Abstract 20439: Sam68 Negatively Modulates Macrophage Autophagy in Atherosclerosis
Background: Macrophage autophagy regulates the pathogenesis of atherosclerosis. The Src-associated substrate during mitosis of 68 kDa (Sam68) belongs to the family of KH domain RNA binding protein; its role in macrophage autophagy and atherosclerosis has not been studied.
Methods & Results: In Raw264.7 macrophages, lentivirus-shRNA mediated knockdown of Sam68 (Sam68-KDn) led to a reduced protein level of total LC3, LC3-II, and p62/sqstm1 under basal culture condition, however, to a greater accumulation of LC3 in the presence of autolysosome inhibitor Bafliomycin A1, suggesting that Sam68 is physiologically a repressor of autophagy flux. Consistently, in HeLa/GFP-LC3 cells with Sam68-KDn, Bafliomycin A1 treatment also led to a greater autophagy flux than in control HeLa/GFP-LC3 cells, as revealed by autophagosome punctae counting (immuno-fluorescent microscopy) and median fluorescent intensity (flow cytometry). Notably, the mRNA levels of well-known transcriptional targets of autophagy related genes (MAP1LC3B, SQSTM1, BECN1, and Atg12) were not perturbed. In addition, Transmission electron microscopy revealed more autophagosome and autolysosome in the Raw264.7/Sam68-KDn cells than control Raw264.7 cells. Interestingly, the increase in autophagy flux induced by Sam68-KDn in Raw264.7 and HeLa/GFP-LC3 cells persist when autophagy was induced by starvation and Rapamycin (100nM). When treated with oxLDL (50μg/ml), both Raw264.7/Sam68-KDn cells and Sam68-null (Sam68 -/-) murine peritoneal macrophages demonstrated faster autophagy flux, formed less foam cells and produced lesser amounts of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-a), than control cells with an intact Sam68. Furthermore, ApoE-/-Sam68-/- mice developed a significantly smaller area of atherosclerotic lesions in the aorta as compared to ApoE-/- mice after feeding with Western diet for 8-12 weeks.
Conclusions: Our data reveal that Sam68 suppresses macrophage autophagy, which may contribute to the development of atherosclerosis.
Author Disclosures: J. Shi: None. S. Han: None. J. Zhou: None. L. Yang: None. B. Arnone: None. C. Boriboun: None. C. He: None. G. Qin: None.
- © 2016 by American Heart Association, Inc.