OBJECTIVE: To uncover the mechanism by which hypoxia enhances cartilage matrix synthesis by human articular chondrocytes. METHODS: The hypoxic response was investigated by exposing normal (nonarthritic) human articular chondrocyte cultures to 20% oxygen and 1% oxygen. Induction of the differentiated phenotype was confirmed at the gene and protein levels. In its first reported application in human articular chondrocytes, the RNA interference method was used to directly investigate the role of specific transcription factors in this process. Small interfering RNA directed against hypoxia-inducible factor 1alpha (HIF-1alpha), HIF-2alpha, and SOX9 were delivered by lipid-based transfection of primary and passaged human articular chondrocytes. The effect of each knockdown on hypoxic induction of the chondrocyte phenotype was assessed. RESULTS: Hypoxia enhanced matrix synthesis and SOX9 expression of human articular chondrocytes at both the gene and protein levels. Although HIF-1alpha knockdown had no effect, depletion of HIF-2alpha abolished this hypoxic induction. Thus, we provide the first evidence that HIF-2alpha, but not HIF-1alpha, is essential for hypoxic induction of the human articular chondrocyte phenotype. In addition, depletion of SOX9 prevented hypoxic induction of matrix genes, indicating that the latter are not direct HIF targets but are up-regulated by hypoxia via SOX9. CONCLUSION: Based on our data, we propose a novel mechanism whereby hypoxia promotes cartilage matrix synthesis specifically through HIF-2alpha-mediated SOX9 induction of key cartilage genes. These findings have potential application for the development of cartilage repair therapies.
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Adolescent, Adult, Basic Helix-Loop-Helix Transcription Factors, Cartilage, Articular, Cell Culture Techniques, Child, Chondrocytes, Female, High Mobility Group Proteins, Humans, Hypoxia, Male, Middle Aged, Phenotype, SOX9 Transcription Factor, Transcription Factors, Up-Regulation