Multipotent hematopoietic cell lines derived from C/EBPα(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte-colony-stimulating factor, and retinoic acid-induced granulocytic differentiation

The transcription factor C/EBP alpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (GCSE) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBP alpha 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/EBP alpha knockout animals (C/EBP alpha(-/-)) and their heterozygous littermates (C/EBP alpha(+/-)). Surprisingly, the C/EBP alpha(-/-) 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/EBP alpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBP alpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBP alpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBP alpha -responsive genes can occur in the absence of C/EBP alpha, 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. (C) 2001 by The American Society of Hematology.