The Mechanism Of ERK5 Signalling Pathway In The Fluid Shear Stress-mediated Osteoblast Mechanotransduction

Background The stress environment of bone plays an important role in the process of bone formation and bone remodeling. Thus, studying the mechanism of the biomechanics signal transduction of osteoblasts under fluid shear stress helps to understand and look for the treatment the osteoporosis and other reducing bone diseases. Fluid shear stress is one of the main model of mechanical stress to stimulate the metabolism of bone cells in the body. Our research group has found that fluid shear stress promoted the activation of ERK5, however, the specific mechanism of ERK5 signaling pathway in the osteoblastic mechanotransduction is still rarely studied.Purpose The mouse MC3T3-E1 osteoblastic cells were subjected to fluid shear stress. We detected the effect of fluid shear stress on osteoblastic mechanotransduction related signalling pathway of COX-2/PGE2 expression to investigate the role of ERK5 signaling pathway in the regulation of above genes. Then, we further studied the effect of fluid shear stress on the NO/c GMP/PKG signaling pathway, to investigate the role of NO/c GMP/PKG signaling pathway in fluid shear stress mediated ERK5 activation.In order to further research on fluid shear stress-mediated ERK5 activation, we study the effect of NO/c GMP/PKG signaling pathway on ERK5 phosphorylation and nuclear translocation.MethodsPart I: This part is divided into four groups, blank control group, fluid shear stress group, ERK5 si RNA group, fluid shear stress + ERK5 si RNA group. The MC3T3-E1 osteoblast was subjected to 12 dyne/cm2 fluid shear stress for 45 min, and si RNA was used for silencing ERK5 gene. Then, PGE2 ELISA kit was used to detect the release of PGE2 in osteoblast, RT-PCR were used to detect the expression of ERK5 and COX-2 gene, Western blot was observe the ERK5, COX-2, CREB and NF-κB protein expression.Part II: This part was divided into blank control group, the fluid shear stress group, fluid shear stress + L-NAME group, fluid shear stress +ODQ group, fluid shear stress +(RP)-8-p CPT-PET-c GMPS group, fluid shear stress +PKG-I si RNA group and fluid shear stress +PKG-II si RNA group. The mouse osteoblasts was subjected to 12 dyne/cm2 fluid shear stress and NOS inhibitor L-NAME, s GC inhibitor ODQ, PKG inhibitor(Rp)-8-p CPT-PET-c GMPS were used to inhibit the NO/c GMP/PKG signalling pathway. Western blot was used to detect the expression of NOS, s GC, PKG-I, PKG-II and ERK5 activation. In addition, using si RNA to silence PKG-I and PKG-II gene to observe ERK5 activation and its nuclear translocation.ResultsPart I: Compared with control group, fluid shear stress significantly increased ERK5 protein phosphorylation, COX-2 m RNA/protein expression and PGE2 synthesis(p<0.05). At the same time, fluid shear stress stimulated the phosphorylation of transcription factor CREB and NF-κB(p<0.05). Compared with fluid shear stress group, application of 60 nm ERK5 si RNA for 48 h significantly decreased ERK5 protein expression, COX-2 expression, PGE2 synthesis and the phosphorylation of CREB and NF-κB(p<0.05). Silencing the CREB gene and blocking the expression of NF-κB can inhibit the expression of COX-2 protein(p<0.05).Part II: Compared with control group, fluid shear stress stimulated NO and c GMP synthesis, p-NOS、s GC、PKG-I、PKG-II and p-ERK5 protein expression(p<0.05). Compared with fluid shear stress group, application of L-NAME, ODQ and(Rp)-8-p CPT-PET-c GMPS significantly reduced the phosphorylation of ERK5(p<0.05). Silencing the PKG-II gene using si RNA significant decreased the expression and nuclear translocation of p-ERK5(p<0.05) while silencing the PKG-I gene had no effect on the expression and nuclear translocation of p-ERK5.Conclusion ERK5 signaling pathway mediated fluid shear stress promotes the expression of COX-2/PGE2 through CREB and NF-κB. In addition, the fluid shear stress can promote ERK5 activation and nuclear translocation via NO/c GMP/PKG-II signalling pathway.