Functional And Mechanism Study Of FGF9 On Joint,Tooth Development And Bone Metabolism

Fibroblast growth factor 9（Fgf9）is a member of FGF family.It plays important roles in inheritant disease,organ development and homeostasis.However,the underlying regulatory mechanism is unknown,especially in joint development,tooth development and bone homeostasis.Human Multiple Synostoses Syndrome（SYNS）is an autosomal dominant disorder with involvement in proximal interphalangeal,carpal tarsal,humeroradial and cervical spine joints.Mutation（Ser99Asp）on FGF9 has been identified to be responsible for SYNS3.To investigate the role of FGF9 in SYNS3,we generated a mouse model with S99N mutation(Fgf9^mt/mt)which exhibited similar multiple joint synostosis symptom as in human SYNS3 patients.We find that Fgf9inhibits mesenchymal cell differentiating into chondrocytes through downregulating chondrogenic transcriptional factor Sox6 and Sox9,and up-regulates the expression level of Gdf5 through activating its promoter activity.It plays important roles in the matainance of interzone.Molecular simulation and in vitro binding assays suggest that S99N mutation in Fgf9 leads to a conformational change which shows higher affinity to heparin,resulting in a binding competition between Fgfr and heparin binding site,which impedes the Fgf9 signaling transduction during the early stage of joint development.During exploring the mechanism of FGF9 S99N in SYNS3,we accidentally discovered Fgf9 plays crucial role in bone homeostasis.In the bone of mutant mice,the accounts of osteoblast cells increased along with the enforcement of the cell functions.In addition,the osteoclast cells decreased in cell number and size.All these lead to the increase of BMD in mutant mice.In vitro differentiation assay shows that the S99N mutation not only significantly enhanced the ability of BMSC osteigenic differentiation and calcification,but also inhibited osteoclast cells differentiation and maturation.At the same time,we found that Fgf9 could significantly down regulated the osteogenic genes in osteoblast cell through MAPK and PI3K/AKT signaling pathways.These results indicates that Fgf9 is a negative regulator of bone homeostasis.Besides,we also find that Fgf9 plays important roles in tooth development.Mice with S99N mutation exhibited deflection of upper and lower incisors and enamel dysplasia.Afterwards,the inner enamel epithelium in mutant mice prominently decreased and disorderly arranged.And also,enamel formation related genes were significantly down regulated in mutant mice.In addition,we proved that FGF9signaling transduction was significantly attenuated in the tooth of mutant mice.It not changes the ameloblast cells proliferation and apoptosis,but affects the expression level of ameloblast regulatory gene Shh,which implies that Fgf9 might regulate the ameloblast cells differentiation and maturation.