Mechanism of FGF23 processing in fibrous dysplasia

Abstract

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G(s) α that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G(s) α mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients.

Fig. 1.

The ratio of total FGF23 to intact FGF23 in human plasma. (A) The ratios of total FGF23 (intact+C-terminal FGF23) to intact FGF23 (iFGF23) are shown in subjects with fibrous dysplasia (FD), other disease states, and controls. Other disease states include tumor-induced osteomalacia (TIO), X-linked hypophosphatemia (XLH), and renal failure (RF), as well as normal control subjects (Normal). Significant (p < 0.05) and nonsignificant (p = NS) are indicated. Of note, the difference between the FD group and the XLH group was not significant. (B) The total (intact+C-terminal) FGF23 values, the relationship between the two as defined by the regression line, and the slope and p value of the line are shown for the subjects with fibrous dysplasia. (C) The total and intact FGF23 values, the relationship between the two as defined by the regression line, and the slope and p value of the line are shown for all other subjects with TIO, XLH, RF, and Normal (NV). FGF23 was measured by two different ELISA assays; one that detects total FGF23 (C-terminal+intact FGF23) and one that detects only iFGF23. Differences between groups were assessed by one-way ANOVA using Dunn’s multiple comparison test, and linear regression was assessed by best fit.

Fig. 2.

cAMP levels, FGF23, O-linked glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3), and furin expression in human bone marrow stromal cells (BMSCs). (A) Media and cell lysate cAMP levels were measured by ELISA in BMSCs that had been derived from a subject with fibrous dysplasia (FD). Because patients with FD are mosaics for mutations in G_sα, primary cultures of cells from a bone specimen were subcloned, and pure colonies bearing WT G_sα sequence R201R or FD-causing G_sα sequence R201H (FD) were studied. cAMP levels were higher in lysates from mutant cells (error bar=1 SD, p < 0.05). (B) Products of RT-PCR amplification reactions performed with total RNA isolated from cultured BMSCs from the WT and FD cells are shown and demonstrate the presence of FGF23, ppGaNAcT3, and furin transcripts in BMSCs.

Fig. 3.

Colocalization of FGF23 and O-linked glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) or furin in normal (WT) and fibrous dysplasia bone marrow stromal cells. Confocal microscopy was performed for FGF23, ppGalNAcT3 (A,B), and furin (C,D) in wild-type (WT; A,C) and fibrous dysplasia (FD; B,D) bone marrow stromal cells. Because patients with FD are mosaics for mutations in G_sα, primary cultures of cells from a bone specimen were subcloned, and pure colonies bearing WT G_sα sequence R201R or FD-causing G_sα sequence R201H (FD) were studied. Cells were grown on glass coverslips, fixed, and immunostained. Throughout, FGF23 is stained in green, ppGalNAcT3 or furin in red, and nuclei in blue (DAPI). Merged images are indicated. FGF23 is colocalized with ppGalNAcT3/furin in a perinuclear, Golgi-associated pattern. Representative data from two separate experiments are shown.

Fig. 4.

O-linked glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and furin enzymatic activities in wild-type and fibrous dysplasia bone marrow stromal cells. (A) ppGalNAcT3 enzymatic activity was measured in vitro from equal amounts of wild-type (WT) and fibrous dysplasia (FD) cell lysates using a ppGalNAcT3-specific HIV gp 120-specific peptide as a substrate. Because patients with FD are mosaics for mutations in G_sα, primary cultures of cells from a bone specimen were subcloned, and pure colonies bearing WT G_sα sequence R201R or FD-causing G_sα sequence R201H (FD) were studied. FD BMSCs demonstrated significantly less ppGalNAcT3 activity than WT cells (p < 0.05), consistent with underglycosylation leading to increased furin susceptibility and FGF23 processing. Substrate specificity for HIV gp120 peptide was tested by using purified ppGalNacT1, T2, and T3 (right panel) (see Supplemental Fig. S2 for additional controls). (B) Furin enzymatic activity was measured in equal numbers of WT and FD BMSCs using a fluorogenic substrate. FD cells had significantly greater furin activity than WT cells (p < 0.05). Furin activity in WT cells was increased to that of FD cells by treatment with the nonhydrolyzable analogue of cAMP, dibutylryl cAMP (dbcAMP), or adenylyl cyclase activator forskolin.

Fig. 5.

FGF23 production by HEKF cells transfected with wild-type or mutant G_sα. FGF23-producing HEKF cells were transiently transfected with either wild-type (WT) or fibrous dysplasia-causing G_sα mutants (R201C or R201H) to assess for the effect of increased cAMP caused by these mutations on FGF23 processing. (A) Cells transfected with mutated G_sα constructs had significantly higher levels of cAMP. (B) Transfected cells produced very high levels of FGF23 with relatively higher levels of total (intact + C-terminal FGF23) in all transfected cells. (C) However, the relative levels of total FGF23 to intact (iFGF23) were not higher in the cells bearing the mutated G_sα constructs, nor were the levels of total FGF23 different between cells transfected with WT versus mutant G_sα constructs. (D) Regression analysis of the relationship between total FGF23 versus intact FGF23 revealed there were significantly higher levels of total FGF23 than intact FGF23, consistent with greater processing of intact FGF23 in HEKF cells overexpressing G_sα and cAMP.

Fig. 6.

O-linked glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and furin enzymatic activities in HEKF cells expressing activating G_sα mutants. (A) ppGalNAcT3 enzymatic activity was measured in vitro from equal amounts of cell lysates from parental HEKF cells and HEKF cells transfected with equal levels of wild-type (WT) or constructs bearing the fibrous dysplasia-causing activating mutations of G_sα (R201C and R201H). The ppGalNAcT3-specific HIV gp 120-specific peptide was used as a substrate. All cells transfected with additional G_sα demonstrated significantly less ppGalNAcT3 activity than parental HEK cells (p < 0.05), and cells with activating mutations demonstrated less activity than cells transfected with additional WT G_sα, consistent with underglycosylation leading to increased furin susceptibility and FGF23 processing. (B) Furin enzymatic activity was measured in equal numbers of HEKF and HEKF with G_s constructs using a fluorogenic substrate. Cells with either the R201C- or R201H-activating mutations of G_sα had significantly greater furin activity than cells with WT G_sα (p < 0.05).