title: Hsp90β inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes.
abstract: BACKGROUND
  Hsp90β is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90β in key aspects of OA pathogenesis.
  
  
  METHODS
  Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90β in stimulated cells.
  
  
  RESULTS
  Hsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes.
  
  
  CONCLUSIONS
  The present results show how Hsp90β modulates NO production and NO-mediated cellular death in human OA chondrocytes.
authors: Rheumatology Division, ProteoRed/ISCIII Proteomic Group, INIBIC - Hospital Universitario A Coruña, As Xubias S/N, 15006 - A Coruña, Spain.
authors: Valentina
authors: V
authors: Calamia
authors: Maria C
authors: MC
authors: de Andrés
authors: Natividad
authors: N
authors: Oreiro
authors: Cristina
authors: C
authors: Ruiz-Romero
authors: Francisco J
authors: FJ
authors: Blanco
mesh_terms: D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D016207:Cytokines / Q000494:pharmacology; D005260:Female; D055785:Gene Knockdown Techniques; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D009675:Novobiocin / Q000494:pharmacology; D020370:Osteoarthritis, Knee / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection
chemical_list: D016227:Benzoquinones; D016207:Cytokines; D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; D047029:Lactams, Macrocyclic; C118603:NOC 12; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D034741:RNA, Small Interfering; D009599:Nitroprusside; D009675:Novobiocin; D009569:Nitric Oxide; C001277:geldanamycin