Phosphorylated Proteomic Analysis Of Rat Chondrocytes Under Periodic Mechanical Stress

Background:In recent years,a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress.In our previous studies,we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway.Despite these results,the effects and mechanisms of periodic mechanical stress are still not fully understood,so in this study we extended our study to include proteomic techniques.Aims:We screened the differentially phosphorylated proteins before and after the stress by phosphoproteomic techniques to further explore the effects of periodic mechanical stress on chondroytesMethods:In this study,we used proteomic analysis to detect quantitative changes and modifications(phosphorylation)in chondrocytes under periodic mechanical stress.The results were obtained by direct cell counting,cell counting kit-8(CCK-8)assay,use of a phosphorylation inhibitor,quantitative real-time PCR(qPCR)analysis,western blot analysis and immunofluorescence assay.Results:First,from phosphoproteomics data analysis,a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified.The number of significant differentially expressed proteins was 97 and the number of phosphopeptides was 108(ratio>1.20 or<0.83 and p<0.05).Second,periodic mechanical stress increased GSK3-beta(Y216)phosphorylation,promoted the phosphorylation of beta-catenin,decreased beta-catenin levels and suppressed the expression of type I collagen.In contrast,inhibition of GSK3-beta by TWS119,which particularly inhibits the phosphorylation of Y216,suppressed the phosphorylation of beta-catenin,which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen.Conclusions:First,we successfully constructed the differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress.Second,we discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of glycogen systhase kinase 3-beta(GSK3-beta)and beta-catenin.