown allowed nearly complete 32Dcl3 granulocytic maturation. Cebpa knockdown also generated lineage-negative blasts with increased colony replating capacity but unchanged cell cycle parameters, likely reflecting complete differentiation block. The shRNA having the greatest effect on lineage skewing reduced Cebpa 3-fold in differentiating cells but 6-fold in accumulating blasts. Indicating that Cebpa is the relevant shRNA target, shRNA-resistant C/EBP?-ER rescued marrow myelopoiesis. Cebpa knockdown in murine marrow cells also increased in vitro erythropoiesis, perhaps reflecting 1.6-fold reduction in PU.1 leading to GATA-1 derepression. Global gene expression analysis of lineage-negative blasts that accumulate after Cebpa knockdown demonstrated reduction in Cebpe and Gfi1, known transcriptional regulators of granulopoiesis, and also reduced Ets1 and Klf5. Populations enriched for immature granulocyte or monocyte progenitor/precursors were isolated by sorting Lin-Sca-1-c-Kit+ cells into GCSFR+MCSFR- or GCSFR-MCSFR+ subsets. Cebpa, Cebpe, Gfi1, Ets1, and Klf5 RNAs were increased in the c-Kit+GCSFR+ and Klf4 and Irf8 in the c-Kit+MCSFR+ populations, with PU.1 levels similar in both. In summary, higher levels of C/EBP? are required for granulocyte and lower levels for monocyte lineage specification, and this myeloid bifurcation may be facilitated by increased Cebpa gene expression in granulocyte compared with monocyte progenitors.

SUBMITTER: Ma O

PROVIDER: S-EPMC3994156 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Granulopoiesis requires increased C/EBPα compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells.

Ma Ou O Hong SunHwa S Guo Hong H Ghiaur Gabriel G Friedman Alan D AD

PloS one 20140421 4

C/EBPα is required for the formation of granulocyte-monocyte progenitors; however, its role in subsequent myeloid lineage specification remains uncertain. Transduction of murine marrow with either of two Cebpa shRNAs markedly increases monocyte and reduces granulocyte colonies in methylcellulose or the monocyte to neutrophil ratio in liquid culture. Similar findings were found after marrow shRNA transduction and transplantation and with CEBPA knockdown in human marrow CD34+ cells. These results ...[more]

PMID: 24752325

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Project description:The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of β-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates β-catenin-TCF/LEF interaction. Disruption of the β-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of β-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of β-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the β-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the β-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Dataset Information

Granulopoiesis requires increased C/EBP? compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells.

Publications

Granulopoiesis requires increased C/EBPα compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells.

Similar Datasets

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