Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.
The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes.
Collins
S J
SJ
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. scollins@fhcrc.org
Ulmer
J
J
Purton
L E
LE
Darlington
G
G
D000818:Animals; D015152:Blotting, Northern; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D005838:Genotype; D016179:Granulocyte Colony-Stimulating Factor / Q000097:blood*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000097:blood*; D006098:Granulocytes / Q000166:cytology*; D008099:Liver / Q000166:cytology / Q000196:embryology; D051379:Mice; D018345:Mice, Knockout; D012313:RNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D012016:Reference Values; D012190:Retroviridae / Q000235:genetics; D015854:Up-Regulation; D016662:Virus Integration
D022763:CCAAT-Enhancer-Binding Protein-alpha; D016179:Granulocyte Colony-Stimulating Factor; D012313:RNA; D016178:Granulocyte-Macrophage Colony-Stimulating Factor