The Effect Of MTORC2 And Sema3A On Bone Metabolism

Objective:The purpose of our current study was to investigate the effect of mTORC2 signaling on skeleton in mice;and the associations between serum Sema3A and bone metabolism in women.Methods:Mice with conditionally deletion of rictor in osteoblasts(Rictor_ob^-/-)was used.Dual energy x-ray absorptiometry,micro-CT and histophomormetry analyses were employed to investigate changes in bone phenotypes.Three-point bending test was used to measure the biomechanical properties of the femur.Dynamic and static histomorphometric analyses,serum biomarkers of bone turnover,and mRNA changes of the bone tissues were measured to examine changes of bone turnover.Primary osteoblasts were isolated from mice with homogynous rictor flox and infected with adenovirus expressing cre recombinase to delete rictor,real-time PCR was used to detect the osteoblast differention,and western blot was explored to examine the changes in mTORC2 signaling activity.In order to investigate the association between serum Sema3A and bone metabolism,we enrolled a total of 1012 pre-and postmenopausal women.Questionnaires were filled in by all subjects to collect baseline data including history of osteoporotic fractures.Dual-energy X-ray absorptiometry was performed to determine the BMDs at the lumbar spine and femoral neck.Fasting serum were allocated and frozen in-80℃untile use.Serum Sema3A were detemined using ELISA kit.Osteocalcin and cross-linked C-telopeptide of type 1 collagen were measured by ECL.Results:We found that Rictor_ob^-/-mice showed a significant reduction in bone mineral density by dual energy x-ray absorptiometry.Micro-computed tomography analyses revealed a marked decline in femoral mid-shaft cortical bone mass,cortical area fraction and cortical thickness of the Rictor_ob^-/-mice;correspondingly,cortical bone porosity and marrow area unusually increased.While,the distal femoral trabecular bone mass,bone volume fraction,trabecular thickness,trabecular number and trabecular separation were similar between the two groups by micro-CT.Thinner trabeculae were found in the L4 vertebrae with relatively normal structural indices of trabecular numbers and separation by histomorphometric analysis.Furthermore,these changes were associated with significantly decreased bone biomechanical properties of the femur,with significantly reduced peak load and stiffness of the Rictor_ob^-/-mice.Dynamic and static histomorphometric analyses of the L4 vertebrae demonstrated reduced bone formation rate,mineral apposition rate,mineralization area per bone area and osteoid thickness with normal osteoblast area per bone area.Meanwhile,serum bone formation marker osteocalcin was also reduced in Rictor_ob^-/-mice.For bone resorption,although osteoclast number were comparable by histomorphpmetry at L4 vertebrae,reduced serum CTx as well as lowered mRNA expression of calcition receptor in calvaira and tibia of Rictor_ob^-/-mice were observed.Osteoblast differentiation was reduced,with a sharp decline in phosphoralation of Akt at S473,the well-characterized biomarker of mTORC2 activity,in primary cultures of osteoblasts that did not contain rictor.In our population study,a significant positive association between Sema3A and Ocn levels was demonstrated（r=0.077,P=0.025）when age was adjusted in postmenopausal women（n=860）.The serum Ocn level was significantly higher in the highest quartile of serum Sema3A as compared with the lowest quartile（21.09?0.56 ng/mL vs 19.45?0.44 ng/mL,P=0.018）.Serum Sema3A concentrations were similar in subjects with normal BMD,osteopenia or osteoporosis,and those with and without osteoporotic fractures.Multivariate stepwise regression analysis revealed that cross-linked C-telopeptide of type 1 collagen,body mass index,creatinine,Sema3A,L1–4 BMDs,and age were determinants of Ocn（adjusted R² for the model=0.532,P<0.001）.Conclusion:Using genetically modified mice,we find that deletion of rictor in mature osteoblasts induce a significant decrease in osteoblast and osteoclast activities.The net effect of these changes is a substantial impairment of cortical bone and a minor influence on trabecular bone.The mTORC2 signaling pathway presumably mediates these phenotypes.For the first time in humans,we provided clues to a possible link between serum Sema3A and the bone formation marker in postmenopausal women.However,serum Sema3A was in no association with bone resorption marker,BMD or osteoporotic fracture.