G-CSF signaling can differentiate promyelocytes expressing a defective retinoic acid receptor: evidence for divergent pathways regulating neutrophil differentiation

Several lines of investigation suggest that granulocyte colony-stimulating factor (G-CSF) augments all-trans retinoic acid (ATRA)-induced neutrophil differentiation in acute promyelocytic leukemia (APL). We sought to characterize the relationship between G-CSF- and ATRA-mediated neutrophil differentiation. We established a G-CSF receptor-transduced promyelocytic cell line, EPRO-Gr, derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent EPRO cell line harboring a dominant-negative retinoic acid receptor alpha (RARalpha). In EPRO-Gr, neutrophil differentiation occurs either in GM-CSF upon addition of ATRA or upon induction with G-CSF alone. Transient transfection of EPRO-Gr cells with a RARE-containing reporter plasmid demonstrates increased activity in the presence of ATRA, but not G-CSF, while STAT3 phosphorylation occurs only in response to G-CSF. This suggests that ATRA-mediated differentiation of EPRO-Gr cells occurs via a RARE-dependent, STAT3-independent pathway, while G-CSF-mediated differentiation occurs via a RARE-independent, STAT3-dependent pathway. ATRA and G-CSF thus regulate differentiation by divergent pathways. We characterized these pathways in the APL cell line, NB4. ATRA induction of NB4 cells resulted in morphologic differentiation and up-regulation of C/EBPepsilon and G-CSFR, but not in STAT3 phosphorylation. The addition of G-CSF with ATRA during NB4 induction resulted in STAT3 phosphorylation but did not enhance differentiation. These results may elucidate how G-CSF and ATRA affect the differentiation of primary and ATRA-resistant APL cells.