Aggregated Low-Density Lipoprotein Induces LRP1 Stabilization Through E3 Ubiquitin Ligase CHFR Downregulation in Human Vascular Smooth Muscle Cells

Abstract

Objective-Low density lipoprotein retention and aggregation in the arterial intima are key processes in atherogenesis. Aggregated LDL (agLDL) is taken up through low-density lipoprotein receptor-related protein 1 (LRP1) by human vascular smooth muscle cells (VSMC). AgLDL increases LRP1 expression, at least in part, by downregulation of sterol regulatory element-binding proteins. It is unknown whether agLDL has some effect on the ubiquitin-proteasome system, and therefore on the LRP1 receptor turnover. The objective of this study was to analyze the effect of agLDL on the degradation of LRP1 by the ubiquitin-proteasome system in human VSMC. Methods and Results-Human VSMC were isolated from the media of human coronary arteries. Ubiquitinylated LRP1 protein levels were significantly reduced in human VSMC exposed to agLDL (100 μg/mL) for 20 hours (agLDL: 3.70±0.44 a.u. versus control: 9.68±0.55 a.u). Studies performed with cycloheximide showed that agLDL prolongs the LRP1 protein half life. Pulse-chase analysis showed that LRP1 turnover rate is reduced in agLDL-exposed VSMC. Two-dimensional electrophoresis shows an alteration in the proteomic profile of a RING type E3 ubiquitin ligase, CHFR. Real-time PCR and Western blot analysis showed that agLDL (100 μg/mL) decreased the transcriptional and protein expression of CHFR. CHFR silencing increased VSMC, but not macrophage, LRP1 expression. However, CHFR silencing did not exert any effect on the classical low-density lipoprotein receptor protein levels. Furthermore, immunoprecipitation experiments demonstrated that the physical interaction between CHFR and LRP1 decreased in the presence of agLDL. Conclusion-Our results demonstrate that agLDL prolongs the half life of LRP1 by preventing the receptor ubiquitinylation, at least in part, through CHFR targeting. This mechanism seems to be specific for LRP1 and VSMC.

Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans

“…As expected, LRP1 WT levels decreased progressively with a half-life of >24 hours. 26 In contrast, the half-life of p.Val3244Ile mutant was ≈50% and of the p.Glu3983Asp ≈85% shorter compared with WT (Figure 1 E). In an attempt to restore the mutant LRP1 expression, transiently transfected cells were cultured at lower temperature (30°C), which has been previously shown to improve protein folding and subsequently stability.…”

“…Such studies may benefit from a focus on those exons encoding for the domains involved in the uptake of CTSD (ie, exons 83–84). 26 Further support may come from in vitro studies showing that decreased ABCA1 at the cell membrane because of LRP1 downregulation is associated with reduced cholesterol efflux to apoA1. In addition, downregulation of LRP1 in human hepatic cell lines may help to study the anticipated increased SR-B1–mediated uptake of cholesteryl ester from HDL.…”

High Concentrations of Rosiglitazone Reduce mRNA and Protein Levels of LRP1 in HepG2 Cells

“…Previous studies have demonstrated that the pharmacological activation of PPARγ promotes its own proteolytic degradation via UPS ( Hauser et al, 2000 ; Kilroy et al, 2009 ). In addition, it has been proposed that UPS is the key regulator for LRP1 degradation in a number of cell lines, including HepG2 ( Melman et al, 2002 ; Cal et al, 2013 ). The use of MG132, a potent UPS inhibitor, in HepG2 cells treated with RGZ (30 μM) for 24 h did not prevent LRP1 protein degradation, as shown in Figure 2C .…”

Molecular Biology of Atherosclerosis

“…Interestingly, LRP1 can mediate uptake of aggregated LDL and subsequent cholesterol accumulation in VSMC. Aggregated LDL seems to stabilize LRP1 and increase its expression by decreasing ubiquitin-mediated degration (240). VSMC-specific deficiency of LRP1 in LDLR-null mice resulted in thickening of the media, dilation of the aorta, aneurysm formation, and accelerated atherosclerosis (191).…”