title: Granulopoiesis requires increased C/EBPα compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells.
abstract: 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 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/EBPα-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/EBPα 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.
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Ou
authors.initials: O
authors.lastname: Ma
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: SunHwa
authors.initials: S
authors.lastname: Hong
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
authors.affiliation: Division of Hematologic Malignancies, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Ghiaur
authors.affiliation: Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Friedman
mesh_terms: D000818:Animals; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006098:Granulocytes / Q000166:cytology* / Q000378:metabolism*; D006410:Hematopoiesis / Q000235:genetics / Q000502:physiology; D000090062:Kruppel-Like Factor 4; D051379:Mice; D008810:Mice, Inbred C57BL; D038042:Myelopoiesis / Q000235:genetics / Q000502:physiology; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D022762:CCAAT-Enhancer-Binding Proteins; C530718:CEBPA protein, mouse; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D013926:Thrombopoietin; D016186:Receptor, Macrophage Colony-Stimulating Factor
references.citation: Blood. 1992 Oct 1;80(7):1725-35
references.pmid: 1391942
references.citation: Cell. 1996 Oct 18;87(2):307-17
references.pmid: 8861914
references.citation: Blood. 2006 Jun 1;107(11):4308-16
references.pmid: 16469877
references.citation: Nat Genet. 2004 Jun;36(6):624-30
references.pmid: 15146183
references.citation: Blood. 2007 Nov 1;110(9):3136-42
references.pmid: 17671233
references.citation: Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8705-10
references.pmid: 10411939
references.citation: J Immunol. 2009 May 1;182(9):5757-62
references.pmid: 19380823
references.citation: J Exp Med. 2005 May 2;201(9):1487-502
references.pmid: 15867096
references.citation: Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):569-74
references.pmid: 9012825
references.citation: Blood. 2012 May 10;119(19):4408-18
references.pmid: 22451420
references.citation: Cancer Res. 2010 Oct 15;70(20):7949-59
references.pmid: 20924107
references.citation: Nat Genet. 2008 Jan;40(1):51-60
references.pmid: 17994017
references.citation: Nat Immunol. 2003 Oct;4(10):1029-36
references.pmid: 12958595
references.citation: Clin Cancer Res. 2009 Sep 1;15(17):5303-7
references.pmid: 19706798
references.citation: J Clin Invest. 2012 Dec;122(12):4490-504
references.pmid: 23160200
references.citation: Oncogene. 2008 Apr 24;27(19):2772-9
references.pmid: 18026136
references.citation: Mol Cell Biol. 2001 Jun;21(11):3789-806
references.pmid: 11340171
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):672-7
references.pmid: 16407117
references.citation: Genes Dev. 2012 Jul 15;26(14):1587-601
references.pmid: 22759635
references.citation: Blood. 2011 Aug 25;118(8):2266-74
references.pmid: 21725048
references.citation: Science. 1991 Jan 18;251(4991):288-92
references.pmid: 1987644
references.citation: Nat Commun. 2013;4:1660
references.pmid: 23552075
references.citation: Blood. 2011 Jul 28;118(4):1077-86
references.pmid: 21636860
references.citation: Blood. 2009 Sep 3;114(10):2172-80
references.pmid: 19587381
references.citation: Immunity. 2004 Dec;21(6):853-63
references.pmid: 15589173
references.citation: Leuk Res. 2012 Jan;36(1):110-6
references.pmid: 21993314
references.citation: J Immunol. 2002 Aug 1;169(3):1261-9
references.pmid: 12133947
references.citation: Mol Cell Biol. 1998 Jul;18(7):4301-14
references.pmid: 9632814
references.citation: Blood. 2003 Mar 15;101(6):2206-14
references.pmid: 12406909
references.citation: Genes Dev. 1999 Jun 1;13(11):1398-411
references.pmid: 10364157
references.citation: J Leukoc Biol. 2011 Oct;90(4):643-51
references.pmid: 21543584
references.citation: Blood. 1999 Jul 15;94(2):560-71
references.pmid: 10397723
references.citation: J Immunol. 1987 Jun 1;138(11):3829-35
references.pmid: 2438328
references.citation: Mol Cell. 2010 May 28;38(4):576-89
references.pmid: 20513432
references.citation: Blood. 2006 Aug 15;108(4):1223-9
references.pmid: 16645168
references.citation: Blood. 2012 May 24;119(21):4878-88
references.pmid: 22474248
references.citation: Cell. 2004 May 28;117(5):663-76
references.pmid: 15163413
references.citation: Leuk Res. 2011 Jul;35(7):909-13
references.pmid: 21470678
references.citation: Cell Res. 2005 Feb;15(2):92-6
references.pmid: 15740636
references.citation: Mol Cancer Res. 2005 Oct;3(10):585-96
references.pmid: 16254192
references.citation: J Leukoc Biol. 2003 Sep;74(3):464-70
references.pmid: 12949251
references.citation: Blood. 1996 Aug 15;88(4):1234-47
references.pmid: 8695841