Rictor/mTORC2 Deficiency In Osteoclasts Increases Bone Mass By Inhibiting Osteoclasts Differentiation In Mice

1.BackgroundBones are constantly in the balance of bone resorption and bone formation,and once this balance is broken,it can lead to bone metabolic diseases such as osteoporosis and osteoarthritis and so on.Osteoclasts are giant multinucleated cells differentiated from mononuclear macrophages.It exerts unique bone resorption function by forming a special absorption ring structure.Studying the regulation mechanism of osteoclast proliferation,differentiation and function is of great significance for elucidating the pathogenesis of bone metabolic diseases and finding effective targets of drug.The mTOR is a conserved serine/threonine kinase.Signaling pathway of the mTOR is a key signaling pathway for the body to experience nutrition and stress signals and regulate the cell growth,proliferation and metabolism.The mTOR performs functions by interacting with other proteins in the cell to form two different complexes,including mTORC1 and mTORC2 whose specific component protein is Rictor.The mTORC2 can regulate actin cytoskeleton reorganization,cell survival and cell differentiation through downstream substrates such as Akt,and promote bone cell adhesion and survival so as to affect the bone metabolism.However,the role and mechanism of the Rictor/mTORC2 in osteoclast proliferation,differentiation and functional regulation have not yet been studied.In this study,we found that the Rictor/mTORC2 played an important role in the proliferation and differentiation of osteoclasts from the mice and the cells whose Rictor is deficient which provided a new viewpoint for the study of the mechanism of bone metabolism regulation.2.MethodsThe Rictor deficiency in osteoclasts of mice was confirmed through constructing the mouse models whose Rictor was deficient in osteoclasts and by genotype identification and protein level testing of immunohistochemistry,immunofluorescence and western blotting.The effect of Rictor deficiency in osteoclasts on bone masses and osteoclast number in mice was determined by detecting the growth and development of the mice,measuring the bone mineral density by microCT,observing the staining slids with histological experiment.The function of osteoblasts was detected by ELISA and calcein assay.The changes of the ultrastructures,especially the structures of the absorption rings,were observed by transmission electron microscopy.The induced osteoclast differentiation test was carried out to verify the in vivo experimental results.The effect of Rictor deficiency in osteoclasts on osteoclast mobilization and bone loss was detected by a low calcium diet model.3.Results1)During the process of osteoclast differentiation,the expression of Rictor was up-regulated.2)The mice of Rictor deficiency in osteoclasts were successfully constructed.3)Rictor deficiency in osteoclasts had no significant effect on the growth and development of mice.4)The microCT analysis results showed that the bone mineral density of the 20-week-old RictorKO mice increased compared with those control mice compared in the same litter.5)HE staining showed that the bone trabeculas of the RictorKO mice increased.6)Rictor deficiency in osteoclasts had no significant effect on the number of osteoblasts and bone formation in mice,but it could inhibit bone resorption.7)Analysis of the transmission electron microscopy showed that morphological structures of osteoclasts were not affected by Rictor deficiency.Trap staining and osteoclast counting showed that the number of osteoclasts in RictorKO mice decreased significantly.8)In vitro experiments showed that Rictor deficiency lead to the decreased ability of osteoclast differentiation and bone resorption.9)The number of osteoclasts of 6-week-old and 20-week-old RictorKO mice with low calcium for 1 or 4 weeks increased compared with that of the control group,and the bone loss was significantly reduced,which showed that the Rictor deficiency attenuated osteoclast mobilization and low calcium diet induced bone loss.4.Conclusion1)Rictor/mTORC2 in osteoclast is unnecessary for bone growth and development in mice.2)The number of osteoclasts and the ability of bone resorption decreases in the mice with the Rictor deficiency,but the bone formation ability is not affected,which eventually leads to an increase in bone mass.3)Rictor/mTORC2 plays an important role in the proliferation and differentiation of osteoclast precursors.4)Rictor/mTORC2 deficiency can resist osteoclast mobilization and bone loss induced by low calcium and thus,targeting Rictor/mTORC2 can be a novel strategy for the prevention and treatment of bone loss related diseases.