Abstract

Recruitment of monocytes is essential for effective control and clearance of invading pathogens by migrating to the sites of infection, but recruited monocytes also contribute to the pathogenesis of chronic inflammatory and degenerative diseases, such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling monocytes migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of monocytes to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate monocytes motility is therefore important for gaining a better understanding of monocytes function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on monocytes and the cellular mechanism for these effects by using small-interfering RNA to knockdown GMFG in human primary monocytes. Knockdown of endogenous GMFG results in significantly reduced (220.6 ± 9.4 to 89.0 ± 3.2, p<0.003) chemotactic migration toward SDF-1 and enhanced adhesion on fibronectin, VCAM-1 and ICAM-1 compared with control siRNA transfected cells. Flow cytometry analysis shows that knockdown of GMFG enhances the expression of β1-, β2−integrin on the cell surface compared with control cells. Confocal microscopy analysis exhibited that GMFG is colocalized with internalized β1-, β2-integrin in early endosomes in primary monocytes. However, an internalization assay shows that β1- and β2-integrin were internalized with similar kinetics during the initial uptake time points both in control or GMFG knockdown primary monocytes. These data demonstrated that internalization of β1- and β2-integrin was not impaired in GMFG knockdown cells, suggesting that GMFG regulates β integrins recycling. Antibody-based recycling assays showed that GMFG knockdown cells resulted in enhanced plasma membrane exocytosis of β1- and β2-integrin compared with control cells. Western blot analysis revealed that SDF-1 stimulated the phosphoryalation of FAK (Tyr397) is moderately (∼ 40%) increased in GMFG knockdown monocytes compared with control cells. These results suggest that impaired monocytes migrated toward SDF-1 stimulation in GMFG depletion monocytes is due to enhanced adhesion, which is caused by accelerated recycling of β1- and β2-integrin to the surface. Taken together, these results indicate that GMFG is required to maintain the proper expression levels of β1- and β2-integrin on the plasma membrane and is fundamental for integrin-mediated monocytes motility.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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