Role Of FGFR2in Calvarial Defect Healing And Bone Formation

Fibroblast growth factors and their receptors (FGFs/FGFRs) play important roles inbone development and growth. Mutations of FGFRs can lead to many human skeletongenetic disorders，such as achondroplasia (ACH), craniosynostosis (CS) and so on. FGFRsbelong to the tyrosine kinase receptor family, which is consist of three parts: extracellularligand binding domain, transmembrane domain and highly conservative intracellular signaltransduction domain. Like other tyrosine kinase receptors, after combined with FGFs,FGFRs undergo dimerization to activate the intracellular tyrosine kinase activity, and thentransfer the signals through Ras-MAPK, PI3K-AKT, PLC-γ signaling pathways.FGFR2is an important memberof the FGFRs family. Multiple studies have shown thatmutations of FGFR2can affect bone development and result in craniosynostosis. Twogain-of-function mutations of FGFR2(S252W, P253R) are responsible for Apert syndrome,which is characterized by craniosynostosis, short head deformities and craniofacialdysplasia with serious syndactylia. Loss-of-function mutions of FGFR2causes Bent BoneDysplasia, which is characterized by craniosynostosis, dysmorphic facial features and bentlong bones. Fgfr2knockout mice die in embryonic day10.5, and have multiple organdevelopmental disorders including limbs. Conditional knockout of Fgfr2mediated byDermo1-cre results dwarfism and decreased bone mass in mice.Calvarial defect healing is a complicated regeneration process which is similar withskull development to some degree. A variety of molecules involved in skull developmentalso play important roles during calvarial defect healing such as Wnts, FGFs, BMPs, etc.Given that FGFR2plays an important role in skull development, we speculate that FGFR2may participate in the regeneration process after calvarial defect, but its specific functionand related mechanisms are not clear. So in the first part of the experiment, we inducedgain-of-function mutation of FGFR2(inducible expression P253R mutation) in adultmice, observed the influence of FGFR2on calvarial defect healing through establishing ofcalvarial defect model and discussed the possible mechanisms. Mechanical bone marrow ablation (BMX), which causes injury inducedintramembranous bone formation, is a very useful experimental model to study genes orgrowth factors involved in bone formation and determine their roles in bone turnover. Inthe view of that both of the process of bone formation after BMX and calvarial defect areintramembranous ossification, we further used the BMX model to study the role of FGFR2in bone formation.Methods:PartⅠ: The role of FGFR2in calvarial defect healing1．calvarial defects were made in FGFR2P253R/+mice;2．Micro-CT was used to dynamically observe the healing process2weeks,4weeks,8weeks after calvarial defect; defect healing area were also Quantitatively measured;3．X-Ray was used to analyze the calvarial defect healing8weeks after calvarialdefect; H.E staining was used to observe the new bone formation around defect edge8weeks after calvarial defect;4．Quantitative PCR was used to detect the mRNA levels of Runx2、ColI、OP、OC2weeks after calvarial defect;5．Osteoblast cell culture was used to examine the role of FGFR2on osteoblastproliferation and differentiation; the protein level of phosphorylated ERK1/2was detectedby western blot;6．Quantitative PCR was used to detect mRNA levels of canonical Wnt/β-cateninpathway genes Dvl2, β-catenin, Tcf-1in osteoblasts.PartⅡ: The role of FGFR2in bone formation after mechanical bone marrowablation1．X-Ray and Micro-CT were used to analyze newly formed bone tissue1and2weeksafter BMX;2．Histomorphometric measurement and quantitative PCR were used to examine boneformation after BMX;3．Trap staining and quantitative PCR were used to examine trabecular osteoclasticbone resorption. Results:PartⅠ: FGFR2P253R/+mice showed accelerated defect healing and enhancedosteogenesis.1．Micro-CT, Radiography and H.E staining results showed that FGFR2P253R/+micehad accelerated defect healing ability.2．FGFR2P53R/+osteoblasts showed accelerated proliferation and differentiation; Theexpressions of bone markers including Runx2, ColI and OP were increased, and the activityof ERK1/2pathway was enhanced in FGFR2P253R/+osteoblasts.3．The expression levels of Dvl2, Tcf-1mRNA were increased in FGFR2P253R/+osteoblasts.PartⅡ: FGFR2P253R/+mice showed increased bone formation and delayed boneresorption after BMX.1．Radiography and Micro-CT results showed that FGFR2P253R/+mice had increasedbone formation1and2weeks after BMX.2．Osteoblast number and osteoblast differentiation transcriptional regulator Runx2were increased in FGFR2P253R/+mice after BMX.3．Osteoclast surface area was reduced in FGFR2P253R/+mice1weeks after BMX,while osteoclast surface area and the ratio of RANKL to OPG mRNA levels were increasedin FGFR2P253R/+mice2weeks after BMX.Conclusions:1．FGFR2promotes calvarial defect healing.2．FGFR2accelerates the proliferation of osteoblasts, and promotes the differentiationof osteoblasts partially by up-regulating Runx2and through ERK1/2MAPK pathway.3．FGFR2accelerates osteoblasts differentiation probably through activating thecanonical Wnt/β-catenin signaling pathway.4．FGFR2increases trabecular bone formation and delays trabecular bone resorptionafter BMX.