Overexpression Of Bmi-1 In Lymphocytes Stimulates Osteogenesis By Improving Osteogenetic Microenvironment

Bmi1 is a member of the PcG?Polycomb group?family.Bmi1 plays a key role in self-renewal and/or expansion of normal adult hematopoietic,neural,mammary epithelial,intestinal and bone marrow mesenchymal stem cells?BM-MSC?.Bmi1gene knockout homozygous mice exhibited neurological abnormalities,severe hematopoietic defects,bone growth disorder and premature osteoporosis.Previous study has demonstrated that overexpression of Bmi1 in lymphocytes results in transformation of axial skeleton,however,it is unknown whether overexpression of Bmi1 in lymphocytes can stimulate osteogenesis by improving osteogenetic microenvironment.To determine whether overexpression of Bmi1 in lymphocytes can stimulate osteogenesis by improving osteogenetic microenvironment,maintain the self-renewal capacity of the bone marrow mesenchymal stem cells and promote their differentiation into osteoblasts.We compared the skeletal phenotype of the eight-week-old wild-type?WT?and E?Bmi1 transgenic mice.We first confirmed a high expression level of Bmi1 gene and protein in thymus,spleen and bone marrow in E?Bmi1 transgenic mice using real-time quantitative RT-PCR and western blot.The size of the body,thymus and spleen and the body weight were increased in E?Bmi1 transgenic mice compared to the WT littermates.To investigate the effect of the overexpression of Bmi1 in lymphocytes in osteogenesis and bone formation,we used radiography,micro-CT scanning and three-dimensional reconstruction,histological,histochemical and immunohisto-chemical methods to analysis the bone phenotype differences of the 8-week-old WT and E?Bmi1transgenic mice.The results showed that:the length of the long bones and vertebrae,width of the growth plate,PCNA?Proliferating cell nuclear antigen?-positive chondrocytes,bone mineral density?BMD?,trabecular bone volume,osteoblasts number,alkaline phosphatase?ALP?and type I collagen?COL-I?positive areas increaesd dramatically in E?Bmi1 transgenic mouse compared to the WT littermates.Consistent with the morphological changes,the bony tissue expression levels of osteoblast-related genes including Runx2,ALP,Col-I and osteocalcin?OCN?mRNA were increased significantly in E?Bmi1 transgenic mice compared to WT mice.However,the tartrate-resistant acid phosphatase?TRAP?-positive osteoclast surface were also increased significantly in E?Bmi1 transgenic mice compared to WT mice.Above results showed that overexpression of Bmi1 in lymphocytes enhanced the bone turnover and promoted the endochondral bone formation.To determine whether overexpression of Bmi1 in lymphocytes can stimulated the osteogenesis by promoting the proliferation and osteoblastic differentiation of the bone marrow mesenchymal stem cells?BM-MSCs?,we cultured the bone marrow cells and compared the differences of the proliferation and osteoblastic differentiation capacity of the BM-MSCs in WT and E?Bmi1 transgenic mice using cytochemistry staining and image analysis.The results showed that:The numbers of the total colony-forming unit-fibroblastic?CFU-f?and ALP positive CFU-f?CFU-fap?generated by bone marrow cell cultures were increased significantly in E?Bmi1transgenic mice and were similarly increased in WT mouse-derived bone marrow cell co-cultures with the conditioned medium from bone marrow cells or spleen lymphocyte cultures of E?Bmi1 transgenic mice.These results indicated that overexpression of Bmi1 in lymphocytes can promote BM-MSC proliferation and osteoblast differentiation,and this role may also be related to other factors which lymphocytes secrete.To identify which factors secreted by Bmi1 overexpression lymphocytes can stimulate the osteoblastic bone formation and the osteoclastic bone resorption,we examined the serum protein expression differences in 8-week-old WT and E?Bmi1mice using protein assay analysis.The results showed that:the serum expression level of interleukin-15?IL-15?,follistatin-like protein 3?FLRG or FSTL3?and insulin-like growth factor binding protein?IGFBP-7?in E?Bmi1 transgenic were increased 7.2,7.6 and 3.5 times compared with WT mice.The effects of these proteins in osteoblastic bone formation and osteoclastic bone resorption still to be discussed.Previous studies showed that Bmi1 deficiency lead increasing oxidative stress,however,whether overexpression of Bmi1 in lymphocytes can promote the growth and development of mice by inhibiting the oxidative stress still unknown.To answer this question,we examined the changes of oxidative stress related factor in 8weeks-old E?Bmi1 transgenic mice and WT littermates.The results showed that:ROS?reactive oxygen species?level in bone marrow,thymus,spleen and kidney cells of E?Bmi1 transgenic mouse significantly reduced compared to WT mouse,detected by DCF-DA incorporation and flow cytometry.We also examined the activity of the superoxide dismutase?SOD?,total antioxidant capacity?T-AOC?levels and malondialdehyde?MDA?levels,results showed that SOD activity and T-AOC levels in bone marrow,thymus,spleen,kidney cells and blood plasma were significantly increased in E?Bmi1 transgenic mice compared to WT mice,while malondialdehyde?MDA?levels,the end products of the lipid oxidative reaction,were significantly decreased.These results indicated that overexpression of Bmi1 in lymphocytes can promote the growth and development of mice by increasing the antioxidant capacity and inhibiting the oxidative stress in multiple organs.Our previous study had demonstrated that,in PTHrP1-84 knock-in?Pthrp KI?mice,which are missing the nuclear localization sequence?NLS?and the C-terminal region of PTHrP,expression of the polycomb protein Bmi1 is reduced and potentially can mediate its more seriously premature osteoporosis and premature aging phenotypes than Bmi1 deficiency mice.To determine whether overexpression of Bmi1 in lymphocytes can correct or improve the premature aging and osteoporosis phenotype of the Pthrp KI mice,we have therefore crossed E?Bmi1 transgenic mice with Pthrp KI^+/-mice to generate a Bmi1 lymphocytes overexpressed Pthrp KI mice?E?KI mice?and compared them with Pthrp KI and WT littermates.Results showed that overexpression of Bmi1 in lymphocytes extended the average lifespan of Pthrp KI mice.Compared with the Pthrp KI mice,body weight,size and weight of the thymus and spleen increased significantly in E?KI mice,although E?KI mice were not normalized.These results indicated that overexpression of Bmi1 in lymphocytes can prolong the lifespan of Pthrp KI mice and improve their growth and development.To determine whether overexpression of Bmi1 in lymphocytes can correct or improve the skeletal growth retardation and osteoporosis phenotype of the Pthrp KI mice,we used Imaging,histological,histochemical,immunohistochemical and Real-time quantitative RT-PCR methods to analysis the bone phenotype differences of the 2-week-old four genotype mice.Results showed that:the length of the long bones,width of the growth plate,PCNA-positive chondrocytes,BMD,trabecular bone volume,osteoblasts number,ALP,TRAP and COL-I positive areas increaesd dramatically in E?KI mice compared to the Pthrp KI littermates.Consistent with the morphological changes,the bony tissue expression levels of osteoblast-related genes including Runx2,ALP,Col-I and OCN mRNA were increased significantly in E?KI mice compared to Pthrp KI littermates,although E?KI mice were not normalized.These results indicated that overexpression of Bmi1 in lymphocytes can improve the skeletal growth retardation and osteoporosis phenotype of the Pthrp KI mice.To determine whether overexpression of Bmi1 in lymphocytes improved the premature osteoporosis phenotype of the Pthrp KI mice by inhibited their oxidative stress,we used 2,7-dichlor odihydrofluorescein diacetate?DCF-DA?incorporation and flow cytometry analysis,Real-time quantitative RT-PCR and Western blot methods to examine the ROS levels of bone marrow,spleen and thymus cells and antioxidant enzyme gene and protein expression levels in bony tissue of four genotypes mice.Results showed that:ROS levels of bone marrow,spleen and thymus cells were decreased,expression of antioxidant enzyme gene including including SOD1,2 and 3,Glutathione reductase?GSR?and glutathione peroxidase 1?GPX1?and antioxidant protein including SOD1 and Sirt1 were dramatically increased in bony tissue of E?KI mice compared to Pthrp KI littermates.These results indicated that overexpression of Bmi1 in lymphocytes can inhibited the increasing of the oxidative stress in Pthrp KI mice.To further determine whether overexpression of Bmi1 in lymphocytes also can inhibite the DNA damage response in Pthrp KI mice,changes of DNA damage response related index in bony tissue of four genotypes mice were demonstrated by immunohistochemical and Western blot analysis.Results showed that:?H2AX-positive cells,expression levels of?H2AX,p16 and p53 protein were dramatically decreased in E?KI mice compared to Pthrp KI littermate,while,expression level of Bmi1 protein was increased.These results indicated that overexpression of Bmi1 in lymphocytes also can inhibited the increasing DNA damage response occured in Pthrp KI mice.This study for the first time demonstrated that overexpression of Bmi1 in lymphocytes can stimulate osteogenesis by stimulating the proliferation of BM-MSCs and promoting their osteoblastic differentiation.We also confirmed that over-expression of Bmi1 in lymphocytes can partially correct the premature aging phenotype and premature osteoporosis of the Pthrp KI mice by inhibiting its oxidative stress and DNA damage response.This study demonstrated that overexpression of Bmi1 in lymphocytes can increase the osteogenesis by improving the osteogenesis microenvironment and stimulating the BM-MSCs'proliferation and osteoblastic differetiation.The results of this study provide an experimental and theoretical basis for a new strategy for prevention and treatment of osteoporosis by improving the osteogenesis microenvironment.