Abstract

Background: LDL receptor-related protein 1 (LRP1) in mouse macrophages protects against atherosclerosis. One possible mechanism is by maintaining normal cholesterol homeostasis. Cholesterol efflux from foam cells, mediated by ABCA1, is essential for protection from atherosclerosis. An important factor for cholesterol homeostasis is glycosphingolipid (GSL) trafficking. Prosaposin, precursor of sphingolipid activator proteins (saposins), is necessary for normal GSL metabolism and consequently cholesterol trafficking. It is also a ligand of LRP1. Our objective was to determine whether the deletion of macrophage LRP1 promotes atherosclerosis by altering cholesterol homeostasis. Methods: Peritoneal macrophages from MɸLRP+/+ and MɸLRP-/- mice were incubated with acetylated LDL for cholesterol loading. The media and the cell lysates from both cell types were used for immunoblotting, RT-PCR, cholesterol efflux assays, and fluorescence microscopy. Cholesterol efflux assay showed impaired efflux by LRP1-/- macrophages. However, the level of ABCA1, the major cholesterol transporter, and its mRNA transcript were elevated, indicating that cholesterol may not be available for export by ABCA1. The mRNA levels of prosaposin were not different between the two cell types, but immunoblotting showed accumulation of intracellular and extracellular prosaposin by the LRP-/- macrophages. Even with elevated intracellular prosaposin, LRP-/- macrophages contained less saposin A, as shown by immunoblotting. The accumulations likely contribute to a lesser proportion of prosaposin getting routed to lysosomes for maturation into saposins. RT-PCR and immunoblotting suggested that other receptors for prosaposin were not downregulated with LRP1 deletion, thus they do not seem to contribute to the accumulation of extracellular prosaposin. Fluorescence microscopy for prosaposin and lysosomes again confirmed prosaposin accumulation with LRP1deletion. Free cholesterol and GSL stains reflected their accumulation in LRP-/- macrophages, and the peripheral punctate vesicular patterns of prosaposin and free cholesterol in LRP-/- macrophages suggest their localization in early endosomes. Conclusion: MɸLRP1 deletion leads to abnormal GSL metabolism by altering prosaposin trafficking for maturation to saposins. Normal GSL trafficking is important to maintain normal cholesterol homeostasis, and thus alteration of the former will affect the latter. This could explain the impaired cholesterol efflux by LRP-/- macrophages despite overexpression of ABCA1. Hence, macrophage LRP1 protects against atherosclerosis by maintaining normal cholesterol homeostasis.