| Backgrounds:The Wnt canonical signaling pathway is mediated by β-catenin and relies on itsstabilization. To activate the Wnt canonical signaling pathway, Wnt proteins first bind toFrizzled family receptor and LRP5/6co-receptor complex, then cause disruption of GSK-3βinhibitory complex and stabilisation of β-catenin, which is transmited to nucleus andactivates transcriptions of related factors. In recent years, a number of studies haveconfirmed that activation of Wnt/β-catenin signaling pathway in osteoblasts at differentperiods could increase bone volume, showing promising prospect in the treatment ofosteoporosis.Osteocytes make up more than95%of all bone cells in the adult skeleton. Recentstudies have shown that osteocyte can function as endocrine cell, and affect osteoblast andosteoclast activities, thus osteocyte plays an important role in regulating bone metabolismand bone development. However, the effect of constitutive activation of β-catenin inosteocytes on bone homeostasis is unknown currently. Therefore, our first aim was toobserve the effect of constitutive activation of β-catenin in osteocytes on bone mass andbone growth.Sclerostin (SOST), specifically expressed in osteocytes, is a strong inhibitor ofWnt/β-catenin signaling pathway, and can negatively regulate bone formation. Recently,multiple basic researches and clinical studies have found that, SOST neutralizing antibodyexerted a certain effect on treating osteoporosis. However, the underlying mechanism andpossible side effect are still unclear currently. Dentin matrix protein1(DMP1) isspecifically expressed in dentin and osteocytes, and is essential for bone mineralization andphosphorus metabolism. Dmp1knockout mice developed a typically phenotype withshorter vertebral body and decreased bone volume. In this study, we tried to observe that whether SOST neutralizing antibody could improve the low-bone-mass phenotype in DMP1knockout mice, and see whether it had side effects.Although it is now confirmed that Wnt/β-catenin signaling pathway play a vital rolein regulation of bone mass and bone growth, the downstream transcription factorsinteracting with this pathway and the possible mechanism is still unclear. Osterix (Osx), anosteoblast-specific transcription factor, is essential for osteoblast differentiation and boneformation. One study has proved that Osx could decrease osteoblast proliferation as aconsequence of the Osx-mediated inhibition of Wnt/β-catentin activity. Thus we speculatethat Osx may be one of the downstream transcription factors of Wnt/β-catenin signalingpathway.Therefore, in the present study, we aimed:1. To observe the effect of constitutiveactivation of β-catenin in osteocytes on bone mass and bone growth;2. To see whetherSOST neutralizing antibody could improve the low-bone-mass phenotype in DMP1knockout mice, and whether it had side effects;3. To investigate the role of Osterix inregulating bone mass and bone development by means of gene knockout and geneoverexpression.Methods:Part I: the effect of constitutive activation of β-catenin in osteocytes on bone massand bone growth1. Mice were observed for their body size, meanwhile the body weight and length offemur and tibia were measured;2. Histological sections of mice were stained with H&E, X-Ray and Micro-CT wereused to observe bone structure and analyze bone mass.3. TRAP staining was used to observe osteoclast activities, BrdU staining was used toassay osteoblasts proliferation, TUNEL assay was performed to evaluate cell apoptosis, andconcentration of serum biomarkers of osteoblasts and osteoclasts was measured by ELISA;4. Immunohistochemisty and reverse transcription PCR (RT-PCR) were used toanalysis osteoblastogenesis and osteoclastogenesis in vivo and in vitro;5. Three-point bending test was used to test biomechanical properties of femur,undecalcified skeleton sections were stained with Von Kossa to observe bonemineralization. The concentrations of serum ALP, phosphate and calcium were measured. 6. Cell culture system was used to evaluate the differentiation of osteoblasts andAlizarin red staining was used to observe osteocytes mineralization.Part II: Study of the effect of SOST neutralizing antibody on bone mass in normaland DMP1knockout mice.1. SOST neutralizing antibody (Scl-Ab) was administrated to DMP1knockout miceand their wild-type littermates at4or12weeks of age, and lasted for eight consecutiveweeks. Untreated animals were treated with same volume of saline alone as control.2. The vertebral structure and bone mass were analyzed by X-Ray, MicroCT andhistological section H&E staining.3. Safranin O staining and TRAP staining were employed to evaluate cartilagecomponent content and osteoclasts activities respectively.4. Expression levels of bone formation and bone resorption related proteins invertebral body were analysed by immunohistochemistry.Part III: The role of Osterix in regulating bone mass and bone development1. Osx conditional knockout and over-expression mice were obtained by adapting theCre-LoxP recombination system, and survived mice were recorded. we observed the bodysize, length and vertebrate morphology.2. The change of vertebral form and bone mass were evaluated by X-Ray radiologyand H&E staining.3. TUNEL assay was used to evaluate hypertrophic cell apoptosis in growth plate.The cartilage component content and osteoclast formation in vertebral cancellous bonewere measured by safranin O staining and TRAP staining respectively.4. The nissl staining was used to observe neuronal injury in spinal cord.5. Immunohistochemistry was used to assay osteoblast and osteoclast markers in vivo.Results:Part I: constitutive activation of β-catenin in osteocytes increased cancellous bonemass, impaired bone strength and bone growth.1. The constitutive activation of β-catenin (CA-β-catenin) mice showed decreasedbody size and body weight, mostly died at5to6weeks after birth. Linear growth of longbone was stunted in CA-β-catenin mice2. The cancellous bone volume significantly increased in CA-β-catenin mice, almost filled the entire bone marrow cavity.3. Increased bone resorption and bone formation activities were observed inCA-β-catenin mice.4. Bone strength decreased in CA-β-catenin mice, which exhibited thinner and moreporous cortical bone together with impaired mineralization.Part II: SOST neutralizing antibody treatment effectively improved loss of bonemass in DMP1knockout mice by activate wnt/β-catenin signaling.1. The radiodensity and bone mass of vertebrae in DMP1knockout mice wassignificantly decreased. After8weeks treatment with SOST neutralizing antibody, boneloss was effectively improved, and reached a level higher than that of normal wild-typemice.2. SOST neutralizing antibody could facilitate osteoblast differentiation andmaturation, meanwhile inhibit osteoclast activity of vertebra in DMP1KO mice.3. SOST neutralizing antibody could activate wnt/β-catenin signaling pathway invertebra of DMP1KO mice.4. SOST neutralizing antibody had no effect on retarded growth of vertebra inDMP1KO mice.Part III: Conditional knockout of Osx in osteoblasts could increase bone massand lead to severe spine deformities.1. The Osx conditional knockout (Osx-KO) mice showed excessive immature bonetissue in vertebrae, and the possible mechanism might be activation of Wnt/β-cateninsignaling pathway.2. Osx-KO mice showed thoracolumbar spine deformities including wedged vertebralbody, spinal stenosis, and scoliosis, this is consistent with the performance of congenitalscoliosis in humans.3. Severe spinal cord injuries were observed in Osx-KO mice, due to abnormalformation of vertebral arches.4. Over-expression of Osx in osteoblasts had no obvious effect on spine development.Conclusions:1. Constitutive activation of β-catenin in osteocytes could increase cancellous bonemass, however, with impaired bone strength and bone growth.Wnt/β-catenin signaling pathway in osteocytes plays an important role in controlling bone mass and maintainingbone homeostasis, which is achieved by adjusting the balance of oesteoblasts andosteoclasts.2. SOST neutralizing antibody could effective increase vertebral bone volume innormal and DMP1knockout mice，via activation of Wnt/β-catenin signaling pathway.3. Deletion of Osx in osteoblasts will increase bone mass by activating Wnt/β-cateninsignaling. It could also cause congenital scoliosis，and over-expression of Osx has nosignificant impact on bone development. |
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