Takeyoshi Yamashita

Klotho converts canonical FGF receptor into a specific receptor for FGF23

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 level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, 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.

FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.

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.

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

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.

Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism

Inorganic phosphate is essential for ECM mineralization and also as a constituent of important molecules in cellular metabolism. Investigations of several hypophosphatemic diseases indicated that a hormone-like molecule probably regulates serum phosphate concentration. FGF23 has recently been recognized as playing important pathophysiological roles in several hypophosphatemic diseases. We present here the evidence that FGF23 is a physiological regulator of serum phosphate and 1,25-dihydroxyvitamin D (1,25[OH]2D) by generating FGF23-null mice. Disruption of the Fgf23 gene did not result in embryonic lethality, although homozygous mice showed severe growth retardation with abnormal bone phenotype and markedly short life span. The Fgf23(-/-) mice displayed significantly high serum phosphate with increased renal phosphate reabsorption. They also showed an elevation in serum 1,25(OH)2D that was due to the enhanced expression of renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) from 10 days of age. These phenotypes could not be explained by currently known regulators of mineral homeostasis, indicating that FGF23 is essential for normal phosphate and vitamin D metabolism.

Expression of Vascular Endothelial Growth Factors and Their Receptors during Osteoblast Differentiation

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like...

Direct evidence for a causative role of FGF23 in the abnormal renal phosphate handling and vitamin D metabolism in rats with early-stage chronic kidney disease

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with early chronic kidney disease (CKD) and are postulated to cause low blood levels of 1,25-dihydroxyvitamin D, as well as normal phosphate levels. In order to provide more direct evidence for the pathophysiological role of FGF23 in the settings of mineral ion homeostasis typically seen in early CKD, we studied rats with progressive CKD treated with anti-FGF23 neutralizing antibody. Without antibody treatment, rats with CKD exhibited high circulating levels of FGF23 and parathyroid hormone, low 1,25-dihydroxyvitamin D, and normal serum phosphate levels, accompanied by increased fractional excretion of phosphate. Antibody treatment, however, lessened fractional excretion of phosphate, thus increasing serum phosphate levels, and normalized serum 1,25-dihydroxyvitamin D by increased 1α-OHase and decreased 24-OHase expressions in the kidney. These antibody-induced changes were followed by increased serum calcium levels, leading to decreased serum parathyroid hormone. Hence, our study shows that FGF23 normalizes serum phosphate and decreases 1,25-dihydroxyvitamin D levels in early-stage CKD, and suggests a pathological sequence of events for the development of secondary hyperparathyroidism triggered by increased FGF23, followed by a reduction of 1,25-dihydroxyvitamin D and calcium levels, thereby increasing parathyroid hormone secretion.

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

BACKGROUND Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation. OBJECTIVES The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. RESULTS We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls. CONCLUSION We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

Therapeutic Effects of Anti‐FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

X‐linked hypophosphatemia (XLH), characterized by renal phosphate wasting, is the most common cause of vitamin D‐resistant rickets. It has been postulated that some phosphaturic factor plays a causative role in XLH and its murine homolog, the Hyp mouse. Fibroblast growth factor 23 (FGF23) is a physiological phosphaturic factor; its circulatory level is known to be high in most patients with XLH and Hyp mice, suggesting its pathophysiological role in this disease. To test this hypothesis, we treated Hyp mice with anti‐FGF23 antibodies to inhibit endogenous FGF23 action. A single injection of the antibodies corrected the hypophosphatemia and inappropriately normal serum 1,25‐dihydroxyvitamin D. These effects were accompanied by increased expressions of type IIa sodium‐phosphate cotransporter and 25‐hydroxyvitamin‐D‐1α‐hydroxylase and a suppressed expression of 24‐hydroxylase in the kidney. Repeated injections during the growth period ameliorated the rachitic bone phenotypes typically observed in Hyp mice, such as impaired longitudinal elongation, defective mineralization, and abnormal cartilage development. Thus, these results indicate that excess actions of FGF23 underlie hypophosphatemic rickets in Hyp mice and suggest a novel therapeutic potential of the FGF23 antibodies for XLH.

Anti‐FGF23 Neutralizing Antibodies Show the Physiological Role and Structural Features of FGF23

Fibroblast growth factor (FGF)23 is proposed to play a physiological role in the regulation of phosphate and vitamin D metabolism; deranged circulatory levels of FGF23 cause several diseases with abnormal mineral metabolism. This paper presents a novel approach to analyze the mechanism of action of FGF23 using anti‐FGF23 monoclonal antibodies that can neutralize FGF23 activities both in vitro and in vivo. We developed two antibodies (FN1 and FC1) that recognize the N‐ and C‐terminal regions of FGF23, respectively. Both FN1 and FC1 inhibited FGF23 activity in a cell‐based Klotho‐dependent reporter assay. Their administration caused marked increases in serum phosphate and 1,25D levels in normal mice. These changes were accompanied by altered expression in the kidney of type IIa sodium‐phosphate cotransporter, 25‐hydroxyvitamin‐D‐1α‐hydroxylase, and 24‐hydroxylase. Thus, this study using neutralizing antibodies confirms that FGF23 is a physiological regulator of phosphate and vitamin D metabolism. We addressed the mechanism of action for these neutralizing antibodies. Structural analysis of the FGF23/FN1‐Fab complex showed that FN1 masked putative FGF receptor‐binding sites in the N‐terminal domain of FGF23, whereas biochemical analyses showed that FC1 interfered with the association between FGF23 and Klotho by binding to the C‐terminal domain of FGF23. Taken together, our results suggest that the N‐ and C‐terminal domains of FGF23 are responsible for association with cognate FGF receptors and Klotho, respectively, and that these interactions are indispensable for FGF23 activity.

Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23.

Two hyperphosphatemic patients with mutations in GALNT3 showed low intact FGF23 levels with marked increase of processed C‐terminal fragments. FGF23 protein has three O‐linked glycans and FGF23 with incomplete glycosylation is susceptible to processing. Silencing GALNT3 resulted in enhanced processing of FGF23. Decreased function of FGF23 by enhanced processing is the cause of hyperphosphatemia in patients with GALNT3 mutation.