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Identification of clock as a mechanosensitive gene by large-scale DNA microarray analysis: downregulation in osteoarthritic cartilage

The chondrocytes of cartilage have so far been reported to include three mechanoresponsive genes: cartilage matrix protein (CMP, matrilin-1), type X collagen, and Indian hedgehog (Ihh). In fact, all of the genes identified in these chondrocytes are associated solely with mechanical stress. In this study we examined the how mechanical stress influenced the patterns of gene expression of chondrocytes in three-dimensional (3D) sponges in order to better understand the mechanisms that control the chondrocyte phenotype during the development of osteoarthritis. We cultured mouse chondrocytes in 3D sponge as a model of mechanical stress and isolated total RNA for a large-scale DNA microarray analysis covering 12000 genes. We analyzed the pattern of gene expression in relation to gene localization in cellular components such as the cytoplasm, cytoskeleton, and nucleus. Immunoblotting of osteoarthritis cartilage were performed using polyclonal anti-clock antibody. The biological rhythm of mRNA of clock and clock-related genes was analyzed by real-time polymerase chain reaction (PCR). Clock gene was confirmed by real-time PCR to validate the microarray data. Other clock-related genes such as Per1 and Per2 were also expressed in chondrocytes, exhibiting biological rhythm after serum shock. The large-scale DNA microarray indicated that clock and other genes functionally associated with mechanical stress play an essential role in regulating the biological rhythm of chondrocyte metabolism in osteoarthritis cartilage.