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Am J Physiol Renal Physiol 293: F1577-F1583, 2007. First published August 15, 2007; doi:10.1152/ajprenal.00463.2006
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Fibroblast growth factor 23 impairs phosphorus and vitamin D metabolism in vivo and suppresses 25-hydroxyvitamin D-1{alpha}-hydroxylase expression in vitro

Farzana Perwad,1 Martin Y. H. Zhang,1 Harriet S. Tenenhouse,2 and Anthony A. Portale1

1Pediatrics, University of California San Francisco, San Francisco, California; and 2Pediatrics and Human Genetics, McGill University, Montreal, Quebec, Canada

Submitted 20 November 2006 ; accepted in final form 9 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fibroblast growth factor-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/Pi) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 µg/g body wt) induced significant hypophosphatemia, 20 and 29% decreases (P < 0.001) in brush-border membrane (BBM) Na/Pi cotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/Pi cotransporter protein in BBM. Multiple injections (6, 12, and 24 µg/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1{alpha}-hydroxylase mRNA (P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1{alpha}-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1{alpha}-hydroxylase mRNA with a maximum suppression of 37% (P < 0.05). Suppression was detectable after 6 h of exposure and maximal after 21 h. In MCT cells, FGF-23(R176Q) suppressed 1{alpha}-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1{alpha}-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1{alpha}-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.

bone and mineral homeostasis; hypophosphatemic syndromes

FIBROBLAST GROWTH FACTOR-23 (FGF-23), a bone-derived circulating peptide, is an important regulator of phosphorus and 1,25-dihydroxyvitamin D [1,25(OH)2D] metabolism and is required for maintenance of normal bone and mineral homeostasis. An excess of FGF-23 has been linked to the pathogenesis of tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets (4, 12, 21, 29, 33). These hypophosphatemic syndromes share common clinical and laboratory features which include rickets and osteomalacia, hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately low serum 1,25(OH)2D concentrations, and greatly increased serum FGF-23 concentrations. That excess circulating FGF-23 contributes to the pathogenesis of these disorders is supported by observations that administration of recombinant FGF-23 or its overexpression in animals induces hypophosphatemia and inhibition of sodium (Na)-dependent Pi (Na/Pi) cotransport in renal brush-border membrane (BBM) vesicles (1, 19, 29). FGF-23 suppresses the renal production of 1,25(OH)2D by suppressing 25-hydroxyvitamin D 1{alpha}-hydroxylase (1{alpha}-hydroxylase, P450c1{alpha}) mRNA and protein expression and stimulating 24-hydroxylase mRNA expression (1, 19, 29).

1{alpha}-Hydroxylase and 24-hydroxylase are mitochondrial cytochrome P-450 enzymes responsible for the synthesis and degradation of 1,25(OH)2D, respectively. The changes in renal abundance of 1{alpha}-hydroxylase and 24-hydroxylase mRNA induced by administration of FGF-23 are rapid, suggesting a direct action of FGF-23 on expression of their genes (28).

The active form of vitamin D, 1,25(OH)2D, plays a critical role in calcium and Pi metabolism and is required for skeletal growth in children and maintenance of skeletal integrity in adults. 1,25(OH)2D is produced in the kidney by 1{alpha}-hydroxylation of 25-hydroxyvitamin D (25OHD) by the 1{alpha}-hydroxylase enzyme (3, 5–7, 14, 23). Circulating concentrations of 1,25(OH)2D primarily reflect its synthesis in the kidney, as bilateral nephrectomy and chronic renal failure result in low circulating 1,25(OH)2D concentrations (8). Thus 1{alpha}-hydroxylation of 25OHD is the rate-limiting step in the bioactivation of vitamin D. In the mammalian kidney, expression of the gene encoding 1{alpha}-hydroxylase, CYP27B1, and thus activity of this enzyme are primarily regulated by parathyroid hormone (PTH), calcium, Pi, 1,25(OH)2D itself, and based on recent evidence, FGF-23. However, the mechanisms by which FGF-23 regulates vitamin D metabolism are unknown.

In the present study, we sought to determine whether FGF-23 directly regulates renal 1,25(OH)2D metabolism by regulating expression of the enzymes responsible for its synthesis and degradation. We demonstrated the biological potency of recombinant human FGF-23(R176Q) by administering it to wild-type mice and determining its effect on serum Pi concentration, renal Na/Pi cotransport activity, and renal 1{alpha}-hydroxylase and 24-hydroxylase mRNA abundance. We then examined the effects of FGF-23(R176Q) on 1{alpha}-hydroxylase and 24-hydroxylase gene expression in vitro in human and mouse renal proximal tubule epithelial cells. In addition, we examined the signaling pathways involved in the regulation of 1{alpha}-hydroxylase gene expression by FGF-23.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice

C57BL/6J mice (age 78 ± 32 days), purchased from Jackson Laboratory (Bar Harbor, ME), were injected intravenously with human recombinant FGF-23(R176Q) (0.33 µg/g body wt) and killed either 5 or 17 h later. Animals were anesthetized with pentobarbital sodium, blood was obtained for determination of serum Pi concentration, and the kidneys were removed for preparation of renal BBM vesicles. In separate experiments, C57BL/6J mice (age 102 ± 29 days) were injected intraperitoneally with FGF-23(R176Q) (0.07–0.30 µg/g body wt) at 8-h intervals for 4 days. At the time of death, the kidneys were removed and rapidly frozen in liquid nitrogen for subsequent extraction of RNA. In some studies, one kidney was placed in homogenizing medium at 4°C for isolation of renal mitochondria and determination of 1{alpha}-hydroxylase enzyme activity (24). Serum Pi concentration was determined using a kit from Stanbio Laboratories (San Antonio, TX). For most experiments, mice were fed a standard rodent diet containing 1% phosphorus and 1% calcium. In some experiments, mice were fed a diet containing 0.02% phosphorus and 1% calcium (test diet TD 86128, Harlan Teklad, Madison, WI). All procedures were approved by the Committee on Animal Research, University of California San Francisco and the Canadian Council on Animal Care.

Cell Culture

Human (HKC-8) and mouse (MCT) cells are SV-40-transformed renal epithelial cells that exhibit features characteristic of proximal renal tubule epithelia (2, 9, 22, 26). In preliminary experiments, we determined that HKC-8 cells express 1{alpha}-hydroxylase enzyme activity and mRNA and 24-hydroxylase mRNA. Exposure to forskolin (1 µM) induced a sixfold increase in 1{alpha}-hydroxylase enzyme activity and threefold increase in mRNA abundance (A. A. Portale, unpublished results), as others observed (2, 15). MCT cells also express 1{alpha}-hydroxylase and 24-hydroxylase mRNA, and 1{alpha}-hydroxylase mRNA is increased by forskolin and PTH and decreased by 1,25(OH)2D (22). HKC-8 cells were maintained in DMEM (DME-H21/F12 50% mix; GIBCO) with 5% fetal bovine serum (Hyclone), 1% penicillin-streptomycin, and 1% fungizone and plated at 1 x 105–1.3 x 106 cells/well in six-well plates for all experiments. Forty-eight hours later, culture media were replaced with serum-free media and the following day cells were treated with FGF-23(R176Q) or other reagents. MCT cells were maintained in DME-H21 (GIBCO) with 10% fetal bovine serum (Hyclone), 1% penicillin-streptomycin, and 1% fungizone, plated at 1 x 105–1.3 x 106 cells/well, and studied in a manner identical to that of HKC-8 cells. Cells were treated with FGF-23(R176Q) for 21 h except for experiments performed to detect phosphorylation of signaling molecules, in which the treatment time ranged from 1 to 120 min.

FGF-23

Recombinant human FGF-23(R176Q) (Genzyme, Framingham, MA) contains a mutation in its proprotein convertase (furin) proteolytic cleavage site in which arginine at position 176 is replaced by glutamine. This mutation, which is identical to that in patients with autosomal dominant hypophosphatemic rickets, renders the FGF-23(R176Q) protein resistant to proteolytic processing (30). FGF-23(R176Q) has enhanced biological potency in vivo and in vitro when compared with that of native FGF-23 (1).

Renal BBM Preparation and Pi Uptake Studies

BBM vesicles were prepared from kidney cortex (2 kidneys from each mouse were used for each BBM prep) by the MgCl2 precipitation method as described (32), and aliquots were used for Pi transport studies, protein determination (Lowry method), and Western blot analysis. Enrichment of 8- to 10-fold with respect to alkaline phosphatase activity was routinely obtained. The transport of [32P]phosphate (0.1 mM), [35S]sulfate (0.1 mM), and [3H]glucose (0.01 mM) was measured at 6 s (initial rate) at room temperature in the presence of 100 mM mannitol, 10 mM Tris·HEPES, pH 7.4, 100 mM KCl, or 100 mM NaCl by rapid filtration as described (32). The Na+-dependent component of transport was obtained by the difference in uptake in NaCl and KCl, respectively, and is expressed as picomoles per milligram of BBM protein per 6 s.

Western Blot Analysis

For Western blot analysis, BBMs (10–50 µg protein) were suspended in gel buffer, heated at 55°C for 3 min, fractionated on 10% polyacrylamide gels in the presence of SDS, transferred to nitrocellulose membranes, and probed with rabbit polyclonal antibodies generated against rat Na/Pi cotransporter Npt2a (32) and actin (Sigma, St. Louis, MO). Antigen antibody complexes were visualized by chemiluminescence (ECL kit, Amersham Biosciences, Montreal, Quebec). The abundance of Npt2a cotransporter protein, relative to that of actin, was quantitated using FujiScan software as described (10). Activation of the mitogen-activated protein kinase (MAPK) pathway was determined by detection of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 proteins in cell lysates from MCT cells treated with FGF-23(R176Q) (100 ng/ml) for 1–120 min. In some experiments, cells were treated with the MAPK/ERK1/2 kinase (MEK1/2) inhibitor PD98059 (50 µM; Calbiochem, San Diego, CA) for 30 min before treatment with FGF-23(R176Q). Cell lysates were prepared by using modified RIPA buffer (1 mM PMSF, 1x protease inhibitor, 1 mM activated sodium orthovanadate, 1 mM sodium fluoride, 150 mM sodium chloride, 1% NP-40, 50 mM Tris·HCl, pH 7.4, 1 mM EDTA, and 0.25% sodium deoxycholate). Phosphorylated proteins were detected using rabbit anti-phospho-ERK1/2 (1:200 dilution) and anti-phospho-p38 (1:200 dilution) antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) and visualized by chemiluminescence (ECL kit, Pierce Biotechnology, Rockford, IL). Equal protein loading was determined using anti-ERK2 antibodies (Santa Cruz Biotechnologies).

Renal 1{alpha}-Hydroxylase and 24-Hydroxylase mRNA

Total RNA was isolated from the kidney using TRIzol reagent (Invitrogen). cDNA was synthesized using 1x PCR buffer, 7.5 mM MgCl2, 1 mM dNTP, 5 µM random primers, and 2.5 U/µl MMLVRT enzyme at the following temperatures: 25°C for 10 min, 48°C for 40 min, and 95°C for 5 min. Human and mouse 1{alpha}-hydroxylase, 24-hydroxylase, and beta-glucuronidase (Gus) Taqman probes and primers were purchased from Integrated DNA Technologies (Coralville, IA), and the abundance of 1{alpha}-hydroxylase and 24-hydroxylase mRNA, relative to that of Gus mRNA, was quantitated by real-time PCR using the ABI Prism 7700 Sequence Detection System (Applied Biosystems) as described (24). Two hundred fifty nanograms of template cDNA were used per PCR reaction, and the samples were amplified with an initial melt at 95°C for 12 min followed by 45 cycles at 95°C for 15 s and at 60°C for 1 min. The threshold cycle (Ct) at which a statistically significant increase in signal above background fluorescence was determined, and the Ct values for the 1{alpha}-hydroxylase and 24-hydroxylase transcripts were normalized to Ct values for Gus. A passive reference dye, ROX, was used to normalize for variations in volume or dye concentration between sample wells. Renal mitochondrial 1{alpha}-hydroxylase activity was determined as described (24).

RT-PCR Analysis

Total RNA (1 µg) was isolated from cultured HKC-8 cells, and RT-PCR was performed using the one-step RT-PCR kit (Qiagen). Conditions for amplification were as per manufacturer's protocol using specific primers for human klotho cDNA (5'-GGTCAAGTAC TGGATCACCA-3', and 3'-AACTGCTGACTTTTTTGCTC-5') and human FGF receptors (FGFR) 1-4 (FGFR1: 5'-ATCAACCTGCTGGGGGCCTGCACGCAGGAT-3' and 3'-TCCCGTGCGAGGCCAAAGTCT GCTATCTTCAT-5'; FGFR2: 5'-TGTCTGGTCCTT CGGGGTGTTAATGTGGGA-3' and 3'-TCATGTTTTAACACTGCCGTTTATGTGTGGA-5'; FGFR3: 5'-TCCTGCTCTGGGAGATCTT CACGCTGGGGGGCT-3' and 3'-AGCAGGTCG TGGGCAAACACGGAGTC GTCCCCT-5'; FGFR4: 5'-TGTGGTCTTTTGGGATCCTGCT ATGGGAGAT-3' and 3'-TCATGTCTGCACCC CAGACCCGAAGGGGAA-5'). The human klotho primers detect both transmembrane and secreted forms of klotho (416-bp PCR product). As a positive control, HKC-8 cells were transiently transfected with a plasmid expressing the transmembrane form of klotho (a kind gift from Dr. M. Kuro-o). The PCR products were electrophoresed on 2% agarose gels containing ethidium bromide (0.5 mg/ml) and visualized by UV transillumination.

Statistical Analysis

Data are expressed as means ± SE. The significance of differences between two groups was analyzed by Student's t-test or between multiple groups by ANOVA and post hoc testing was performed using the Bonferroni t-test. A P value <0.05 was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Experiments

Serum Pi concentrations and renal BBM Na/Pi cotransport. To characterize the effects of FGF-23 on Pi metabolism in vivo, we administered FGF-23(R176Q) to normal mice fed a 1% Pi diet. A single intravenous injection (0.33 µg/g body wt) of FGF-23(R176Q) induced a 30% decrease in serum Pi concentration 17 h after injection (P < 0.05; Fig. 1A). In BBM vesicles prepared from kidney cortex of FGF-23-treated mice, Na/Pi cotransport activity was significantly reduced by 20 and 29% at 5 and 17 h, respectively (P < 0.001), compared with that in vehicle-treated controls (Fig. 1B). No change was detected in the transport of sulfate or glucose in FGF-23-treated mice compared with that in the control group (not shown). Western blot analysis revealed that the decrease in Na/Pi cotransport activity in FGF-23-treated mice was associated with a 41 and 37% decrease in the abundance of type IIa Na/Pi cotransporter protein at 5 and 17 h (P < 0.05), respectively (Fig. 2, A and B).


Figure 1
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  		Fig. 1. Effect of FGF-23(R176Q) on serum Pi concentration (A) and renal brush-border membrane (BBM) Na/Pi cotransport activity (B) in normal mice. Mice were injected intravenously with human recombinant FGF-23(R176Q) (0.33 µg/g body wt) and killed either 5 or 17 h later. BBM vesicles were prepared from kidney cortex as described in MATERIALS AND METHODS. Bars depict means ± SE (n = 2–10 mice per group). *P < 0.05 compared with the vehicle group.

 

Figure 2
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  		Fig. 2. Effect of FGF-23(R176Q) on Npt2a protein abundance in normal mice. Mice were injected intravenously with human recombinant FGF-23(R176Q) (0.33 µg/g body wt) and killed either 5 or 17 h later. BBM vesicles were prepared from kidney cortex as described in MATERIALS AND METHODS. A: BBMs were fractionated on 10% SDS-PAGE and probed with anti-Npt2a and actin antibodies. Antigen antibody complexes were visualized by immunoblotting. B: abundance of Npt2a cotransporter protein, relative to that of actin, is expressed as a percent relative to vehicle. Bars depict means ± SE (n = 2–10 mice per group). *P < 0.05 compared with the vehicle group.

 
Renal 1{alpha}-hydroxylase and 24-hydroxylase mRNA. To determine the effects of FGF-23 on renal vitamin D metabolism, we injected FGF-23(R176Q) (6, 12, or 24 µg/day) intraperitoneally at 8-h intervals for 4 days into normal mice fed a 1% Pi diet. FGF-23(R176Q) induced dose-dependent decreases in renal 1{alpha}-hydroxylase mRNA abundance of 38, 63, and 75%, respectively (P < 0.05), as determined by real-time PCR, relative to that in vehicle-treated mice (Fig. 3A). Conversely, FGF-23(R176Q) induced a dose-dependent increase in 24-hydroxylase mRNA abundance of 4- to 12-fold (P < 0.05; Fig. 3B).


Figure 3
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  		Fig. 3. Effect of FGF-23(R176Q) on renal P450c1{alpha} (1{alpha}-hydroxylase) and P450c24 (24-hydroxylase) mRNA abundance in normal mice fed a 1% Pi diet. Mice were injected intraperitoneally with FGF-23(R176Q) (0.07–0.30 µg/g body wt) at 8-h intervals for 4 days, killed, and total RNA was isolated from the kidney. The abundance of renal P450c1{alpha} mRNA (A) and P450c24 mRNA (B) was quantitated by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle. Bars depict means ± SE (n = 3–4 mice per group). *P < 0.05 compared with the vehicle group.

 
To examine the effect of dietary Pi intake on the response to FGF-23, we injected FGF-23(R176Q) (3, 6, or 12 µg/day) at the above schedule into normal mice fed a Pi-restricted (0.02%) diet. In mice fed the Pi-restricted diet, FGF-23(R176Q) induced a greater suppression of 1{alpha}-hydroxylase mRNA abundance (97% suppression with 12 µg/day) than induced in mice fed the 1% Pi diet. FGF-23(R176Q) induced a dose-dependent 41–87% decrease in renal mitochondrial 1{alpha}-hydroxylase activity, and the decrease in enzyme activity correlated directly with the 51–98% decrease observed in 1{alpha}-hydroxylase mRNA abundance (R = 0.95, P < 0.01). The increase in 24-hydroxylase mRNA abundance induced by FGF-23 was also greater in mice fed the Pi-restricted diet (38-fold increase with 12 µg/day) than in mice fed the 1% Pi diet.

In Vitro Experiments

1{alpha}-Hydroxylase mRNA. To determine whether the effect of FGF-23(R176Q) to suppress renal 1{alpha}-hydroxylase gene expression is direct, we treated human (HKC-8) and mouse (MCT) proximal renal tubule cells with FGF-23(R176Q) at varying doses (1 to 200 ng/ml) for 21 h in vitro. In HKC-8 cells, FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1{alpha}-hydroxylase mRNA abundance with a maximum suppression of 37% (P < 0.05; Fig. 4A), relative to that in cells treated with vehicle. The magnitude of suppression of 1{alpha}-hydroxylase mRNA increased with time of exposure to FGF-23(R176Q) (10 ng/ml) and was significant at 6 to 21 h of exposure (P < 0.05; Fig. 4B). Similar findings were observed in MCT cells, in which FGF-23(R176Q) induced a dose-dependent suppression of 1{alpha}-hydroxylase mRNA abundance after 21 h of exposure (P < 0.05; Fig. 5A). However, the concentration of FGF-23 required to suppress 1{alpha}-hydroxylase mRNA expression in these cells was ~10-fold higher than that required in HKC-8 cells.


Figure 4
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  		Fig. 4. Effect of FGF-23(R176Q) on P450c1{alpha} mRNA abundance in HKC-8 cells. HKC-8 cells were cultured under standard conditions and treated with vehicle or FGF-23(R176Q) (1–10 ng/ml) for 21 h (A) and vehicle on FGF-23(R176Q) (10 ng/ml) for varying periods of time (B). Total RNA was isolated and P450c1{alpha} mRNA abundance was determined by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle. Bars depict means ± SE (n = 2–4 experiments, each performed in triplicate). *P < 0.05 compared with the vehicle group.

 

Figure 5
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  		Fig. 5. Effect of FGF-23(R176Q) on P450c1{alpha} (A) and P450c24 mRNA abundance (B) in MCT cells. MCT cells were treated with vehicle or FGF-23(R176Q) (10–200 ng/ml) for 21 h. P450c1{alpha} and P450c24 mRNA abundance was determined by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle. Bars depict means ± SE (n = 2–6 experiments, each performed in triplicate). *P < 0.05 compared with the vehicle group.

 
24-Hydroxylase mRNA. To determine whether the effect of FGF-23(R176Q) to increase renal 24-hydroxylase gene expression is direct, we treated MCT cells with FGF-23(R176Q) at varying doses for 21 h. At concentrations of 10 and 50 ng/ml, FGF-23(R176Q) induced a twofold increase in 24-hydroxylase mRNA abundance (P < 0.05; Fig. 5B) compared with that in cells exposed to vehicle. These findings parallel the effects of FGF-23 to increase renal 24-hydroxylase mRNA in mice injected with FGF-23, although the magnitude of stimulation was several-fold lower in vitro.

MAPK Signaling Pathway

Recent experiments in mice and in cultured renal epithelial cells suggested that activation of the MAPK signaling pathway plays an important role in mediating the renal response to FGF-23 (34, 35). To determine whether FGF-23(R176Q) activates the MAPK signaling pathway in MCT cells, we performed Western blot analyses to detect phosphorylation of ERK1/2 and p38 proteins, both of which are downstream signaling components of the MAPK pathway. In MCT cells, FGF-23(R176Q) induced phosphorylation of ERK1/2 protein within 5 min of exposure, and the effect was observed for up to 2 h (Fig. 6A). However, when cells were pretreated with the inhibitor PD98059, FGF-23(R176Q)-induced phosphorylation of ERK1/2 was completely blocked (Fig. 6A). PD98059 specifically inhibits MEK1/2, preventing phosphorylation of its downstream target, ERK1/2. FGF-23(R176Q) did not induce phosphorylation of p38 protein (data not shown).


Figure 6
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  		Fig. 6. Effect of FGF-23(R176Q) on signal transduction via the MAPK pathway. A: MCT cells were treated with vehicle or FGF-23(R176Q) (100 ng/ml) for 1–120 min. For lane 7, samples were incubated with PD98059 (50 µM) 30 min before exposure to FGF-23(R176Q). Cell lysates were prepared in the presence of phosphatase inhibitors, and phosphorylated proteins were detected by chemiluminescence using rabbit anti-phospho-ERK1/2 antibodies (top). Equal protein loading was monitored by detection of total ERK2 protein (bottom). B: MCT cells were treated with PD98059 (50 µM) 30 min before exposure to FGF-23(R176Q) (100 ng/ml) or vehicle and samples were subsequently incubated for 21 h. P450c1{alpha} mRNA abundance was quantitated by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle. Bars depict means ± SE (n = 4 experiments, each performed in triplicate).

 
To determine whether activation of the MEK/ERK1/2 signaling pathway is necessary for the regulation of 1{alpha}-hydroxylase gene expression by FGF-23(R176Q), we measured 1{alpha}-hydroxylase mRNA abundance in MCT cells treated with FGF-23(R176Q), with and without prior treatment with PD98059. In MCT cells pretreated with PD98059, the suppression of 1{alpha}-hydroxylase mRNA induced by FGF-23(R176Q) was completely blocked (Fig. 6B).

Expression of Klotho and FGFR 1-4 mRNA

Recent studies showed that the aging suppressor gene Klotho encodes a protein that directly binds to multiple FGFRs (17, 34). Cell proliferation assays demonstrate that klotho augmented FGF-23-induced phosphorylation of FGFRs and thus activation of the ERK1/2 pathway, providing evidence that klotho acts as a critical cofactor essential for FGF-23 signaling (17, 34). To determine whether klotho and FGFRs are expressed in HKC-8 cells, we performed RT-PCR analysis using specific primers. We detected mRNA for klotho and for FGFR 1–4 in HKC-8 cells and observed an increase in klotho expression in cells transfected with a klotho expression plasmid (Fig. 7).


Figure 7
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  		Fig. 7. Detection of klotho and fibroblast growth factor receptor (FGFR) mRNA. RT-PCR analysis was performed on total RNA isolated from HKC-8 cells using specific primers for human klotho (A) and human FGFR 1-4 (B). HKC-8 cells transfected with transmembrane klotho expression plasmid were used as positive control (A). The PCR products were electrophoresed on 2% agarose gels and visualized by UV transillumination.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrate that injection of recombinant mutant FGF-23(R176Q) into normal mice induces hypophosphatemia, suppression of renal Na/Pi cotransport activity, dose-dependent suppression of the renal abundance of 1{alpha}-hydroxylase mRNA, and stimulation of 24-hydroxylase mRNA. In cultured renal proximal tubule cells treated with FGF-23(R176Q), we demonstrate direct suppression of the abundance of 1{alpha}-hydroxylase mRNA and stimulation of 24-hydroxylase mRNA. We provide evidence that FGF-23 induces activation of the MAPK signaling pathway via ERK1/2 and that such activation is necessary for FGF-23 to induce suppression of 1{alpha}-hydroxylase gene expression in proximal tubule cells.

To determine the effect of FGF-23 on renal handling of Pi, we administered recombinant mutant FGF-23(R176Q) to normal mice fed a normal-Pi diet. FGF-23(R176Q) induced hypophosphatemia and suppression of Na/Pi cotransport activity in BBM vesicles at 5 and 17 h after injection. The decrease in BBM Na/Pi cotransport activity was accompanied by a corresponding decrease in the abundance of Npt2a protein. These findings confirm previous reports that hypophosphatemia and renal Pi wasting are induced in mice by administration of a single or multiple doses of recombinant human wild-type or mutant FGF-23 (28, 30).

FGF-23 plays a critical role in the regulation of vitamin D metabolism. Administration of FGF-23 or its overexpression in mice induces a significant reduction in serum concentrations of 1,25(OH)2D (28, 29). To determine the effect of FGF-23 on the enzymes responsible for the synthesis and catabolism of 1,25(OH)2D, we administered multiple intraperitoneal injections of FGF-23(R176Q) to wild-type mice fed a normal-Pi diet. FGF-23(R176Q) induced a dose-dependent suppression of renal 1{alpha}-hydroxylase mRNA abundance of 38 to 75% compared with that in mice injected with vehicle. Conversely, renal abundance of P450c24 mRNA increased by 4- to 12-fold in FGF-23-treated mice. The magnitude of suppression of 1{alpha}-hydroxylase mRNA and of stimulation of 24-hydroxylase mRNA was greater in FGF-23-injected mice fed a Pi-restricted diet. Moreover, under these conditions, FGF-23 elicited comparable changes in 1{alpha}-hydroxylase activity. These findings confirm other reports that FGF-23 can regulate renal expression of the enzymes responsible for the production and catabolism of 1,25(OH)2D in vivo (1, 28, 29).

Although FGF-23 is currently thought to play a critical role in regulating the renal reabsorption of Pi and the production of 1,25(OH)2D, little is known about the molecular mechanisms of its action in target tissues. The rapid changes in renal 1{alpha}-hydroxylase and 24-hydroxylase mRNA expression we and others observed after a single injection of FGF-23 in mice (28) support the hypothesis that FGF-23 acts directly on the kidney to regulate renal 1,25(OH)2D production. In the present study, we show that FGF-23(R176Q) induces a direct, dose-dependent suppression of 1{alpha}-hydroxylase mRNA in human and mouse renal proximal epithelial cells in vitro. In HKC-8 cells, the suppression of 1{alpha}-hydroxylase mRNA abundance was detected as early as 6 h with a maximum suppression observed at 21 h of treatment with FGF-23(R176Q). In MCT cells, the magnitude of suppression of 1{alpha}-hydroxylase mRNA induced by FGF-23(R176Q) was comparable to that of HKC-8 cells at 21 h of exposure, although higher doses of FGF-23(R176Q) were required to induce this effect in MCT cells. In contrast to the suppressive effect on 1{alpha}-hydroxylase mRNA, the abundance of 24-hydroxylase mRNA increased by twofold in MCT cells treated with FGF-23(R176Q). These novel findings provide evidence that FGF-23 directly regulates 1{alpha}-hydroxylase and 24-hydroxylase gene expression in renal tubule cells, thereby directly regulating the production and catabolism of 1,25(OH)2D in the mammalian kidney.

Recent studies showed that FGF-23 activates the receptors for the FGFs in vitro (35, 36). FGFRs are encoded by four genes, FGFR 1–4, which generate seven isoforms by alternate splicing (16, 25, 27, 31). Activation of FGFR by ligand binding induces tyrosine autophosphorylation, stimulation of intrinsic receptor tyrosine kinase activity, and subsequent activation of downstream signaling pathways. Although little is known about FGF-23-induced signal transduction, studies in opossum kidney cells have shown that recombinant mouse FGF-23 binds specifically to FGFR receptor isoforms 2c and 3c and activates the MAP kinase signaling pathway, leading to phosphorylation of p38 and ERK1/2 and inhibition of sodium-dependent Pi uptake (35). To determine the role of the MAP kinase pathway in the regulation of vitamin D metabolism by FGF-23, we treated MCT cells with FGF-23(R176Q) and determined phosphorylation of ERK1/2 and p38. FGF-23(R176Q) induced rapid phosphorylation of ERK1/2, and this effect was successfully blocked by prior treatment with the MEK inhibitor PD98059. Phosphorylation of p38 was not observed. Simultaneous experiments demonstrated that activation of the ERK1/2 pathway was necessary for FGF-23 to induce suppression of 1{alpha}-hydroxylase gene expression, as the suppression was completely blocked in MCT cells pretreated with PD98059. Thus these novel findings demonstrate that FGF-23 can regulate 1{alpha}-hydroxylase gene expression in proximal tubule MCT cells via activation of the MAPK/ERK1/2 signaling pathway and provide evidence that FGF-23 acts directly on the kidney in vivo to suppress the renal production of 1,25(OH)2D.

The Klotho gene which encodes a 130-kDa type I membrane protein is essential for FGF-23 signaling (17, 34). The extracellular domain of klotho protein is shed and secreted into the blood (11, 18), potentially functioning as a humoral factor. Although little is known about the functions of the transmembrane or secreted forms of klotho, it has been shown that klotho binds to multiple FGFRs and increases their affinity for FGF-23. Whether the transmembrane or the secreted form of klotho is the cofactor essential for FGF-23 signaling remains unknown. In mice, klotho is predominantly expressed in the distal convoluted tubule and was not detected by in situ hybridization or immunohistochemistry in the proximal tubule (13, 20). Since renal Pi reabsorption and 1,25(OH)2D production occur predominantly in the proximal tubule, it was proposed that klotho functions in a paracrine/endocrine fashion to mediate the actions of FGF-23 on Pi transport and 1,25(OH)2D production in this segment of the nephron. In the present study, we demonstrate that klotho and FGFR 1–4 mRNA are expressed in HKC-8 cells, a renal proximal tubule epithelial cell line. Further studies are needed to identify the specific receptors that mediate the actions of FGF-23 on renal vitamin D metabolism.

In summary, the present study demonstrates that FGF-23 plays an important role in regulating Pi and vitamin D metabolism in vivo and acts directly to regulate 1{alpha}-hydroxylase and 24-hydroxylase gene expression in renal epithelial cells in vitro.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-54433 (to A. A. Portale) and DK-073092 (to F. Perwad), the March of Dimes Birth Defects Foundation (to A. A. Portale), the Canadian Institutes of Health Research (MT-14107 to H. S. Tenenhouse), the National Kidney Foundation (to F. Perwad), and gifts from the David Carmel Trust (to A. A. Portale).


   ACKNOWLEDGMENTS
 
We thank S. Schiavi (Genzyme) for providing recombinant FGF-23(R176Q) protein used in the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Portale, 533 Parnassus Ave. Rm U-585, San Francisco, CA 94143 (e-mail: aportale{at}peds.ucsf.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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