title: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cardiovascular Research Center of Barcelona, CSIC-ICCC, IIB-SantPau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
authors.forename: Roi
authors.initials: R
authors.lastname: Cal
authors.forename: Maisa
authors.initials: M
authors.lastname: García-Arguinzonis
authors.forename: Elena
authors.initials: E
authors.lastname: Revuelta-López
authors.forename: José
authors.initials: J
authors.lastname: Castellano
authors.forename: Teresa
authors.initials: T
authors.lastname: Padró
authors.forename: Lina
authors.initials: L
authors.lastname: Badimon
authors.forename: Vicenta
authors.initials: V
authors.lastname: Llorente-Cortés
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000201:enzymology; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047468:Immunoprecipitation; D008077:Lipoproteins, LDL / Q000378:metabolism*; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D055550:Protein Stability; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D018797:Cell Cycle Proteins; C475476:LRP1 protein, human; D008077:Lipoproteins, LDL; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; C412002:CHFR protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex