Bmi1 Plays A Criticle In P27 Defeciency Induced Activation Of Shh Signal Pathway

To determine the effect of p27 defeciency on skeletal phenotypes and development,we compared the difference between the wild-type and p27-/-mice at 4 weeks of age by using morphological,cellular and molecular techniques.The length of long bones was longer,the bone mineral density was increased in p27-/-mice demonstrated by radiography,micro-CT scanning and 3D reconstruction.The trabecular bone volume,osteoblast number,positive area of alkaline phosphatase(ALP)and type I collagen were all increased significantly,the number of adipocytes within bone marrow was reduced in 4-weeks-old p27-/-mice.Consistent with the morphological alterations,the mRNA expression levels of Runx2,ALP,type I collagen and osteocalcin(OCN)were up-regulated in the bone tissue derived from p27-/-mice.The tartrate-resistant acid phosphatase(TRAP)positive osteoclast number was decreased in the p27-/-mice compared to their WT littermates,but not quite significant.And the gene expression levels of both RANKL and osteoprotegerin(OPG)were similar to the morphological alterations in the bone tissue derived from p27-/-mice detected by real-time RT-PCR.To determine the mechanism of p27 defeciency promoted osteogenesis,microarray analyses of differential gene expression profiles were performed in long bone extracts from p27-/-mice and their wild-type littermates.Results revealed that the expression levels of molecules in the sonic hedgehog(Shh)signaling pathway,including Shh,Smoothened(Smo),Gli 1/2/3 and Bmi1 were up-regulated,whereas suppressor of fused(Sufu),a negative regulator of this pathway was down-regulated in p27-/-mice.Their up-regulation or down-regulation at mRNA and protein levels was confirmed by real-time RT-PCR and Western blots.We then employed Shh antagonist KAAD-Cyclopamine to treat BM-MSCs derived from wild-type or p27-/-mice,respectively.Results showed that the forming efficiency of the colony-forming unit-fibroblastic(CFU-f)and alkaline phosphatase positive CFU-f(CFU-fap)given rise by KAAD-Cyclopamine-treated BM-MSCs derived from wild-type or p27-/-mice were decreased significantly compared to control group,respectively.To confirm above results in vivo,we treated WT and p27-/-mice with Shh antagonist Vismodegib,then we detected the change after the treatment.We confirmed Shh signaling pathway was inhibited after the treatment,and the mRNA expression levels of Runx2,ALP,type I collagen and osteocalcin(OCN)were down-regulated in Vismodegib treated mice.The bone mineral density was decreased in Vismodegib treated mice demonstrated by radiography,micro-CT scanning and 3D reconstruction.The trabecular bone volume,osteoblast number,positive area of alkaline phosphatase(ALP)and type I collagen were all increased significantly,the number of adipocytes within bone marrow was also reduced in Vismodegib treated micePrevious studies determined the promoter region of Shh gene,and located this region in 701 bp(-550～+151 bp).To determine the mechanism of p27 regulates Shh signal pathway,we adopted bioinformatic approaches and found a consensus E2F4 binding site(-81～-100 bp)in the promoter region of shh gene.Results from ChIP assay and EMSA Luciferase assay indicated that transcription factor E2F4 could bind to Shh promoter.Luciferase assay and western blot results showed that over-expression of E2F4 gene inhibited shh promoter activity and protein levels,and the change vice versa.Sufu is an important suppressor of Shh signal pathway.To determine whether p27 can affect stability of Sufu protein to regulate the Shh signal pathway,we used the protein turn over experiments to detected,and results showed that Sufu degraded rapidly in p27-/-mice cells,and over-expression can decreases the rate of Sufu degradation in the cells.To determine whether Bmil plays a criticle role in p27 defeciency induced Shh activation on promoting osteogenesis,we generated Bmi1-/-p27-/-double knockout mice and compared them with p27-/-,Bmi1-/-,and wild-type littermates.Body size,body weight,bone mineral density,trabecular bone volume,osteoblast numbers,ALP and type I collagen positive areas were increased significantly in p27-/-mice,but decreased significantly in Bmi1-/-mice compared to wild-type mice.The double mutant Bmi1-/-p27-/-mice however closely resembled the phenotype of the mice,including growth retardation and premature osteoporosis.Consequently,we asked whether the signaling pathway activated by p27 deletion and leading to increased skeletal size could be blocked by the deletion of Bmi-1.To examine this question,microarray analyses of differential gene expression profiles were performed in long bone extracts from p27-/-mice and their wild-type littermates.Results revealed that the expression levels of molecules in the sonic hedgehog(Shh)signaling pathway,including Shh,Smoothened(Smo),Glil/2/3 and Bmi1 were up-regulated,whereas suppressor of fused(Sufu)a negative regulator of this pathway was down-regulated in p27-/-mice.Their up-regulation or down-regulation at mRNA and protein levels was confirmed by real-time RT-PCR and Western blots.We then employed Shh-N and the Shh antagonist KAAD-Cyclopamine to treat BM-MSCs derived from wild-type,p27-/-,Bmi1-/-or Bmi1-/-p27-/-mice,respectively.Results showed that cell proliferation was accelerated in a dose dependent manner in Shh-N-treated BM-MSCs derived from wild-type or p27-/-mice,but not in those derived from Bmi1-/-or Bmi1-/-p27-/-mice.In contrast,cell proliferation was inhibited in a dose dependent manner in KAAD-Cyclopamine-treated BM-MSCs derived from wild-type or p27-/-mice,but not in those derived from Bmi1-/-or Bmi1-/-p27-/-mice.To determine whether Bmi1 deficiency could inhibit hedgehog activated osteosarcoma growth,we created Saos2、MG63 and U20S,three stable Bmil shRNA transfected osteosarcoma cell lines.Then we used single cell clone and wound healing test to examie the proliferation and migration ability of Bmil shRNA transfectd osteosarcoma cell lines.The protein expression levels of p27 and Caspase3,were up-regulated,Shh were down-regulated.To observe tumor formation after Bmi1 knockown,vector or stable Bmi1 shRNA transfected osteosarcoma cell lines were injected in armpit of 6 week-old nude mice,we find that Bmi1 knockdown could inhibit the tumor size in the three osteosarcoma cell lines,and immunohistochemical analysis of Ki67 positive cells showed that Bmi1 knockdown could inhibit the tumor proliferation.Our results demonstrated that deletion of Bmi1 can block effects of p27 deficiency induced activation of Shh signal pathway.Bmi1 therefore is a critical effective molecule for p27 defeciency induced activation of Shh signal pathway.