Glia Maturation Factor-γ Regulates Autophagy in Murine Macrophages

Abstract

Macrophages are centrally involved in both innate and adaptive immunity by secretion of regulatory immune mediators, phagocytosis, and presentation of antigens. Autophagy is an essential intracellular quality control process with multiple roles in normal physiological homeostasis. In macrophages, autophagy pathway also plays an important role in facilitating proper immunological functions by secretion of diverse proteins involved in inflammation, intercellular signaling, and pathogen infection. Accordingly, perturbations in autophagy have been linked to inflammatory diseases and defective immune responses against pathogens. It has been shown that actin cytoskeleton dynamics and actin regulatory proteins play an important role in the regulation of autophagosome formation and maturation. Here, to determine whether Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, regulates autophagy, we silenced GMFG in murine bone marrow-derived macrophages (BMDM) and Raw-264.7 macrophages. We found that GMFG is required for autophagy activation. Immunoblot analysis demonstrated that knockdown of GMFG decreased the LC3-II expression levels in basal condition, starvation- or rapamycin-induced autophagy in murine Raw-264.7 macrophages. Knockdown of GMFG did not prevent autophagic flux, as treatment with lysosomal V-ATPase inhibitor bafilomycin A1 was able to enhance LC3-II levels to comparable level of control siRNA treatment cells. Immunofluorescence analysis also exhibited the number of LC3 puncta reduced per cell during the starvation- or rapamycin-induced autophagy in GMFG knockdown cell compared with control cells, consistent with a defective autophagy process. These observations suggest that GMFG acts as an early stage in autophagosome formation. The serine/threonine kinase mTOR is a central regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR. Therefore, we decided to investigate the effect of GMFG knockdown on upstream of autophagy pathway. GMFG knockdown decreased the phosphorylation of AMPK and mTOR levels, but did not remarkably decrease Atg5, Atg7, or Atg16L1 levels (autophagy pathway core proteins in the early steps of autophagosome biogenesis) in GMFG knockdown murine macrophage. Moreover, we observed that Atg9 did not colocalization with EEA in both GMFG knockdown and control cells under basal condition. Whereas, after rapamycin treatment for 4 hours, Atg9 translocated to EEA and Atg9 colocalization with EEA decreased in GMFG knockdown macrophages compared with control cells. We also observed increase in the appearance of lysosomes by staining with lysotracker and increased staining for Lamp1-positive compartments by immunofluorescence in GMFG knockdown of macrophages. Furthermore, knockdown of GMFG enhanced the levels of the autophagy substrate p62 and reduced the lysosomal protease cathepsin D and cathepsin C maturation and activity. These observations suggest that GMFG knockdown induced the insufficient autophagy associated with compromised lysosomal function. Importantly, we found that knockdown of GMFG remarkably decreased the phosphorylation of YAP, LATS1 and the levels of RhoA/Rock, suggesting that downregulation of GMFG affected the autophagy associated with YAP-RhoA/Rock signaling pathway. Together, our finding shows that GMFG is an important molecular in regulating autophagy, further study the mechanisms responsible for GMFG's function will be of great value in the understand the autophagy related disease in immune system. Disclosures No relevant conflicts of interest to declare.