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INTRODUCTION |
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 1
,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 1
-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.
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EXPERIMENTAL PROCEDURES |
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
-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 DH5
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
1
,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.
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RESULTS |
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.
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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.
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).
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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 1
,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.
1 ,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 1 ,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.
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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.
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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.
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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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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.
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DISCUSSION |
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 1
,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.