Mutant hFGF23(A12D) stimulates osteoblast differentiation through FGFR3.

Fibroblast growth factor (FGF) 23 is a member of the FGF family involved in bone development by interacting with FGFRs. In a previous study, we discovered a mutant human FGF (hFGF) 23 (A12D) in the mandibular prognathism (MP) pedigree. However, the exact role of hFGF23(A12D) during bone formation remains unclear. The aim of this study was to identify the function of hFGF23(A12D) in bone formation. We infected isolated rat calvaria (RC) cells with the recombinant lentivirus containing mutant hFGF23(A12D) and WT hFGF23 respectively. Real-time PCR, western blot and enzyme-linked immunosorbent assay confirmed that hFGF23(A12D) failed to be secreted. We measured cell growth via the CCK-8 assay based on Zsgreen expression, detected cell differentiation ability via alkaline phosphatase staining, performed RT-PCR and found that hFGF23(A12D) inhibited proliferation of RC cells and stimulated the differentiation of RC cells to osteoblasts. Through RNA sequencing, RT-PCR and western blot, we found increased expression of FGFR3. Through co-immunoprecipitation assays and immunofluorescence staining, we revealed that hFGF23(A12D) activated the mitogen-activated protein kinase signalling pathway through interactions with the intracellular domain of FGFR3. In summary, we determined the mechanisms of hFGF23(A12D) involved in osteoblast generation and formation which is specifically due to its interaction with FGFR3.