The Mechanism Of Deptor Controls The Mesenchymal Stem Cells Differentiation And Facilitates The Process Of Osteoporosis

BackgroudOsteoporosis is one of the most serious skeletal diseases in the postmenopausal women and elderly population.It is characterized by the imbalance between bone formation and bone resorption,resulting in low bone density and high risk of fracture.Fat accumulation in the bone marrow occurs in osteoporosis patients.Previous studies showed that Deptor inhibits osteogenic differentiation while promotes adipogenic differentiation of BMSCs.However,the mechanism of Deptor controls the balance between adipogenic and osteogenic differentiation is unlcear.ObjectiveTo investgate the mechanism of Deptor controls the balance between adipogenic and osteogenic differentiation in BMSCs.MethodsThe model of osteoporosis induced by OVX was established using C57BL/6 mice.Micro-CT and H&E staining were used to detect the rate of bone formation and fat accumulation.Constructed mice with Deptor specific knockout in osx using cre-loxp system followed by OVX surgery.The influence of Deptor kncokout were analysed by Micro-CT,H&E staining,immunochemistry and calcein double-labeling.In vitro,qPCR、Western blot were utilized to evaluate the level of Deptor during adipogenic and osteogenic differentiation.BMSCs were co-cultured with Deptor plasmids and Deptor siRNA plasmids respectively,then oeteogenic and adipogenic differentiation for 5days.Analyse the effect of Deptor on BMSCs differentiation with qPCR、Western blot and Luciferase reporter system.Screened the Deptor interacting proteins with Proteomicanalysis.Certified the interaction between TAZ and Deptor by immunoprecipitation.PDZ domain plasmids、DEPdomain plasmids、full length Deptor plasmids co-cultrued with TAZ plasmids and TAZ lack of PDZ-binding motif plasmids in HEK293T cells respectively.Identify the binding sites between Deptor and TAZ by immunoprecipitation.To explore whether Deptor controls BMSCs differentiation via inhibits transactivation properties of TAZ.Transfected TAZ-GST+Deptor plasmids and Deptor plasmids with C3H10T1/2 respectively,then oeteogenic and adipogenic differentiation for 3days.qPCR,western blot,ALP staining,and oil red staining were used to detect whether the increased of TZA reverse the effect of Deptor on BMSCs differentiation.ResultsBone loss and fat accumulation were observed in C57BL/6 OVX mices.More bone trabecula and and less adipocytes were formed in Deptor knockout mice after OVX surgery.Higher level of Deptor were expressed during osteogenic differentiation,while reduced Deptor accured during adipogenic differentiation.Deptor did not directly targets Runx2 and PPARy to control the osteogenic and adipogenic differentiation of BMSCs.PDZ domain of Deptor directly targeted PDZ binding motif of TAZ.Deptor inhibited TAZ transactivation properties of TAZ,which upregulated PPARy transcription activity and downregulated Runx2 transcription activity.Conclusion1.Deptor is upregulated during the adipogenic differentiation while downregulated in osteogenic differentiation of BMSCs 2.OVX BMSCs show elevated Deptor expression 3.Deptor knockout alleviates bone loss and marrow fat accumulation in OVX mice 4.Deptor impairs osteogenic differentiation while facilitates adipogenic differentiation of BMSCs 5.Deptor inhibits Runx2 while promotes PPARy transcription activity in BMSCs 6.Deptor directly targets TAZ 7.Deptor controls BMSCs differentiation by inhibiting transactivation properties of TAZ.