The Study Of Fluid Shear Stress Promoting The Proliferation Of MC3T3-E1 Cells Via ER-α-ERK5 Signaling Pathway

Background: With the change of modern social life,the incidence of osteoporosis,delayed union and nonunion of fracture has been increasing year by year.Mechanical stress plays an important role in bone formation,bone remodeling and bone dynamic balance maintenance,but this process cannot do without synergistic effect of estrogen and related signaling molecules on the mechanical signal conduction.Therefore,it is important to study and elucidate the specific mechanisms of mechanical stress and estrogen action on osteoblasts for the prevention and treatment of osteoporotic fracture,delayed union and nonunion.Fluid shear stress(FSS)is one of the most studied mechanical stress models.Previous studies in our laboratory have shown that FSS can promote osteoblast proliferation by activating ERK5,but little is known about its molecular mechanism.Objective: In this experiment,the fluid shear stress(FSS)of 12dyn/cm2 was loaded on MC3T3-E1 cells,in order to verify that FSS promotes osteoblast proliferation by activating ERK5,explore the loading time of FSS in later experiments,and study the effect of estrogen receptor alpha(ER-α)on the activation of extracellular signal-regulated kinase 5(ERK5)and the specific molecular mechanism of ER-α-ERK5 signaling pathway in the proliferation of osteoblasts promoted by the FSS.Methods: MC3T3-E1 cells were treated with specific ER-α antagonist MPP(methyl-piperidino-pyrazole,1 μmol/L),highly selective ERK5 inhibitor XMD8-92(5 μmol/L),or loading 12 dyn/cm2 FSS.The proliferation activity of osteoblasts was detected by MTT assay,and the protein expression levels of ER-α,ERK5,p-ERK5,Cyclin D1 and CDK4 protein were detected by Western blotting.Results: Physiological FSS exposure for 60 min could promote MC3T3-E1 cell proliferation significantly and increase the expression of Cyclin D1 and CDK4;Meanwhile,the expression of ER-α and p-ERK5 increased significantly.The expression of p-ERK5 decreased significantly,after being treated with MPP,so were the expression levels of Cyclin D1 and CDK4.After incubation with XMD8-92 to inhibit the ERK5 activity,the expression levels of Cyclin D1 and CDK4 in osteoblasts promoted by FSS were significantly decreased.Conclusion: ER-α mediates FSS promoting the activation of ERK5 in osteoblast MC3T3-E1 cells.FSS up-regulates CyclinD1 and CDK4 expression and results in MC3T3-E1 cells proliferation via ER-α-ERK5 signaling pathway.