Inactivation of the LRP1 Intracellular NPxYxxL Motif in LDLR-Deficient Mice Enhances Postprandial Dyslipidemia and Atherosclerosis

Abstract

Objective-The purpose of this study was to determine the significance of the intracellular NPxYxxL motif of LRP1 for the atheroprotective role of this multifunctional receptor. Methods and Results-LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with LDLR-deficient mice, a model for atherosclerosis. In this LDLR Ϫ/Ϫ background the mutated mice showed a more atherogenic lipoprotein profile, which was associated with a decreased clearance of postprandial lipids because of a compromised endocytosis rate and reduced lipase activity. On an atherogenic diet LRP1 mutant mice revealed a 50% increased development of atherosclerosis. This aggravation was accompanied by an increase in smooth muscle cell (SMC) and collagen content and apoptotic cells in the lesions. The mutation showed, however, a limited impact on basal PDGFR-␤ expression and signaling and the antimigratory property of apoE on PDGF-BB-stimulated SMCs. Additionally, levels of LRP1 atherogenic ligands, like MMP2, t-PA, FVIII, and the inflammatory ligand TNF-␣ showed to be significantly elevated.Conclusion-These findings demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1. This motif is relevant for normal control of lipid metabolism and of atherogenic and inflammatory ligands, but has no pronounced effect on regulating PDGF-BB/PDGFR-␤ signaling in SMCs. Key Words: atherosclerosis Ⅲ growth factors Ⅲ immune system Ⅲ lipases Ⅲ lipids T he large endocytic receptor low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a multifunctional protein that binds multiple extracellular ligands including apolipoprotein E (apoE) containing lipoproteins, lipoprotein lipase, complexes of proteinases-proteinase-inhibitors, hormones, matrix proteins, and growth factors like plateletderived growth factor (PDGF; reviewed by Herz 1 ). This 600 kDa receptor is proteolytically cleaved by a furin-like endoprotease into 2 subunits of 515 kDa and 85 kDa. The very large extracellular ␣-subunit contains the ligand-binding domains and is noncovalent associated with the smaller ␤-subunit, containing an extracellular part, the membrane spanning domain and the cytoplasmatic or intracellular domain (LRP1-ICD).In several studies using conditional inactivation of LRP1 in mice, the receptor has been associated with a clear role in the pathogenesis of atherosclerosis. In the liver it has been shown that the receptor is important for the removal of atherogenic lipoproteins and other proatherogenic ligands from the circulation. 2,3 Boucher et al, moreover, showed that LRP1 has a cholesterol-independent role in atherosclerosis by modulating the activity and cellular localization of the PDGF receptor-␤ (PDGFR-␤) in vascular smooth muscle cells (SMCs). 4 Finally, 2 studies argue that LRP1 in macrophages has an effect on atherosclerosis through the modulation of the extracellular matrix and inflammatory responses. 5,6 In all these studies, the overall function of LRP1 in a specific cell type was disrupted. These app...

Inactivation of the proximal NPXY motif impairs early steps in LRP1 biosynthesis

“…3b, the uptake of a 2 M by the NPXY mutant MEFs is significantly reduced in comparison with wild-type MEFs. As already reported recently [34], the internalization of a 2 M is only to a limited extent reduced in NPXY2. However, internalization by the NPXY1 and NPXY1?2 mutants was almost reduced to background levels observed in LRP1-deficient MEFs and wild-type MEFs, in which the uptake by LRP1 was inhibited by the specific inhibitor RAP.…”

“…Only recently, it was shown in an LDLR-deficient background that this motif contributes to the protective role of LRP1 with regard to atherosclerosis [34]. However, we hypothesized that combined inactivation of both motifs might result in a phenotype at least as dramatic as the single inactivation of the NPXY1 motif.…”

Opposing Effects of Apolipoprotein M on Catabolism of Apolipoprotein B–Containing Lipoproteins and Atherosclerosis

“…Mice with a knock-in mutation in the NPxYxxL motif of LRP1 designed to attenuate LRP1-mediated lipoprotein internalization (Lrp1 n2/n2 ) also had increased plasma apoM Ϸ50% (PϽ0.005) despite less pronounced elevation of the plasma cholesterol concentration ( Figure 1A and 1B). 32 Combined deficiency of the LDL receptor and LRP1 caused a further increase of plasma apoM ( Figure 1B). In contrast, total plasma apoM was not significantly increased in ApoE Ϫ/Ϫ mice irrespective of the pronounced elevation of VLDL/LDL cholesterol ( Figure 1A and 1B).…”

Molecular Biology of Atherosclerosis

“…LRP1 internalization defective knock-in also had a 49% increase in atherosclerosis (643). Hepatocyte-specific LRP1 deficiency was atherogenic independent of effects on plasma lipids (489).…”