Study On The Mechanism Of Action Of Gene Recombinant Parathyroid Hormone Related Peptide 87-139 In Preventing Aging

Parathyroid hormonr related peptide(PTHrP)is not only distributed in various tissues,but also plays multi-functions.PTHrP plays different functions with different fractions.Its nuclear localization sequence(NLS),amino acids 87-107,can transfer into nuclei and plays an intracrine action.To investigate the function of NLS of PTHrP in vivo,we generate an animal model with disruption of the NLS of PTHrP by introducing a premature termination codon TGA into the PTHrP gene and created a "knock-in" mouse expressing PTHrP(1-84),a form of the protein missing the NLS and its carboxyl terminus(PTHrP KI).Mice homozygous for the knock-in mutation displayed features of impaired growth and early senescence.Therefore we proposed that PTHrP NLS and C-terminus play a critical role in preventing aging.To determine whether PTHrP NLS and C-terminus play a critical role in preventing aging,the expression plasmid pGEX-2TK/PTHrP87-139 which included PTHrP NLS and C-terminus was constructed using gene enginnering technique.The plasmid was transformed into BL21 and the protein expression was induced by IPTG.The protein was purified using a GST purification systerm.The purified PTHrP87-139 appered immunoactivity demonstrated by Western blot.To determine whether exougrous PTHrP87-139 can translocate into nuclei,bone marrow mesenchymal stem cells(BM-MSCs)derived from PTHrP KI mice were treated with or without the recombinant PTHrP87-139 and the subcellular localization of PTHrP87-139 was detected by immunofluorence staining using antibodies against PTHrP C-terminus.Results showed that positive fluorence signals were detected in BM-MSCs at 3 hours after the treatment with exougrous PTHrP87-139,but not in cells from control cultures.To further verify this result,PTHrP87-139 labeled with FITC was synthesized and was used for the treatment of BM-MSCs.subcellular localization was examined under flourence microscope.Results show that positive flourence signals were detected in nuclei of BM-MSCs at 15 min and reached to maxima at 3 hours after the treatment of PTHrP87-139-FITC。These results demonstrated that exougrous PTHrP87-139 can translocate into nuclei.To determine whether the exougrous PTHrP87-139 can stimulate the proliferation and differentiation of BM-MSC,bone marrow cells derived from wild type or PTHrP KI mice were treated with or without the recombinant PTHrP87-139,its effects on the forming efficency of colony forming unit-fiberblastic(CFU-f)and alkaline phosphatase(ALP)positive CFU-f(CFU-fap),the proliferation and apoptosis of BM-MSCs and the expression of cell cycle regulating moleculars were assesed by cytochemical staining,PI staining or PI and Annexin V double staining,Western blot and real-time RT-PCR.Results showed that the treatment of exougrous PTHrP87-139 increased the forming efficiency of CFU-f and CFU-fap,stimulated cell proliferation,inhibited cell apotosis,upregulated Runx2,Bmi-1 and downregulated p16,p21 and p27 at both mRNA and protein levels in BM-MSCs resulting bone marrow cell cultures.These results indicate that exougrous PTHrP87-139 can stimulate the proliferation,osteoblastic differentiation and inhibit the apoptosis of BM-MSCs by upregulating Runx2,Bmi-1 and downregulating p16,p21 and p27.To determine whether the administration of exougrous PTHrP87-139 can rescue the senescence phenotypes of PTHrP KI mice,PTHrP KI mice was injected subcutaneously daily with the 80 μg/Kg recombinant PTHrP87-139 for 10 days started from day 4 after birth.The wild-type and PTHrP KI mice were injected with the vehicle as control.The body weight was measured and the lifespan was recorded.Alterations of the morphology of thymus and spleen,the senescence and proliferation of thymocytes and spleen cells and the expression of cell cycle regulating molecules were analyzed in thymus and spleen at 2 weeks of ages by techniques of histopathology,cell biology and molecular biology.The phenotypes of skeletons were also analyzed using described techniques above to determine effect of exougrous PTHrP87-139 on skeletal growth,osteoblastic bone formation and the expression of osteoblast-related regulating molecules.Our results showed that the body weight was increased and the lifespan was longer in PTHrP87-139-treated PTHrP KI mice compared with untreated PTHrP KI mice.The size of thymus and spleen was increased significantly,SA-β-Gal positive cells were reduced,Ki67 positive cells were increased significantly,the repression levels of Bmi-1 wereupregulated,whereas the expression levels of p16,p21 and p27 were downregulated in thymus and spleen from PTHrP87-139-treated PTHrP KI mice compared with untreated PTHrP KI mice,however,these parameters were not normalized.Bone mineral dentisy,length of tieba,trabecular bone volume,osteoblast number,positive areas of type I collagen and osteocalcin were increased significantly,the expression levels of type PTH receptor,Runx2 and Bmi-1 were upregulated,whereas the expression levels of p16,p21 and p27 were downregulated PTHrP87-139-treated PTHrP KI mice compared with untreated PTHrP KI mice,however,these parameters were not normalized.These results indicate that PTHrP87-139 plays an role in preventing aging through its function to inhibit cell senescense and stimulate skeletal growth and bone formation by upregulating Bmi-1 expression and downregulating p16,p21 and p27 expression.In this study,we prepared purified recombinant PTHrP87-139 successfully using gene engineering approach,and demonstrate that it plays important roles in stimulating BM-MSC proliferation and differentiation into osteoblasts in vitro and in preventing aging in vivo.Results from our study were not only revealed the mechanism of PTHrP87-139 in stimulating bone formation and in preventing aging,but also provided experimental and theorical evidence for clinical application of PTHrP87-139 in preventing osteoporosis and aging.