ERK5 Signaling Pathway Mediates The Effects Of Fluid Shear Stress On The Expression Of MMPs And TIMPs In MC3T3-E1 Osteoblasts

Objective:to observe matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)expression in MC3T3-E1 osteoblastic cells induced by fluid shear stress,as well as to study the effect of ERK5 signaling pathway in it.Methods:MC3T3-E1 cells were treated with FSS,XMD8-92 alone,or in combination,XMD8-92group using the concentration of 5umol/L,MC3T3-E1 osteoblasts were loaded with FSS for 0,15,30,45 and 60 min respectively to determine the optimal FSS loading time for MMPs and TIMPs in MC3T3-E1 osteoblasts,the protein levels of MMPS,TIMPs,phospho-ERK5(P-ERK5)and ERK5 were detected by Western blotting.Results:The changes of MMP-1 and TIMP-1 protein were time-dependent in MC3T3-E1 cells at12 dyn/cm~2 FSS for 0,15,30,45 and 60 min respectively,MMP-1 gradually increased from 15min,the difference was statistically significant(P<0.01),reached a peak at 45 min,60 min has begun to decline,but still higher than the blank control group,the difference was statistically significant(P<0.01),The expression of TIMP-1 began to decrease at 15 min,which was the lowest at 45 min(P<0.01).The expression level of TIMP-1 was up-regulated at 60 min.In subsequent experiments,FSS was loaded for 45 min.FSS loading 12dyn/cm~2 for 45min,Can significantly promote the phosphorylation of ERK5 in MC3T3-E1 cells,that is,the proportion of P-ERK5/ERK5 increased significantly(P<0.01),MC3T3-E1 cells were pretreated with 5 uM XMD8-92 for1 h,Western blotting showed that XMD8-92 could inhibit the activation of ERK5 in normal MC3T3-E1 cells(P<0.05)and inhibit the expression of P-ERK5 induced by FSS.The expression of MMP-1 and MMP-13(P<0.001)and MMP-3 expression in MC3T3-E1 cells were significantly up-regulated by FSS at 12 dyn/cm~2 for 45 min(P<0.05).Western blotting showed that XMD8-92could down-regulate the expression of MMP-1,MMP-3(P<0.001)and the expression of MMP-13(P<0.05)in normal MC3T3-E1 cells and down-regulated the expression of MMP-1 and MMP-13induced by FSS(P<0.001)and the expression of MMP-3(P<0.05),The expression of TIMP-1,TIMP-2 and TIMP-3 in MC3T3-E1 cells was down-regulated by FSS for 12 dyn/cm~2(P<0.05).MC3T3-E1 cells were pretreated with 5 uM XMD8-92 for 1 h,The results showed that XMD8-92could up-regulate the expression of TIMP-1 and TIMP-2(P<0.05)and TIMP-3(P<0.001)in normal MC3T3-E1 cells,and up-regulate the expression of TIMP-1 TIMP-2,TIMP-3 induced by FSS(P<0.05).Conclusions:Physiological strength(12 dyn/cm~2)of fluid shear force on MC3T3-E1 osteoblasts after 45min can significantly up-regulate the expression of MMPs,down-regulation of TIMPs expression,but this effect can be ERK5 high selectivity inhibitor XMD8-92 Block,This indicates that ERK5 signaling pathway regulates fluid shear stress on the expression of MMPs and TIMPs in osteoblasts.