The Effect Of Hedgehog Signaling Pathway On The Regulation Of In Vivo Osteoclasts And Overall Bone Mass

BackgroundIn recent years,the incidence of osteoporosis has increased with the aging of the population.Osteoporotic fracture is the most dangerous consequence.Its high mortality and disability rate not only seriously affect the quality of life and survival time of patients,but also bring heavy psychological and economic burden to families and society.Therefore,the study of the pathogenesis of osteoporosis is a particularly urgent topic at present.Hedgehog signaling pathway plays an important role in the development of bone,and the abnormal signal intensity often leads to various diseases.The role of Hedgehog signaling pathway in osteoporosis has also been widely studied.It is found that Hedgehog signaling pathway promotes the proliferation and differentiation of osteoblasts and the ability of bone formation in vivo and in vitro.Moreover,Hedgehog signaling pathway can regulate osteoclasts indirectly by regulating the ratio of RANKL and OPG secreted by osteoblasts in vivo,thus affecting the overall bone mass.However,compared with the full study of the mechanism of osteoblast regulating by the Hedgehog signaling pathway,the study of osteoclast by the Hedgehog signaling pathway is scarce.At present,only in vitro studies have shown that the Hedgehog signaling pathway regulates the proliferation and differentiation of osteoclasts and the ability of bone resorption.Whether Hedgehog signaling pathway plays a positive role in regulating osteoclasts and how it affects the overall bone mass is still an open question.ObjectiveObjective to study the role of Hedgehog signaling pathway in osteoclasts,observe its effects on the whole bone mass and study its mechanism,so as to provide references for the treatment of osteoporosis.Methods1,A gene tool mouse model that specifically up-regulated or inhibited Hedgehog signaling pathway in osteoclasts was constructed.CTSK-Cre mice was obtained by using knock in technology.The PTC^（F/F）mice who’s PTC locus labeled by LoxP was obtained by transgene technology.The SMO^（F/F）mice that SMO locus labeled by LoxP was obtained by transgene technology.CTSK-Cre mice were hybridized with PTC mice and SMO mice respectively.CTSK-Cre,PTC^（F/F）mice,which are specific up regulation of Hedgehog signaling pathway in osteoclasts in vivo.And CTSK-Cre;SMO^（F/F）mice that specifically inhibit the Hedgehog signaling pathway in the osteoclasts in vivo.It ensures that it can be normally bred without fatal embryo and is used for follow-up test.2,Compare the bone mass between CTSK-Cre;PTC^（F/F）mice group and PTC^（F/F）mice group,and between CTSK-Cre;SMO^（F/F）mice group and SMO^（F/F）mice group.Observe the effects of specific regulation Hedgehog signaling pathway in osteoclasts on osteoclasts and overall bone mass in vivo.The mice were grown to the observation time point and were fixed,and the total bone mass was evaluated by microscopic CT scan.The number of osteoclasts in the body was observed by TRAP staining.The osteogenic ability of osteoblasts was observed by double labeled and I collagen immunohistochemical staining.The serum bone resorption and bone formation markers were detected to evaluate the state of bone transformation in mice.3,The bone marrow mononuclear cells from experimental group CTSK-Cre,SMO^（F/F）mice and the control group SMO^（F/F）mouse were tested in vitro.We detected the proliferation ability of bone marrow mononuclear cells.Detect the differentiation ability from bone marrow mononuclear to osteoclasts,the bone absorption ability of osteoclasts and RNA expressed in those progress.Observe the effect of hedgehog signal pathway in osteoclast proliferation and differentiation and the bone resorption function in vitro.Osteoclast osteoblast co culture experiment was performed.Then we use RT-PCR,alizarin red and ALP staining to detect bone formation ability of those osteoblasts.Assessment the indirect regulation effect of Hedgehog signal on osteoblasts by regulating osteoclasts.Using RT-PCR to screen cytokines expressed by osteoclasts which may regulate osteoblasts.Result1,CTSK-Cre,PTC^（F/F）mice,CTSK-Cre,SMO^（F/F）were successfully obtained that specifically regulate the Hedgehog signaling pathway in osteoclasts in vivo.The mice can be born normally,the ratio of birth sex and the proportion of genotypes conform to the law of Mendel genetics.2,When completely knocking out the PTC gene of osteoclasts in vivo,the bone mass of CTSK-Cre,PTC^（F/F）mice group was significantly reduced,and spontaneous fractures appeared in the femoral shaft,and the activity was limited.The number of osteoclasts in the body increased significantly,and the ability of the osteoblast cells to secrete type I collagen was enhanced.Due to some reasons such as the lack of nutrition,CTSK-Cre;PTC^（F/F）mice died in about 10 days.We used CTSK-Cre;PTC^（F/+）mice to observe the changes of bone resorption and bone formation.We found that half knockout PTC gene in osteoclasts failed to change the bone absorption ability and bone formation ability.3,The bone mass of CTSK-Cre;SMO^（F/F）mice that SMO gene was completely deleted in osteoclast decreased compared with control group.We also found that the osteoclast number and the bone resorption was reduced in CTSK-Cre;SMO^（F/F）mice.At the same time the osteoblastic osteogenic ability also decreased significantly in CTSK-Cre;SMO^（F/F）mice.Bone resorption and bone formation markers also indicated that the bone conversion rate decreased in CTSK-Cre;SMO^（F/F）mice.Because we only inhibit the Hedgehog signaling pathway in osteoclasts,it is not difficult to see that these results indicate that Hedgehog signaling pathway plays an indirect role in regulating osteoblasts by regulating osteoclasts.4,In vitro studies showed that the inhibition of Hedgehog signaling pathway had no significant effect on the proliferation of bone marrow mononuclear cells in vitro,but the differentiation of osteoclasts was significantly impaired.PT-PCR showed that the expression of genes related with osteoclast differentiation and fusion decreased.Although osteoclasts of the experimental group can form normal pseudopodia,but bone resorption experiment results showed that its bone resorption ability decreased.Through co-culture experiments of osteoclasts and osteoblasts,we found that osteogenic factors related to osteoblasts were significantly reduced in experimental group.Alizarin red and ALP results showed that osteoblast bone formation ability was also significantly reduced.The results of RT-PCR detection on the related factors of osteoclast regulating osteoblasts showed that factors which could promote osteoblast differentiation secreted by osteoclasts decreased significantly.ConclusionTo sum up,we have found that the Hedgehog signaling pathway plays an important role in the regulation of osteoclasts in vivo.As for osteoclasts,the up regulation of Hedgehog signaling pathway will increase the number of osteoclasts and enhance their bone resorption function.Down regulation of Hedgehog signaling pathway will reduce the number of osteoclasts and decrease their bone resorption function,which is consistent with in vitro experiments.For the whole bone mass,when the Hedgehog signaling pathway is completely knocked out,osteoclast bone resorptio n is active,and the bone remodeling balance is severely damaged.Severely damaged bone causes spontaneous fracture in mice and endangers the survival of mice.When inhibiting Hedgehog signaling pathway in osteoclasts,in addition to inhibited bone resorption,osteoblast bone formation ability is also significantly reduced,bone remodeling is in a negative balance,and ultimately resulted bone loss in mice.In combination with in vitro data,Hedgehog signaling pathway has a positive regulatory effect on osteoclasts,and plays an indirect role in regulating osteoblasts by regulating osteoclasts.This study suggests that further search for osteoblast induci ng factor secreted by osteoclasts is nice candidate for the prevention and treatment of osteoporosis.