Abstract
In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the athero-sensitive DBA/2 strain compared to the athero-resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we hypothesized that the lysosomal response to acetylated LDL (AcLDL) loading may be defective in DBA/2 vs. AKR foam cells. For all our studies, we cultured AKR and DBA/2 macrophages with or without AcLDL for 24h. AcLDL loaded DBA/2 vs. AKR cells exhibited a 40 to 50% decrease in lysosome number as measured by staining cells with Lysotracker or an anti-Lamp1 antibody, respectively. Lysosomal degradation capacity was assayed by cell incubation with Alexa647-DQ-ovalbumin, and we observed that AcLDL loading led to a 15% decrease in lysosomal capacity in DBA/2 foam cells (p<0.001) while it had no effect in AKR cells. Also, unloaded and loaded AKR cells had higher degradation capacity than DBA/2 cells (31% and 43%, respectively, p<0.001) which couldn’t be explained by a change in lysosomal pH. As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05). AKR and DBA/2 macrophages were labeled with a TFEB antibody and the nucleus to cytoplasm fluorescence intensity ratio was assessed. AcLDL loading led to a 37% increase in TFEB nuclear localization in AKR foam cells vs. unloaded cells (p<0.001) with no effect in DBA/2 macrophages. These results were confirmed by western blot after cellular fractionation. In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain decreased lysosome number and function and impaired autolysosome formation in DBA/2 cells.
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