DNA Damage Checkpoint Pathway Modulates The Regulation Of Skeletal Growth And Osteoblastic Bone Formation By Parathyroid Hormone-Related Peptide

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 role of PTHrP NLS and C-terminus in regulating skeletal development still remains unclear.To determine whether oxidative stress and DNA damage response pathways are involved in the regulation of skeletal growth and development by PTHrP,we examined the alterations of oxidative stress and DNA damage response related molecules in PTHrP KI skeletal tissues in vivo.These results revealed that compared with wild-type controls,the levels of ROS and the DNA damage-related molecules including?-H2AX,p-Chk2,and p53 were significantly increased,while the expression of mRNA in anti-oxidative enzymes including SOD1 and 2,catalase,glutathione reductase?GSR?,and glutathione peroxidase 4?GPX4?was decreased in PTHrP KI skeletal tissues.Furthermore,we disrupted the DNA damage response pathway by Chk2 deletion in PTHrP KI mice(Chk2^-/-KI).Their phenotypes were compared with WT,Chk2^-/-,and PTHrP KI littermates using imageology,histochemistry,immunohistochemistry,Real time RT-PCR,and Western blot.To assess whether the deletion of Chk2 can rescue the growth retardation and premature senescence caused by PTHrP KI,we examined the effect of the deletion of Chk2 on lifespan,habitus,and body weight in PTHrP KI mice.These results showed that the mean lifespan of Chk2^-/-KI mice was extended to 3 weeks?12-24days?from 2 weeks?5-18 days?in PTHrP KI mice.The habitus and body weight were significantly increased in Chk2^-/-KI mice compared with PTHrP KI mice,However,they were both reduced in PTHrP KI and Chk2^-/-KI mice compared with wildtype mice.These results demonstrated that the deletion of Chk2 prolong the lifespan and increase the body size and weight in PTHrP KI mice.To determine whether the deletion of Chk2 can rescue skeletal growth retardation caused by PTHrP KI,we analyzed the bone phenotypes of the 2-week-old four genotype mice using X-ray and histology.Our results showed that the length of long bones,the width of cartilage growth plate and proliferation area were increased in Chk2^-/-mice,which,however,were significantly reduced in both PTHrP KI and Chk2^-/-KI mice compared to WT mice.Notably,these parameters were increased significantly in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that the deletion of Chk2 improve skeleton growth defect displayed in PTHrP KI mice.To determine whether the deletion of Chk2 can rescue the loss of bone mineral density and the decrease in bone volume caused by PTHrP KI,we examined the effect of deletion of Chk2 on bone mineral density and bone volume of PTHrP KI mice using Micro-CT,3D reconstruction,and total collagen histochemistry.Results showed that the number of BMD,BV/TV,trabecular and cortical bone volume and trabecular bone were significantly higher in Chk2^-/-mice than in both PTHrP KI and Chk2^-/-KI mice.However,these parameters were significantly increased in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that deletion of Chk2 can partly improve the loss of bone mineral density and bone volume in PTHrP KI mice.To test whether the improvement in bone mineral density and bone volume in PTHrP KI mice by deletion of Chk2 was related to osteoblastic bone formation,we examined the effect of deletion of Chk2 on osteoblastic bone formation of PTHrP KI mice using histology,immunohistochemistry,and molecular biology.Results showed that the number of osteoblasts,ALP and type I collagen?COL-I?-positive bone areas,the mRNA expression of osteoblast related genes including Runx2,ALP,OCN,and COL-I were significantly increased in Chk2^-/-mice,which,however,were significantly reduced in both PTHrP KI and Chk2^-/-KI mice compared to WT mice,Moreover,these parameters were markedly increased in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that deletion of Chk2 can improve the decline in bone mineral density and bone volume by enhancing osteoblastic bone formation in PTHrP KI mice.To assess whether the effects of deletion of Chk2 on osteoblastic bone formation of PTHrP KI mice was associated with the elevation of proliferation and differentiation in bone marrow mesenchyme stem cells?BM-MSC?,we performed colony forming units-fibroblast?CFU-f?assay and cytochemical staining for ALP.Results showed that compared to PTHrP KI mice,CFU-f and ALP-positive area were significantly increased in Chk2^-/-KI mice.These results indicated that deletion of Chk2 in PTHrP KI mice can improve osteoblastic bone formation by promoting the proliferation and differentiation of BM-MSCs into osteoblasts.To explore whether the improvement in bone mineral density and bone volume caused by deletion of Chk2 in PTHrP KI mice was associated with osteoclastic bone absorption,we examined the effects of deletion of Chk2 on osteoclastic bone absorption of PTHrP KI mice using histochemistry and molecular biology.Results showed that Oc.S/B.S,RANKL/OPG,and the mRNA expression of osteoclast-related genes TRAP and RANKL were significantly decreased in Chk2^-/-mice,which,however,were markedly increased in both PTHrP KI and Chk2^-/-KI mice compared to WT controls.Additionally,these parameters were significantly lower in Chk2^-/-KI mice than in PTHrP KI mice.These results indicated that deletion of Chk2 can improve premature osteoporosis by inhibiting osteoclastic bone absorption in PTHrP KI mice.To determine whether the deletion of Chk2 improved the premature osteoporosis phenotype of the PTHrP KI mice through inhibiting their oxidative stress,Real-time RT-PCR and Western blot were performed to examine the expression of antioxidant enzyme genes in bony tissue of grouped mice.Results showed that the expression of antioxidant enzyme genes including SOD1/2,CAT,GSR and GPX4 mRNA and SOD1,Bmi1 protein was remarkably increased in bony tissues of Chk2^-/-KI mice compared with PTHrP KI littermates.These results indicated that the deletion of Chk2 can rescue the premature osteoporosis exhibited in PTHrP KI mice by inhibiting the oxidative stress.To evaluate whether the improvement in the premature osteoporosis caused by deletion of Chk2 in the PTHrP KI mice was associated with the decline of DNA damage,we examined the DNA damage and response-related parameters in bony tissues of indicated group of mice using Real-time RT-PCR and Western blot.These results showed that the expression of?-H2AX,Caspase-3,p53 and cyclin dependent kinase inhibitors including p16,p19,p53,p21,and p27 was dramatically decreased in bony tissues of Chk2^-/-KI mice compared to PTHrP KI littermates.These results indicated that deletion of Chk2 can rescue the premature osteoporosis of PTHrP KI mice by inhibiting the DNA damage and response.These results indicated that deletion of PTHrP nuclear localization sequence and C terminal result in an increase in oxidative stress and DNA damage,whereas deletion of Chk2 can rescue the early senescence and growth retardation caused by PTHrP KI.Furthermore,deletion of Chk2 can improve osteoblastic bone formation,promote the proliferation and differentiation of BM-MSC into osteoblasts,and suppress osteoclastic bone absorption by inhibiting the oxidative stress and DNA damage response pathway.Our data demonstrated that the DNA damage checkpoint pathway may be involved in the role of PTHrP in regulating skeletal growth and development.This study might provide experimental and theoretical evidence for the application of PTHrP NLS and the C-terminus in the prevention and treatment of osteoporosis.