Overexpression Of Bmi1 In Mesenchymal Stem Cells Mediates Intracrine Actions Of PTHrP In Regulating Skeletal Growth And Development

Bmi1,a member of the Polycomb group family,can inhibit cyclin dependent kinase inhibitors including p16 and p19,and plays a key role in self-renewal and/or expansion of normal adult hematopoietic,neural,mammary epithelial,and bone marrow mesenchymal stem cells?BM-MSCs?.To determine whether overexpression of Bmi1 in mesenchymal stem cells can influence skeletal growth and development,we generated the mouse model with the overexpression of Bmi1in mesenchymal stem cells(Bmi1^Tg).To determine the effects of the overexpression of Bmi1 in mesenchymal stem cells on skeletal growth and development,we analyzed the phenotypes of Bmi1^Tgg mice compared with their wildtype littermates using X-ray,micro-CT and 3D reconstruction,histological,immunohistochemistry,Real-time RT-PCR,and Western blot.Results showed that the mRNA and protein expression of Bmi1 were significantly increased in diverse tissues of Bmi1^Tg mouse,especially BM-MSCs.Meanwhile,the habitus and body weight,the length of long bones,and the width of cartilage growth plate,BMD,BV/TV,trabecular and cortical bone volume and trabecular bone numbers,osteoblast numbers,ALP and type I collagen?COL-I?positive bone areas,BM-MSC CFU-f and ALP positive area were increased significantly in Bmi1^Tgg mice.Additionally,the mRNA expression of osteoblast related genes including Runx2,ALP,COL-I,and OCN was significantly increased in Bmi1^Tgg mice,while the osteoclastic bone absorption of Bmi1^Tg mice was significantly decreased compared to WT mice.These results indicated that Bmi1overexpression in mesenchymal stem cells promote the skeletal growth and development by stimulating the proliferation of BM-MSCs and differentiation into osteoblasts,and inhibiting osteoclastic bone absorption.Previous study has demonstrated that Bmi1 knockout mice exhibited severe bone growth defect and premature osteoporosis by inhibiting the proliferation of BM-MSCs and differentiation into osteoblasts.To determine whether Bmi1overexpression in mesenchymal stem cells can rescue the growth retardation and premature senescence caused by Bmi1 deficiency,we examined the effects of Bmi1overexpression in mesenchymal stem cells on the bone phenotypes of Bmi1^-/-mice byusingX-ray,Micro-CTand3Dreconstruction,histological,immunohistochemistry,Real-time RT-PCR,and Western blot.These results showed that the mean lifespan of Bmi1^TgBmi1^-/-mice was significantly extended compared with Bmi1^-/-mice.The habitus and body weight,the length of long bones,the width of cartilage growth plate and proliferation area,BMD,BV/TV,trabecular and cortical bone volume and the number of trabecular bone and osteoblast,ALP and type I collagen?COL-I?positive bone areas,BM-MSCs CFU-f and ALP positive area were significantly increased in Bmi1^TgBmi1^-/-mice compared to Bmi1^-/-mice.However,Bmi1^TgBmi1^-/-mice showed a significant decrease in the osteoclastic bone absorption compared to Bmi1^-/-mice.These results indicated that Bmi1overexpression in mesenchymal stem cells rescued the osteoporotic phenotypes of Bmi1^-/-mice,but also significantly prolonged their lifespan by stimulating the proliferation of BM-MSCs and differentiation into osteoblasts,and inhibiting osteoclastic bone absorption.We previously generated a parathyroid hormone related peptide?PTHrP?knock-In?PTHrP KI?mouse model by introducing a premature termination codon TGA in PTHrP in ES cells and that expresses PTHrP?1–84?,a truncated form of the protein that is missing NLS?the nuclear localization sequence?and the C-terminal region of the protein.PTHrP KI mice displayed early senescence,skeletal growth retardation and defective osteoblastic bone formation.However,the mechanism underlying the involvement of PTHrP NLS and C-terminus in skeletal development remains unclear.PTHrP KI mice exhibited a decline in the expression of Bmi1 and premature osteoporosis.We found that Bmi1 and PTHrP were colocalized to cellular nuclei and interacted physically.To determine whether Bmi1overexpression in mesenchymal stem cells can rescue the growth retardation and premature senescence caused by PTHrP KI,we crossed Bmi1^Tgg mice with PTHrP KI^+/-mice to generate Bmi1mesenchymal stem cells overexpression mice(Bmi1^TgKI),and compared them with PTHrP KI littermates by using X-ray,micro-CT and 3D reconstruction,histology,immunohistochemistry,Real-time RT-PCR,and Western blot.Results showed Bmi1^TgKI mice displayed an extended lifespan,an increase in body weight,and an improvement in skeletal growth and osteoblastic bone formation in the PTHrP KI mice,with these defects not completely rescued.The length of long bones,the width of cartilage growth plate and proliferation area,BMD,BV/TV,trabecular and cortical bone volume and the number of trabecular bone and osteoblast,ALP and type I collagen?COL-I?positive bone areas,BM-MSC CFU-f and ALP positive area were significantly increased in Bmi1^TgKI mice compared to PTHrP KI mice.The expression of osteoblast related genes including Runx2,ALP,COL-I and OCN and anti-oxidative enzymes including SOD1 and 2,catalase,glutathione reductase and glutathione peroxidase 1 was up-regulated in bony tissues of the Bmi1^TgKI mice compared to PTHrP KI mice.However,the osteoclastic bone absorption,RANKL/OPG,the mRNA expression of osteoclast related genes TRAP and RANKL,and the expression of tumor suppressor genes like p16,p19,p53,and p21 was decreased in bony tissues of the Bmi1^TgKI mice compared to PTHrP KI mice.In addition,the protein expression of proliferating cell nuclear antigen?PCNA?and anti apoptotic protein Bcl-2 was dramatically increased in bony tissue of Bmi1^TgKI mice compared to PTHrP KI littermates.Taken together,these results demonstrated that the overexpression of Bmi1 in BM-MSCs partly rescued the defects in skeletal growth and osteogenesis in the PTHrP KI mice by inhibiting oxidative stress and inactivating p16 and p19 signaling.Therefore,these studies indicated that the PTHrP-Bmi1 signaling axis might play an essential role in the regulation of skeletal growth and development.Our study firstly established the mouse model of Bmi1 overexpression in mesenchymal stem cells,which is an important animal model for studying the mechanism of Bmi1 in the regulation of skeletal growth.Bmi1 Overexpression in mesenchymal stem cells promoted the skeletal growth and development by stimulating the proliferation of BM-MSCs and differentiation into osteoblasts,and inhibiting osteoclastic bone absorption.These results demonstrated that Bmi1overexpression in BM-MSCs partly rescued the defects in skeletal growth and osteogenesis in PTHrP KI mice by inhibiting oxidative stress and inactivating p16and p19 signaling.Therefore these studies indicated that the PTHrP-Bmi1 signaling axis might play an essential role in regulating skeletal growth and development.This study might provide an experimental and theoretical evidence for the application of PTHrP NLS and the C-terminus in the prevention and treatment of osteoporosis.