The Role And Mechanism Of PDK1 In The Regulation Of Osteoclasts In Osteolysis-related Diseases And Fracture Healing

Bone homeostasis is a dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption,and the imbalance of bone homeostasis can lead to a variety of diseases,such as osteopetrosis,osteoporosis,and osteolysis.Osteolysis and osteoporosis are common disorders associated with imbalance of bone metabolism in the elderly population,and their treatment mainly revolves around two aspects: promoting bone formation and inhibiting bone resorption..The formation of osteoclasts responsible for bone resorption is regulated by two key cytokines,M-CSF and RANKL,which,in combination with the receptor RANK,initiates a series of downstream signaling responses leading to the differentiation of monocytes and their fusion into mature osteoclasts with bone resorption activity.The PI3K-AKT signaling cascade is one of the important pathways in response to RANKL activation,while PDK1 is the main upstream lipid kinase of the PI3K-AKT cascade,and its main function is to activate AKT by means of phosphorylation,triggering the cascade and further activating effector factors downstream of AKT.However,the regulatory role of PDK1 in osteoclasts has not been clarified.Aim: To explore the molecular mechanism of PDK1 gene involved in the regulation of osteoclast differentiation and maturation,and to find a new drug regulatory target for the treatment of osteoporosis or osteolysis-related diseases.Methodes: 1.Mice of conditional knockout PDK1 in osteoclast were constructed by Cre-Lox P system driven by Cathepsin K promoter.The changes of skeletal phenotype and osteoclast activity in mice were observed by micro-CT scanning and histomorphological staining to analyze the effect of osteoclasts regulated by PDK1 gene on bone homeostasis in mice.2.BMMs were extracted from mice and induced to culture in vitro under the stimulation of RANKL and M-CSF.The effects of knocking out PDK1 gene on osteoclast differentiation,maturation and apoptosis were observed by TRAP staining,hydroxyapatite coating absorption assay,CCK-8 proliferation activity assay,TUNEL apoptosis staining,Real-time PCR and Western blot,and the molecular mechanism of PDK1 involved in the regulation of osteoclast differentiation was elucidated.3.On the basis of elucidating the molecular mechanism of PDK1 involved in the regulation of osteoclast differentiation,UHMWPE particle induced mice calvarial osteolysis and OVX-induced mice osteoporosis models were constructed to observe and analyze the protective effect of PDK1 gene regulated osteoclasts on the imbalance of bone homeostasis by micro-CT scanning,serological detection by ELISA,histomorphological staining and biomechanical testing.4.Mice model tibial fracture was constructed to observe and analyze the effect of PDK1 gene regulated osteoclasts on bone injury repair by X-ray radiography,micro-CT scanning,histomorphological staining and biomechanical testing.Results:1.PDK1-c KO mice exhibited a macroscopically smaller body size,resembling a dwarf phenotype compared to WT mice.Micro-CT scanning showed that bone mass was significantly increased in PDK1-c KO mice,presenting a petrous bone phenotype.TRAP staining of tibial tissue sections showed that the number of osteoclasts in vivo was significantly reduced in PDK1-c KO mice compared with WT mice.The results of serological analysis showed that serum levels of bone resorption markers were significantly reduced in PDK1-c KO mice.However,the basal mineral appositional rate(MAR),the time of appearance of secondary ossification centers,and the levels of bone formation markers fluorescently labeled with osteocalcin-alizarin red in the femoral cortex were not significantly different between the two groups.2.BMMs were extracted from mice and cultured with RANKL and M-CSF in vitro,and the results showed that the number of TRAP-positive osteoclasts generated by differentiation of BMMs was significantly reduced in PDK1-c KO group compared with WT group,and also the ability to absorb hydroxyapatite coating was significantly attenuated.FITC-Phalloidin staining revealed that BMMs in the PDK1-c KO group had a reduced ability to form pseudopodial body actin bands,a diminished ability to fuse with each other to form multinucleated osteoclasts,and a significantly reduced average number of nuclei in osteoclasts.RT-PCR results showed that osteoclast related gene expression levels were significantly decreased in PDK1-c KO group,and further western blot quantitative analysis showed that the expression levels of PDK1,p-AKT(Thr308)and p-GSK3? and NFATc1 were significantly reduced in osteoclasts in PDK1-c KO group.3.In the calvarial osteolysis model,the results of serological analysis showed that the expression levels of serum inflammatory cytokines TNF-? and IL-1? were significantly decreased in PDK1-c KO group compared with WT group.Micro-CT scanning showed that the degree of skull osteolysis was significantly reduced,and both the perforation rate and pore area were significantly reduced in PDK1-c KO group compared with WT group.TRAP staining of calvarial tissue showed that a large number of TRAP-positive osteoclasts accumulated on the calvarial surface in WT group,and the number of TRAP-positive cells was significantly reduced in PDK1-c KO group.In the OVX induced osteoporosis model,the results of Micro-CT scanning of the lumbar showed that the WT group mice had significantly reduced bone mass and more severe osteoporosis compared with PDK1-c KO group.The results of TRAP staining in lumbar tissue were similar to those in the calvarial osteolysis model,with a large number of TRAP-positive osteoclasts accumulating on the surface of the trabecular in WT group,while the number of TRAP-positive cells was significantly reduced in PDK1-c KO group.The results of lumbar compression stress testing showed that the maximum stress,elastic modulus,compression displacement,and energy absorption of the lumbar were significantly increased in PDK1-c KO group compared with WT group.4.In the tibial fracture model,the results of X-ray radiography and Micro-CT scanning showed that the PDK1-c KO group had later bone bridge formation and delayed callus resorption and reconstruction compared with WT group.The results of TRAP staining and SO/FG staining showed that a large number of TRAP positive osteoclasts were formed in the callus of the WT group,and TRAP positive osteoclasts were significantly reduced in the PDK1-c KO group compared with WT group.The expression of osteoclast marker genes in the callus tissue of PDK1-c KO group was also significantly lower than that of WT group,while the expression of osteoblast marker genes was not significantly different between the two groups.The maximum torque and torsional stiffness of the callus were significantly lower in PDK1-c KO group compared with WT group.Conclusion:1.The PDK1 gene regulates bone homeostasis by regulating the differentiation and activity of osteoclasts in vivo,which affects the bone remodeling process and bone tissue structure in growing mice.2.The PDK1 gene positively regulates the differentiation and maturation and bone resorption function of osteoclasts through the AKT-GSK3?-NFATc1 signal transduction pathway.3.The PDK1 gene compensates for the imbalance in bone homeostasis by regulating the differentiation and resorptive activity of osteoclasts and has the potential to be used as a drug target for the treatment of osteoporosis and osteolysis related diseases.4.Deletion of the PDK1 gene excessively inhibits osteoclast differentiation,which may delay callus resorption and remodeling and affect the process of bone injury repair.