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 upregulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBPe 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 Epo/G-CSF receptor consisting of the Epo receptor extracellular domain fused to the G-CSF receptor 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 p27kip1 levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBPe. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via non-redundant G-CSF receptor signals.