title: Proteasome inhibition decreases inflammation in human endothelial cells exposed to lipopolysaccharide.
abstract: BACKGROUND
  The proteasome degrades ubiquitinated proteins and is the major pathway for intracellular protein degradation. The role of the proteasome in endothelial dysfunction observed in septic shock remains unknown. We stimulated primary cultures of human umbilical vein endothelial cells with lipopolysaccharide (LPS) and investigated effects on the proteasome. We hypothesized that proteasome inhibition would decrease endothelial cell activation, oxidative stress, and alter the proteome.
  
  
  METHODS
  Endothelial cells were exposed to LPS (100 ng/mL) for 6 hours with or without lactacystin (5 mM), a proteasome inhibitor. Proteasome content and ubiquitinated proteins were measured by enzyme-linked immunosorbent assay and immunoblot, respectively. Markers of cellular activation, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, were measured by immunoblot and immunoassay. Superoxide anion production was determined by dihydroethidium assay, and nitrotyrosine (a marker of peroxynitrite) was visualized by immunofluoresence. The endothelial cell proteome was analyzed by 2D gel electrophoresis.
  
  
  RESULTS
  LPS stimulation of endothelial cells significantly increased proteasome content, whereas the total levels of ubquitinated proteins decreased. This suggests that LPS activates the proteasome system in endothelial cells. LPS increased total content and cell surface expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, whereas proteasome inhibition ameliorated these increases. LPS increased both superoxide anion production and nitrotyrosine staining. Proteasome inhibition decreased both markers of cellular oxidative stress. Proteomic analysis identified two novel proteins upregulated by LPS and normalized with proteasome inhibition as follows: guanine nucleotide binding protein-1 and heterogeneous ribonucleoprotein K transcript variant.
  
  
  CONCLUSIONS
  These results suggest that inhibition of the proteasome diminishes a number of markers of cellular stress induced by LPS. The proteasome may be a promising therapeutic target in clinical situations of severe pro-inflammatory stress.
authors: Departments of Obstetrics and Gynecology, Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada. manojlalu@gmail.com
authors: Manoj M
authors: MM
authors: Lalu
authors: Han
authors: H
authors: Xu
authors: Sowndramalingam
authors: S
authors: Sankaralingam
authors: Sandra T
authors: ST
authors: Davidge
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000473:pathology; D006801:Humans; D007249:Inflammation / Q000188:drug therapy* / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects* / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D012261:Ribonucleoproteins / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C516790:GNB1 protein, human; D044387:GTP-Binding Protein beta Subunits; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D008070:Lipopolysaccharides; D061988:Proteasome Inhibitors; D012261:Ribonucleoproteins; C067713:lactacystin; C072074:HNRNPK protein, human; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine