The P27 Pathway Modulates The Regulation Of Skeletal Growth And Osteoblastic Bone Formation By Parathyroid Hormone-related Peptide

We previous 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 but can still signal through its cell surface receptor.PTHrP KI mice displayed growth retardation and premature senescence and osteoporosis.However,the action and mechanism of PTHrP NLS and C-terminus on skeletal development and osteoblastic bone formation are unclear.To determine whether the p27 pathway modulates the regulation of skeletal growth and osteoblastic bone formation by PTHrP NLS and the C-terminus,compound mutant mice which are homozygous for both p27 deletion and the PTHrP KI mutation(p27-/-PTHrP KI)were generated.Their skeletal phenotypes were compared with p27-/-,PTHrP KI and WT mice by techniques of imageology,histochemistry,immunohistochemistry,real-time RT-PCR and Western blots.To determine whether the deletion of p27 can rescue the growth retardation and premature senescence caused by PTHrP KI,we examined the effect of deletion of p27 on lifespan,habitus and body weight of PTHrP KI mice.Our results showed that p27-/-mice and WT mice were survival for more than 2 years,however,the mean survival time was about 2 weeks in PTHrP KI mice and was prolonged to about 3 weeks in p27-/-PTHrP KI mice.The habitus and body weight were increased significantly in p27-/-mice,but were reduced in both PTHrP KI and p27-/-PTHrP KI mice compared to their wild-type littermates,however they were increased significantly in p27-/-PTHrP KI mice compared to PTHrP KI mice.These results indicate that the deletion of p27Kip1 in Pthrp KI mice resulted in a longer lifespan and an improvement in growth development.To assess whether the deletion of p27 can correct skeletal growth retardation caused by PTHrP KI,we examined the effect of deletion of p27 on the length of long bones and the proliferation of chondrocytes of PTHrP KI mice using techniques of imageology,histology and immunohistochemistry for proliferating cell nuclear antigen(PCNA).Results showed that the length of long bones,the width of proliferating zone of cartilage growth plate and the percentage of PCNA positive chondrocytes were increased obviously in p27-/-mice,but were reduced significantly in both PTHrP KI and p27-/-PTHrP KI mice compared to wild-type mice,however these parameters were increased significantly in p27-/-PTHrP KI mice compared to PTHrP KI mice.These results indicate that the deletion of p27 can improve skeleton growth in PTHrP KI mice.To determine whether the deletion of p27 can correct premature osteoporosis caused by PTHrP KI,we examined the effect of deletion of p27 on osteoblastic bone formation of PTHrP KI mice using techniques of micro-CT and 3D reconstruction,histology or immunohistochemistry and molecular biology.Results showed that BMD,trabecular bone volume,osteoblast numbers,and ALP,type I collagen(Col I)and osteocalcin(OCN)positive bone areas,the mRNA expression levels of osteoblast related genes including Runx2,ALP,Col-I and OCN,protein expression levels of PTHR and IGF-1 were increased significantly in p27-/-mice,but were reduced significantly in both PTHrP KI and p27-/-PTHrP KI mice compared towild-type mice,however these parameters were increased significantly in p27-/-PTHrP KI mice compared to PTHrP KI mice.These results indicate that deletion of p27 can improve osteoblastic bone formation in PTHrP KI mice.To further investigate whether the effect of deletion of p27 on osteoblastic bone formation of PTHrP KI mice is associated with alterations of expression levels of cell cycle regulating molecules.We examined protein expressions on bone tissue including protooncogene Bmi-1 and cell cyclin-dependent kinase inhibitors p16 and p53.Results showed that protein expression levels of Bmi-1 were up-regulated in p27-/-mice,but were down-regulated in both PTHrP KI and p27-/-PTHrP KI mice compared to wild-type mice,however they were up-regulated in p27-/-PTHrP KI mice relative to PTHrP KI mice.In contrast,protein expression levels of p53 and p16 were not altered significantly in p27-/-mice,and were up-regulated in both PTHrP KI and p27-/-PTHrP KI mice compared to wild-type mice;however they were down-regulated in p27-/-PTHrP KI mice compared to PTHrP KI mice.These results indicate that the deletion of p27 can improve osteoblastic bone formation in PTHrP KI mice by up-regulating Bmi-1 expression and down-regulating p16 and p53 expression.To assess whether the the effect of deletion of p27 on osteoblasts of PTHrP KI mice was associated with alterations of the recruitment and differentiation of bone marrow mesenchyme stem cells(BM-MSCs),colony forming units-fibroblast(CFU-f)assay was performed.Results showed that CFU-f and ALP positive area were increased significantly in p27-/-mice compared to wild-type mice,but were decreased significantly in PTHrP KI mice compared to WT mice.However,these parameters were increased significantly in p27-/-PTHrP KI mice compared to PTHrP KI mice.These results imply that the deletion of p27 in PTHrP KI mice can improve osteoblastic bone formation by stimulating the proliferation of BM-MSCs and differentiation into osteoblasts.Results from this study suggest that PTHrP NLS and the C-terminus plays roles in stimulating skeletal growth and osteoblastic bone formation by inhibiting p27 mediated through the up-regulation of Bmi-1 which inhibits p16?p53 to stimulate the proliferation of chondrocytes and BM-MSCs and and differentiation into osteoblasts.It will provide experimental and theoretical evidence for the application to PTHrP NLS and the C-terminus to prevent and treat osteoporosis.