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

Abstract

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/EBPε 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-CSFmediated 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 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/EBPε. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via non-redundant G-CSF receptor signals.

Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis

“…Notably, in our earlier studies in which LRG1 was transfected into murine myeloid cell lines and overexpressed, it was found to localize to the MPO-containing primary granules [17]. In this model system, ectopic expression of LRG1 was driven by a constitutive CMV promoter [23]. It is not surprising then that constitutively expressed LRG1 was found to co-localize with MPO, since LRG1 expression occurred concomitant with MPO expression.…”

“…Localization of the human gene for LRG1 by our group to a region on chromosome 19 where the genes for multiple neutrophil granule proteins also map prompted us to postulate that LRG1 might be a neutrophil granule protein. Using murine myeloid cell lines transfected with a tagged LRG1 cDNA construct, LRG1 was found to be packaged into the primary granules of differentiating cells [23]. A drawback of these studies was the use of an overexpression system, which can lead to inappropriate targeting of proteins to subcellular compartments that differ from their native counterparts in primary cells.…”

Autologous Extracellular Cytochrome c Is an Endogenous Ligand for Leucine-rich α2-Glycoprotein and β-Type Phospholipase A2 Inhibitor

“…Recently, mitochondrial DAMPs including formyl peptides and mitochondrial DNA have been shown to activate human polymorphonuclear neutrophils (PMNs) through the pattern recognition receptors and lead to PMN migration and degranulation in vitro and in vivo (36). Because LRG was shown to be up-regulated during neutrophilic differentiation (8) and be localized in the primary (azurophilic) granule compartment (37), it is possible that LRG functions as one of the pattern recognition receptors of PMN and modulates the neutrophilic function through an innate immune pathway.…”

Proteomic Analysis of Coronary Sinus Serum Reveals Leucine-Rich α2-Glycoprotein as a Novel Biomarker of Ventricular Dysfunction and Heart Failure

“…Also, LRG has been shown to be associated with neutrophilic differentiation 14,15 and prevention of lymphocyte apoptosis, 17 whereas in hepatoma cell lines, LRG expression has been shown to be associated with increased susceptibility to TGF-induced growth suppression. 18 Some evidence suggests that LRG may be an acute phase protein.…”

“…LRG was identified in 1977 as a trace component of human serum, 11 and resolution of the primary structure in 1985 indicated that it existed as a single polypeptide chain of approximately 45 kDa. 12 The precise function of LRG has yet to be defined, but evidence to date suggests that it is associated with inflammatory responses and neutrophilic differentiation [13][14][15][16][17] as well as cellular responses to the profibrotic cytokine transforming growth factor (TGF)-␤. 18,19 A growing body of data suggests that myocardial fibrosis is a central abnormality in the pathogenesis of HHD, DD, and the development of HF.…”