Role And Mechanism Of Sirt1 Overexpression In Mesenchymal Stem Cells In Stimulating Skeletal Growth And Development

Sirt1,a family member of histone deactylase?,is homology with silent information regulator 2?Sir2?.It can not only catalyze histones but also plays an important role in deactyling non-histone transcription factor substrates,which are involved in a variety of signaling pathways,such as FoxOs,p53,NF kappa B,and c-myc protein associated with the neuroprotective mechanism,cell senescence,apoptosis,metabolism,inflammation,cancer,and oxidative stress.Previous studies indicate that the specific knockdown of Sirt1 in mesenchymal stem cells inhibited the proliferation and differentiation of bone marrow mesenchymal stem cells and reduced bone volume.However,the effect of Sirt1 overexpression in mesenchymal stem cells on bone formation still remains unclear.To determine the effect of Sirt1 on skeletal development and osteogenesis,we firstly constructed a mouse model with the overexpression of Sirt1 in mesenchymal stem cells using transgenetic engineering.The expression of Sirt1 in bone tissues was significantly higher in transgenic mice than in wild-type controls.Bone phenotypes of Sirt1^TGG mice were compared with their wild-type littermates.To assess the effect of the overexpression of Sirt1 in mesenchymal stem cells on bone formation and osteogenesis,bone phenotypes of 4-week-old WT and Sirt1^TGG mice were analyzed by X-ray photography,micro CT,three-dimensional reconstruction,histology,and immunohistochemistry.These results showed that compared with WT littermates,the size of long bone,cartilage growth plate width,the secretion of collagen type II,bone volume,osteoblasts,alkaline phosphatase?ALP?and type I collagen?Col I?-positive area,osteogenesis and osteogenesis-related gene expression,including Runx2,ALP and Col I and Osteocalcin?OCN?and Wnt3a-LRP5 pathway related gene significantly increased in the Sirt1^TGmice,with tartrate-resistant acid phosphatase?TRAP?positive osteoclast markedly reduced.These results demonstrated that the overexpression of Sirt1 in mesenchymal stem cells increased bone volume by promoting cartilage bone growth and inhibiting bone resorption.To examine whether the overexpression of Sirt1 can promote osteogenesis by enhancing BM-MSC proliferation and osteogenic differentiation,we cultured bone marrow cells in vitro,and examined the proliferation and differentiation of BM-MSC using immunochemistry.These findings revealed that the percentage of CFU-F area,and ALP-positive CFU-F area in SIRT1 transgenic mice significantly increased compared with WT mice.These results demonstrated that overexpression of Sirt1could promote the proliferation and osteogenic differentiation of BM-MSC.Resveratrol,a Sirt1 activator,can scavenge oxygen free radicals and inhibit its production.Thus the overexpression of Sirt1 in mesenchymal stem cells might promote skeletal development by inhibiting oxidative stress.The levels of oxidative stress in bone marrow cells and bone tissues were analyzed in Sirt1^TGmice and their wild-type mice.The results showed that Sirt1^TGmice displayed a significantly higher level of reactive oxygen species?ROS?than wild-type mice.Moreover,the expression of antioxidant enzyme genes including SOD1,SOD2,Gpx1,GSR,TxN,and the expression of SOD1,SOD2,Prdx1,Prdx4 were significantly elevated in Sirt1^TGmice compared with WT mice.These results indicated that the overexpression of Sirt1 could promote skeletal development by inhibiting oxidative stress.Our previous study found that Bmi-1?B cell lymphoma viral insertion site 1?knockout mice exhibited premature aging and osteoporosis,and the expression SIRT1in bone tissues was remarkably decreased.To investigate whether the overexpression of Sirt1 could rescue the premature aging and osteoporosis in Bmi-1 deficient mice,we established the mesenchymal stem cells overexpressing Sirt1 in Bmi-1 knockout mice.These results showed that compared with Bmi-1^-/-mice,the lifespan of Sirt1^TGBmi-1^-/-mice was significantly prolonged and the weight markedly raised.These results indicated that the overexpression of Sirt1 could rescue the premature aging of Bmi-1^-/-mice.To identify whether the overexpression of Sirt1 in mesenchymal stem cells could rescue skeletal growth retardation and premature osteoporosis in Bmi-1deficient mice,skeletal phenotypes of Bmi-1^-/-mice age 4 weeks were analyzed using histology,histochemistry,immunohistochemistry,and Real-time PCR.These results revealed that the size of long bone,cartilage growth plate width,the secretion of collagen type II,bone volume,bone cells,alkaline phosphatase?ALP?,the percentage of type I collagen?Col I?positive-area and osteogenesis-related gene expression including Runx2,ALP and Col I,Osteocalcin?OCN?and Wnt3a-LRP5 pathway-related genes was significantly elevated in the Sirt1^TGBmi-1^-/-mice,with TRAP positive osteoclasts significantly declined.These findings demonstrated that the overexpression of Sirt1 could prevent bone growth retardation and osteoporosis in Bmi-1 deficient mice.To determine whether Sirt1 overexpression in mesenchymal stem cells can prevent premature osteoporosis in Bmi-1 deficient mice by inhibiting oxidative stress,we analyzed ROS levels by using flow cytometry,and examined the expression of cyclins and protein kinase inhibitors in bone marrow cell and bone tissue in the four indicated mice using Real time PCR and Western blot.These results showed that compared with WT mice,Bmi-1^-/-mice exhibited a significantly higher level of reactive oxygen species?ROS?,and an increase in the expression of antioxidant enzyme genes including SOD1,SOD2,FOXO3a,Gpx1,GSR and TxN and in the expression of antioxidase proteins including SOD1,SOD2,FOXO3a expression,with the expression of CDKI,p53 and p21 significantly decreased.These results showed that the overexpression of Sirt1 in mesenchymal stem cells promote the antioxidant capacity of skeleton,inhibit oxidative stress,and prevent skeletal aging,thus playing a key role in skeletal growth and development.These results demonstrated that the overexpression of Sirt1 in mesenchymal stem cells might promote the proliferation of BM-MSC,enhance the development of cartilage and inhibit bone turnover.Additionally,we found that the overexpression of Sirt1 inhibited oxidative stress,and prevented the premature aging and osteoporosis in Bmi-1 deficient mice.This study not only investigate the mechanism underlying the involvement of Sirt1 in skeletal development,but might provide theoretical and experimental basis for Sirt1 activation in the prevention and treatment of osteoporosis.