title: Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes.
abstract: OBJECTIVE
  To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1beta (IL-1beta). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-kappaB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor beta (TGFbeta) and TGFbeta receptors, and production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)).
  
  
  METHODS
  Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O(2) (hypoxia) or 21% O(2) (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1beta. For reoxygenation experiments, cells were treated in the same way in 5% O(2), except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction.
  
  
  RESULTS
  In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1beta-induced DNA binding of NF-kappaB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1beta treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1beta in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1beta in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1beta in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1beta only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1beta in reoxygenated cultures. Stimulation of TGFbeta1 expression by IL-1beta was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1beta on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1beta-induced PGE(2) production.
  
  
  CONCLUSIONS
  Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1beta. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1beta on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1beta-induced NO production is associated with a significant decrease in PGE(2) synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.
authors.affiliation: Laboratory of Connective Tissue Biochemistry, Faculty of Medicine, 14032 Caen Cedex, France.
authors.forename: G
authors.initials: G
authors.lastname: Martin
authors.forename: R
authors.initials: R
authors.lastname: Andriamanalijaona
authors.forename: S
authors.initials: S
authors.lastname: Grässel
authors.forename: R
authors.initials: R
authors.lastname: Dreier
authors.forename: M
authors.initials: M
authors.lastname: Mathy-Hartert
authors.forename: P
authors.initials: P
authors.lastname: Bogdanowicz
authors.forename: K
authors.initials: K
authors.lastname: Boumédiene
authors.forename: Y
authors.initials: Y
authors.lastname: Henrotin
authors.forename: P
authors.initials: P
authors.lastname: Bruckner
authors.forename: J-P
authors.initials: JP
authors.lastname: Pujol
mesh_terms: D053674:Aggrecans; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis; D016326:Extracellular Matrix Proteins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D006706:Homeostasis; D000860:Hypoxia / Q000235:genetics*; D051793:Hypoxia-Inducible Factor 1; D007375:Interleukin-1 / Q000494:pharmacology*; D037181:Lectins, C-Type; D045726:Metalloproteases / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D009687:Nuclear Proteins / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D051793:Hypoxia-Inducible Factor 1; D007375:Interleukin-1; D037181:Lectins, C-Type; D016328:NF-kappa B; D009687:Nuclear Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D019714:Tissue Inhibitor of Metalloproteinases; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D009569:Nitric Oxide; D004247:DNA; D020929:Mitogen-Activated Protein Kinase Kinases; D045726:Metalloproteases; D015232:Dinoprostone; D010100:Oxygen