Involvement Of ERK5 Signaling Pathway In Osteoblasts Apoptosis And Osteoporosis Development In Mice

Objective: Fluid shear stress(FSS) is a potent mechanical stimulus and prevents osteoblast from apoptosis. However, little is known about the role of ERK5 signaling pathway in FSS-mediated anti-apoptotic effects in osteoblast, and the effects of ERK5 signaling on bone homeostasis in vivo have not yet been described. Thus, the identification of new cellular and molecular mechanisms of ERK5 involved in osteoblast apoptosis and osteoporosis helps to provide important insight for novel therapeutic targets.Methods: In this sthdy, mechanical stimulation of FSS can activates ERK5, and the activity of ERK5 was blocked by XMD8-92 or ERK5-siRNA. Then, MC3T3-E1 cells were incubated with XMD8-92 or ERK5-siRNA, exposed to FSS or left static, and stimulated with TNF-α. Cell apoptosis was assessed based on nuclei staining with Hoechst 33258, TdT-UTP nick end labeling(TUNEL) array, or flow cytometric staining. Relative levels of gene expression related to ERK5 were detected using PCR array. Caspase-3 activity was detected by cleavage of chromogenic caspase substrates. The protein expression was detected by western blotting. Mice were injected intraperitoneally with XMD8-92 and/or DEX. We further detected the function of XMD8-92 in histology, biomechanics, proliferation, apoptosis, and adipogenesis in vivo and in vitro.Results: Although 1 ng/mL of TNF-α had no effect on osteoblast apoptosis, high concentrations of TNF-α promoted apoptosis in a dose dependent manner. Physiological FSS(12dyn/cm2) for 1h significantly stimulated ERK5 phosphorylation, and and phosphorylated ERK5 can shuttle from the cytosol to the nucleus. Activation of ERK5 by FSS is effectively blocked by 5μM of XMD8-92 or ERK5-siRNA. FSS inhibits osteoblast apoptosis induced by TNF-α(from 34.2% to 13.8%). XMD8-92 or ERK5-siRNA promotes TNF-α-induced cells apoptosis and blocks FSS-mediated antiapoptotic effect. Furthermore, XMD8-92 significantly decreased phosphorylation of Bad in osteoblast and blocked the phosphorylation of Bad by FSS. Compared to TNF-α-treated group, the levels of foxo3 a, fasl, bim, and caspase-3 transcripts were significantly increased in ERK5-siRNA-transfected cells with TNF-α, whereas akt expression was dramatically decreased. The activation of ERK5, AKT and FoxO3 a by FSS are blocked in ERK5-siRNA-transfected osteoblast. LY294002 was also sufficient to attenuate the phosphorylation of AKT and FoxO3 a induced by FSS, but no significant change was observed for the phosphorylation level of ERK5. ERK5 silencing increases the expression of FasL and Bim and activity of caspase-3 induced by TNF-α in osteoblast. However, activation of ERK5 by FSS down-regulates the expression of FasL and Bim and activity of caspase-3 induced by TNF-α in osteoblast. For the in vivo experiments, phosphorylated ERK5 levels were significantly suppressed by XMD8-92 in femoral samples. The results of histology and biomechanics indicated mice with XMD8-92 treatment exhibited reduced bone mass and accelerates DEXinduced osteoporosis. We also found that XMD8-92 up-regulates RANKL/OPG rate and FasL expression, but down-regulates Cyclin B1 and CKD1 expression in vitro and in vivo, then osteoblasts proliferation viability is reduced and osteoblast apotosis is increased, resulting in osteoporosis in the end. More adipocytes are detected in mice treated with XMD8-92. Interestingly, XMD8-92 also accelerates the changes of abovementioned indexes in DEX-treated mice.Conclusion: 1. Fluid shear stress inhibits osteoblast apoptosis via ERK5 signaling pathway. FSS generates biochemical signals that transduce to the cytoplasm of osteoblasts to activate ERK5, and phosphorylated ERK5 can shuttle from the cytosol to the nucleus: a, activation of ERK5 leads to phosphorylation of Bad, and phosphorylation of Bad is sequestered by the 14-3-3 protein in the cytoplasm, which prevents it from translocating to mitochondria where it can induce activation of caspase-3; b, activated ERK5 activates AKT. FoxO3 a is phosphorylated through an ERK5-AKT-dependent mechanism. Phosphorylated FoxO3 a is sequestered by the 14-3-3 protein in the cytoplasm, and prevents it from translocating to nucleus where it can increase the protein expression FasL and Bim. The decrease of FasL and Bim expression reduces the activity of caspase-3, and ultimately exerts a protective effect that prevents osteoblasts from apoptosis.2. The mechanisms whereby attenuated ERK5 activity leads to osteoporosis appear to be involved: a, inactivation of ERK5 in mice bone tissue up-regulates RANKL/OPG rate; b, suppresses osteoblasts vitality by reducing Cyclin B1 and CDK1 expression; c, increases osteoblasts apoptosis due to up-regulation of FasL; d, augments adipocytes number. The above alterations eventually leads to osteopenia and more severe DIO.