The Mechanism Study Of Osteoporosis Treatment Through Regulating EIF2? Signaling Pathway

BackgroundOsteoporosis is a major skeletal disease with low bone mineral density,which leads to an increased risk of bone fracture.Bone remodeling is a continuous bone resorbing and rebuilding process,undertaken mainly by bone-resorbing osteoclasts and bone-forming osteoblasts.Endoplasmic reticulum(ER)stress in osteoblasts causes apoptosis in the development of osteoporosis.Disturbance to normal functions of the ER leads to an evolutionarily conserved stress response,the unfolded protein response,which is primarily aimed at damage compensating but may eventually trigger cell death if the dysfunction is severe or prolonged.However,upregulation of autophagy is an important mechanism by which cells remove unfolded or misfolded proteins and organelles.When endoplasmic reticulum stress activates,the unfolded protein response is not sufficient to maintain cell homeostasis,and autophagy is activated.PKR-like ER kinase(PERK)is a sensor of endoplasmic reticulum stress that stimulates eukaryotic translation initiation factor 2?(eIF2?)phosphorylation and raises ATF4 levels.The PP1 complex dephosphorylates p-eIF2?.ATF4 can induce autophagy and the expression of the proapoptotic factor CCAAT/enhancer binding protein homologous protein(CHOP).Salubrinal preserves eIF2? phosphorylation as a selective inhibitor of a phosphatase complex of eIF2?,PP1,providing the possibility of drug treatment for ER stress-induced diseases.However,the mechanism of salubrinal regulates osteoporosis by eIF2? signaling pathway remains to be elucidated.ObjectivesTo study the mechanism by which salubrinal regulates osteoporosis through regulating eIF2? signaling pathway,we used the unloading mouse model to detect the role of endoplasmic reticulum stress in the development of osteoporosis.To further clarify the mechanism,we used salubrinal as a tool to regulate the ER stress signaling pathway and explored the effect of regulating ER stress on unloading mouse.In addition,the ovariectomized(OVX)mice were used to investigate the effect of salubrinal on the pathogenesis of postmenopausal osteoporosis,the effect on the differentiation of bone marrow mesenchymal stem cells,and the regulation of ER stress and autophagy in osteoblasts by regulating eIF2? and regulation of nuclear factor of activated T-cells cytoplasmic 1(NFATc1)and Rac1 in osteoclasts.MethodsExperiment 1: In the first set of experiments,fifty-four mice were used to evaluate the role of ER stress in the development of unloading-induced osteoporosis.These mice were divided into nine groups: the age-matched control(AC;n = 6)and eight hindlimb unloading groups(HU,based on the duration for unloading such as: 3h,6h,12 h,1d,2d,3d,7d,and 14d;n = 6).At different time points,histological and bone marrow cell experiments were performed to examine the effects of unloading on osteoblast differentiation and osteoclast development.Western blot was performed to analyze the expression of endoplasmic reticulum stress-related proteins at different time points.Correlation analysis verified the association between endoplasmic reticulum stress and osteoblast differentiation and osteoclast development.In the second set of experiments,eighty-one mice were divided into three groups(n = 27 for each group): the age-matched control group(AC),hindlimb unloading group(HU),and salubrinal-treated hindlimb unloading group(US).All animals were sacrificed at 2 weeks.Bone mineral density(BMD),micro-computed tomography(?CT),and hematoxylin-eosin(H&E)staining were used to analyze changes in bone mass.Histology and bone marrow cell assays examined the effects of salubrinal on osteoblast differentiation and osteoclast development.Transmission electron microscopy,immunohistochemistry,TdT-mediated dUTP Nick-End Labeling(TUNEL)staining and Western blot were used to analys the mechanism of salubrinal regulation of endoplasmic reticulum stress to reduce bone loss.Experiment 2: One hundred and twenty-three C57BL/6 female mice were randomly divided into 3 groups,sham control group,ovariectomized group,and salubrinal-treated ovariectomized group(n = 41).At 4 weeks,after surgery,the OVX+Sal group received subcutaneous injection of salubrinal,mice were sacrificed at week 8.Transmission electron microscopy,Western blot and immunofluorescence analysis were performed to evaluate the changes of endoplasmic reticulum stress and autophagy in osteoblasts by salubrinal.The mechanism of salubrinal on osteoblasts and osteoclasts was examined using MC3T3-E1,RAW264.7 cell line and bone marrow cells.BMD,?CT and histological analysis were used to detect the effect of salubrinal on OVX mice.ResultsCompared to the age-matched control,unloaded mice reduced the B.Ar/T.Ar as well as the number of osteoblasts,and they increased the osteoclasts number on the trabecular bone surface in a time-dependent way.Unloading-induced disuse osteoporosis significantly increased the expression of Bip,p-eIF2? and ATF4 in short-term within 6h of tail suspension,but time-dependent decreased in HU 2 d to HU 14 d.Furthermore,a significant correlation of ER stress with the differentiation of osteoblasts and osteoclasts was observed.Administration of salubrinal suppressed the unloading-induced decrease in bone mineral density,B.Ar/T.Ar and mature osteoclast formation,migration and adhesion.Salubrinal also increased the colony-forming unit-fibroblasts and osteoblasts.Salubrinal increased the expression of Bip,p-eIF2? and ATF4,and the abnormal ER expansion was significantly improved.A TUNEL assay together with CHOP expression indicated that ER stress-induced osteoblast apoptosis was rescued by salubrinal.Collectively,the results support the notion that ER stress plays a key role in the pathogenesis of disuse osteoporosis,and salubrinal attenuates unloading-induced bone loss by altering proliferation and differentiation of osteoblasts and osteoclasts via eIF2? signaling.In the OVX model,salubrinal regulates the activation of endoplasmic reticulum stress and autophagy in osteoblasts in vivo,and promotes osteogenic differentiation by regulating the expression of LC3 p62 and Atg7 through eIF2? signaling pathway,and promoting the osteoblasts differentiation.Salubrinal promotes differentiation and mineralization of osteoblasts in vitro and in vivo,and inhibits the expression of NFATc1 and Rac1 GTPase in osteoclasts.Partial silencing of Rac1 by siRNA indicated that salubrinal inhibited mRNA levels of TRAP and cathepsin K.Salubrinal significantly inhibits the effects of osteoclast formation,migration and adhesion in vitro,while inhibiting the development of osteoclasts in vivo.BMD,?CT,H&E staining and calcein experiments showed that Salubrinal significantly improved OVX-induced reduction of bone mineral density and bone volume fraction.In conclusion,our results demonstrate the efficacy of osteoporosis treatment by regulating eIF2? signaling pathway.ConclusionsThis study demonstrates that endoplasmic reticulum stress plays an important role in the development of osteoporosis.Salubrinal significantly improves bone loss induced by unloading and OVX by regulating bone marrow cell differentiation.Salubrinal promotes osteoblast formation by regulating eIF2?-mediated endoplasmic reticulum stress-autophagy axis and inhibits osteoclast bone resorption by regulating the expression of NFATc1 and Rac1.