title: A peptidyl-glucosamine derivative affects IKKalpha kinase activity in human chondrocytes.
abstract: BACKGROUND
  Nuclear factor-kappaB (NF-kappaB) transcription factor regulates several cell signaling pathways, such as differentiation and inflammation, which are both altered in osteoarthritis. Inhibitor kappaB kinase (IKK)alpha and IKKbeta are kinases involved in the activation of the NF-kappaB transcription factor. The aim of the present study was to determine the effects of glucosamine (GlcN), which is administered in the treatment of osteoarthritis, and of its 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-beta-D-glucose (NAPA) derivative on IKK kinases and, consequently, on NF-kappaB activation in human chondrocytes.
  
  
  METHODS
  The human chondrosarcoma cell line HTB-94 and human primary chondrocytes were stimulated with tumor necrosis factor (TNF)alpha after pre-treatment with GlcN or NAPA. Gene mRNA expression level was evaluated by real-time PCR. Inhibitor kappaB protein (IkappaB)alpha phosphorylation and p65 nuclear re-localization were analyzed by Western blotting; IKKalpha nuclear re-localization was also investigated by immunocytochemistry and Western blotting. IKK kinase activity was studied by in vitro kinase assay.
  
  
  RESULTS
  After TNFalpha stimulation, the mRNA expression level of some of the genes under NF-kappaB control, such as interleukin (IL)-6 and IL-8, increased, while treatment with GlcN and NAPA reverted the effect. We investigated the possibility that GlcN and NAPA inhibit IKK kinase activity and found that NAPA inhibits the IKKalpha kinase activity, whereas GlcN does not. Interestingly, both GlcN and NAPA inhibit IKKalpha nuclear re-localization.
  
  
  CONCLUSIONS
  Our results demonstrate that glucosamine and its peptidyl derivative can interfere with NF-kappaB signaling pathway by inhibiting IKKalpha activity in human chondrocytes. However, the mechanism of action of the two molecules is not completely overlapping. While NAPA can both specifically inhibit the IKKalpha kinase activity and IKKalpha nuclear re-localization, GlcN only acts on IKKalpha nuclear re-localization.
authors.affiliation: Department of Biochemical Sciences, Sapienza University of Roma, P,le Aldo Moro, 5, 00185 Roma, Italy. anna.scottodabusco@uniroma1.it
authors.forename: Anna
authors.initials: A
authors.lastname: Scotto d'Abusco
authors.forename: Laura
authors.initials: L
authors.lastname: Politi
authors.forename: Cesare
authors.initials: C
authors.lastname: Giordano
authors.forename: Roberto
authors.initials: R
authors.lastname: Scandurra
mesh_terms: D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D019902:Chondrocytes / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects* / Q000502:physiology; D020869:Gene Expression Profiling; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016328:NF-kappa B; D012333:RNA, Messenger; C496559:CHUK protein, human; D051550:I-kappa B Kinase; D005944:Glucosamine