title: Glucosamine modulates TNF-α-induced ICAM-1 expression and function through O-linked and N-linked glycosylation in human retinal pigment epithelial cells.
abstract: OBJECTIVE
  The purpose of this article was to investigate the effects of glucosamine (GlcN) on the TNF-α-induced expression of intercellular adhesion molecule 1 (ICAM-1) and the function of ICAM-1 in ARPE-19 cells in vitro.
  
  
  METHODS
  We quantified protein levels of TNF-α-induced ICAM-1 in ARPE-19 cells with Western blotting. The effects of GlcN on O-linked glycosylation, and therefore on ICAM-1 expression, were compared after the addition of alloxan, an inhibitor of O-linked N-acetylglucosamine transferase (OGT), or O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase (O-GlcNAcase [OGA]), or after OGT gene overexpression. The effect of GlcN on the N-linked glycosylation of ICAM-1 was evaluated by the change in its molecular mass on Western blotting. The effect of O-linked glycosylation on the nuclear factor κB (NF-κB) signaling pathway was examined using an NF-κB reporter gene assay. The effect of GlcN on ICAM-1 adhesion activity was examined using an ICAM-1 adhesion assay.
  
  
  RESULTS
  GlcN, PUGNAc, and OGT overexpression inhibited TNF-α-induced ICAM-1 expression and NF-κB activity in ARPE-19 cells. Alloxan increased ICAM-1 expression and NF-κB activity in TNF-α-induced ARPE-19 cells. GlcN and tunicamycin reduced the molecular mass of TNF-α-induced ICAM-1 in ARPE-19 cells. The proteasome inhibitor MG-132 suppressed the GlcN-induced reduction in the molecular mass of TNF-α-induced ICAM-1. GlcN also attenuated the adhesion activity of TNF-α-induced ICAM-1.
  
  
  CONCLUSIONS
  GlcN inhibits ICAM-1 expression and functions by modulating the O-linked glycosylation of factors involved in NF-κB signaling and by reducing the N-linked glycosylation of TNF-α-induced ICAM-1 in ARPE-19 cells. These effects may contribute to the GlcN-mediated anti-inflammatory effects in the eye.
authors.affiliation: Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Ching-Long
authors.initials: CL
authors.lastname: Chen
authors.forename: Chang-Min
authors.initials: CM
authors.lastname: Liang
authors.forename: Yi-Hao
authors.initials: YH
authors.lastname: Chen
authors.forename: Ming-Cheng
authors.initials: MC
authors.lastname: Tai
authors.forename: Da-Wen
authors.initials: DW
authors.lastname: Lu
authors.forename: Jiann-Torng
authors.initials: JT
authors.lastname: Chen
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000496:Alloxan / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005944:Glucosamine / Q000494:pharmacology*; D006031:Glycosylation; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology; D048448:Phenylcarbamates / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000201:enzymology; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D010091:Oximes; D048448:Phenylcarbamates; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D000496:Alloxan; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D001619:beta-N-Acetylhexosaminidases; D005944:Glucosamine; D000117:Acetylglucosamine