Interrupting TGF-β1/CCN2/integrin-α5β1 signaling alleviates high mechanical-stress caused chondrocyte fibrosis

Y.-Z. Huang, L. Zhao, Y. Zhu, S.-J. Tian, W. Zhang, S. Liu, J.-F. Ge

Department of Orthopedics, The Affiliated Zhangjiagang Hospital of Soochow University, Zhangjiagang, China. gejianfei0271@tom.com

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• Orthopedics

OBJECTIVE: Mechanical-stress has been reported to trigger cartilage fibrosis, in which transforming growth factor (TGF)-β and connective tissue growth factor (CCN2) are involved. However, the function of integrin-α5β1, a cytomembrane receptor, on mechanical-stress related fibrosis has not yet been elucidated. This study aims to reveal the interaction of TGF-β1/CCN2/integrin-α5β1 in the mechanical-stress induced chondrocyte (CH) fibrosis.

PATIENTS AND METHODS: We used different levels (5% and 10%) of cyclic tension simulation (CTS) to stretch CHs and observed the gene expression of TGF-β1/CCN2/integrin-α5β1 as well as the fibrous related genes containing collagen I/II/III, Runx2, MMP13, and ADAMTS-5 by real-time polymerase chain reaction (RT-PCR) or immunofluorescence. We used the siRNA or the corresponding antagonist of TGF-β1, CCN2, integrin-α5β1 during the CTS to clear the effect of them in the fibrosis progress. In addition, to verify the crosstalk between TGF-β1, CCN2, and integrin-α5β1, we used the recombinant human (rh)-TGF-β1 and CCN2 to culture CHs without CST.

RESULTS: 24 hours-10% CTS was sufficient to induce a decrease of collagen II and increase the collagen I/III, Runx2, MMP13, and ADAMTS-5 gene expression. Under CTS, TGF-β1 silencing resulted in a decline of CCN2, integrin-α5β1, and alleviated the CHs fibrosis. Apart from this, blocking CCN2 or integrin-α5β1 expression also contributed to the suppression of 10% CTS induced CHs fibrosis. Meanwhile, the exogenic protein supplement raised the cellular TGF-β1 or CCN2 expression and increased the integrin-α5β1 mRNA level. However, the downregulation of TGF-β1 or CCN2 did not affect integrin-α5β1 expression, whether the CTS exited or not.

CONCLUSIONS: High mechanical-stress induces CHs fibrosis via the activation of TGF-β1/CCN2/integrin-α5β1 signaling, and interrupting the TGF-β1, CCN2, or integrin-α5β1 expression can alleviate the fibrous process.

 

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