Effects Of Constitutive Activation Of β-catenin On Bone Mass And Growth At Different Postnatal Stages In Mice

Background and Objective: Osteoporosis is a progressive bone disease that ischaracterized by a decrease in bone mass and density which can lead to an increased riskof fracture. It is becoming a serious problem for the public economy and health, because ofthe increase in elderly population in the near future. So,it is meaningful and valuable toprevent and cure this bone disease. Pharmacotherapeutic options, include bisphosphonates,calcitonin, estrogen or hormone therapy, estrogen agonist/antagonist, and parathyroidhormone (PTH) medications. They are effective in a certain degree，but they are easy tocause side effects.Wnt signaling pathway plays an important role in controlling bone formation andresorption, thus presenting a potential target for the development of novel bone-buildingdrugs. However, controversies exist with regard to several issues, including safety, dosage,duration of treatment, and mechanisms of intervention on osteoblastogenesis andosteoclastogenesis. Bone mass is influenced by the balance achieved between bone-formingcells (osteoblasts) and bone-resorbing cells (osteoclasts). Several lines of evidence suggestthat Wnt/β-catenin signaling is essential for bone formation and resorption. Loss-of-function mutations in human LRP5are associated with osteoporosis-pseudogliomasyndrome, which is characterized by low bone mineral density and skeletal fragility. Toexamine the importance of β-catenin later in osteoblast development, constitutively activeβ-catenin was overexpressed in osteoblasts expressing collagen αI-CRE,these micemanifest an osteopetrotic phenotype. However, controversies exist regarding the role of theWnt/β-catenin signaling pathway in osteoblastogenesis and osteoclastogenesis. Using amouse model lacking the β-catenin, Holmen et al. showed that mutant mice have low bonemass, owing to accelerated bone resorption. Similar results were in Kramer’s observation.However, controversies exist in Cawthorn and Riddle’s reports. Chen’s experimentsindicate that stabilization of β-catenin in both early and late postnatal stages significantlyincreased trabecular bone volumes through increasing vertebral bone formation and decreasing bone resorption.In the present study, we investigated the effects of CA-β-catenin on vertebral bonegrowth and remodeling in mice at different stages using pro-collagen I Cre-ERTMas thepromoter, which could be activated at designated time-points via injection of tamoxifen.Methods:Part I Effects and mechanism of constitutive activation of β-catenin on bonemass at different postnatal stages in miceCatnblox(exon3) mice were crossed with mice expressing the TM-inducible Cre fusionprotein, which could be activated at designated time points via injection of tamoxifen.β-catenin was stabilized by tamoxifen injection3days, and2,4,5, and7months after birth,and the effects lasted for one month.1. Physical examination was carried out including:body height, height of limbs etc;2. X-Ray and Micro-CT were used to analyze the bone mass and bone structure ofmice;3. HE staining and Safranine O staining were used to analyze the bone Microscopicmorphological changes;4. TRAP staining was used to evaluate the number of osteoclast;5. IHC staining was used to observe the expression of BSP、RUNX2、TNAP;6. Real-time quantitative RT-PCR was used to evaluate the mRNA expression levels ofTNAP，Runx2，BSP，TRAP，RANKL，OPG;7. Serum ELISA for PINP、OC、CTX-I、TRAcP5b were used to investigate boneformation and resorption.Part II Effects and mechanism of constitutive activation of β-catenin on bonegrowth at different postnatal stages in mice1. Generation of mice（like above）;2. HE staining and Safranine O staining were used to observe the growth platemorphology;3. The concentration of serum calcium and phosphorus were measured;4. IHC staining was used to observe the expression of FGF23; 5. TUNEL was used to assay vertebral growth plate chondrocyte apoptosis.Results:1. Constitutive activation of β-catenin at both early (day3) and late stages (months2,4,5and7) significantly increased trabecular bone volumes in mice：at early stage，bone massincreased300%；at late stage，180—200%;2. The location and extent of maturation of excessive formed bone varied withdifferent stages of β-catenin stabilization. Upon induction of β-catenin stabilization fromday3to month1of life, excessive formed bone was located in close proximity to thegrowth plate; while mice with β-catenin stabilization at the late stages (2,4,5and7monthsafter birth) displayed even distribution of excessive bone in the vertebral body;3. IHC analyses showed higher expression of TNAP protein in ca-β-catenin thanwild-type mice, indicative of increased bone formation ability; Runx2and BSP are markersfor osteoblasts at the early and late stages, respectively.Notably, the numbers of bothRunx2and BSP-positive cells in CA-β-catenin mice were greater than those in wild-typemice in both early and late postnatal stages. The results of real-time PCR and ELISA furthersupported the histology findings.4. When β-catenin was stabilized with Col1-CreERTMas a promoter from day3of life,mice displayed shorter stature in their first month of life, compared with wild-type mice.H&E and Safranin O staining showed that growth plates in ca-β-catenin mice were longerthan that in wild-type mice,The hypertrophic layer of the growth plate was the mostenlarged part. And there is no obvious apoptosis in the growth plate. In contrast, β-cateninstabilization at months4,5, and7of life had no evident effect on growth plate length andstatures.5. When-catenin was stabilized at early stage, CA--catenin mice demonstratedhypophosphatemia, but the concentration of serum calcium was not affected, IHC analysesshowed higher expression of FGF23protein in ca-β-catenin than wild-type mice. Whilemice with β-catenin stabilization at the late stages (2,4,5and7months after birth), theconcentration of serum phosphorus was still lower than that of wild-type mice. But theconcentration of serum calcium and expression of FGF23protein were not affected. Conclusions:CA-β-catenin at different stages after birth can increase mice vertebral bone volume.This increased bone volume was the result of increased bone formation and decreased boneresorption caused by CA-β-catenin. However, stabilization of β-catenin at early stages afterbirth hinders linear growth in mice. The possible mechanism is that hypophosphatemia andhigher expression of FGF23protein hindered the development of growth plate.