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Objective. Autologous chondrocyte implantation requires expansion of cells ex vivo, leading to dedifferentiation of chondrocytes (loss of aggrecan and type II collagen to the profit of type I and type III collagens). Several approaches have been described for redifferentiation of these cells. Among them, low oxygen tension has been exploited to restore the differentiated chondrocyte phenotype, but molecular mechanisms of this process remain unclear. However, under conditions of hypoxia, one of the major factors involved is hypoxiainducible factor 1␣ (HIF-1␣). The purpose of this study was to investigate the role of HIF-1␣ during human chondrocyte redifferentiation.Methods. We used complementary approaches to achieving HIF-1␣ loss (inhibition by cadmium ions and dominant-negative expression) or gain (ectopic expression and cobalt ion treatment) of function. Expression of chondrocyte, as well as fibroblast-like, phenotype markers was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. Binding activities of HIF-1␣ and SOX9, a pivotal transcription factor of chondrogenesis, were evaluated by electrophoretic mobility shift assays and by chromatin immunoprecipitation assay.Results. We found that hypoxia and HIF-1␣ not only induced the expression of SOX9, COL2A1, and aggrecan, but they simultaneously inhibited the expression of COL1A1, COL1A2, and COL3A1. In addition, we identified the binding of HIF-1␣ to the aggrecan promoter, the first such reported demonstration of this binding.Conclusion. This study is the first to show a bimodal role of HIF-1␣ in cartilage homeostasis, since HIF-1␣ was shown to favor specific markers and to impair dedifferentiation. This suggests that manipulation of HIF-1␣ could represent a promising approach to the treatment of osteoarthritis.

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Objective. Extracellular matrix deposition is tightly controlled by a network of regulatory cytokines. Among them, interleukin-1␤ (IL-1␤) and transforming growth factor ␤1 (TGF␤1) have been shown to play antagonistic roles in tissue homeostasis. The purpose of this study was to determine the influence of IL-1␤ on TGF␤ receptor type II (TGF␤RII) regulation and TGF␤1 responsiveness in human articular chondrocytes.Methods. TGF␤1-induced gene expression was analyzed through plasminogen activator inhibitor 1 and p3TP-Lux induction. Receptor-activated Smad (RSmad) phosphorylation, TGF␤ receptors, and Smad expression were determined by Western blotting and real-time reverse transcription-polymerase chain reaction techniques. Signaling pathways were investigated using specific inhibitors, messenger RNA (mRNA) silencing, and expression vectors.Results. IL-1␤ down-regulated TGF␤RII expression at both the protein and mRNA levels and led to inhibition of the TGF␤1-induced gene expression and Smad2/3 phosphorylation. Moreover, IL-1␤ strongly stimulated the expression of inhibitory Smad7.TGF␤RII overexpression abolished the loss of TGF␤1 responsiveness induced by IL-1␤. The decrease in TGF␤RII required de novo protein synthesis and involved both the NF-B and JNK pathways.Conclusion. We demonstrate that IL-1␤ impairs TGF␤1 signaling through down-regulation of TGF␤RII, which is mediated by the p65/NF-B and activator protein 1/JNK pathways, and secondarily through the up-regulation of Smad7. These findings show that there is cross-talk in the signaling of 2 regulatory cytokines involved in inflammation.

Level III; case-control study.

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Objective. To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS).Methods. The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1␤ (IL-1␤). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by realtime polymerase chain reaction analysis. The effect of CS and IL-1␤ on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography.Results. CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1␤ increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1␤-induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1␤-induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1␤-treated FLS than in FLS treated with IL-1␤ alone.Conclusion. CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1␤-enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint.Hyaluronan (HA) is a glycosaminoglycan polymer of repeated N-acetylglucosamine ␤(1-4) glucuronic acid ␤(1-3) disaccharide units (1). It is a major component of the extracellular matrix and can attain a molecular mass of 20 ϫ 10 6 daltons (2). It traps a large amount of water, giving rise to solutions of high viscosity and elasticity (3). HA participates in tissue remodeling, normal tissue homeostasis, and disease, including osteoarticular pathology, immune and inflammatory disorders, pulmonary and vascular diseases, and cancer (4,5). In joints, fibroblast-like synoviocytes (FLS) are the main source of HA. Its concentration in the synovial fluid is ϳ3 gm/liter, providing most of the properties of synovial fluid (6) and protecting cartilage from overload peaks (7). The molecular weight of synovial fluid HA is ϳ6 ϫ 10 6 daltons (8,9).In osteoarthritis (OA) synovial fluid, both the concentration and size of HA are reduced, possibly by the release of reactive oxygen species and enzymes (10).

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