Human Fibroblast Growth Factor-23 Mutants Suppress Na+-dependent Phosphate Co-transport Activity and 1alpha ,25-Dihydroxyvitamin D3 Production*

Hitoshi SaitoDagger §, Kenichiro KusanoDagger , Masahiko Kinosaki, Hirotaka Ito, Michinori HirataDagger , Hiroko Segawa||, Ken-ichi Miyamoto||, and Naoshi FukushimaDagger

From the Dagger  Pharmaceutical Laboratory II and  Technology Laboratory, Chugai Pharmaceutical Co., Ltd., Shizuoka 412-8513 and the || Nutritional Science, Department of Nutrition, School of Medicine, Tokushima University, Tokushima 770-8503, Japan

Received for publication, August 2, 2002, and in revised form, October 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human fibroblast growth factor 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a 20-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/Pi co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1alpha ,25-dihydroxyvitamin D3 to less than 15 pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The FGF23 protein is a novel, secreted protein that consists of 251 amino acids, including a putative N-terminal signal peptide (residues 1-24) (1). Patients with autosomal dominant hypophosphatemic rickets (ADHR),1 a rare renal phosphate-wasting disorder, have been found to have three missense mutations in two arginine residues, R176Q, R179Q, and R179W in the FGF23 gene (2, 3). Also, the FGF23 was identified as a causative factor of oncogenic osteomalacia (OHO) (4). It was highly expressed in the tumor isolated from OHO patients (5), whereas, it was expressed at a very low level in normal tissues. Patients with OHO share similar manifestations with ADHR such as hypophosphatemia, decreased or inappropriately normal 1alpha ,25-dihydroxyvitamin D concentrations, and osteomalacia. Recent studies indicated administration of the recombinant FGF23 protein reduced serum phosphate without affecting serum calcium, as well as increasing renal phosphate excretion in mice. Mice bearing FGF23-expressing CHO cells showed a suppressed 25-hydroxyvitamin D 1alpha -hydroxylase mRNA expression in the kidney (4). These results suggested FGF23 might inhibit renal sodium-dependent phosphate (Na/Pi) co-transporter activity. However, according to the results from the in vitro studies using opossum kidney cells, the FGF23 effects on Na/Pi co-transporter are still controversial (4, 6). Yamashita et al. reported that heparin was required to activate FGF23·FGFR3c complex and phosphate reabsorption in opossum kidney cells. Also, the mitogen-activating protein kinase signal pathway was a major pathway for the FGF23 signaling (7). Shimada et al. and White et al. reported that ADHR mutants became resistant to the intracellular proteolytic cleavage by the subtilisin-like protein convertases in vitro, so that a large amount of the mutant protein consequently may be secreted in an intact form into the blood circulation (8, 9). To investigate the mechanisms of the FGF23 action on phosphate homeostasis in vivo, we transiently introduced the FGF23 mutant expression plasmids into rodents by the naked DNA injection, and examined changes in biochemical parameters, parathyroid hormone, and vitamin D metabolites. Also, we examined the phosphate uptake of brush border membrane vesicles (BBMVs) isolated from the kidney and intestine of the naked DNA-injected animals to assess the effect of FGF23 mutant on Na/Pi co-transport activity in kidney and intestine. Furthermore, we investigated target organs of hFGF23 by using the thyroparathyroidectomized rat model and the adenine-induced rat nephritis model.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of FGF23 cDNA-- Full-length human FGF23 cDNA was cloned from commercially available human heart cDNA library (BD Biosciences Clontech, Palo Alto, CA) by PCR, using the forward primer (5'-GGAATTCTCGAGCCACCATGTTGGGGGCCCGCCTCAGGCTCTG-3') and the reverse primer (5'-GGAATTCTCGAGCTACTAGATGAACTTGGCGAAGG-3'). The PCR conditions were: an initial 2-min denaturation at 95 °C, followed by 35 cycles of 60 s at 60 °C, 40 s at 94 °C, and a final 7-min extension at 72 °C. The 750-bp PCR product was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) plasmid. The identity of each cDNA was confirmed by DNA sequence analysis (model 377 Genetic Analyzer, Applied Biosystems, Foster City, CA).

Construction of Plasmids Expressing FGF23 and Its Mutants-- Mutagenesis was performed according to a method called "recombinant polymerase chain reaction" (10). To generate the expression vector for FGF23 mutants, DNA fragments amplified by PCR were inserted into a TA cloning vector, pCR2.1-TOPO (BD Biosciences Clontech). The DNA sequences of the obtained cDNAs were confirmed by DNA sequencing. The plasmids were digested with EcoRI and then the fragments were inserted into a unique EcoRI site between the CAG promoter and a 3'-flanking sequence of the rabbit beta -globin gene in the pCAGGS3 expression plasmid, which was kindly provided by Dr. Miyazaki (11). The hFGF23, hFGF23R176Q, hFGF23R179Q, hFGF23R179W, hFGF23R176QR179Q, and hFGF23R176QR179W expression plasmids were named pCGF23, pCGFM1, pCGFM2, pCGFM3, pCGFM4, and pCGFM5, respectively. The empty pCAGGS3 plasmid was used as a MOCK control. All the plasmids were grown in Escherichia coli DH5alpha cells, and prepared with a Qiagen EndFree Plasmid Giga kit (Qiagen, Tokyo, Japan). The quantity and the quality of the plasmids were determined by measuring absorbance at 260 and 280 nm. Naked DNA injection solution containing 10 µg of pCGF23, pCGFM1, pCGFM2, pCGFM3, pCGFM4, and pCGFM5 or MOCK plasmid was prepared using the TransIT® in vivo gene delivery system (TaKaRa, Osaka, Japan) immediately before injection.

Production of C-terminal FLAG-tagged hFGF23R179 Mutant Protein-- 5 × 106 of COS cells suspended in 400 µl of Dulbecco's modified minimal essential medium (Invitrogen, Rockville, MD) containing 10% fetal bovine serum was transfected with 10 µg of pCGFM2-F plasmid, the expression plasmid of C-terminal FLAG (residues DYKDDDDK)-tagged hFGF23R179Q, by electroporation. Transfected cells were grown in the 10% fetal bovine serum/Dulbecco's modified minimal essential medium overnight and then incubated in serum-free CHO-S-SFMII (Invitrogen) medium for 3 days. The conditioned media were collected and centrifuged at 3000 rpm for 15 min to remove the cell pellets. The supernatant was applied to the anti-FLAG antibody affinity column equilibrated with phosphate-buffered saline containing 0.05% Tween 80 and eluted with 0.5 M glycine/HCl (pH 3.5). Purified C-terminal FLAG-tagged hFGF32R179Q protein was analyzed by SDS-PAGE and the Western blot using anti-FLAG monoclonal antibody (Sigma, St. Louis, MO).

Western Blot Analysis of N-terminal FLAG-tagged hFGF23 and hFGF23R179Q Mutant-- COS cells (5 × 106) were seeded on 100-mm culture dishes and transiently transfected in serum-free medium with the FuGENETM 6 reagent (Roche Applied Science, Indianapolis, IN) using 1 µg of plasmid DNA according to the manufacturer's instructions. After transfection, the incubation was continued for an additional 48 h. Conditioned media were concentrated ~20 times with Centricon-10 columns (Millipore Corp., Bedford, MA) at 4 °C. The concentrated culture media were applied to 12% SDS-PAGE and transferred to an Immobilon P membrane (Millipore) by semi-dry electroblotting. The expressed N-FLAG-tagged proteins were detected by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate system (KPL Inc., Gaithersburg, MD) with anti-FLAG M2 monoclonal antibody (Sigma) used as a first antibody and alkaline phosphatase-conjugated anti-mouse IgG antibody (ZyMed Laboratories, South San Francisco, CA) as a second antibody.

Naked DNA Injection into Mice and Rats-- Seven-week-old male Sprague-Dawley rats weighing 180 g were purchased from Charles River (Tokyo, Japan) and maintained under specific pathogen-free conditions with a 12-h light/dark cycle. All rats were allowed unlimited access to a normal rodent food (CE-2, CREA Japan Inc., Tokyo, Japan) and tap water. After acclimating for 1 week, animals were intravenously injected with 12 ml of a DNA solution containing 10 µg of each expression plasmid, pCGF23, pCGFM1, pCGFM2, pCGFM3, pCGFM4, pCGFM5, pCGF23NF, pCGFM2NF, as described above, or MOCK plasmid under light anesthesia. Four days after the naked DNA injection, blood samples were obtained from the vena cava under ether anesthesia. Serum calcium, serum inorganic phosphorus, urea nitrogen, and serum creatinine were measured by an automatic analyzer (Type 7170E, Hitachi Corp., Japan). Serum concentration of the C-terminal portion of hFGF-23 was determined by the Human FGF-23 (C-term) ELISA Kit (Immunotopics Inc., San Clemente, CA). Purified C-terminal FLAG-tagged human FGF-23 protein was used as a standard. Serum 1alpha ,25-dihydroxyvitamin D was determined by the radioreceptor assay (Mitsubishi BCL, Tokyo, Japan). Serum 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were measured by competitive protein binding assay (Mitsubishi BCL). Serum PTH was determined by the Rat Intact PTH ELISA Kit (Immunotopics, Inc., San Clemente, CA). In the case of mice, 7-week-old male CD-1 mice (Charles River Japan) weighing ~30 g were maintained under the same conditions as the rats described above. 3 ml of DNA solution containing 10 µg of expression plasmid was intravenously injected into the mice. All animal procedures were conducted in accordance with Chugai Pharmaceutical's ethical guidelines of animal care, and all experimental protocols were approved by the Animal Care Committee of the institution.

Quantitative Analysis of FGF23 mRNA Expression-- Liver total RNA in naked DNA-injected animals was extracted with ISOGEN (Nippon Gene, Tokyo, Japan). cDNA was synthesized using Moloney murine leukemia virus H- reverse transcriptase (Superscript, Invitrogen), oligo(dT)12-18 primer. The relative amount of FGF23 cDNA compared with glyceraldehyde-3-phosphate dehydrogenase cDNA was determined by a Model 7700 sequence detector (Applied Biosystems).

Preparation of BBMVs and Transport Measurements-- BBMVs were prepared from the rat kidney by the Ca2+ precipitation methods as previously described (12-14). The uptake of radiolabeled Pi was measured by the rapid filtration technique. Reaction was initiated by mixing 20 µl of the vesicle suspension with 80 µl of incubation solution (100 mM mannitol, 125 mM NaCl, 20 mM HEPES/Tris (pH 7.4), 0.1 mM KH2PO4) and incubated at 25 °C for 1 min. The measurement of Na+-dependent and Na+-independent Pi uptake was performed as described previously. The transport reaction was terminated by adding 1 ml of an ice-cold stop solution (100 mM mannitol, 100 mM choline chloride, 20 mM MgSO4, 20 mM HEPES/Tris (pH 7.4), and 5 mM KH2PO4). The reaction mixture was immediately transferred to a premoistened filter (0.45 µm) maintained under vacuum.

Naked DNA Injection into Adenine-induced Nephritis Model Rats-- Seven-week-old male Wistar rats (CLEA Japan Inc.) were maintained with a CE-2 diet containing 1.18% calcium and 1.03% phosphorus. To induce adenine-induced nephritis (15), rats were administered orally 240 mg/kg body weight of adenine dissolved in carboxymethyl cellulose, 5 days per week for 3 weeks. At Day 21, rats were intravenously injected 12 ml of DNA solution containing 10 µg of each expression plasmid, pCGF23, pCGFM2, or MOCK. From then, rats were administered the adenine solution everyday. Blood samples were obtained at Day 28 from the jugular vein under ether anesthesia.

Infusion of FGF23R179Q Protein or PTH-(1-34) into TPTX Rats-- Eight-week-old male Sprague-Dawley rats purchased from Charles River (Tokyo, Japan) were thyroparathyroidectomized (TPTX) under ether anesthesia. After confirming that hypocalcemia was induced, rats were fixed in Ballman cages and administered either vehicle, PTH-(1-34) at a constant rate of 0.3 nmol/ml/h, or C-terminal FLAG-tagged hFGF23R179Q protein at a constant rate of 6 µg/ml/h for 6 h through an intravenous catheter (PE-50, IntramedicTM, Becton Dickinson, Sparks, MD) placed into the femoral vein (16). The vehicle solution contained 0.05% Tween 80 in saline. Total blood was collected at 6 h via the vena cava under ether anesthesia. Serum inorganic phosphorus and serum calcium were determined by an automatic analyzer.

Statistical Analysis-- Data were expressed as means ± S.E., and statistical significance was determined using SAS and Student's t test. A p value of <0.05 was considered statistically significant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human FGF23 Mutants Induced Hypophosphatemia but Not Native Human FGF23-- Eight-week-old Sprague-Dawley rats were intravenously administered 12 ml of the naked DNA solution containing 10 µg of pCGF23, pCGFM2, or MOCK plasmid. Animals were sacrificed to collect blood samples at Day 4. Rats injected with hFGF23R179Q mutant expression plasmid, pCGFM2, demonstrated a significantly low serum inorganic phosphorus concentration at an average of 6.2 mg/dl, compared with MOCK control of 8.5 mg/dl. However, administration of native human FGF23 expression plasmid, pCGF23, did not induce hypophosphatemia. Serum inorganic phosphorus remained at a normal value of 8.6 mg/dl (Fig. 1).


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Fig. 1.   Effect of the hFGF23 and hFGF23R179Q mutant on serum inorganic phosphorus. 10 µg of either pCAGGS3 (MOCK), hFGF23 or hFGF23R179Q expression plasmid was administered intravenously with 12 ml of the delivery solution within into male Sprague-Dawley rats. Blood samples were taken at Day 4 from vena cava under ether anesthesia. Serum inorganic phosphorus was determined according to the Fiske-Sabbarow method. Data are representative of at least three independent experiments and shown with means ± S.E. *, statistically significant between MOCK group and either hFGF23 or hFGF23R179Q group; p < 0.05.

Hypophosphatemic activity was compared among all ADHR mutants (R176Q, R179Q, R179W) and double mutants (R176Q/R179Q, R176Q/R179W) using naked DNA injection of mice. There was no difference in the hypophosphatemic activity among the three ADHR mutants. Also, no synergistic and/or additional effect was observed in the double mutants, suggesting one missense mutation is sufficient to produce hypophosphatemic activity (Table I). Other serum biochemical parameters such as serum calcium, serum urea nitrogen, and serum creatinine were within a normal range among all mice, as shown in Table I. Therefore, naked DNA injection of the hFGF23 mutant expression plasmids induced hypophosphatemia in both rats and mice, and it was not due to a physical impairment of the nephron caused by the rapid and transient blood volume expansion.

                              
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Table I
Serum biochemical parameters of the FGF23 or the FGF23 mutants injected mice

Native Human FGF23 Was Not Secreted as an Intact Form-- Previous reports indicated injected naked DNA plasmids were mainly incorporated in the liver and remained expressed for 2-4 weeks (17). To assess an expression level of the injected plasmids, liver total RNA in the injected rats was isolated using ISOGEN. Expression ratio of hFGF23 or hFGF23R179Q mRNA in comparison with endogenous glyceraldehyde-3-phosphate dehydrogenase mRNA in the liver was determined by the quantitative RT-PCR. Both hFGF23 and hFGF23R179Q mutant mRNA were highly expressed in the injected animals 100-fold more than in MOCK control animals on Day 4 (Fig. 2A). However, as shown in Fig. 2B, the C-terminal portion of human FGF-23 protein was detected only in the serum from the FGF23R179Q-injected rat (1109 pg/ml). The C-terminal hFGF23 level was below the detection limit in the sera from the hFGF23-injected rat and the MOCK-injected rats. The Western blot analysis revealed that the N-terminal FLAG-tagged human FGF23 was not secreted as an intact form with a putative molecular mass of 32 kDa but rather as a smaller 20-kDa peptide (Fig. 2C) in the COS cell-conditioned media, whereas, the N-terminal FLAG-tagged human FGF23R179Q mutant was secreted in the conditioned media as an unchanged 32-kDa protein. These observations suggest that native hFGF23 protein was cleaved into a 20-kDa peptide by intracellular protease(s), presumably at the RXXR motif from residues Arg-176 to Arg-179, and then lost its hypophosphatemic activity.


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Fig. 2.   A, hFGF23 and hFGF23R179Q mRNA expression in the liver. Approximately 100 mg of liver samples were excised 4 days after the naked DNA injection, and immediately homogenized with ISOGEN on ice. Total RNA from each sample was prepared according to the manufacturer's instructions. Expression ratio of the MOCK, hFGF23, or hFGF23R179Q mRNA versus endogenous glyceraldehyde-3-phosphate dehydrogenase mRNA was determined using the quantitative RT-PCR. The PCR products were monitored by the fluorescence of the SYBR Green. B, serum concentration of C-terminal portion of hFGF23. The serum concentration of C-terminal hFGF23 of the MOCK-, hFGF23-, or hFGF23R179Q-injected rat was determined by the ELISA described in the text. C, Western blot analysis of the transfected cell-conditioned media. COS cell was transfected with the MOCK, N-terminal FLAG-tagged hFGF23 or N-terminal FLAG-tagged hFGF23R179Q expression plasmid using the FuGENE 6 reagent. The condition media were collected after a 48-h incubation and concentrated ~10 times using the Centricon-10. The FLAG epitope was detected by the ECL detection kit (Amersham Biosciences), using the anti-FLAG monoclonal antibody as a first antibody and the horseradish peroxidase-conjugated anti-mouse IgG antibody as a second antibody. Lane 1, N-terminal FLAG-tagged human FGF23; lane 2, N-terminal FLAG-tagged hFGF23R179 mutant; lane 3, MOCK control. Right arrows indicate molecular weights determined using the Broad-Range molecular weight marker (Bio-Rad).

Human FGF23R179Q Inhibited Vitamin D Hydroxylation-- Serum concentration of vitamin D metabolites in the rat serum was measured by radioreceptor assay using purified calf thymus vitamin D receptor. Serum 1alpha ,25-dihydroxyvitamin D3 in the FGF23R179Q-injected rats was decreased to less than the detection limit at 15 pg/ml. The hFGF23 or the MOCK-injected rats remained within the normal range at 132 and 115 pg/ml, respectively (Fig. 3). Serum 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 concentrations were also low in the hFGF23R179Q-injected rats. Thus, hFGF23R179Q inhibited vitamin D hydroxylation both in the kidney and liver.


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Fig. 3.   1alpha ,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 concentrations in the naked DNA injected rat serum. Either MOCK (n = 6; open columns), hFGF23 (n = 6; shaded columns), or hFGF23R179Q (n = 6; filled columns) mutant expression plasmid was injected intravenously into 8-week-old male rats. Serum concentration of 1alpha ,25-dihydroxyvitamin D3 at Day 4 was determined by the radioreceptor assay. Similarly, serum 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were measured by the competitive protein binding assay. Each column represents the mean ± S.E.

Renal and Intestinal Na/Pi Co-transport Activity-- The sodium-dependent phosphate co-transport activities were determined in the isolated BBMVs from the kidney and the small intestine (jejunum). Na+-dependent phosphate uptake of the BBMVs from the kidney was suppressed ~45% in the hFGF23R179Q-injected rats (Fig. 4A). In the small intestine, hFGF23R179Q injection reduced Na+-dependent phosphate uptake of the BBMVs by about 30%, as shown in Fig. 4B.


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Fig. 4.   Effect of hFGF23 on Na+-dependent Pi co-transport activity in rat kidney and intestine. BBMVs were prepared from rat kidney and small intestine (jejunum) 4 days after the naked DNA injection by the Ca2+ precipitation method. The Pi uptake was determined as described in the text. Results are mean ± S.E. (n = 6); *, p < 0.05, Na+-dependent: filled columns; Na+-independent: open columns.

Effects of hFGF23R179Q on Serum Inorganic Phosphorus in Adenine-induced Nephritis Rats-- To investigate the contribution of the intestinal Na/Pi co-transporters to the hypophosphatemic activity of hFGF23R179Q, we used the adenine-induced nephritis rat model. Adenine-induced nephritis rats were developed by oral administration of adenine for 3 weeks, which resulted in severe renal dysfunction with extremely elevated serum inorganic phosphorus (Fig. 5A) and creatinine (Fig. 5B). hFGF23R179Q had no effect on serum inorganic phosphorus of adenine-induced nephritis rats. This observation indicated that hypophosphatemic activity of the hFGF23R179Q is mainly due to the inhibition of the renal Na/Pi co-transporter, so that the hFGF23R179Q loses its hypophosphatemic activity when renal function was diminished.


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Fig. 5.   Effect of hFGF23R179 on serum inorganic phosphorus and creatinine of adenine-induced rat nephritis. Either MOCK or hFGF23R179Q expression plasmid was injected adenine-induced nephritis model rats. Blood samples were obtained at Day 4, and determined serum inorganic phosphorus (A) and serum creatinine (B) using automatic analyzer. Rats maintained with a normal rat chow were used as a control. Data represent mean ± S.E. (n = 6). *, p < 0.05 by Student's t test.

Hypophosphatemic Activity of hFGF23R179Q Was Independent of PTH-- As shown in Table II, the serum PTH of hFGF23R179Q rats was slightly lower than that of MOCK or hFGF23 rats; however, the difference was not statistically significant. Thyroparathyroidectomized (TPTX) rats were administered either vehicle, PTH-(1-34) (0.3 nmol/ml/h) or C-FLAG hFGF23R179Q protein (6 µg/ml/h) for 6 h through an intravenous catheter into the femoral vein, and then serum inorganic phosphorus and serum calcium were determined. Hyperphosphatemia induced by TPTX was cured by the infusion of hFGF23R179Q protein as well as PTH-(1-34) (Fig. 6A). The PTH-(1-34) administration also restored serum calcium; however, hFGF23R179Q did not affect serum calcium (Fig. 6B), suggesting that hFGF23R179Q reduces phosphate reabsorption, independent of PTH action.

                              
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Table II
Serum PTH concentration of the hFGF23 or the FGF23R179Q mutant-injected rats


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Fig. 6.   Effect of hFGF23R179 on serum inorganic phosphorus and calcium of thyroparathyroidectomized rats. Either vehicle (n = 3), PTH-(1-34) (n = 3), or C-terminal FLAG-tagged hFGF23R179 protein (n = 3) was infused via femoral catheter into thyroparathyroidectomized rats for 6 h. Serum inorganic phosphorus (A) and serum calcium (B) were determined by the Fiske-Sabbarow method and by the OCPC method, respectively. Data represent mean ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gene transfer by in vivo intravenous injection of hFGF23 expression plasmid into rodents increases the level of mRNA expression in the liver more than 100-fold compared with the MOCK control. Expression of exogenous DNA remains for 2-4 weeks (17). We have demonstrated that hFGF23R179Q mutant decreases serum inorganic phosphorus and inhibits low phosphate-induced 1alpha ,25-dihydroxyvitamin D3 production in proximal tubules by naked DNA injection in vivo. Also, we demonstrated that hFGF23R179Q inhibits the renal phosphate reabsorption without affecting parathyroid hormone secretion (18-20). However, injection of the native hFGF23 expression plasmid failed to show these effects, which may be due to the intracellular degradation of the hFGF23 protein during the secretion.

Previous reports (4) demonstrated that the C-terminal 6xHis- or V5/6xHis-tagged hFGF23 proteins were secreted into the cell-conditioned media partly as an intact form. Injection of purified C-His-hFGF23 proteins in mice resulted in lower serum inorganic phosphorus, as is observed in the mice bearing the CHO cells overexpressing C-His-hFGF23. White et al. (8, 9) reported that the FGF23R176Q mutant becomes resistant to the protein processing in the secretion machinery. It was also true for the C-His-FGF23, which is partially secreted as a 32-kDa protein to the conditioned media. On the other hand, N-FLAG-tagged hFGF23 was mostly secreted as a 20-kDa degraded form (Fig. 2C). In our study, the C-FLAG-hFGF23 was partly secreted as a 32-kDa form and had a hypophosphatemic activity (data not shown). According to these results, hFGF23 looses the hypophosphatemic activity when cleaved at a position between Arg-176 and Arg-179. Tumors isolated from patients with OHO overexpressed native hFGF23 but not the mutant form. Inoculation of the extracted tumor into nude mice induced hypophosphatemic rickets-like phenotype (21). These observations indicate FGF23 in the OHO tumor cells is not fully cleaved somehow during the secretory process. It is unlikely that an excess amount of hFGF23 overwhelms abundant intracellular subtilisin-like protein convertases, thus, it remains possible that one or more other factors expressed in the tumor cell inhibit FGF23 degradation.

The hFGF23R179Q mutant suppressed the Na/Pi co-transport activity both in the kidney and in the small intestine in vivo. The hFGF23R179Q lost its ability to reduce serum phosphorus when the proximal tubules in the kidney were impaired by adenine administration, suggesting that the hFGF23R179Q-induced hypophosphatemia occurs mainly through inhibition of the phosphate reabsorption in the proximal tubules, and thus, the renal Na/Pi co-transport activity seems more important for the overall Pi homeostasis in vivo.

Dietary Pi, vitamin D, and parathyroid hormone are three major physiological regulators of phosphate homeostasis. In particular, parathyroid hormone (PTH) induces phosphaturia by inhibiting the Na/Pi co-transport activity in kidney. Thyroparathyroidectomy leads to a moderate hyperphosphatemia with an increase in the Na/Pi co-transport activity. Whereas, the human FGF23 mRNA was expressed relatively highly in the thyroid/parathyroid tissue determined by RT-PCR, it is plausible that hFGF23 works as an autocrine/paracrine factor in the thyroid/parathyroid, and regulates hormone secretion such as PTH, thyroid hormone, and calcitonin. Our study indicates that hFGF23R179Q can act as a systemic factor, by normalizing serum phosphorus without changing serum calcium. Moreover, the hFGF23R179Q-naked DNA injection did not affect serum PTH (Table II). Therefore, the mechanism of the FGF23 action in regulating phosphate homeostasis is independent of PTH, thyroid hormone, and calcitonin.

The naked DNA injection was successfully applied to evaluate the in vivo biological activity of the FGF23, because the FGF23 was a secreted protein and required only small amount to influence phosphate metabolism. This technique may be applied to other humoral factors such as peptide hormones and cytokines (22-24). This naked DNA technique can exclude the risk of phenotypic abnormalities during development, which frequently occurred in the transgenic mice (25-27). hFGF23R179Q expression plasmid-injected animals remain hypophosphatemic for more than 2 weeks. Thus, a small amount of FGF23 protein is sufficient to induce hypophosphatemia in rodents. This technique would also make further in vivo studies possible with regard to investigating the relationship between FGF23 and PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and/or between FGF23 and FGF receptors. Both identifying the true target organs/cells and clarification of the detailed action of FGF23 will give us new insights in understanding phosphate homeostasis, and it will lead to the development of novel diagnostic or therapeutic reagents for diseases accompanying abnormal phosphate homeostasis.

    ACKNOWLEDGEMENTS

We thank Drs. Koh-Ichi Jishage and Kiyoshi Habu (Technology Laboratory, Chugai Pharmaceutical Co., Ltd.) for providing technical information concerning the naked DNA injection. We also thank Dr. Jun-Ichi Miyazaki (Osaka University) for providing us the pCAGGS3 expression vector. We also thank Dr. Paul Langman (Chugai Pharmaceutical Co., Ltd.) for his assistance with English usage.

    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: Pharmaceutical Laboratory II, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan. Tel.: 81-550-87-6735; Fax: 81-550-87-5326; E-mail: saitohts@chugai-pharm.co.jp.

Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207872200

    ABBREVIATIONS

The abbreviations used are: ADHR, autosomal dominant hypophosphatemic rickets; hFGF23, human fibroblast growth factor 23; PTH-(1-34), parathyroid hormone (1-34); OHO, oncogenic osteomalacia; CHO, Chinese hamster ovary cells; BBMV, brush border membrane vesicle; ELISA, enzyme-linked immunosorbent assay; TPTX, thyroparathyroidectomized; RT, reverse transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. The ADHR Consortium. (2000) Nat. Genet. 26, 345-348[CrossRef][Medline] [Order article via Infotrieve]
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