Mutant hFGF23(A12D) stimulates osteoblast differentiation through FGFR3

Abstract 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.

reported genome-wide linkage and whole-exome sequencing analyses on an MP pedigree, and in three unrelated MP patients, we discovered a mutation of FGF23 c.35C>A. 12 The treatment of mandibular prognathism in clinical includes orthodontic therapy and orthognathic surgery. Both of them involve bone remodelling which lead us to consider how mutant hFGF23 would affect bone remodelling during treatment.
In this study, we used osteoblasts to evaluate the direct function of FGF23 (A12D) in bone development. We determined that hFGF23(A12D) inhibited proliferation of RC cells and stimulated the differentiation of RC cells to osteoblasts through activating the mitogen-activated protein kinase (MAPK) signalling pathway via interactions with the intracellular domain of FGFR3.

| Isolation, infection and induction of RC Cells
Male Sprague Dawley (SD) rats were purchased from the Shanghai

| Enzyme-linked immunosorbent assay
After the cells were infected and osteogenically induced, conditioned media were collected and stored at −80°C until use. FGF23 was determined using the human FGF23 enzyme-linked immunosorbent assay (ELISA) kit (BiotechWell, Shanghai, China), according to the manufacturer's instructions.

| Alkaline phosphatase staining and quantification
Alkaline phosphatase staining was performed using the Alkaline

| Alizarin red staining
Cells were stained in Alizarin red staining solution (Cyagen Biosciences, Suzhou, China), according to the manufacturer's instructions, and images were taken with a stereomicroscope.
Subsequently, cells were submerged in 1 mL 10% hexadecylpyridinium chloride for 30 minutes at 37°C. Finally, a microplate reader was used to measure the absorbance at 562 nm. The products were verified by melting curve analysis, and the relative gene expression levels were calculated by the 2 −ΔΔCT method.

| RNA sequencing
Total RNA was extracted with the TRIzol reagent on the ninth day of osteogenic induction. First, we checked total RNA quality.
Subsequently, mRNA was purified with oligo-dT, and the retrieved RNA was fragmented to obtain 100-300 bp fragments. The first and second cDNA strands were synthesized. Double-strand cDNA was purified with magnetic beads and resolved in elution buffer for end repair and adenylated 3′ ends. After ligating adapters and performing size selection, the products were amplified to build the cDNA library. Sequencing was performed by the Illumina Hiseq 2500 (Illumina Inc, CA, USA) after a library quality check. Cuffdiff software was used to analyse the differential expression, and P < 0.05, |log2 (fold change)|>1 and q value <0.05 were considered significant.

| Transient transfection of 293 T cells
The

| Western blotting
Cells were lysed in cold RIPA buffer with protease inhibitors After washing three times with TBST, membranes were incubated for 1 hour at room temperature with secondary antibodies. Finally, membranes were washed three times with TBST, and data were captured with a chemiluminescence detection system (GE AI600, Boston, MA, USA).

| Statistics analysis
All data were analysed by one-way ANOVA and independent t tests.
P < 0.05 was considered significant.

| Mutant hFGF23(A12D) inhibits proliferation ability of RC cells
Given that RC cells can be induced to differentiate into osteoblasts, 9 we considered whether mutant hFGF23(A12D) would have an influence on RC cells. In order to determine the effect of mutant hFGF23(A12D) on cell proliferation ability, we performed the CCK-8 assay in rLV-mCMV-ZsGreen, hFGF23(A12D) and hFGF23-WT cells.
As shown in Figure 2A, on the first and second day, cell growth ability was not different among the three groups. From the third day up to the sixth day, cell proliferation was significantly decreased in the hFGF23(A12D) group, whereas the hFGF23-WT group showed no significant difference from the control, except on day 4 when the proliferation of hFGF23-WT cells was significantly higher than that of control cells ( Figure 2B).
Taken together, these results indicate that mutant hFGF23(A12D) inhibits the proliferation ability of RC cells, while hFGF23-WT does not. The inhibition could owe to cell transformation and differentiation ( Figure S1).

| Mutant hFGF23(A12D) stimulates differentiation from RC cells to osteoblasts and inhibits matrix mineralization
To address whether hFGF23(A12D) effects cell differentiation from RC cells to osteoblasts, we performed osteoblast induction in RC cells, including the rLV-mCMV-ZsGreen, hFGF23(A12D) and hFGF23-WT groups. First, we analysed ALP activity in three groups by ALP staining. As shown in Figure 2C, Furthermore, we detected matrix mineralization by Alizarin red staining (stains calcium nodules) on day 21 in induced osteoblasts from these groups. As shown in Figure 2E, the number of Alizarin In contrast, hFGF23-WT can inhibit both differentiation and matrix mineralization.

| Mutant hFGF23(A12D) activates MAPK signalling pathway and increases FGFR3 expression during osteoblast differentiation in RC cells
In order to determine how mutant hFGF23(A12D) influences osteoblast differentiation, we performed RNA-sequence analysis of the induced osteoblasts. As shown in Figure 3A and Table 2  Together, these observations suggest that mutant hFGF23(A12D) activates the MAPK signalling pathway and increases FGFR3 expression during osteoblast differentiation from RC cells.

| Mutant hFGF23(A12D) activates MAPK signalling pathway through interaction with intracellular domain of FGFR3
Given that our results showed that mutant hFGF23(A12D) can activate the FGFR3-dependent MAPK signalling pathway, and mutant hFGF23(A12D) failed to be secreted by osteoblasts, we considered whether hFGF23(A12D) may function through the FGFR3 intracellular domain. First, we performed a bioinformatics analysis to predict whether the hFGF23(A12D) protein could bind to the intracellular domain of FGFR3. As shown in Figure 4A, results showed that the predicted value of Delta G (binding free energy) for the binding of the

| D ISCUSS I ON
It is well known that hFGF23 belongs to a subfamily of mammalian endocrine FGFs, which can reduce phosphate reabsorption and suppress the production of 1,25(OH)2D through reduction of 1α-hydroxylase. 13 However, the function of mutant FGFs has not been clearly defined. Here, we clearly demonstrate that mutant hFGF23(A12D) stimulates the differentiation of RC cells to osteoblasts through the MAPK signalling pathway via FGFR3 activation, which interacts with hFGF23(A12D) through its intracellular cytoplasmic tyrosine kinase domain ( Figure 4D). In this study, for the first time, we have identified a non-secretory mutant hFGF23(A12D), which plays an important role in osteoblast differentiation.  This suggests that the function of mutant hFGF23(A12D) during RC cell differentiation is different from wild-type FGF23. We postulated that this is related to the mutation of A12D, which is located in the hydrophobic core of the signal peptide, and blocks hFGF23(A12D) secretion. Conversely, a previous study showed that hFGF23 also inhibited osteoblast mineralization through FGFR1c, 9 while our results showed that compared to hFGF23, mutant hFGF23(A12D) less slightly inhibited mineralization in osteoblasts, which suggests that mutant hFGF23(A12D) is involved in osteoblast mineralization through a different pathway than hFGF23-WT.
Our study also suggests that mutant hFGF23(A12D) inhibited in the presence of alpha-Klotho. 10 We predicted that the difference in secondary structure ( Figure S4) between hFGF23-WT and hFGF23(A12D) may result in novel functions. Therefore, we suggested that mutant hFGF23(A12D) can bind to certain intracellular signalling molecules. Notably, the results of bioinformatics analysis, co-IP and immunofluorescence staining revealed that mutant hFGF23(A12D) interacts with the intracellular domain of FGFR3. Therefore, we have reason to consider that the non-secretory mutant hFGF23(A12D) functions through intracellular action.
FGFR3 is one of a family of four membrane-bound tyrosine kinase receptors, largely expressed in cartilage and bone, 17  However, heterozygous mice showed no significant differences in appearance, 8  In conclusion, we demonstrated that compared to hFGF23-

CO N FLI C T S O F I NTE R E S T
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

AUTH O R S' CO NTR I B UTI O N
Y. Tu, contributed to conception, design, acquisition, analysis, interpretation, drafted manuscript and critically revised manuscript; T. Qu, contributed to design, analysis, drafted manuscript; F Chen, contributed to conception, design and critically revised manuscript.
All authors gave final approval and agrees to be accountable for all aspects of work ensuring integrity and accuracy.