title: MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.
abstract: OBJECTIVE
  Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro.
  
  
  METHODS
  HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9.
  
  
  RESULTS
  Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone.
  
  
  CONCLUSIONS
  Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yan-ling
authors.initials: YL
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yu-zhou
authors.initials: YZ
authors.lastname: Gui
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Shu-mei
authors.initials: SM
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Xin-bo
authors.initials: XB
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yi-ping
authors.initials: YP
authors.lastname: Wang
mesh_terms: D000924:Anticholesteremic Agents / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017035:Pravastatin / Q000494:pharmacology; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000924:Anticholesteremic Agents; D007976:Leupeptins; D008077:Lipoproteins, LDL; D061988:Proteasome Inhibitors; D011973:Receptors, LDL; D011493:Protein Kinase C; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D017035:Pravastatin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde