MTOR/Raptor-S6k1 Signaling Pathway Promote Skeletogenesis And Bone Development Via Runx2 In Mice

Osteoblasts are the bone-making cells during bone development and bone remodeling.Osteoblasts are deprived from bone marrow stromal cells(BMSCs),which are multi potent progenitor cells and can differentiate into a number of lineages including bone,cartilage and adipocyte.The differention of osteoblasts are co-regulated by several extracellular signals.mTOR signals integrating many extracellular signals,is important for differention of many cell types.But it is unclear that what is the role of mTOR signaling pathway in osteoblast and bone development.The present study aim at illuminating the function and molecule mechanism of mTOR signaling pathway in osteoblast and bone development in mice.Results: 1.mTOROsx mice exhibited dwarfism,shrinked appendicular skeleton,abnormal shape and size of clavicle and craniofenestria.2.Micro-CT determined osteoporosis in femur of mTOROsx mice 3.RapOsx mice had dwarfism phenotype,shrinked appendicular skeleton,abnormal shape and size of clavicle and craniofenestria.4.Osteoporosis was observed in femur of RapOsx mice.5.MAR and BFR decreased in RapOsx mice compared with WT mice.6.There was a smaller number of mature osteoblasts in RapOsx mice.7.Deletion of Raptor with Ad-CRE impaired osteogenenic differention and Runx2 expression in Rapfl/fl BMSCs in vitro.8.There were less mature osteoblasts in RapOsx mice when compared with WT mice.9.Calvarial cells from P5 mTOROsx mice exhibited decreased osteogenic differention when compared with WT mice.10.Bone resorption decreased in both mTOROsx and RapOsx mice.11.The phosphorylation level of S6K1 and S6 decreased due to deletion of Raptor both in vivo and in vitro.12.The phosphorylation level of S6K1 and S6 decreased due to deletion of mTOR both in vivo and in vitro.13.CAS6K1 rescued impaired osteogenic differention in calvarial cells from P5 RapOsx mice.14.CAS6K1 promoted Runx2 expression in in calvarial cells from P5 RapOsx mice.15.Runx2 and its downstream Col1,Ocn decreased in both mTOROsx and RapOsx mice.16.Runx2 rescued impaired osteogenic differention in calvarial cells from RapOsx mice.17.CAS6K1 promote the activity of Runx2 enhancer but not its promoter.18.CASK1 promote DLX5-drived Runx2 enhancer activity,but they do not interact with each other.19.CASK1 promote ER?-drived Runx2 enhancer activity.20.ER?promote DLX5-drived Runx2 enhancer activity and ER?interact with DLX5.21.CAS6K1 promote ER?and DLX5 drived Runx2 enhancer activity.Conclsion: 1.mTOR deletion in preosteoblasts induced dwarfism,cleidocranial dysplasia and osteoporosis in mice.2.Raptor deletion in preosteoblasts replicate all most all the phenotype of mTOR deletion,which indicate that mTOR mainly function inmTOR/Raptor complex in osteoblast.3.Impaired osteogenic differerntion and skeletogenesis were the main reason of bone defects in mTOROsx and RapOsx mice.4.S6K1 regulate the expression of Runx2 though its enhancer.