Abstract

Cellular chemotaxis is a fundamental process in which cells migrate directionally when they are exposed to external chemical gradients. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity and chemotaxis, including the contribution of p38 MAPK and PTEN dependent migration of neutrophils toward their end target chemoattractants. However, it remains unclear how chemoattractant stimulation regulates F-actin dynamics and PTEN localization in migrating neutrophils. Glia maturation factor (GMFG) is a highly conserved protein expressed mainly in the blood and immune systems. Our lab previously reported that GMFG is upregulated in granulocyte-colony stimulating factor (G-CSF)-induced differentiated myeloid cells (Shi Y, et al, Genomics Proteomics Bioinformatics. 2006 Aug;4(3):145–55). Furthermore, while recent studies indicate that GMFG is involved in the regulation of actin cytoskeleton reorganization in microendothelial cells, little is known about the role of GMFG in hematopoietic myeloid cells. In this study, we investigated the role of GMFG in the chemotaxis of differentiated neutrophil-like HL-60 cells and how GMFG mediated neutrophils chemotaxis occurs in response to chemotactic signals. We have now shown that small interfering RNA-mediated depletion of GMFG expression in differentiated neutrophil-like HL-60 cells exhibit greater than 75% inhibition of basal cell migration, that could not be rescued in the presence of chemoattractant FMLP, but could rescued by IL-8. These data suggest that an endogenous expression of GMFG is required for chemotaxis toward end target chemoattractant FMLP, but not for toward intermediate chemokines IL-8. Moreover, immunofluorescence assay shows that gene silencing of GMFG abolished FMLP-induced the phosphorylation of p38 MAPK, localization of phosphatase PTEN, and F-actin reorganization. In contrast, transient overexpression of GMFG in K562 cells significantly enhanced basal cell migration by 2 fold (P=0.01) and chemotaxis toward FMLP by 2.2 fold (P=0.04), but there was no change with IL-8 stimulation. The overexpression of GMFG caused the activation of p38 MAP kinase and increased F-actin expression in the absence of fMLP stimulation. Pretreatment with p38 MAPK inhibitor SB203580 completely blocked the GMFG-dependent activation of p38 MAPK and enhanced expression of F-actin as well as cell migration. These results suggest that GMFG-induced cell migration in K562 cells is depending on p38 MAPK via F-actin reorganization. Taken together, our findings identify GMFG as a novel factor that mediates the FMLP-induced p38-dependent signal pathway leading to PTEN redistribution and F-actin reorganization during migrating neutrophils toward end target stimulation. These data, if confirmed, implicates GMFG as a potential target for therapeutic intervention in disorders associated with enhanced and pathological neutrophil infiltration.

Disclosures: No relevant conflicts of interest to declare.

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