The Mechanisms Of Rat Chondrocyte Proliferation And Matrix Synthesis Under Periodic Mechanical Stress

Objective To explore the effect of ERK1/2on rat chondrocyte proliferation andmatrix synthesis under periodic mechanical stress.Methods The experiment consisted of two steps. In the first step, rat chondrocytesderived from the second generation were divided into a pressure group and a controlgroup and incubated under a periodic mechanical stress field (200Kpa,0.1Hz) and ina simple-cell culture device, respectively. Cells were maintained under periodicmechanical stress or static conditions for0,0.5,1, and2h prior to Western blotanalysis, respectively. Cells were maintained under periodic mechanical stress orstatic conditions for8h prior to quantitative real-time PCR analysis, respectively.Cells were cultured for3days under periodic mechanical stress or static conditions8h per day prior to direct cell counting and CCK-8assay, respectively. In the secondstep, rat chondrocytes derived from the second generation were divided into apretreatment group and a control group and treated with50μM PD98059(PD98059pretreatment group) or an equivalent amount of DMSO (0.1%, v/v, Control group) fora1h period of pretreatment, or were pretreated with shRNA targeted to ERK1/2(ERK1/2shRNA pretreatment group) or control scrambled shRNA (Control group),respectively. Cells were maintained under periodic mechanical stress or static conditions for1h prior to Western blot analysis and for8h prior to quantitativereal-time PCR analysis, respectively. Cells were cultured for3days under periodicmechanical stress or static conditions8h per day prior to direct cell counting andCCK-8assay, respectively.Results Under periodic mechanical stress, both rat chondrocyte proliferation andmatrix synthesis were significantly increased (p <0.05for each) and were associatedwith increases in the phosphorylation levels of ERK1/2-Thr202/Tyr204(p <0.05foreach). And pretreatment with the ERK1/2selective inhibitor, PD98059, and shRNAtargeted to ERK1/2reduced periodic mechanical stress-induced chondrocyteproliferation and matrix synthesis (p <0.05for each).Conclusions Periodic mechanical stress promotes chondrocyte proliferation andmatrix synthesis through ERK1/2. Objective To explore the effects of PLCγ1and Rac1on rat chondrocyte proliferationand matrix synthesis under periodic mechanical stress and the relationship amongPLCγ1, Rac1and ERK1/2.Methods The experiment consisted of two steps. In the first step, rat chondrocytesderived from the second generation were divided into a pressure group and a controlgroup and incubated under a periodic mechanical stress field (200Kpa,0.1Hz) and ina simple-cell culture device, respectively. Cells were maintained under periodicmechanical stress or static conditions for0,0.5,1, and2h prior to Western blotanalysis and for1h prior to Rac1GTPase activity assay, respectively. In the secondstep, rat chondrocytes derived from the second generation were divided into apretreatment group and a control group and treated with10μM U73122(U73122pretreatment group) or an equivalent amount of DMSO (0.1%, v/v, Control group) fora1h period of pretreatment, or were treated with50μM NSC23766(NSC23766pretreatment group) or an equivalent amount of DMEM-F12(Control group) for a1hperiod of pretreatment, respectively. Cells were maintained under periodicmechanical stress or static conditions for1h prior to Western blot analysis and for8hprior to quantitative real-time PCR analysis, respectively. Cells were cultured for3days under periodic mechanical stress or static conditions8h per day prior to directcell counting and CCK-8assay, respectively.Results Under periodic mechanical stress, the phosphorylation levels ofPLCγ1-Tyr783and Rac1-Ser71and Rac1activation were significantly increased (p <0.05for each). Pretreatment with the PLCγ1selective inhibitor, U73122, reducedperiodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (p<0.05for each), and attenuated the phosphorylation of ERK1/2-Thr202/Tyr204(p < 0.05), while the phosphorylation level of Rac1-Ser71was not inhibited (p>0.05).Proliferation, matrix synthesis and ERK1/2activation were inhibited afterpretreatment with the Rac1inhibitor NSC23766in chondrocytes in response toperiodic mechanical stress (p <0.05for each), while the phosphorylation site ofPLCγ1-Tyr783was not affected (p>0.05).Conclusions Periodic mechanical stress-initiated PLCγ1and Rac1act in parallel witheach other and mediate rat chondrocyte proliferation and matrix synthesis throughERK1/2. Objective To explore the effect of Src on rat chondrocyte proliferation and matrixsynthesis under periodic mechanical stress and the relationship between Src andERK1/2, PLCγ1, Rac1.Methods The experiment consisted of three steps. In the first step, rat chondrocytesderived from the second generation were divided into a pressure group and a controlgroup and incubated under a periodic mechanical stress field (200Kpa,0.1Hz) and ina simple-cell culture device, respectively. Cells were maintained under periodicmechanical stress or static conditions for0,0.5,1, and2h prior to Western blotanalysis, respectively. In the second step, rat chondrocytes derived from the secondgeneration were divided into a pretreatment group and a control group and treatedwith10μM PP2(PP2pretreatment group) or an equivalent amount of DMSO (0.1%,v/v, Control group) for a1h period of pretreatment, or were pretreated with shRNAtargeted to Src (Src shRNA pretreatment group) or control scrambled shRNA(Control group), respectively. Cells were maintained under periodic mechanical stressor static conditions for1h prior to Western blot analysis and for8h prior toquantitative real-time PCR analysis, respectively. Cells were cultured for3daysunder periodic mechanical stress or static conditions8h per day prior to direct cellcounting and CCK-8assay, respectively. In the third step, rat chondrocytes derivedfrom the second generation were divided into a pretreatment group and a controlgroup and treated with10μM U73122(U73122pretreatment group) or an equivalentamount of DMSO (0.1%, v/v, Control group) for a1h period of pretreatment, or weretreated with50μM NSC23766(NSC23766pretreatment group) or an equivalentamount of DMEM-F12(Control group) for a1h period of pretreatment, respectively.Cells were maintained under periodic mechanical stress or static conditions for1hprior to Western blot analysis, respectively.Results Under periodic mechanical stress, the phosphorylation levels of Src-Tyr418 were significantly increased (p <0.05for each). Inhibition of Src activity with PP2and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis(p <0.05for each) and attenuated ERK1/2, PLCγ1and Rac1activation inchondrocytes subjected to periodic mechanical stress (p <0.05for each). Thephosphorylation levels of Src-Tyr418were not inhibited after pretreatment with thePLCγ inhibitor U73122or the Rac1inhibitor NSC23766under periodic mechanicalstress (p>0.05for each).Conclusions Src mediates rat chondrocyte proliferation and matrix synthesis throughRac1, PLCγ1and ERK1/2under periodic mechanical stress. Objective To explore the effects of integrinβ1and integrinβ3on rat chondrocyteproliferation and matrix synthesis under periodic mechanical stress and therelationship between integrinβ1and Src, Rac1, PLCγ1, ERK1/2.Methods The experiment consisted of three steps. In the first step, rat chondrocytesderived from the second generation were divided into a pressure group and a controlgroup and incubated under a periodic mechanical stress field (200Kpa,0.1Hz) and ina simple-cell culture device, respectively. Cells were maintained under periodicmechanical stress or static conditions for8h prior to quantitative real-time PCRanalysis, respectively. In the second step, rat chondrocytes derived from the secondgeneration were divided into a pretreatment group and a control group and treatedwith10μg/mL blocking antibodies against integrinβ1or integrinβ3(Pretreatmentgroup) or an equivalent amount of DMEM-F12(Control group) for a5h period ofpretreatment, respectively. Cells were maintained under periodic mechanical stress orstatic conditions for8h prior to quantitative real-time PCR analysis, respectively.Cells were cultured for3days under periodic mechanical stress or static conditions8h per day prior to direct cell counting and CCK-8assay, respectively. In the third step,rat chondrocytes derived from the second generation were divided into a pretreatmentgroup and a control group and treated with10μg/mL blocking antibody againstintegrinβ1(Pretreatment group) or an equivalent amount of DMEM-F12(Controlgroup) for a5h period of pretreatment, respectively. Cells were maintained underperiodic mechanical stress or static conditions for1h prior to Western blot analysis,respectively.Results Under periodic mechanical stress, the gene expression of integrinβ1wassignificantly increased (p <0.05), while integrinβ3gene expression was not affected(p>0.05). Pretreatment with blocking antibody against integrinβ1reduced periodicmechanical stress-induced chondrocyte proliferation and matrix synthesis (p <0.05 for each), and attenuated the phosphorylation of Src-Tyr418, Rac1-Ser71, PLCγ1-Tyr783and ERK1/2-Thr202/Tyr204(p <0.05for each). Inhibition of integrinβ3did notabrogate chondrocyte proliferation and matrix synthesis under periodic mechanicalstress (p>0.05).Conclusions Under periodic mechanical stress, integrinβ1mediates rat chondrocyteproliferation and matrix synthesis through Src, Rac1, PLCγ1and ERK1/2. In conclusion, this study reveals the mechanisms of signal transduction bywhich periodic mechanical stress promotes rat chondrocyte proliferation andmatrix synthesis. Integrinβ1senses periodic mechanical stress and activates Src.Then Src promotes the activation of Rac1and PLCγ1. Rac1and PLCγ1act inparallel with each other and converge upon ERK1/2. Finally, activated ERK1/2mediates rat chondrocyte proliferation and matrix synthesis. Briefly, periodicmechanical stress enhances rat chondrocyte proliferation and matrix synthesisthrough the integrinβ1-Src-Rac1/PLCγ1-ERK1/2signaling pathway.