Akt phosphorylation in human chondrocytes is down-regulated via p53r2 in response to mechanical stress

Abstract

Purpose: p53 tumor suppressor protein is activated in response to DNA damage. When the DNA is damaged, Ser15 residue is phosphorylated and p53R2 is induced. The role of p53R2 in chondcotytes is unknown. In this study, we evaluated p53R2 expression in chondrocytes in response to mechanical stress. Furthermore, we investigated the function of p53R2 in response to mechanical stress. Methods: OA cartilage samples were obtained from total knee replacement surgery, and normal cartilage samples were from femoral neck fracture. The expression of p53R2 was analyzed by immunohistochemistry. Chondrocytes were isolated from OA and normal cartilage and grown. The expression of p53R2 in chondrocytes was detected by western blotting and real-time PCR. Normal and OA chondrocytes were introduced 5% tensile strain using FX-2000. After the strain, expressions of p53R2, ERK1/2, JNK, Akt, phospho-ERK1/2, phospho-JNK and phospho-Akt were detected by western blotting respectively. OA chondrocytes after transfection of p53R2 siRNA or control siRNA were introduced the same strain. Expressions of p53R2 and the same protein kinases were detected by western blotting. Furthermore, glycosaminoglycan(GAG) quantification was performed after the strain. Results: p53R2 was highly expressed in OA cartilage in comparison with normal cartilage by immunohistochemistry. Western blotting and real-time PCR showed p53R2 expression in chondrocytes was higher in OA chondrocytes than normal chondrocytes, too. (Fig.1) p53R2 expression in OA and normal chondrocytes was increased after 5% tensile strain. On the other hand, Akt phosphorylation was down-regulated after the strain. However other protein kinases were not regulated significantly. (Fig.2) Akt-phosphorylation was up-regulated after transfection of p53R2 siRNA. (Fig.3) GAG was down-regulated by the 5% strain, however up-regulated after p53R2 siRNA transfection. Conclusions: p53R2 could regulate Akt signaling in chondrocyte mechanotransduction. Down-regulation of p53R2 expression might increase production of glycosaminoglycan. We consider that the regulation of p53R2 might be a strategy for OA treatment. View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)