Role Of Low Density Lipoprotein Receptor-related Protein 6 In Parathyroid Hormone-mediated Bone Metabolism

Objective:It is not clear about the role of LRP6 in bone formation and the relationship of PTH mediated bone anabolic activity.Therefore,these studies will reveal the mechanism of LRP6 in PTH mediating the process of bone anabolic activity and the role of LRP6 in osteoblast.MATERIALS AND METHODS:The administration of PTH for mice was made.Male mice were administered a single dose of either acetic acid in sterile PBS or PTH1-34.Mouse recombinant Wnt3a was also injected.Mice were sacrificed at 0.5,2,8,and 24h after injector.In another step detection of temporal dynamics of cAMP signaling in living cells was observed.And cells were collected and sent for western blot analysis using antibody against p-CREB.Furthermore,we did the construction of LRP6 null mice,LRP6^floxp/floxploxp/floxp mice were crossed with Oc-Cre mice to generate Oc-Cre::LRP6^f/f/f progeny,which were used in subsequent mating.The animals that had a genotype of Cre^-/-::LRP6^f/f were considered control（WT）,and Cr^+/-::LRP6^f/f/f were considered LRP6 deletion（KO）.Than did the administration of PTH for LRP6 conditional null mice and analyzed the skeletal phenotypes.Later we did theμCT analysis,tibia and femora were taken from mice,dissected free of soft tissue.And fixed in70%ethanol,and analyzed by a high resolutionμCT.And Western blot and ELISA analysis of sclerostin level in femur bone tissue were checked.And histochemistry,immunohistochemistry and histomorphometric analysis were done.In Statistics,data was analyzed by Student’s t-test and are presented as the mean±SEM.At least three times to repeat each experiment.A p value less than 0.05 was considered significant.Results:PTH inhibited the expression of sclerostin in osteocytes.Formalin-fixed tibia tissue section was processed and then IHC staining for sclerostin.Single dose injection of PTH reduced sclerostin-positive osteocytes at 8 and 24 h after injection.p value less than 0.05 was considered significant.Western blot analysis the sclerostin protein levels in whole-bone matrix in a time-dependent manner.As sclerostin is a specific negative regulator of LRP5/6,we predicted that suppressing the sclerostin would increase the availability of LRP5/6 to facilitate PTH signaling in a positive feedback fashion.We also found DKK1 and sclerostin work as antagonists for PTH stimulated cAMP generation and phosphorylation of CREB by bindling to LRP6.We used the FRET technology to examined whether DKK1 and sclerostin modulate c AMP production through LRP6.We also found bone mass was decreased in adult mice with conditional LRP6 deletion in osteoblast.To determine the role of LRP6 in osteoblastic bone formation,we generated mice lacking LRP6 specifically in mature osteoblasts.Furthermore,bone formation,but not bone resorption,was impaired in mice with Oc-Cre–mediated conditional LRP6 deletion,bone histomorphometric analyses of femora revealed no changes in the number of osteoblasts and osteoclasts in 1-month-old KO mice.A reduced number of osteoblasts but no change in the numbers of osteoclasts was observed in 3-month-old KO mice compared with their WTlittermates.Reduced bone formation in 3-month-old KO mice was also confirmed by Goldner’s Trichrome staining and double calcein labeling analysis.Taken together results demonstrated that deletion of LRP6 in osteoblasts impaired bone formation without affecting osteoclastic bone resorption during bone remodeling in adult mice.We also found Oc-Cre–mediated conditional LRP6 deletion leads to decreased expression of osteoblast differentiation genes and accelerated osteoblast apoptosis during bone remodeling.And Oc-Cre–mediated LRP6-deficient mice exhibit blunted osteoblast differentiation and anti-apoptotic responses to PTH.And PTH failed to stimulateβ-catenin and Gas-PKA signaling in LRP6-deficient osteoblasts.CONCLUSION:In conclusion,our studies demonstrate that LRP6 in osteoblasts is a positive regulator for osteoblastic bone formation during bone remodeling and plays a critical role in osteoanabolic action of PTH.And LRP6 as an important bone formation co-receptor,promote the production of cAMP and P-CREB and PTH mediated bone formation is the molecular mechanism for bone remodeling.