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Keywords:

  • FGF-23;
  • KLOTHO;
  • FGF RECEPTOR INHIBITOR;
  • PHOSPHATE HOMEOSTASIS;
  • VITAMIN D METABOLISM

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

The functional interaction between fibroblast growth factor 23 (FGF-23) and Klotho in the control of vitamin D and phosphate homeostasis is manifested by the largely overlapping phenotypes of Fgf23- and Klotho-deficient mouse models. However, to date, targeted inactivation of FGF receptors (FGFRs) has not provided clear evidence for an analogous function of FGFRs in this process. Here, by means of pharmacologic inhibition of FGFRs, we demonstrate their involvement in renal FGF-23/Klotho signaling and elicit their role in the control of phosphate and vitamin D homeostasis. Specifically, FGFR loss of function counteracts renal FGF-23/Klotho signaling, leading to deregulation of Cyp27b1 and Cyp24a1 and the induction of hypervitaminosis D and hyperphosphatemia. In turn, this initiates a feedback response leading to high serum levels of FGF-23. Further, we show that FGFR inhibition blocks Fgf23 transcription in bone and that this is dominant over vitamin D–induced Fgf23 expression, ultimately impinging on systemic FGF-23 protein levels. Additionally, we identify Fgf23 as a specific target gene of FGF signaling in vitro. Thus, in line with Fgf23- and Klotho-deficient mouse models, our study illustrates the essential function of FGFRs in the regulation of vitamin D and phosphate levels. Further, we reveal FGFR signaling as a novel in vivo control mechanism for Fgf23 expression in bone, suggesting a dual function of FGFRs in the FGF-23/Klotho pathway leading to vitamin D and phosphate homeostasis. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Fibroblast growth factor 23 (FGF-23) is a bone-derived mediator of phosphate homeostasis.1 Among the family of FGF ligands, which signal through FGF receptor (FGFR) tyrosine kinases primarily in a para- and/or autocrine fashion, FGF-23 belongs to a subgroup of FGFs that function as endocrine factors.2, 3 Expression of FGF-23 is observed primarily in osteocytes and is regulated by serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and phosphate.1, 4

Excess levels of FGF-23—owing to impaired degradation or increased protein production— have been identified as the causative factor in several hereditary hypophosphatemic disorders characterized by renal phosphate wasting and impaired 1,25(OH)2D3 production, including X-linked hypophosphatemic rickets (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR).5–8 Transgenic mice overexpressing a stable mutant form of FGF-23 also develop hypophosphatemia and rickets-like phenotypes.9

Subsequent studies have revealed that FGF-23-mediated signaling negatively affects renal vitamin D biosynthesis by transcriptional repression of CYP27B1, which catalyzes the production of the biologically active vitamin D metabolite 1,25(OH)2D3, and by induction of CYP24A1, which converts 1,25(OH)2D3 into a metabolite that is less biologically active.10, 11 Additionally, it has been published that FGF-23 suppresses renal phosphate reabsorption by decreasing the expression of the sodium-phosphate cotransporters NPT2A and NPT2C in the brush-border membrane of proximal tubule epithelial cells.12, 13

In patients suffering from familial tumoral calcinosis (FTC), loss of FGF-23 function is associated with hyperphosphatemia, elevated or inappropriately normal 1,25(OH)2D3 levels, and ectopic calcifications.14 This disease is caused by inactivating mutations in FGF2315 or GALNT3 (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3),16 the product of which O-glycosilates FGF-23 and thereby protects it from proteolytic processing.17, 18 Additionally, FTC can be caused by loss-of-function mutations in the KLOTHO gene encoding a single-pass transmembrane protein primarily expressed in the kidney.19 The strikingly similar phenotypes of Fgf23- and Klotho-deficient mice,20–23 both resembling the human disease FTC, indicated a common pathway for the function of these two genes. In vitro studies demonstrated that Klotho acts as an essential coreceptor for efficient binding of FGF-23 to members of the FGFR family, thereby determining tissue specificity for endocrine FGF-23 signaling.24, 25

The FGFR family comprises four members, FGFR1, FGFR2, FGFR3, and FGFR4, existing in multiple isoforms.3 With the exception of FGFR4, alternative splicing in the third extracellular immunoglobulin-like domains results in isoforms with distinct ligand-binding specificity.3 FGF-23 has been shown to form a complex with FGFR4 or the IIIc isoforms of FGFR1 and FGFR3 in vitro.24, 25 In vivo, single or double depletion of Fgfr3 and Fgfr4 does not lead to defects in phosphate homeostasis.26, 27 Compound deletion of Fgfr3 and Fgfr4 affects vitamin D homeostasis and partially rescues the hypophosphatemic phenotype in the Hyp mouse model of XLH26, 28 but does not induce hyperphosphatemic conditions as in Hyp/Klotho−/− or Hyp/Fgf23−/− double-mutant mice.23, 29 Deletions of Fgfr1 or Fgfr2 in mice are embryonically lethal prior to kidney development,30–33 but recently, a transgenic mouse model with a conditional deletion of Fgfr1 in metanephric mesenchyme was generated.34 Data from that study indicate that the regulation of NPT2A and NPT2C expression on exogenous FGF-23 stimulation is mediated predominantly by FGFR1, but the conditional deletion of Fgfr1 did not result in a hyperphosphatemic phenotype similar to Fgf23- or Klotho-deficient mice.

Since genetic ablation of FGFRs might not account for redundancy of FGFR function or compensatory events during embryonic development, we aimed at providing evidence for the function of FGFRs in phosphate homeostasis in vivo by using pharmacologic inhibition of FGFRs. For this purpose, we have used the selective pan-FGFR inhibitor PD173074.35 We find that FGFR inhibition counteracts the biologic activity of FGF-23 in the kidney, leading to hyperphosphatemia and hypervitaminosis D, thus resembling the phenotypes of Fgf23 or Klotho deficiency. Additionally, the transient block of FGFR function using pharmacologic inhibition allows examination of feedback-regulation events induced on interference with phosphate and vitamin D homeostasis. Furthermore, we identify FGF signaling as a novel regulatory mechanism for the control of Fgf23 expression in bone in vivo, suggesting a dual function for FGFR-dependent signaling in the regulation of phosphate homeostasis.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Mice

Wild-type BALB/c mice were obtained from Janvier (Le Genest-St-Isle, France). Cyp27b1−/− mice were generated by targeted deletion of exon 8 of the Cyp27b1 locus in ES cells derived from BALB/c mice. The targeting vector was constructed from two Cyp27b1 genomic DNA fragments: A 5-kb 5' homology arm containing exon 8 flanked by loxP elements and a 2.1-kb 3' homology arm. Both DNA fragments were obtained by PCR from BALB/c mouse genomic DNA, confirmed by DNA sequencing, and cloned into a vector containing an HSVtk-Neo expression cassette flanked by Frt sites. Following ES cell transfection and positive clone selection, the neomycin cassette was removed by transfection of an Flp expression plasmid into targeted ES cells before injection into C57Bl/6 blastocysts.

All mice were kept in cages under standard laboratory conditions with a constant temperature of 20 to 24 °C and a 12/12-hour light/dark cycle. Mice were fed on a standard rodent diet (3302; Provimi Kliba SA, Kaiseraugst, Switzerland) with water ad libitum. Protocols and handling and care of the mice conformed to the Swiss federal law for animal protection under the control of the Cantonal Veterinary Offices Basel-Stadt and Vaud, Switzerland.

Cell culture

UMR-106 cells were obtained from ATTC (CRL-1661; Manassas, VA, USA) and maintained in DMEM/Ham's F-12 (Amimed, Allschwil, Switzerland) containing 10% fetal calf serum (FCS; Thermo Scientific Hyclone, Rockford, IL, USA), 10 mM Hepes (GIBCO, Carlsbad, CA, USA), 2 mM L-glutamine (GIBCO), and antibiotics. Cells were plated in 6-well tissue culture plates at initial densities of 0.5 × 106/well. Cells were treated with 50 ng/mL of FGF9 (PeproTech, Rocky Hill, NJ, USA), 50 ng/mL of EGF (Invitrogen, Carlsbad, CA, USA), 250 nM PD173074 (Sigma, St Louis, MO, USA), 100 nM PD0325901 (Selleck, Houston, TX, USA), and 500 nM RAF265 (kindly provided by Darrin Stuart, Novartis Institutes for BioMedical Research, Emeryville, CA, USA). Heparin (10 µg/mL; Sigma) was added to all treatments with FGF9.

FGFR inhibitor and 1,25(OH)2D3 treatment

The FGFR inhibitor PD173074 (50 mg/kg; Sigma) or vehicle only (PEG-300 [AppliChem, Darmstadt, Germany]/glucose 5% [B. Braun, Sempach, Switzerland], 2:1 mix) was administered orally to wild-type and Cyp27b1−/− mice. For 1,25(OH)2D3 treatments, 1,25(OH)2D3 (Sigma) was predissolved in ethanol and added to an aqueous solution of 5% mannitol (Sigma), 0.3% sodium citrate (Sigma), and 0.05% Tween-80 (Applichem). 1,25(OH)2D3 (500 ng/kg) or the vehicle only was administered by subcutaneous injections. Mice were treated at 6 to 7 weeks of age with the exception of the PD173074–1,25(OH)2D3 cotreatment study (Fig. 5), in which mice were 12 weeks of age. Mice were anesthetized by isoflurane inhalation, and blood was collected from the caval vein. Mice were euthanized by exsanguinations, and kidneys and tibial and femoral bones were obtained. Concentrations of PD173074 in kidney and bone at various times after treatment were determined by liquid chromatography-tandem mass spectrometry.

Serum and urinary parameters

Serum was separated from whole blood using clot activator centrifugation tubes (SARSTEDT, Nümbrecht, Germany). Then 100 µL of serum was used for determination of phosphate and calcium levels using the VetScan diagnostic profiling system (Abaxis, Union City, CA, USA). Serum concentrations of 1,25(OH)2D3 were analyzed using a radio receptor assay kit (Immundiagnostik, Bensheim, Germany). FGF-23 serum levels were determined by an ELISA detecting intact FGF-23 (Kainos, Tokyo, Japan). Parathyroid hormone (PTH) levels were determined from plasma (Supplemental Fig. S2A) or serum (Supplemental Fig. S2B) using a mouse intact PTH ELISA (Immutopics, San Clemente, CA, USA). Urine samples were collected in individual metabolic cages (Tecniplast, Buguggiate, Italy). Mice were acclimatized to metabolic cages for 3 days. Body weight and food and water intake were monitored daily and were not significantly different between the two groups. On day 4 in metabolic cages, the mice were given vehicle or PD173034 (50 mg/kg) by gavage at time 0, and urine was collected subsequently at time 7, 12, and 24 hours. Urine ionic composition was analyzed in the Laboratoire Central de Chimie Clinique, Centre Hospitalier Universitaire Vaudoise (CHUV) University Hospital (Lausanne, Switzerland).

RNA purification and quantitative real-time PCR (qPCR)

For RNA isolation from mouse tibial and femoral bones, epiphyses were cut off, and bone marrow was removed by centrifugation at 4 °C. Tissue was homogenized using a Precellyis 24 bead homogenizer (Bertin, Montigny-le-Bretonneux, France), and RNA was extracted with TRIzol reagent (Invitrogen). RNA was purified subsequently by chloroform extraction, isopropanol precipitation, and RNeasy Mini Kit (Qiagen, Valencia, CA, USA). For kidney RNA, approximately 60 mg of tissue was homogenized in 1.5 mL of RTL buffer (Qiagen) with a rotor-stator homogenizer (Digitana, Muralto, Switzerland), and RNA was purified with the RNeasy Mini Kit. RNA isolation from UMR-106 cells was performed in a 6-well plate format using approximately 106 cells per sample. Random hexamer-primed cDNA was synthesized with 0.5 to 2 µg of RNA and MultiScribe MuLV Reverse Transcriptase (Applied BioSystems, Foster City, CA, USA). Quantitative real-time PCR was performed in an iQ5 Real-Time PCR Detection System (BioRad, Hercules, CA, USA) using a qPCR core kit for probe assay (Eurogentec, Seraing, Belgium) and an equivalent of 40 or 80 ng of RNA from each sample. The data were normalized to Gapdh expression. TaqMan assays and primer sequences used are indicated in the supplemental material.

Western blot analysis

UMR-106 cells were plated in 6-cm dishes at an initial density of 1.2 × 106 cells/dish. Lysates were prepared with M-PER lysis buffer (Pierce, Rockford, IL, USA) supplemented with complete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail tablets (Roche, Branchburg, NJ, USA). Cellular lysates were separated by SDS-PAGE and transferred to PVDF membranes. Proteins were visualized using antibodies to total-Erk1/2 (9102; Cell Signaling Technology, Beverly, MA, USA), phospho-Erk1/2 (9101, Cell Signaling Technology), phospho-FRS2 (3861, Cell Signaling Technology), appropriate horseradish peroxidase–labeled secondary antibodies (Amersham, Pittsburgh, PA, USA, and Jackson ImmunoResearch, Newmarket, UK), and a chemiluminescence detection reagent (Pierce). Anti-α-tubulin (T4026; Sigma) was used as a loading control.

Statistical analysis

All data shown represent mean ± SEM. Statistical analyses were performed using Student's t tests (two-tailed) or one-way analysis of variance (ANOVA). A significance level of p < 0.05 to the corresponding vehicle group is indicated by an asterisk. Unpaired Student's t tests were used for all in vivo studies; in vitro expression data in UMR-106 cells was analyzed by one-way ANOVA followed by Dunnett's multiple-comparison test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Effect of FGFR loss of function on 1,25(OH)2D3 biosynthesis

FGF-23 regulates serum levels of 1,25(OH)2D3 via the transcriptional regulation of Cyp27b1 and Cyp24a1.10, 11 To demonstrate the role of FGFRs in this signaling process in vivo, we analyzed whether loss of FGFR function by means of pharmacologic inhibition in mice with the selective pan-FGFR inhibitor PD17307435 (Supplemental Table S1) antagonizes FGF-23 activity.

Mice were administered 50 mg/kg of PD173034, a dose that inhibited FGFRs in a pharmacokinetic/pharmacodynamic mechanistic mouse model (Supplemental Fig. S1) and blocks growth of FGFR-dependent tumor cells in vivo,36 and levels of Cyp27b1 and Cyp24a1, 1,25(OH)2D3, phosphate, and calcium were determined over a 24-hour time course.

Compared with vehicle-treated control groups, FGFR inhibition induced a 6- and 15-fold increase of Cyp27b1 transcription levels, respectively, after 3 and 7 hours of treatment (Fig. 1A). In contrast, Cyp24a1 mRNA levels were reduced more than 90% at 3 and 7 hours after dosing (Fig. 1B). After 16 hours, the initial regulation of the two genes was reversed (Fig. 1A, B). At this time, Cyp27b1 transcription was reduced to baseline levels and decreased further after 24 hours of treatment. Cyp24a1 levels increased approximately 6-fold at 16 hours after administration of the FGFR inhibitor and about 75-fold after 24 hours. In line with regulation of the renal FGF-23/Klotho target genes, a pharmacokinetic analysis of PD173074 levels revealed maximal compound exposure in the kidney 3 hours after dosing, whereas concentrations decreased 7 and 16 hours after treatment and were undetectable 24 hours after dosing (Fig. 1D).

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Figure 1. FGFR-dependent signaling controls 1,25(OH)2D3 biosynthesis. Regulation of the renal FGF-23 target genes Cyp27b1 (A) and Cyp24a1 (B) on FGFR inhibition in vivo. Wild-type mice received a single oral dose of the FGFR inhibitor PD173074 (50 mg/kg) or vehicle at time 0 and were studied 3, 7, 16, and 24 hours after administration of the compound. Kidneys were sampled, total RNA was isolated, and gene expression was analyzed by quantitative real-time PCR (qPCR). Expression values were normalized to Gapdh mRNA copies. Data are shown as relative levels to the corresponding vehicle control groups (relative expression of 100) and are given as average with SEM (n ≥ 7). (C) Serum 1,25(OH)2D3 levels of wild-type mice treated as described in A and B were determined by radio receptor assay. Data are shown as relative levels to the corresponding vehicle control groups (relative serum levels of 100) and are given as average with SEM (n ≥ 5). Data were compared by unpaired Student's t test; *p < 0.05 with respect to the corresponding vehicle control group. (D) PD173074 concentrations in kidney during the course of treatment. Values are given as average with SEM (n ≥ 4).

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The transcriptional changes of Cyp27b1 and Cyp24a1 in response to pharmacologic FGFR inhibition favor high serum levels of 1,25(OH)2D3. Indeed, while 1,25(OH)2D3 levels remained unchanged at 3 hours after dosing, serum concentrations of 1,25(OH)2D3 increased about 7-fold 7 hours after FGFR inhibitor treatment (Fig. 1C). 1,25(OH)2D3 levels declined 16 hours after treatment, correlating with the induction of Cyp24a1 and the reduction of Cyp27b1 expression, but were still elevated approximately 3-fold compared with the vehicle control group. After 24 hours of dosing, serum levels of 1,25(OH)2D3 equaled those of vehicle-treated mice (Fig. 1C).

Thus pharmacologic inhibition of FGFRs counteracts the biologic activity of FGF-23 in the kidney. While genetic loss-of-function models of FGFRs to date have failed to completely mimic the effects of impaired renal FGF-23/Klotho signaling, our data illustrate the functional role of FGFRs in FGF-23/Klotho signal transduction in vivo.

Since PTH is a critical regulator of mineral ion homeostasis and vitamin D metabolism37 and is transcriptionally repressed by FGF-23 signaling,38 we also have analyzed PTH plasma levels on PD173074 treatment (Supplemental Fig. S2A). We found that plasma PTH concentrations increase approximately 2.5-fold in the presence of high compound exposure after 3 and 7 hours of treatment. On release of the pharmacologic inhibition at 24 hours after dosing, PTH levels were reduced by about 80% compared with the vehicle-treated control group.

Effect of FGFR inhibition on phosphate homeostasis

In addition to its function in 1,25(OH)2D3 biosynthesis, FGF-23 also affects mineral ion homeostasis.10 Therefore, we monitored serum phosphate and calcium levels on administration of PD173074 and found that FGFR inhibition did not affect serum calcium levels but led to significantly elevated serum phosphate concentrations after 16 and 24 hours of dosing (Fig. 2A, B). It has been proposed that transcriptional suppression of the sodium-phosphate cotransporters Npt2a and Npt2c in proximal tubule epithelial cells by FGF-23/Klotho signaling contributes to the reduction in serum phosphate concentrations in vivo.12, 39 However, in our study, FGFR inhibition did not result in increased renal Npt2a and Npt2c mRNA transcription (Supplemental Fig. S3A, B) or elevated protein levels of NPT2A and NPT2C in brush-border membranes (Supplemental Fig. S3C, D). This was in agreement with our analysis of urinary phosphate excretion, where we did not observe hypophosphaturia on treatment with the FGFR inhibitor (Fig. 2C). With respect to urinary calcium levels, we observed an approximately 2.5-fold increase 24 hours after treatment (Fig. 2D). Thus inhibition of FGFRs affects serum phosphate levels but not through modulation of NPT2A/NPT2C expression.

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Figure 2. Inhibition of FGFRs induces hyperphosphatemia. Effect of pharmacologic FGFR inhibition on serum phosphate (A) and calcium (B) levels in wild-type mice. Mice received a single oral dose of the FGFR inhibitor PD173074 (50 mg/kg) or vehicle at time 0 and were studied 7, 16, and 24 hours after administration. Phosphate and calcium levels were determined from serum. For urinary phosphate and calcium measurements, mice received a single oral dose of the FGFR inhibitor PD173074 (50 mg/kg) or vehicle, and urine was collected in metabolic cages in intervals from 0 to 7 hours, 7 to 12 hours, and 12 to 24 hours after treatment. Urinary phosphate/creatinine (C) and calcium/creatinine (D) ratios are shown. Data are shown as relative levels to the corresponding vehicle control groups (relative levels of 100) and are given as average with SEM (n ≥ 6). Data were compared by unpaired Student's t test; *p < 0.05 with respect to the corresponding vehicle control group

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These findings provide the first evidence for the essential role of FGFR-mediated signaling in phosphate homeostasis in vivo, which was not elucidated in studies using genetic deletion of FGFRs to date.

Regulation of FGF-23 expression in response to FGFR inhibitor treatment

High concentrations of both 1,25(OH)2D3 and phosphate have been reported to induce FGF-23 expression in bone.40–43 We therefore tested whether the induction of hypervitaminosis D and hyperphosphatemia on FGFR inhibition (Figs. 1 and 2) would lead to increased FGF-23 levels via this regulatory feedback loop. Consistently, following 24 hours of PD173074 treatment, an approximately 6-fold increase in FGF-23 protein concentrations was measured in sera of wild-type mice (Fig. 3A). However, the elevation in serum FGF-23 levels was not accompanied by an increase in Fgf23 mRNA expression in bone (Fig. 3A).

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Figure 3. FGFR-dependent signaling regulates FGF-23 expression in bone. (A) Serum and bone mRNA expression levels of FGF-23 24 hours after administration of PD173074 (50 mg/kg) or vehicle. FGF-23 protein levels were determined by ELISA from serum. For analysis of Fgf23 mRNA expression, total RNA was prepared from diaphyseal femur and tibia, and gene expression was analyzed by qPCR. Fgf23 mRNA levels are given with respect to Gapdh levels (arbitrarily set as 100) and SEM. (B) Compound levels in bone on PD173074 (50 mg/kg) treatment for 3, 7, 16, and 24 hours, respectively. Values are given as average with SEM (n ≥ 4). (C, D) Serum and bone mRNA expression levels of FGF-23 during the course of FGFR inhibitor treatment. Data are shown as relative levels to the corresponding vehicle control groups (relative levels of 100) and are given as average with SEM (n ≥ 7). Fgf23 mRNA expression values were normalized to Gapdh mRNA copies. Data were compared by unpaired Student's t test; *p < 0.05 with respect to the corresponding vehicle control group.

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Unexpectedly, shorter intervals of FGFR inhibitor treatment resulted in a strong reduction of FGF-23 serum levels. Thus 3 and 7 hours after administration of PD173074, when the compound achieved maximal tissue concentration (Fig. 3B), serum concentrations of FGF-23 declined to almost undetectable levels (Fig. 3C). This was paralleled by a decrease of more than 90% in Fgf23 mRNA transcripts in bone (Fig. 3D). The repression of FGF-23 expression correlated with the pharmacokinetic profile of the FGFR inhibitor because Fgf23 mRNA and serum protein levels recovered from the initial repression after 16 hours of compound administration, in line with its clearance from bone (Fig. 3B).

These results imply a direct effect of FGFR inhibition on FGF-23 expression in bone, which has not been demonstrated so far, and indicate that in addition to 1,25(OH)2D3 and phosphate, FGFR-mediated signaling controls FGF-23 expression in bone. This previously unidentified link may contribute therefore to 1,25(OH)2D3 and phosphate homeostasis via the transcriptional regulation of Fgf23 in bone.

FGFR-mediated regulation of FGF-23 in the presence of hyperphosphatemia and hypervitaminosis D

To address the relevance of this novel regulatory mechanism, we tested whether persistent FGFR inhibition is able to overrule the feedback induction of FGF-23 presumably mediated by high levels of 1,25(OH)2D3 and/or phosphate (Fig. 3A). To achieve persistent inhibition of FGFRs up to 24 hours, wild-type mice were treated with two doses of the FGFR inhibitor 24 and 12 hours before killing (Fig. 4A). This regime was followed based on the pharmacokinetic profile of PD173074 (Fig. 1D and 3B). Mice treated with the two doses of PD173074 had higher compound exposure at 24 hours compared with a single 24-hour treatment (data not shown). Accordingly, at 24 hours after treatment, twice-daily dosing of PD173074 led to increased Cyp27b1 mRNA levels and blocked Cyp24a1 induction (Fig. 4B, C), indicating that FGFR inhibition was sustained. In contrast, treatment with a single dose of the FGFR inhibitor led to repression of Cyp27b1 and increased Cyp24a1 levels. This is in line with the previous experiments and is presumably the result of a feedback loop following the release of pharmacologic FGFR inhibition (Fig. 1).

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Figure 4. FGFR signaling plays a dominant role in the control of 1,25(OH)2D3 biosynthesis and FGF-23 expression. Wild-type mice were treated 24 and 12 hours before killing with vehicle or PD173074 (50 mg/kg) as indicated in panel A. For mRNA expression analysis of Cyp27b1 (B) and Cyp24a1 (C), kidneys were sampled, total RNA was isolated, and gene expression was analyzed by qPCR. Expression values were normalized to Gapdh mRNA copies. Data are shown as relative levels to the corresponding vehicle control groups (relative expression of 100). 1,25(OH)2D3 (D), phosphate (E), and calcium (F) levels were determined from serum. FGF-23 serum levels (G) were determined by ELISA. All data are given as average values with SEM (n ≥ 7). Data were compared by unpaired Student's t test; *p < 0.05 with respect to the vehicle-only control group.

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In agreement with our previous findings (Fig. 2), treatment with a single dose of PD173074 induced hyperphosphatemia, but 1,25(OH)2D3 serum levels were not elevated after 24 hours (Fig. 4D, E). Both single and twice-daily dosing of PD173074 did not affect serum calcium levels (Fig. 4F). For PTH, we observed reduced serum concentrations after treatment with a single dose of PD173074, in line with our previous analysis (Supplemental Fig. S2A), but also a similar repression on twice-daily dosing (Supplemental Fig. S2B). However, persistent FGFR inhibition resulted in both elevated serum phosphate concentrations and a significant 2-fold increase of 1,25(OH)2D3 serum levels.

Interestingly, in the presence of hyperphosphatemia and hypervitaminosis D, FGFR inhibition efficiently repressed the increase in FGF-23 serum levels observed 24 hours after treatment with a single dose of PD173074 (Fig. 4G). Thus these results suggest that not only is FGFR signaling necessary for FGF-23 expression, but also that receptor activity exerts a dominant function over signaling events induced by 1,25(OH)2D3 and phosphate.

Effect of FGFR inhibition on 1,25(OH)2D3-induced transcription of Fgf23

We also assessed whether FGFR inhibition would be able to counteract immediate induction of FGF-23 by 1,25(OH)2D3. For this, we used Cyp27b1-deficient mice, which allow an analysis of Fgf23 transcriptional regulation independent of endogenous 1,25(OH)2D3 levels. Owing to the absence of 1,25(OH)2D3, these mice have very low levels of Fgf23 mRNA (Fig. 5B) and low FGF-23 serum levels44 (data not shown). Cyp27b1−/− mice received subcutaneous injections of 1,25(OH)2D3 followed by FGFR inhibitor treatment (Fig. 5A). Administration of 1,25(OH)2D3 led to elevated serum phosphate and calcium levels 7 hours after dosing, which was not affected by PD173074 cotreatment (Fig. 5C, D). Fgf23 bone mRNA levels in vehicle-treated mice were very low and not further decreased by treatment with PD173074 (Fig. 5B). As expected, administration of 1,25(OH)2D3 led to an increase in Fgf23 expression, but this induction was completely blocked by subsequent FGFR inhibitor treatment (Fig. 5B).

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Figure 5. FGFR inhibition blocks 1,25(OH)2D3-mediated induction of Fgf23 expression in bone. Cyp27b1-deficient mice were treated with 1,25(OH)2D3 (500 ng/kg) and PD173074 (50 mg/kg) as indicated in panel A. (B) Induction of Fgf23 in diaphyseal femur and tibia was analyzed by qPCR. Data are shown as relative expression with respect to Gapdh levels (arbitrarily set as 100). Phosphate (C) and calcium (D) levels were determined from serum. All data are given as average values with SEM (n ≥ 5). Data were compared by unpaired Student's t test; *p < 0.05 with respect to the vehicle-only control group.

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Taken together, these results show that FGFR inhibition blocks 1,25(OH)2D3-mediated induction of Fgf23 transcription, as well as the phosphate/1,25(OH)2D3-dependent feedback regulation of FGF-23 (Fig. 4), arguing for a dominant function of FGFR-dependent signaling events in the control of FGF-23 expression.

FGF-dependent regulation of Fgf23 transcription in vitro

Next, we investigated FGF-dependent regulation of Fgf23 expression using the rat osteosarcoma cell line UMR-106 as an in vitro osteoblast cell model. These cells had baseline FGFR activity, as measured by FRS2 tyrosine phosphorylation, an immediate downstream substrate of FGFRs (Fig. 6C, D). Baseline expression levels of Fgf23 also were detected and were reduced by more than 50% on treatment with the FGFR inhibitor PD173074 (Fig. 6A, B). Conversely, we found that stimulation with a variety of FGF ligands—FGF-1, FGF-2, FGF-9, and FGF-18—significantly induced Fgf23 transcription, which was blocked by cotreatment with PD173074 (Fig. 6A, B, and data not shown). Stimulation of UMR-106 cells with FGF-9 led to a strong phosphorylation of FRS2 and activation of ERK1/2 (Fig. 6C, D). In line with Fgf23 mRNA expression, FGFR inhibition using PD173074 decreased basal phosphorylation levels of FRS2 and ERK1/2 (Fig. 6C, D). Yet MAPK activation alone was not sufficient to trigger Fgf23 induction because EGF, which strongly activated ERK1/2, did not increase Fgf23 transcription (Fig. 6A, C). Interestingly, we did not detect activation of other signaling cascades commonly induced by FGF ligands, such as PI3K/Akt, PLCγ, or JAK-STAT (data not shown). To test for the dependence of MAPK signaling in the transcriptional regulation of Fgf23, we treated UMR-106 cells with the MEK inhibitor PD0325901 and the RAF inhibitor RAF265 (Fig. 6B, D). We found that inhibition of MEK or RAF alone was not sufficient to fully block FGF-9-mediated phosphorylation of ERK1/2 and the induction of Fgf23 (Fig. 6B, D). In contrast, combined RAF and MEK inhibition resulted in a complete block of MAPK signaling and abolished FGF-9-mediated induction of Fgf23.

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Figure 6. FGF signaling controls Fgf23 expression in vitro. (A) qPCR analysis of Fgf23 induction in UMR-106 cells on treatment with FGF-9, EGF, and PD173074. (B) Effect of MAPK pathway inhibition on FGF-9-mediated induction of Fgf23 expression. (C, D) Western blot analysis of FRS2 and ERK1/2 phosphorylation in UMR-106 cells. Cells were treated for 3 (Western blot) or 24 hours (qPCR analysis) with FGF-9 (50 ng/mL), EGF (50 ng/mL), PD173074 (250 nM), PD0325901 (100 nM), and RAF265 (500 nM) as indicated. Heparin (10 µg/mL) was added to all treatments with FGF-9. Activation of Fgf23 is shown relative to transcript levels in vehicle-treated cells (relative expression of 1). Expression values were normalized to Gapdh mRNA copies and are given as average with SEM (n ≥ 3). Data were compared by one-way ANOVA; *p < 0.05 with respect to vehicle-treated cells.

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These results demonstrate that Fgf23 is a target gene of FGF signaling and indicate that ERK1/2 activation is an essential, albeit not sufficient, downstream event for this process. Therefore, the in vitro data confirm and extend our novel observation that FGFR-dependent signaling regulates Fgf23 transcription in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

In this study we show that FGFR inhibition counteracts renal FGF-23/Klotho signaling leading to the deregulation of Cyp27b1 and Cyp24a1 in the kidney and the subsequent induction of high serum levels of 1,25(OH)2D3 and phosphate. In addition, we reveal that FGFR signaling in bone regulates Fgf23 transcription, ultimately leading to the modulation of systemic FGF-23 protein levels (Fig. 7). This adds a previously unidentified layer of FGF-23 regulation and implies a dual function of FGFR signaling in vitamin D and phosphate homeostasis in kidney and bone.

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Figure 7. FGFR signaling exerts a dual function in the control of 1,25(OH)2D3 and phosphate homeostasis. In the kidney, FGFRs negatively regulate 1,25(OH)2D3 biosynthesis by transducing FGF-23/Klotho signals leading to suppression of Cyp27b1 and induction of Cyp24a1. 1,25(OH)2D3 promotes high serum levels of phosphate by increasing the intestinal phosphate uptake from the diet. In bone, both 1,25(OH)2D3 and phosphate induce Fgf23 expression, resulting in a negative-feedback loop. Additionally, FGFR signaling activates Fgf23 transcription, and baseline FGFR signals are required for 1,25(OH)2D3-mediated induction of Fgf23.

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Our results demonstrate the role of FGFRs in the transduction of renal FGF-23/Klotho signaling in vivo and provide evidence for their involvement in the maintenance of phosphate homeostasis because genetic loss-of-function studies of FGFRs have to date failed to fully mimic the hyperphosphatemic phenotypes of Fgf23- or Klotho-deficient mouse models.26–28, 34 Single or compound deletion of Fgfr3 and Fgfr4 has no effect on phosphate homeostasis, and Fgfr3/Fgfr4 double depletion only partially rescues the hypophosphatemic conditions of Phex-deficient mice expressing high levels of FGF-23,26–28 arguing for a contribution of FGFR3 and FGFR4 to FGF-23/Klotho signaling only in the context of pathologically high FGF-23 serum levels. In contrast, genomic ablation of either Fgf23 or Klotho in a Phex-deficient background leads to a complete reversion of hypophosphatemia and resembles the hyperphosphatemic phenotypes of Fgf23 or Klotho deficiency.23, 29 In mice with a conditional deletion of Fgfr1 in the metanephric mesenchyme, leading to a depletion of the receptor from proximal tubule epithelial cells, FGF-23-mediated repression of NPT2A and NPT2C was blunted, but phosphate and 1,25(OH)2D3 levels were indistinguishable from those of wild-type littermates.34 In vitro, FGF-23 can bind to and signal via FGFR1c, FGFR3c, and FGFR4 in a complex with Klotho.24, 25 Hence it is possible that FGF-23/Klotho signals are not mediated exclusively by one specific FGFR in vivo.

To address the involvement of FGFRs in FGF-23/Klotho signaling in vivo, we used the selective pan-FGFR inhibitor PD173074 and found that FGFR loss of function recapitulated the mineral homeostasis changes described for Fgf23- and Klotho-deficient mice. Specifically, pharmacologic FGFR inhibition led to a rapid and strong transcriptional regulation of the renal FGF-23 target genes Cyp27b1 and Cyp24a1, which closely correlated with the pharmacokinetics of PD173074. The reversion of the initial changes in Cyp27b1 and Cyp24a1 on release of pharmacologic inhibition may reflect the impact of a feedback regulation mediated by high serum levels of FGF-23 and 1,25(OH)2D3.10 Following the initial regulation of Cyp27b1 and Cyp24a1, 1,25(OH)2D3 serum levels increased 7 hours after FGFR inhibitor treatment and declined subsequently in line with the induction of Cyp24a1 and the repression of Cyp27b1.

Further, and as described for Fgf23- and Klotho-deficient mice,20–23 FGFR inhibition resulted in significant hyperphosphatemia. We hypothesize that the elevated serum levels of 1,25(OH)2D3 account for the increase in serum phosphate levels in mice treated with the FGFR inhibitor. In addition, FGF-23 has been proposed to control phosphate homeostasis by regulating the expression of the sodium/phosphate cotransporters NPT2A and NPT2C in the brush-border membrane of proximal tubules.9, 10, 12, 13, 45 While it was demonstrated that treatment of mice with purified recombinant FGF-23 leads to the repression of Npt2a mRNA and protein in the kidney,10 we did not observe increased expression of either NPT2A or NPT2C on treatment with the FGFR inhibitor PD173074. Therefore, we propose that upon transient inhibition of basal FGF-23 signaling, the immediate increase in serum phosphate levels might be mediated predominantly via the induction of high 1,25(OH)2D3 serum concentrations. In contrast, pathologic high FGF-23 serum levels may impact on Npt2a and Npt2c expression. This is supported by the finding that pharmacological MEK inhibition results in increased renal Npt2a mRNA expression only in the Hyp mouse model for XLH but not in wild-type mice.46

Based on these results, we hypothesize that FGFR inhibitors may be of therapeutic value for the treatment of FGF-23-related hypophosphatemia disorders such as XLH, ADHR, ARHR, and tumor-induced osteomalacia (TIO).5–8, 11 Using a different approach, Aono and colleagues have shown that blocking FGF-23/Klotho signaling with neutralizing antibodies to FGF-23 is sufficient to normalize serum phosphate levels and rescue the rickets-like phenotype of the Hyp mouse model resembling the human disease XLH.47 We expect that prolonged pharmacologic FGFR inhibition will have similar beneficial effects on the hypophosphatemic conditions and the resulting bone-related phenotypes in Hyp mice and other mouse models of FGF-23-related hypophosphatemia disorders.

Interestingly, serum calcium concentrations are not affected by FGFR inhibition, whereas genetic ablation of Fgf23 or Klotho leads to both hyperphosphatemia and hypercalcemia.21–23 Likewise, pharmacologic MEK inhibition did not affect serum calcium concentrations,46 implying that transient pharmacologic inhibition of FGF-23 signaling may not be sufficient to trigger changes in calcium homeostasis in contrast to genetic loss-of-function models of Fgf23 or Klotho with persistent hypervitaminosis D. Also, we observed increased urinary calcium excretion on treatment with PD173074, potentially counteracting the expected increase in serum calcium levels in response to elevated intestinal calcium uptake in the presence of high 1,25(OH)2D3 serum concentrations.

Since PTH positively affects calcium reabsorption in distal tubular cells,48 the observed increase in calciuria may be a consequence of the low PTH serum levels and the decline in 1,25(OH)2D3 serum concentrations at 24 hours after FGFR inhibitor treatment. PTH serum levels are increased initially after treatment with PD173074, presumably as a result of counteracting FGF-23 signaling in the parathyroid gland.38 In contrast, at 24 hours, when FGFR inhibition is relieved and in the presence of high FGF-23, PTH levels are repressed. This can be explained by an inhibitory effect of FGF-23 on PTH expression in the parathyroid gland but also can be a consequence of the preceding induction of hypervitaminosis D by FGFR inhibitor treatment.49 Indeed, on twice-daily treatment of PD17034, leading to persistent FGFR inhibition and FGF-23 downregulation in the presence of hypervitaminosis D, PTH serum concentrations are repressed, pointing at an inhibitory effect of 1,25(OH)2D3 on PTH expression. Conversely, elevated PTH serum levels at 3 and 7 hours after dosing of PD173074 might contribute to the subsequent increase in 1,25(OH)2D3 serum concentrations, whereas subsequently low PTH levels may support the induction of hyperphosphatemia.37

By using a small-molecular-weight inhibitor to precisely follow the course of events induced on blocking FGF-23/Klotho signaling, we provide insight into feedback mechanisms involved in the control of phosphate homeostasis. We show that FGFR inhibition ultimately leads to significantly higher FGF-23 serum levels, most likely in response to hyperphosphatemia and hypervitaminosis D40–43 induced by the inhibition of FGF-23/Klotho signaling in the kidney. Likewise, FGF-23 serum levels are elevated in Klotho-deficient mice.50, 51 Interestingly, the increase in serum FGF-23 was not paralleled by a concomitant upregulation of Fgf23 mRNA expression in bone, suggesting that nontranscriptional events contribute to the elevated serum FGF-23.

In addition to the functions of FGFRs in phosphate homeostasis in the kidney, we have identified a novel role for FGF signaling in the control of Fgf23 transcription in bone. We show that in vivo, pharmacologic inhibition of FGFRs leads to repression of Fgf23 mRNA expression in bone, correlating with the pharmacokinetics of the compound in this tissue. Fgf23 mRNA repression translated directly into dramatically reduced serum FGF-23, down to almost undetectable levels. FGFR-dependent regulation of Fgf23 also was observed in vitro in UMR-106 osteosarcoma cells, which was confirmed during the submission process in a different in vitro model.52

Owing to the absence of Klotho expression in bone,53 the effect of FGF signaling on Fgf23 expression is unlikely to be mediated by a positive-feedback loop involving FGF-23 itself. Instead, it is more likely that para- or autocrine signaling of other FGF ligands expressed in bone affects Fgf23 transcription, thus constituting an additional layer of FGF-23 regulation. Moreover, using the in vitro model system UMR-106 osteosarcoma cells, we show that FGFR ligands, but not an unrelated receptor tyrosine kinase ligand such as EGF, are effective in inducing Fgf23 expression and that MAPK activation is essential but not sufficient for this event. Since other MAPK target genes were induced by both FGF and EGF treatment in the UMR-106 cell model (eg, Egr2; data not shown), FGF signaling may trigger additional events specific for the regulation of Fgf23, which have yet to be identified.

The impact of FGF signaling on Fgf23 expression in bone was dominant over 1,25(OH)2D3- and/or phosphate-mediated processes. Both hypervitaminosis D and hyperphosphatemia transcriptionally activate Fgf2340, 43 but were not able to do so in the presence of persistent FGFR inhibition. In addition, 1,25(OH)2D3-induced Fgf23 expression in the Cyp27−/− mouse was completely abolished on inhibiting FGFRs. These results indicate that in bone, vitamin D signaling depends on an intact FGFR pathway in order to efficiently induce Fgf23.

Conflicting reports exist with respect to the mechanism of 1,25(OH)2D3-mediated induction of Fgf23. While reporter gene assays imply a direct regulation via a vitamin D–responsive element (VDRE) within the Fgf23 promoter,42 other studies suggest that activation of Fgf23 by 1,25(OH)2D3 occurs indirectly and independent of VDREs.43, 54, 55 Stimulation with 1,25(OH)2D3 induced Fgf23 in UMR-106 cells but, unlike FGF treatment, did not activate the MAPK pathway (data not shown), which indicates differential signaling pathways for FGF- and 1,25(OH)2D3-dependent regulation of Fgf23. Given the dominant repressive effect of FGFR inhibition on Fgf23 transcription, we hypothesize that a basal FGF-dependent signal is essential for maintenance of Fgf23 expression, yet it is unclear how this dependency fits into the network of FGF-23 regulation known so far. Recently, PTH has been described as an additional agent inducing Fgf23 expression,56 but induction of FGF-23 serum levels at 24 hours after PD173074 treatment occurs in the presence of low PTH serum concentrations.

The FGF-dependent induction of Fgf23 could be responsible for hypophosphatemic conditions that are observed in a subset of patients with activating mutations in FGFR1 and FGFR3. Hypophosphatemia and elevated serum levels of FGF-23 have been reported in epidermal nevus syndrome, a rare congenital neurocutaneous disorder consisting of epidermal nevi in association with abnormalities in the cerebral, ocular, skeletal, cardiac, and renal systems57, 58 and activating mutations of FGFR3 are observed frequently in epidermal nevi.59 Likewise, osteoglophonic dysplasia, a genetic disorder involving craniosynostosis and short stature caused by activating mutations of FGFR1, is associated with increased serum concentrations of FGF-23 and phosphate.60, 61 In view of our novel observation that FGFR-dependent signaling controls FGF-23 expression in bone, it might be interesting to assess a potential pathophysiologic role of constitutively activated FGFRs in the mediation of hypophosphatemia in these diseases—respectively, a general impact on FGF-23 levels in conditions with aberrant FGF signaling—in future studies.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

SW, NB, SG, CS, MS, MM, BK, AT, JB, WRS, FH, and DGP are employees of Novartis Institutes for BioMedical Research. NEH and OB state that they have no conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank Ramona Rebmann, Flavia Reimann, Timothy Smith, and Candice Stoudmann for excellent technical assistance. We are grateful to Herbert Schmid, Michaela Kneissel, and Ina Kramer for helpful discussions and critical reading of the manuscript. We thank Darrin Stuart for providing RAF265.

Authors' roles: Study design: SW, WRS, FH, and DGP. Study conduct and data collection: SW, OB, NB, CS, MS, MM, BK, and AT. Data analysis and interpretation: SW, OB, SG, JB, NEH, WRS, FH, and DGP. Drafting manuscript: SW and DGP. Revising manuscript content: SW, OB, NEH, FH, and DGP. All authors approved the final version of manuscript. SW and DGP take responsibility for the integrity of the data analysis.

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  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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JBMR_478_sm_Supp_Info.docx675KSupplementary Information

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