Role Of TFEB SUMOylation In Macrophage Foam Cell Formation

BackgroundAtherosclerosis is known to be the major pathological basis to induce cardiovascular diseases, and macrophage foam cell formation is a typical pathological change in the early stage of atherosclerosis. Recent findings revealed that the autophagy-lysosome in macrophages has been linked to cholesterol metabolism and foam cell formation during atherosclerosis. Therefore, efficient regulation of macrophage autophagy is essential to prevent the formation of foam cells and attenuate the development of atherosclerotic lesions. Recently, covalent posttranslational modification with small ubiquitin-like proteins, particularly SUMO-1, has been recognized to play an important role in diverse physiological and pathological processes, such as embryonic development and the growth of malignant tumors. Transcription factor EB(TFEB) is a master transcriptional regulator of autophagylysosome biogenesis. Moreover, our previous studies demonstrated that TFEB could be modified by SUMO-1. We therefore propose that SUMOylation of TFEB plays an important role in foam cell formation through regulating autophagy-lysosome biogenesis during atherosclerosis.ObjectiveOur present study aims to investigate the effect of SUMOylation of TFEB on autophagylysosome biogenesis, and further explore the role of TFEB SUMOylation in macrophage foam cell formation during atherosclerosis. These findings will help us to better understand the biological function of protein SUMO modification and may serve as a novel potential mechanism for regulating macrophage foam cell formation in the progression of atherosclerosis.Methods1. THP-1 macrophages were treated with 50 μg/m L ox-LDL for 24 h, and the level of TFEB SUMOylation was analyzed by immunoprecipitates.2. Construct THP-1 macrophage cell lines stably expressing human TFEB(TFEB) and SUMOylation site mutated-TFEB(TFEB K316R) by lentivirus-mediated gene delivery. Transmission electron microscopy was performed to observe autophagosomes biogenesis. The expression of autophagy marker LC3 was determined by immunofluorescence and western blot.3. Culture TFEB and TFEB K316 R macrophages, Transmission electron microscopy and LysoTracker red staining were performed to observe lysosomes biogenesis. The expression of lysosome biogenesis marker LAMP1 was determined by immunofluorescence and western blot.4. Culture TFEB and TFEB K316 R macrophages, the expression of autophagy and lysosome biogenesis related gene was determined by real-time quantitative PCR(q RT-PCR).5. Culture TFEB and TFEB K316 R macrophages with or without ox-LDL(50μg/m L) for 24 h. The total cholesterol(TC), cholesterol esters(CE) levels were evaluated using fluorescent enzymatic assay. Cholesterol efflux to lipid-free apo A-I or HDL was measured by liquid scintillation counting.6. Culture TFEB and TFEB K316 R macrophages with or without ox-LDL(50μg/m L) for 24 h.Lipid droplets(LDs) were visualized using Oil Red O and Nile red staining.Results1. TFEB SUMOylation is decreased in THP-1 macrophages treated by ox-LDLWe observed that TFEB could be modified by SUMO-1 in THP-1 macrophages; Moreover, SUMO modification of TFEB was significantly decreased after 24 h of 50μg/m L ox-LDL treatment. These data suggested that TFEB SUMOylation is downregulated in macrophages under pro-atherogenetic conditions, and it may play a critical role in regulating the formation of macrophage foam cells.2. SUMOylation of TFEB induces macrophage autophagyElectron microscopy showed an increase in autophagosome biogenesis in TFEBmacrophages while in TFEB K316 R macrophages this increase was abolished. Moreover,the protein level of LC3 was significantly higher in TFEB WT macrophages than that inTFEB K316 R macrophages. These results clearly indicated that TFEB SUMOylationinduces macrophage autophagy.3. SUMOylation of TFEB induces macrophage lysosome biogenesisElectron microscopy and LysoTracker staining showed an increase in lysosome number and lysosomal compartment in TFEB macrophages but not in TFEB K316 R macrophages. Furthermore, immunofluorescence and western blot showed an increased amount of LAMP1 in TFEB macrophages compared to TFEB K316 R group. These results clearly indicated that TFEB SUMOylation can enhance macrophage lysosome biogenesis.4. SUMOylation of TFEB affects macrophage autophagy and lysosome biogenesis genes expressionTFEB regulates not only autophagy related genes(LC3, ULK1, WIP1, ATG9 B and Becline1), but also lysosome biogenetic genes(LAMP1, CTSB, CLCN7, and ATP6V1A). We observed an increased expression of these genes in TFEB macrophages; however, they were markedly reduced after K316 mutation. These results clearly indicated that TFEB SUMOylation can affect its downstream genes expression, which was essential to autophagy and lysosome biogenesis genes expression in macrophages.5. SUMOylation of TFEB enhances macrophage cholesterol effluxTFEB macrophages exposed to ox-LDL had lower TC and CE levels than those of the control group; whereas, compared to the TFEB group, the TFEB K316 R macrophages exposed to ox-LDL had an increased level of TC content and CE levels. Importantly, overexpression of TFEB in macrophages resulted in a significant increase in CE hydrolysis, whereas overexpressing TFEB K316 R had no such efficacy. In consistence with previous data, the apo A-I-mediated cholesterol efflux levels were increased in the TFEB group, compared to the control group, while TFEB K316 R group exhibited significantly reduced cholesterol efflux by apo A-I compared to the TFEB group. These results indicated that TFEB SUMOylation promotes macrophage LDs hydrolysis and enhances cholesterol efflux.6. SUMOylation of TFEB decreases macrophage foam cell formationOil red O and Nile red staining showed that overexpression of TFEB in macrophages significantly decreased the intracellular LDs accumulation, whereas, these phenotypic changes were largely reversed in TFEB K316 R macrophages. Collectively, these data indicated that TFEB SUMOylation plays a protective role in the formation of macrophage foam cells.Conclusions1. TFEB SUMOylation is downregulated in macrophages by ox-LDL, and it may play a critical role in regulating the formation of macrophage foam cells.2. TFEB SUMOylation induces macrophage lysosome biogenesis and autophagy process by regulating the expression of lysosome and autophagy biogenesis genes.3. TFEB SUMOylation promotes macrophage CE hydrolysis, enhances cholesterol efflux, and ultimately prevents macrophage foam cell formation.