LRG-accelerated differentiation defines unique G-CSFR signaling pathways downstream of PU.1 and C/EBPε that modulate neutrophil activation

Expression of leucine-rich alpha 2 glycoprotein (LRG), a member of the leucine-rich repeat family of proteins, was recently shown to be up-regulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBP epsilon that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter. We also demonstrate that LRG localizes to the same cytoplasmic compartment as myeloperoxidase and that G-CSF treatment of the 32Dcl3 myeloid cell line induces nuclear translocation of LRG. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated, G-CSF-mediated neutrophil differentiation and induction of CD11b expression. In contrast, constitutive expression of LRG in 32Dwt18 cells, expressing a chimeric erythropoietin (Epo)/G-CSFR consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains, failed to induce accelerated neutrophil differentiation and CD11b expression in response to Epo stimulation. LRG-mediated accelerated differentiation and CD11b expression were found to correlate with an increased level of phospho-Stat3 but not with PU.1 or p27(kip1) levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBP epsilon. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via nonredundant G-CSFR signals.