Inactivation of the LRP1 Intracellular NPxYxxL Motif in LDLR-Deficient Mice Enhances Postprandial Dyslipidemia and Atherosclerosis
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−/− 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-β 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-α 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-β signaling in SMCs.
The 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 platelet-derived growth factor (PDGF; reviewed by Herz1). 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 α-subunit contains the ligand-binding domains and is noncovalent associated with the smaller β-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-β (PDGFR-β) 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 approaches, however, meet their limits when they are used for studies of large multidomain proteins where different individual domains are involved in diverse functions, as in the case of LRP1. It has been demonstrated that many motifs in the complex LRP1-ICD, being the 2 NPxY motifs, a YxxL motif, 2 di-leucine motifs, and a protein kinase A (PKA) consensus motif, are potentially involved in directing LRP1 in a cargo transporting or signaling function.7,8 The YxxL motif has been recognized as being the dominant endocytosis signal next to the distal NPxY and distal di-leucine motifs.7 The distal NPxY motif overlapping with the YxxL motif, forming the NPxYxxL double motif, is also capable of interacting with many cytoplasmatic adaptor and scaffold proteins, which in addition can be modulated by the phosphorylation of the tyrosine residue.9,10
In the present study we investigated the role of the NPxYxxL motif in the LRP1-ICD in the development of atherosclerosis in vivo. To tackle this question we made use of mice carrying a knock-in mutation of the NPxYxxL motif into the endogenous Lrp1 gene, which were generated by a recombinase-mediated cassette exchange (RMCE) strategy.11 The LRP1 knock-in mice were crossed into the LDLR−/− background to study the effect of the LRP1-ICD mutation on the pathology in this atherosclerosis model. Our data demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1, not only via control of the lipid metabolism but also through regulation of levels of atherogenic factors like MMP2, tissue-type plasminogen activator (t-PA), and coagulation factor VIII (FVIII) and secretion of the proapoptotic cytokine, TNF-α. Furthermore, there was no significant effect of the NPxYxxL inactivation on LRP1 regulated PDGFR expression and signaling.
An expanded Methods section can be found in supplemental material (available online at http://atvb.ahajournals.org).
Statistical significance between groups was determined by Student t, 1-way ANOVA, Mann–Whitney rank sum, and Kruskal–Wallis tests using STATISTICA version 6 software (StatSoft Inc). P<0.05 was regarded statistically significant.
Inactivation of the LRP1 NPxYxxL Motif Causes an Increase of Remnant Particles
The previously described NPxYxxL mutant11 (LRP1n2/n2) was crossed with LDLR−/− mice to study the impact of the LRP1 knock-in mutation on the phenotype of this LDLR−/− mouse model for atherosclerosis. Because LRP1 is involved in the clearance of lipoproteins from the circulation, serum was obtained. The LDLR−/−LRP1n2/n2 mice show a significant 1.5-fold and 1.6-fold increase in their total serum cholesterol as well as triglyceride concentrations, respectively, compared with the LDLR−/− mice (respectively 486.7±29 mg/dL versus 320±31 mg/dL cholesterol and 265.4±20 mg/dL versus 162.0±17 mg/dL triglycerides; P<0.005). Analysis of serum samples by size exclusion chromatography revealed a higher cholesterol and triglyceride content in the CR, VLDL, and LDL fractions in LDLR−/− LRP1n2/n2 mice (Figure 1A and 1B). This was confirmed by the significant rise in the apoB48 and apoE concentrations seen in plasma of the LDLR−/−LRP1n2/n2 mice (supplemental Figure I). Altogether, the results suggest an accumulation of triglyceride rich lipoprotein particles (TRLs).
LDLR−/−LRP1n2/n2 Mice Have a Delayed Postprandial Lipid Clearance
A possible contributing factor to the observed increase of TRLs in the LDLR−/−LRP1n2/n2 mice is a difference in hepatic VLDL production rates. In contrast to the increase in TRLs the production rate of hepatic VLDL was significantly reduced in the LDLR−/−LRP1n2/n2 mice to 320 mg/dL/h compared to 560 mg/dL/h for the LDLR−/− mice (Figure 1C). Because there was no increased production of hepatic apoB containing particles—on the contrary a decrease was observed—one can assume that an altered clearance of postprandial lipoproteins might be a possible explanation. Therefore, we compared the postprandial response of LDLR−/− and LDLR−/−LRP1n2/n2 mice. We found that the plasma triglyceride clearance in LDLR−/−LRP1n2/n2 mice after an oral fat load was significantly impaired and marked by an increased accumulation of triglycerides up to 4 hours after administration (Figure 1D). The rate of postprandial triglyceride accumulation is both determined by the rate of LpL-mediated triglyceride hydrolysis and the rate of receptor-mediated clearance of remnant lipoprotein particles. In postheparin plasma, however, there was no difference in lipase activity (supplemental Figure II). Therefore, lipase activity in total homogenates and heparin extracts (surface bound activity) from liver, white adipose tissue (WAT), and muscle were evaluated (Table). The results revealed no difference in lipase activity for liver and muscle tissue in both extracts. Contrary wise in WAT the amount of LpL activity was significantly reduced in both heparin releasable and total extracts indicating a decreased expression of LpL. To determine the impact on receptor-mediated clearance the endocytosis rate of the LRP1 ligand apoE was evaluated in peritoneal macrophages derived from LDLR−/− and LDLR−/−LRP1n2/n2 mice (Figure 1E). This analysis illustrated a significant 20% reduction in the clearance rate when the NPxYxxL motif was inactivated. Evaluation of the endocytosis rate of α2M, a specific LRP1 ligand, in mouse embryonic fibroblasts (MEFs) revealed a significant 30% reduction in the uptake for LRP1n2/n2 MEFs and almost no uptake for LRP1−/− MEFs (PEA1312) compared to their wild-type controls (Figure 1F). To substantiate this impact on clearance further, 2 atherogenic LRP1 ligands, t-PA and FVIII, levels were determined in plasma. In the LDLR−/− LRP1n2/n2 t-PA and FVIII plasma levels were, respectively, significantly 1.4-fold and 1.6-fold increased compared to the LDLR−/− controls (supplemental Figure III). The data indicate that inactivation of the NPxYxxL motif of LRP1 in vivo leads to a delayed clearance of postprandial TRLs, via both decreased LpL activity in adipose tissue and a reduced endocytosis rate of the receptor.
Increased Atherosclerotic Lesion Area and Smooth Muscle Cell and Collagen Content
To investigate the impact of the NPxYxxL mutation on the development of atherosclerosis, 12-week-old LDLR−/− (n=23) and LDLR−/−LRP1n2/n2 (n=15) mice were fed an atherogenic diet for 12 weeks. On the atherogenic diet, LDLR−/−LRP1n2/n2 mice had increased triglyceride levels compared to the levels in LDLR−/− mice, however cholesterol levels were not different (Figure 2A and 2B). Similar to the chow diet, an accumulation of TRLs was also reflected in their lipoprotein and apolipoprotein profile (supplemental Figure IV). En face analysis of these mice showed a significant 1.5-fold increase for the development of atherosclerosis in LDLR−/−LRP1n2/n2 mice compared to the LDLR−/− control (respectively 18.8±1.9% versus 12.6±0.6%; P<0.01, Figure 2C). Thus, inactivation of the NPxYxxL motif in LRP1 leads to an increased atherosclerosis development, indicating that this motif is relevant for the atheroprotective properties of the receptor. To evaluate the impact of the LRP1 knock-in mutations on the composition of atherosclerotic lesions, we determined the percentage of SMC, macrophage and collagen content in lesions of LDLR−/−(n=6) and LDLR−/− LRP1n2/n2 (n=6) mice. As shown, the percentage of SMC lesion content at the level of the individual lesions for the LDLR−/−LRP1n2/n2 mice was significantly 2-fold higher compared to the control LDLR−/− mice (Figure 2D through 2F). Furthermore, the mean percentages of collagen content in individual lesions of LDLR−/−LRP1n2/n2 mice were also significantly increased compared to the LDLR−/− mice (Figure 2D, 2G, and 2H). In contrast, results obtained with the antimouse macrophage antibody indicated no differences for macrophages (Figure 2D). Lesion classification indicated no significant differences in the low amount of early lesions but revealed a significant shift from less moderate to more advanced lesions in LDLR−/−LRP1n2/n2 mice compared to the LDLR−/− controls (supplemental Figure V). These results suggest that inactivation of the NPxYxxL motif is essential for an increased SMC content in the lesion area, which is associated concomitantly with an increased collagen content in the atherosclerotic lesions, likely reflecting a phenotypic shift of the lesions.
No Pronounced Impact on LRP1 Mediated PDGFR-β Regulation In Vivo
In the search for a potential mechanism contributing to the increased SMC content in the atherosclerotic lesions of mice bearing the knock-in mutation, we wanted to evaluate whether the NPxYxxL inactivation had an effect on the expression of the PDGFR-β and its signaling as seen for the vascular specific LRP1 knock-out mice.4 Therefore, protein extracts from aortas were checked for expression and phosphorylation of PDGFR-β and ERK by immunoblotting. Determination of the relative optical density of the bands revealed no significant increase in PDGFR-β expression and phosphorylation of PDGFR-β and ERK (supplemental Figure VI). Because these data did not suggest a strong effect on PDGFR-β expression and signaling, we wanted to assess whether the LRP1 mutation had an effect on the LRP1 regulated apoE-mediated inhibition of PDGF-BB–induced SMC migration. The role of the LRP1 knock-in mutation was assessed in vascular SMCs isolated from wild-type (LRP1+/+) and LRP1n2/n2 mice (Figure 3A). The results in the SMCs showed for the NPxYxxL mutation at low concentrations of apoE a loss of the antimigratory effect, whereas higher concentrations of apoE were reducing the migratory property of PDGF-BB as comparable to the wild-type control cells. Similar results were obtained using the derived MEFs (supplemental Figure VII). Thus overall, the NPxYxxL mutation seems to have only a limited impact on the antimigratory property of apoE in vitro. Among ligands for LRP1 involved in migration of SMCs are matrix-metalloproteinases and t-PA, which was already shown to be increased in the plasma of the LDLR−/− LRP1n2/n2 mice. Therefore, MMP2 and MMP9 activity in aortic lysates were evaluated in 12-week-old mice via zymography. The mice having the NPxYxxL inactivation had a significant 2.7-fold increase of MMP2 activity in the aortas, whereas no differences were seen for MMP9 activity (Figure 3B). Altogether, these results suggest that inactivation of the NPxYxxL motif leads to increased levels of aortic MMP2 and plasma t-PA, both LRP1 ligands with promigratory properties.
Proapoptotic Effects via Increased Secretion of TNF-α in Macrophages
Because it has been shown that LRP1 is involved in the regulation of apoptosis,13 the amount of apoptotic cells in the atherosclerotic lesions were determined via TUNEL staining (Figure 4A and 4B). As illustrated in Figure 4C, there was a significant 2-fold increase in the amount of apoptotic cells relative to the plaque size in LDLR−/−LRP1n2/n2 mice. The apoptosis rate in atherosclerotic plaques is largely on the account of the oxysterols present in oxidized LDL particles. Therefore we examined the impact of the NPxYxxL inactivation on apoptosis in mouse peritoneal macrophages after a 20-hour treatment with 0.5 μg/mL staurosporine (STS), 10 μg/mL 7-ketocholesterol or 20 μg/mL 25-hydroxycholesterol (supplemental Figure VIII). No significant difference in apoptosis was observed between LDLR−/−LRP1n2/n2 and LDLR−/− macrophages for STS and both oxysterols. In search for an alternative explanation, the secretion of the proapoptotic inflammatory molecule TNF-α was evaluated in the peritoneal macrophages. It has been shown that LRP1 influences TNF-α levels through its binding to α2M and subsequent internalization of the complex.6 We quantified the amount of TNF-α accumulating in media from lipopolysaccharide (LPS)-stimulated macrophages over time. In comparison to LDLR−/− macrophages, the NPxYxxL inactivation increased the accumulation of TNF-α 1.8-fold and 2.2-fold after 4 and 20 hours incubation, respectively (Figure 4D). Because it has also been shown that macrophage LRP1 influences monocyte chemotactic protein 1 (MCP-1) secretion,6 we next investigated the effect of the NPxYxxL inactivation on its secretion. No effect was observed in LPS stimulated macrophages (supplemental Figure IX). Therefore the data suggest that inactivation of the NPxYxxL motif leads to the activation of a proapoptotic inflammatory response via increased TNF-α levels.
In the present study previously described NPxYxxL knock-in mice, showing no distinct phenotype, were crossed into the LDLR−/− background to elucidate the role of the modified LRP1-ICD motif in the pathogenesis of atherosclerosis. On the LDLR−/− background, the NPxYxxL knock-in mutation resulted in a significant increase in total serum cholesterol and triglyceride levels compared to the background control. It has been described that LRP1 in the liver is involved in clearance of postprandial lipoproteins from the circulation,2 mediated via insulin induced LRP1 translocation14 and subsequent binding of apoE and lipoprotein lipase to LRP1.15 In vitro studies using LRP1 minireceptors with mutated LRP1-ICDs clearly determined that the YxxL motif, comprising the NPxYxxL motif, serves as the dominant endocytosis signal in the LRP1-ICD.7 Our in vivo observations support these in vitro results as shown by increased apoE, apoB48, CR, VLDL, and LDL concentration in the circulation of LDLR−/−LRP1n2/n2 mice, which are not linked to increased hepatic triglyceride production. This was substantiated further in vivo by the increased levels of LRP1 atherogenic ligands like t-PA and FVIII and in vitro via evaluating the clearance of apoE in macrophages on the LDLR−/− background and the clearance of α2M in MEFs on a wild-type background, where we found a significant reduction in the endocytosis capacity of LRP1 by inactivation of the NPxYxxL motif. Nevertheless, next to the reduced endocytosis rate of LRP1, the decreased lipase activity in adipose tissue observed in the knock-in mice likely also contributes to the delayed postprandial lipid clearance. The decreased lipase activity is most likely attributable to a decreased expression of lipases instead of cell surface retention of the enzymes, because no differences in activity were observed between the total and heparin extract in white adipose tissue. These findings are in contrast to the observation in adipocyte specific LRP1 knock-out mice.16 They are, however, in agreement with a recent study where was shown that LRP1-deficiency results in reduced LpL and hormone-sensitive lipase (HSL) expression and activity in adipocytes differentiated from MEFs.17 These results, together with our findings, could imply that the NPxYxxL motif is involved in the regulation of LpL and HSL expression in adipocytes.
The more atherogenic lipoprotein profile seen for the LDLR−/−LRP1n2/n2 mice on chow diet renders these mice more prone to develop atherosclerosis, and as such this correlated with significant higher atherosclerosis development in the aortas on a high-cholesterol diet. The difference in atherosclerosis on the high-cholesterol diet between knock-in mice and control mice can in part be attributed to the altered lipoprotein profile even if only triglyceride levels were significantly higher and cholesterol levels were not different. Accumulation of lipoprotein remnants and, therefore, higher levels in the vessel wall could explain the differences in atherogenesis as seen for patients having type III hyperlipidemia. Hence, these results corroborate the atherogenic potential of triglyceride-rich lipoproteins.
LRP1, however, is not only expressed in liver and adipocytes, therefore it is conceivable that loss of a functional NPxYxxL motif could have an effect on signaling functions of LRP1 in other tissues which can contribute to the atherogenic potential. As reported, mice lacking LRP1 in vascular SMCs have higher PDGFR-β expression and signaling activity.4 Additionally it has been shown that incubation of vascular SMC in vitro with apoE inhibits the stimulatory effect of PDGF-BB on cell migration, and loss of LRP1 expression is preventing this inhibitory activity of apoE.18 Evaluation of the plaque phenotype showed for the LDLR−/− LRP1n2/n2 mice a 2-fold increase in relative SMC content associated with increased collagen content in the lesions. It is known that SMCs in the atherosclerotic lesion are involved in matrix deposition by the production of molecules like collagen, hence explaining the concomitant increase.19 The relative increased SMC content in the lesions of the LDLR−/− LRP1n2/n2 mice could have been caused by a stimulatory effect of the mutations on the migratory properties of SMCs out of the tunica media. Nevertheless, we observed in vitro only a limited decrease in sensitivity for the apoE-mediated inhibition of PDGF-stimulated cell migration in SMCs. So, based on these analyses, only subtle impairment of regulation of PDGFR-β signaling by LRP1 could possibly contribute to the change of plaque phenotype. Unfortunately, a possible impact of the NPxYxxL inactivation on PDGFR-β expression and signaling in the aorta seems be too subtle to measure in our analyses. Alternatively, maybe other LRP1 intracellular motifs are involved in this intracellular crosstalk between LRP1 and PDGFR-β and can compensate for loss of the functional NPxYxxL motif.
The results impelled us to assess the levels of other LRP1 ligands which are shown to be involved in migration of SMCs. Therefore, the activity of MMPs are good candidates because some of them can facilitate cells to migrate by degrading the extracellular matrix,20,21 and LRP1 is involved in their metabolism. In the SMC-specific LRP1 knock-out study by Boucher et al it was shown that MMP2 and MMP9 activities are regulated by LRP1 in the aortic wall.4 In our study we could see a significant increased MMP2 activity in aortic lysates of the LDLR−/−LRP1n2/n2 mice, which seems to explain the observed increase of SMC migration. However, no effect was seen on MMP9 activity. It must be noted that LRP1 regulates MMP9 not only via catabolism of the enzyme,20 but also via triggering MMP9 gene expression via tyrosine phosphorylation of the β-subunit.22 A possible explanation is that inactivation of the NPxYxxL motif impairs both the endocytosis property of LRP17 and tyrosine phosphorylation, reducing both MMP9 clearance and production resulting in unaltered levels in the aorta.
It has been suggested in recent publications that LRP1 is involved in the regulation of inflammatory responses23,24 and apoptotic pathways.13 Because atherosclerosis is an inflammatory disease and apoptosis is attributed to lesion vulnerability,21 the amount of apoptosis in atherosclerotic lesions was quantified and showed a significant 2-fold increase in apoptotic cells for the lesions of LDLR−/−LRP1n2/n2 mice. Because sensitivity of peritoneal macrophages for induction of apoptosis by oxysterols was unchanged, the LRP1-mediated secretion of the proapoptotic cytokine, TNF-α, was evaluated in these macrophages in search of an alternative explanation. Our results demonstrated that disruption of the NPxYxxL motif significantly increases the LPS-induced secretion of TNF-α, and this might indeed explain the increase in apoptotic cells in lesions of the LDLR−/−LRP1n2/n2 mice. These data are corresponding to the results obtained in the LRP1-deficient macrophages, where a similar increased TNF-α secretion was speculated to be due to reduced clearance of TNF-α bound to α2M.6 However no effect was seen on the secretion of the monocyte recruitment factor MCP-1. This observation is consistent with the unaltered macrophage content observed in lesions of LDLR−/−LRP1n2/n2 mice.
So, in conclusion, the obtained results support the hypothesis by which loss of the NPxYxxL motif is affecting LRP1 internalization properties negatively leading to a raise in apoE containing lipoprotein particles, MMP2, t-PA, FVIII, and TNF-α and consequently in a more atherogenic lipoprotein profile and increased atherosclerosis development in the LDLR−/−LRP1n2/n2 mice. These findings are relevant in relation to reports where polymorphisms in the LRP1 gene, LRP1 mRNA, and LRP1 protein expression levels are associated with an increased risk of coronary artery diseases (CAD).25–27 Furthermore, there is evidence that LRP1 polymorphisms might effect the plasma lipid concentrations in the postprandial period.28 Altogether, our study provides experimental evidence in an animal model that subtle partial loss of LRP1 functionality leads to the accumulation of small defects at different levels, all contributing to an increased susceptibility for the development of CAD. In the human population the underlying mechanism is more likely the direct impact of LRP1 allelic differences itself on the expression level of LRP1 or indirectly the impact of modifier genes on the expression level of LRP1, rather than mutation of the protein coding domains. In this view our results emphasize the importance for further investigations toward LRP1 function in relation to postprandial dyslipidemia and the other risk factors for CAD.
We thank Nathalie Feyaerts and Vincent Pruniau for technical assistance.
Sources of Funding
This work was supported by “Fonds voor Wetenschappelijk Onderzoek Vlaanderen” (to A.R.). The research was also funded by PhD grants of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen; to P.G. and S.R.).
Received November 10, 2008; revision accepted June 25, 2009.
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