Epsins Regulate Cholesterol Uptake and Efflux in Macrophages

Abstract

Rationale The transition from a stable lesion to vulnerable atherosclerotic plaque is pivotal in producing heart attacks and strokes. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are crucial for the development of atherosclerosis. However, the molecular mechanisms of epsin-regulated cholesterol uptake, efflux, and reverse cholesterol transport (RCT) in macrophagesduring the progress of atherogenesis remain unknown. In this project, we tested how epsins promote atherosclerosis progression by potentiating scavenger receptor-mediated endocytosis required for lipid uptake, and by targeting ATP-binding cassette sub-family G member 1 (ABCG1) for degradation in atherogenic environment. ABCG1 belongs to a family of proteins critical for the transport of cholesterol across the membrane that is important for macrophage cholesterol efflux, and subsequent RCT. Methods and Results To study the roles of macrophage epsins in controlling scavenge receptor endocytosis and lipid uptake in macrophage, we created macrophage-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. ApoE-/-/Wild type (WT) and ApoE-/-/LysM-DKO mice were intraperitoneally injected with 4% thioglycollate. Peritoneal macrophages were isolated and treated with 10 μg/mL oxLDL for 24 hours and subjected to Bodipy and phalloidin-594 Immunofluorescent (IF) staining. We observed reduced foam cell formation, a lack of actin stress fibers and inhibited CD36-mediated oxLDL uptake by epsin loss. To determine the molecular mechanisms of epsin-dependent, CD36-mediated lipoprotein uptake, we performed flow cytometry, western blot (WB) and immunoprecipitation and detected that epsin binds to CD36 through epsin ENTH domain to facilitate the internalization of CD36. In addition, peritoneal macrophages isolated from WTand LysM-DKO mice fed with western diet were analyzed by WB, IP and flow cytometry, we also observed an accumulation of ABCG1 on the cell surface of DKO macrophages. Further, epsin interacts with ABCG1 under atherogenic conditions and the binding is mediated by the UIM of epsin. Thus, epsins bind to ABCG1 and facilitate the internalization and degradation of ABCG1, implying a role of epsin in cholesterol efflux and RCT. Conclusions Macrophage epsins promote atherogenesis by facilitating CD36-mediated oxLDL uptake and internalization and degradation of ABCG1, suggesting that targeting epsins in macrophage may offer a novel therapeutic strategy against atherosclerosis.