The Effect Of PLC-independent PKC Signaling Pathway In PTH Peptide On Bone Metabolism

Background:Parathyroid Hormone(PTH)is an available recognized drugs to promote bone formation.Previous studies showed that PTH analogue with nonPLC/PKC signaling pathway could augment cancellous bone mass,modify the microstructure of the trabecular bone.On the basis of various signal molecules,more nonPLC/PKC analogues could be designed,in order to improve the efficiency of drugs,and enlarge a solid foundation for PTH application.Previous studies found that this signaling pathway could be divided into cAMP/PKA dependent and independent pathways,but the function of two pathways were not clear.Objective:With PTH analogues,the function of cAMP/PKA dependent nonPLC/PKC was explored in cell proliferation,apoptosis and differentiation.With MY-1 and MY-2,the signaling pathway was detected and the anti-apoptotic effect of new peptide in cells was explored,then osteogenic and osteoclastic effect was compared.In the ovariectomized mice model,with subcutaneous injection of peptide,the anti-osteoporosis effect was observed by microCT reconstruction,evaluating the effect of MY-1 and MY-2 peptide.Method:With MC3T3E1 cell,the ability of cAMP/PKA dependent nonPLC/PKC in proliferation was measured supplied with 2%FBS medium.Then the ability of anti-apoptosis was measured supplied with a-MEM medium.After 14 days the ALP staining and quantitation were performed,then alizarin red staining and calcium quantitation were performed after 28 days.With one-day old C57BL mice,the skull osteoblast cells was isolated,then cAMP and PLC signaling pathway in MY-1 and MY-2 peptides was verified,then the effect of anti-apoptosis.With BMSCs extracted from 6-week old C57BL mice,with osteoblast-induction medium,then the effect of osteogenesis analysis was performed.And the TRAP staining and enzyme quantitation were analyzed after 7 days with 10%FBS medium.Taking 6-week old C57BL female mice,making ovariectomized osteoporosis model,subcutaneous injections of PTH was performed.Sacrificing the mice in 4th and 8th weeks,isolating the tibias,trabecular bone structure was evaluated by microCT analysis.Result:The cAMP/PKA dependent nonPLC/PKC could promoted osteoblast cell proliferation,anti-apoptosis and differentiation.Signal dectection showed that MY-1 and MY-2 could not significantly activate cAMP and PLC signaling pathway.MY-1 and MY-2 had effect in anti-apoptosis.In intermitent PTH administration,BMSCs osteoblast differentiation showed that MY-1 and MY-2 could significantly promote osteoblast differentiation,with slightly activation of osteoclasts differentiation.Continuous PTH administration also show that MY-1 and MY-2 could significantly promote osteoblast differentiation,but osteoclast differentiation was still unobvious.Using the ovariectomized 6-week old C57BL female mice,we found that the bone mass of ovariectomized mice decreasing significantly comparing with Sham group,while the bone mass of administration with MY-1 and MY-2 could reverse the osteoporosis both in 4th week and 8th week.Conclusion:The cAMP/PKA dependent nonPLC/PKC signaling pathway could promote cell proliferation and differentiation,and had the effect in anti-apoptosis.The cAMP/PKA indenpendent nonPLC/PKC signaling pathway was verified in MY-1 and MY-2.They had the effect in anti-apoptosis.The intermitent and continuous PTH administration show osteogenic property with MY-1 and MY-2 but low osteoclastic property.MY-1 and MY-2 showed good effect in anti-osteoporosis.