Study On The Development Of Endochondral Bone Formation Caused By FGFR2Gain-of-fuction Mouse In Early Postnatal Stage

Bones are formed by one of two types of ossification processes, intramembranous orendochondral bone formation. Endochondral bone formation includes cartilage formation andbone formation. Normal development of endochondral bone is dependent on a balancebetween chondrocyte proliferation and differentiation to achieve appositional and interstitialgrowth, and vascular invasion of cartilage to allow for bone deposition. The growth plate notonly is an important place to achieve longitudinal growth of long bone, but also regulates thelongitudinal bone growth rate during endochondral bone development.It is widely accepted that, endochondral bone formation is regulated by interdependentsignal pathways downstream of locally derived growth factors, such as fibroblast growthfactors(FGFs). These FGFs and their receptors are expressed in a time-dependent manner andin spatially restricted patterns in cells of the epiphyseal cartilage of developing bones.Precious researches showed activating mutations in FGFRs results in endochondral boneformation disorders that is characterised by a reduction in proliferating chondrocytes anddefective bone formation.As one of the most severe forms of craniosynostosis, Apert syndrome(AS) is a clinicallydistinct condition characterized by premature fusion of coronal sutures, craniofacial anomalies,and syndactyly of the digits. Remarkably, Apert syndrome is caused by a limited number ofmutations. Ninety-nine percent of reported cases have one of two missense mutations inadjacent amino acids, Ser252Trp and Pro253Arg, of fibroblast growth factor receptor2(FGFR2), The first mutation, S252W, is more common, occurring in67%of patients. Bothmutations affect the highly conservedlinker region between the immunoglobulin-like II and III domains and result in increasedaffinity and altered specificity of fibroblast growth factor(FGF) ligand binding. In addition to the abnormal osteogenesis, chondrogenesis is also affected in patients withApert syndrome. The cranial base abnormalities and epiphyseal dysplasia were observed inApert syndrome patients. Moreover, cartilage abnormalities have been reported previously inseveral mouse models with different mutated FGFR2, including the small body size, retardeddevelopment of the axial and appendicular skeleton and growth arrest of the skull base.FGFR2can activate two primary pathways, the mitogen-activating protein kinase pathwayand protein kinase C pathway. Signaling through FGFR2regulates stem cell proliferation,affecting different lineages such as osteoblasts and chondroblasts.Methods:We analyzed an Apert syndrome mouse model carrying the Fgfr2+/S252Wmutation into themouse genome using gene targeting. We found that the mutant mice exhibitedcraniosynostosis accompanied by short stature, which may be caused by the collective effectsof mutant FGFR2on the chondrocyte and osteoblast lineages. To determine how gain offunction of FGFR2interact during bone development, we generated mutant mice Fgfr2+/S252W,compared the skeletons of newborn pups and the differences of BMSCs bone formationbetween wild and mutant littermates at postnatal stage. Furthermore, we showed that the p38and Erk1/2pathway may play critical roles in the growth retardation of long bone in micewith Fgfr2+/S252Wmutation.Results:1. Fgfr2+/S252Wmouse model exhibited craniosynostosis accompanied by short stature,which phenotypically mimicked that of Apert syndrome patients.2. The growth retardation of long bones were the direct effects of the FGFR2mutation,and the appearance of the secondary ossification center in the tibia of wild-type mouse but notin mutant mouse at P10.Further the density of bone and number of trabecular in metaphysicwere weaker in mutant mice.3. H&E staining showed noticeable shortened zones of proliferating and hypertrophicchondrocytes, and decreased trabecular bone areas in the growth plates of the mutantmice.The decreased both expressions of Col2, Col10in proliferating and hypertrophicchondrocytes were in domain size and intensity, reflecting the decreased length of this zone.4. The proliferation of2-generation BMSCs of mutant Fgfr2+/S252Wwas slower andBMSCs with both Alcian blue and Alizarin red staining showed decreased staining cells in cultured Fgfr2+/S252WBMSCs compared with wild type BMSCs on day14.5. Expressions of the mRNA of chondrocytes differentiation from mutant group weredecreased, while those of osteoblast differentiation level showed an increased trend in mutant.6. The activity of p38and Erk1/2pathway of Fgfr2+/S252Wwas enhanced, which may beinvolved in the effect of FGFR2-regulated chondrocytes and osteoblasts proliferation anddifferentiation. Treatment of the cultured BMSCs with SB203580or PD98059, the p38andErk1/2signaling pathway inhibitor, resulted in increase the expression of osteogenic andchondrogenic marker genes respectively. Especially the levels of both osteogenic andchondrogenic differentiation marker mRNAs were significantly up-regulated in BMSCs fromboth wild-type and Fgfr2+/S252Wmice by SB203580.7. Using in vitro culture of long bones, we found the retardation of total length growth oflong bones has been completely rescued by SB203580treatment, suggesting that p38signaling pathways was more responsible for the retarded long bone development inFgfr2+/S252Wmice.Conclusions:A new Apert syndrome mouse model with Fgfr2+/S252Wmutation was successfullycreated, which was a useful model to research Apert syndrome. Continued enhanced functionof FGFR2mutation acted negative regulator of endochondral bone formation. The mutantmice being were slightly smaller than wild-type littermates and exhibiting dome-shaped skulls.We found abnormal bone epiphyseal growth plates was the key to cuase retarded growth oflong bones. In vitro we cultured BMSCs in chondrogenic differentiation conditions torevealed gain-of-function mutation of FGFR2inhibied the proliferation, chondrogenicdifferentiation, and bone matrix mineralization of BMSCs. However the mutant BMSCsexpressed high osteogenic marker genes comparison with wild-type mice. More importantly,the results indicated that these effects are mediated by the p38and Erk1/2signaling pathway.Furthermore, the retardation of long bones has been completely rescued by SB203580treatment, suggesting that p38signaling pathways was more responsible for the retarded longbone development in Fgfr2+/S252Wmice.