Genetic evidence of serum phosphate-independent functions of FGF-23 on bone

Despina Sitara, Somi Kim, Mohammed S Razzaque, Clemens Bergwitz, Takashi Taguchi, Christiane Schüler, Reinhold G Erben, Beate Lanske, Despina Sitara, Somi Kim, Mohammed S Razzaque, Clemens Bergwitz, Takashi Taguchi, Christiane Schüler, Reinhold G Erben, Beate Lanske

Abstract

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23-/-) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23-/- mice and to examine serum phosphate-independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23-/- mice on phosphate homeostasis and skeletal mineralization. Fgf-23-/-/NaPi2a-/- double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23-/- animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23-/- mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23-/-/NaPi2a-/-, their skeletal phenotype still resembles the one of Fgf23-/- animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23-/- mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Macroscopic phenotype of Fgf-23 −…
Figure 1. Macroscopic phenotype of Fgf-23 − /− /NaPi2a − /− double mutants.
(A) Gross phenotype of control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/− animals at 6 weeks of age. (B) Growth curves and (C) survival ratios for all four genotypes. Data are represented as mean ±SEM (** p<0.01).
Figure 2. Bone mineralization analysis.
Figure 2. Bone mineralization analysis.
(A) Total bone mineral content (BMC; each value obtained for BMC was normalized to the body weight of the corresponding animal). (B) Bone mineral density (BMD) of hind-limbs by Piximus, and (C) pQCT of control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/− animals. (Statistical significance * p<0.05, ** p<0.01,*** p<0.001. Black asterisks represent comparison with control, red with Fgf-23−/−, and blue with Fgf-23−/−/NaPi2a−/−).
Figure 3. Biochemical measurements.
Figure 3. Biochemical measurements.
(A) serum phosphate, (B) serum calcium, (C) calcium-phosphate product, (D) urinary phosphate, (E) fractional renal tubular reabsorption of phosphate (TRP), (F) serum 1,25(OH)2D3, and (G) serum PTH levels in control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/− animals. (H) serum phosphate levels before and after injections with vehicle, PTH (1–34) or PTH (3–34). (Statistical significance *p<0.05, **p<0.01, *** p<0.001. Black asterisks represent comparison with control, red with Fgf-23−/−, and blue with Fgf-23−/−/NaPi2a−/−.)
Figure 4. Histological analysis of bones by…
Figure 4. Histological analysis of bones by von Kossa and Alizarin Red S staining.
(A) Alizarin Red S stained ribs from all genotypes at 6 weeks of age. (B) Three-µm-thick undecalcified sections from 3- and (C) 6 week-old control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/− bones were stained with von Kossa/McNeal. Top panels: tibial growth plate and trabecular bone (magnification x100); lower panels: tibial secondary spongiosa (magnification x400). Black staining represents mineralization. At 6 weeks, more mineral deposition is found in the area below the growth plate (methaphysis) in Fgf-23−/− mice and Fgf-23−/−/NaPi2a−/− double mutants. In addition, areas of unmineralized osteoid (light blue) are found similarly in the secondary spongiosa of Fgf-23−/− and Fgf-23−/−/NaPi2a−/− mice.
Figure 5. In situ hybridization.
Figure 5. In situ hybridization.
Riboprobes for (A) collagen type X (Col X), (B) dentin matrix protein-1 (DMP-1), and osteopontin (OPN) on sections from tibia of control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/−at 3 and 6 weeks.
Figure 6. Histological analysis of soft tissues.
Figure 6. Histological analysis of soft tissues.
Hematoxylin and Eosin-stained sections of intestines and lungs from 6 week-old control, Fgf-23−/−, Fgf-23−/−/NaPi2a−/−, and NaPi2a−/−. Intestinal sections from Fgf-23−/− mice reveal reduced height of intestinal villi and atrophy of intestinal mucosa. In addition, Fgf-23−/− mice exhibit lung emphysema. These features are significantly improved in Fgf-23−/−/NaPi2a−/− mice (magnification ×2.5).
Figure 7. Alizarin Red S staining of…
Figure 7. Alizarin Red S staining of wild-type calvarial osteoblasts treated with either mineralization medium alone or with medium containing hFGF23 protein for 21 days.
Top panels show vehicle treated wild-type cells (n = 15) and bottom panels show wild-type cells treated with hFGF-23 (n = 15).

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