The Role And Mechanism Of Osteoblastic MTORC1 Activation In Inhibiting Age-related Bone Loss

Age-related osteoporosis(senile osteoporosis),one of the most frequent degenerative diseases,has become a hot topic in public health.Increasing age,which influences osteoblast proliferation and differentiation,is associated with a progressive reduction in bone formation and abnormal bone homeostasis.The resultant progressive decline in bone mass may result in osteoporotic fracture,with high incidence of disability and high mortality.Although bone growth ceases once bone mass peaks in adult,bone tissues maintain its metabolic activity and undergo remodelingcontinuously.As age increases,reduction in bone formation mediated by osteoblasts finally results in imbalance between bone formation and resorption,leading to consequent bone loss.Osteoblasts produce ossein and bone matrix during skeletal development.Also,osteoblasts secrete alkaline phosphatase(ALP),osteocalcin(OCN)and osteoprotegerin(OPG)that play important roles in the regulation of bone homeostasis.Increasing evidences link the reduction in bone formation to decrease in osteoblastic differentiation,but increase in adipocyte differentiation of bone marrow mesenchymal stem cells(BMMSCs),reduction in proliferation of osteoblasts,as well as shortened lifespan of osteoblasts.Therefore,it is crucial to explore novel stategiesto promoteosteoblast differentiation and proliferation and to increase the lifespan of osteoblasts,thus reinforcing bone formation in aged people.The mammalian target of rapamycin(mTOR)pathway is the central signaling that regulate both intracellular and extracellular metabolism.mTOR complex 1(mTORC1)modulates cell proliferation,differentiation,apoptosis,and autophagy.However,the role of osteoblastic mTORCl in bone remodeling,especially in bone formation,has not been elucidated in aged people,and the underlying mechanism remains unknown.Objective:(1)to explore the impact of inducible activation of mTORCl specifically in osteoblastson aging mice.(2)Tounravel the mechanism by which osteoblastic mTORCl regulates aged-related bone loss.Methods:inducible osteoblast-specific TSC1 knockout mice were constructed and designated TSC1 OBKO,whereas their littermates were designated control.TSC1ablation specifically in osteoblasts were induced by injecting tamoxifen(once/3 months)afterbone mass peaked at 6 months after birth.micro-CTwas used to analyze bone mass after these mice were sarcrificed at 12 months.Paraffin sections were produced and the relative osteoblastic proliferation,apoptosis and differentiation were analyzed by BrdU staining,Tunel staining and immunohistochemical(IHC)staining,respectively.Next,the role and mechanism of mTORCl in osteoblastic differentiationwere investigated in pre-osteoblast cell line MC3T3-E1 subjected to either rapamycin treatment or TSC1 knockdownmediated by lentivirus,using Alizarin Red S staining,Western Blot,and immunoprecipitation.Results:(1)in vivo experiments:A.Cortical thickness(*P<0.05)and cancellous BMD(**P<0.01)of the femur increased significantlyin TSC1OBKO mice.B.Osteoblast proliferation was enhanced,whereas their apoptosis was reduced in the femoral cancellous bone of TSC1 OBKO mice.C.An increased number of OSX+and OCN+ osteoblasts were observed in cancellous bone sections from femur ofTSC1 OBKO mice by IHC staining(P<0.05).D.A decreased number of osteoclasts were detected by TRAP staining(P<0.01).E.While Runx2 was hard to be detected in osteoblasts of both control and TSC1 OBKO mice,the expression of PAX5,another key transcriptional factor in osteoblastic differentiation,was markedly enhanced in osteoblasts ofTSC1 OBKO mice.(2)Invitro experiments:A.While osteoblastic differentiation of MC3T3-E1 cellswas suppressed by rapamycin,aspecific mTORC1 inhibitor,in a dose-dependent manner,it was enhanced by mTORC1 activation conducted by TSC1 depletion.B.The expression of PAX5 was continuously enhanced during osteoblastic differentiation of MC3T3-E1 cells.C.While the expression of PAX5 was dramatically suppressed by rapamycin treatment,it was markedly enhanced by TSC1 depletion.D.PAX5 interacted with mTOR.Conclusions:(1)Activation of osteoblastic mTORC1 can markedly suppress aged-related bone loss in aging mice.(2)Activation of osteoblastic mTORC1 during aging promotes bone formation through increasing the proliferation and differentiation of osteoblasts while reducing their apoptosis.(3)PAX5 may be involved in osteoblastic differentiation promoted by mTORC1 activation.