We analyzed the effects of an FGF‐23 injection in vivo. FGF‐23 caused a reduction in serum 1,25‐dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF‐23 is a potent regulator of the vitamin D and phosphate metabolism. Introduction: The pathophysiological contribution of FGF‐23 in hypophosphatemic diseases was supported by animal studies in which the long‐term administration of recombinant fibroblast growth factor‐23 reproduced hypophosphatemic rickets with a low serum 1,25‐dihydroxyvitamin D [1,25(OH)₂D] level. However, there is no clear understanding of how FGF‐23 causes these changes. Materials and Methods: To elucidate the molecular mechanisms of the FGF‐23 function, we investigated the short‐term effects of a single administration of recombinant FGF‐23 in normal and parathyroidectmized animals. Results: An injection of recombinant FGF‐23 caused a reduction in serum phosphate and 1,25(OH)₂D levels. A decrease in serum phosphate was first observed 9 h after the injection and was accompanied with a reduction in renal mRNA and protein levels for the type IIa sodium‐phosphate cotransporter (NaPi‐2a). There was no increase in the parathyroid hormone (PTH) level throughout the experiment, and hypophosphatemia was reproduced by FGF‐23 in parathyroidectomized rats. Before this hypophosphatemic effect, the serum 1,25(OH)₂D level had already descended at 3 h and reached the nadir 9 h after the administration. FGF‐23 reduced renal mRNA for 25‐hydroxyvitamin D‐1α‐hydroxylase and increased that for 25‐hydroxyvitamin D‐24‐hydroxylase starting at 1 h. In addition, an injection of calcitriol into normal mice increased the serum FGF‐23 level within 4 h. Conclusions: FGF‐23 regulates NaPi‐2a independently of PTH and the serum 1,25(OH)₂D level by controlling renal expressions of key enzymes of the vitamin D metabolism. In conclusion, FGF‐23 is a potent regulator of phosphate and vitamin D homeostasis.

The klotho gene was first identified as a mutation in a mouse strain that exhibits a syndrome resembling human ageing, and overexpression of klotho extends the lifespan of mice. A possible mechanism of action for the Klotho protein has now been found: it converts FGFR1(IIIc), a receptor for various fibroblast growth factors acting at a local level, into a specific receptor for FGF23, a bone-derived hormone that regulates kidney function. FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level1,2,3,4,5. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney6. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule7, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo . Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

Hypophosphatemic rickets/osteomalacia with inappropriately low serum 1,25-dihidroxyvitamin D level is commonly observed in X-linked hypophosphatemic rickets/osteomalacia, autosomal dominant hypophosphatemic rickets/osteomalacia and tumor-induced osteomalacia. Although the involvement of a newly identified factor, FGF-23, in the pathogenesis of ADHR and TIO has been suggested, clinical evidence indicating the role of FGF-23 has been lacking. We have previously shown that FGF-23 is cleaved between Arg¹⁷⁹ and Ser¹⁸⁰, and this processing abolished biological activity of FGF-23 to induce hypophosphatemia. Therefore, sandwich ELISA for biologically active intact human FGF-23 was developed using two kinds of monoclonal antibodies that requires the simultaneous presence of both the N-terminal and C-terminal portion of FGF-23. The serum levels of FGF-23 in healthy adults were measurable and ranged from 8.2 to 54.3 ng/L. In contrast, those in a patient with TIO were over 200 ng/L. After the resection of the responsible tumor, the elevated FGF-23 level returned to normal level within 1 h. The increase of serum concentrations of 1,25-dihidroxyvitamin D and phosphate, and the decrease of serum 24,25-dihydroxyvitamin D followed the change of FGF-23. In addition, the elevated serum FGF-23 levels were demonstrated in most patients with XLH. It is likely that increased serum levels of FGF-23 contributes to the development of hypophosphatemia not only in TIO but also in XLH.