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Abstract
RGD-CAP (βig-h3), initially cloned as a transforming growth factor (TGF)-β inducible gene in human lung adenocarcinoma cells, was demonstrated to have a negative regulatory function in mineralization in hypertrophic chondrocytes, and the expression was shown to be associated with mechanical stimulation. We hypothesized that mechanical stimulation may regulate the terminal chondrocyte differentiation through the TGF-β pathway by enhancing the RGD-CAP expression. To test this hypothesis, we investigated the effects of mechanical strain on the terminal differentiation and mineralization of growth-plate chondrocytes and assessed the mechanical regulation of TGF-β and RGD-CAP expression. A cyclic mechanical strain of 12% elongation was applied to the cultured prehypertrophic chondrocytes isolated from the rib cartilage of 4-week-old male rats at 30 cycles/min (loading and relaxation on every alternate second). The terminal differentiation and mineralization of chondrocytes were assessed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The gene expressions of TGF-β and RGD-CAP, as well as chondrocytic terminal differentiation markers such as type X collagen and ALP, were examined with real-time RT-PCR. Cyclic mechanical strain decreased the ALP activity and intensity of alizarin red staining in prehypertrophic chondrocytes, as well as the gene expressions of type X collagen and ALP. TGF-β and RGD-CAP were upregulated in the prehypertrophic chondrocytes subjected to mechanical strain, whereas the level of PTHrP receptor mRNA was not affected by the mechanical strain. The neutralizing antibody for TGF-β suppressed the reduction of the mineralization of chondrocyte cultures with the downregulation of RGD-CAP. These results suggest that mechanical strain negatively regulates the terminal differentiation of chondrocytes through the signal pathway of TGF-β with the induction of RGD-CAP.