From the Division of Endocrinology, Department of
Medicine and Lady Davis Institute for Medical Research, Sir
Mortimer B. Davis-Jewish General Hospital, McGill University,
Montréal, Québec H3T 1E2, Canada and ¶ Calcium
Research Laboratory and Department of Medicine, McGill University
Health Centre and Royal Victoria Hospital, McGill University,
Montréal, Québec H3A 1A1, Canada
Received for publication, October 13, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets
(ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg
residues within a subtilisin-like proprotein convertase (SPC) cleavage
site (RXXR motif), leading to protease resistance of FGF23.
The goals of this study were to examine in vivo the
biological potency of the R176Q mutant FGF23 form and to characterize
alterations in homeostatic mechanisms that give rise to the phenotypic
presentation of this disorder. For this, wild type and R176Q mutant
FGF23 were overexpressed in the intact animals using a tumor-bearing
nude mouse system. At comparable circulating levels, the mutant form
was more potent in inducing hypophosphatemia, in decreasing circulating
concentrations of 1,25-dihydroxyvitamin D3
(1,25(OH)2D3), and in causing rickets and
osteomalacia in these animals compared with wild type FGF23. Parameters
of calcium homeostasis were also altered, leading to secondary
hyperparathyroidism and parathyroid gland hyperplasia. However, the
raised circulating levels of parathyroid hormone were ineffective in
normalizing the reduced 1,25(OH)2D3 levels by
increasing renal expression of 25(OH)D3-1 Renal phosphate wasting is associated with a number of hereditary
disorders including X-linked
(XLH)1 and autosomal dominant
(ADHR) forms of hypophosphatemic rickets. In addition to
hypophosphatemia, patients with these two conditions exhibit decreased
or inappropriately normal serum levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), as well as
rickets and osteomalacia. Interestingly, ADHR encompasses not only the
classic presentation of hypophosphatemia and rickets, but also it
displays variable penetrance with delayed onset of the disease and an
even more perplexing feature, spontaneous resolution of the biochemical
defect (1, 2).
The genes responsible for XLH and ADHR have now been identified.
Through positional cloning, a gene that spans the deleted region Xp22.1
in XLH patients or is mutated in nondeletion patients with the disorder
has been identified (designated PEX and subsequently PHEX, for phosphate-regulating gene with
homologies to endopeptidases on the X
chromosome) (3). The predicted human PHEX gene product exhibits structural similarity to a family of neutral endopeptidases involved in either activation or degradation of peptide hormones. Therefore, PHEX probably functions as a protease, and it may act by
processing factor(s) involved in bone mineral metabolism. Extensive mutation analysis of XLH families has revealed a range of defects in
the PHEX gene, all of which appear to be loss of function
mutations (4).
The ADHR gene product, on the other hand, is a new secreted member of
the fibroblast growth factor family of proteins, FGF23 (5, 6). Missense
mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds replace
Arg residues within a subtilisin-like proprotein convertase cleavage
site 176RHTR179
((K/R)Xn(K/R)) motif, where
Xn = 2, 4, or 6 residues of any amino acid) (7).
Whereas native FGF23 protein from culture-conditioned medium
resolves as 32- and 12-kDa forms, the mutated proteins are detected
only as the 32-kDa band (6, 8), suggesting that these mutations cause
the ADHR phenotype possibly by preventing proteolytic cleavage and
thereby enhancing the biological activity of circulating FGF23. In
turn, FGF23 acts either directly or indirectly to decrease phosphate
reabsorption in the proximal nephron, leading to renal phosphate wasting.
Whereas conflicting results have been reported regarding the capacity
of wild type and mutant FGF23 forms to alter phosphate transport
in vitro (6, 9), there has been indirect evidence to support
this action of FGF23. First, FGF23 is abundantly expressed in tumors
associated with tumor-induced osteomalacia or TIO, a paraneoplastic
disease characterized by many of the clinical and laboratory
characteristics reported in patients with ADHR, such as
hypophosphatemia caused by renal phosphate wasting and decreased renal
synthesis of 1,25(OH)2D3 (10). Because removal
of the responsible tumors normalizes phosphate and vitamin D
metabolism, a humoral phosphaturic factor, perhaps FGF23, is thought to
be responsible for this syndrome. Second, overexpression of wild type
FGF23 in an animal model recapitulates the biochemical and skeletal
abnormalities associated with TIO (6). Therefore, it has been
postulated that overproduction of wild type FGF23 causes TIO by
overcoming subtilisin-like proprotein convertase activity, so that the
major fraction of the expressed protein is being secreted as the
full-length molecule. In ADHR, on the other hand, mutations in FGF23
prevent proteolytic cleavage, increase its stability, and enhance the
biological potency of the circulating protein (6, 8). Experimental
proof, however, is lacking.
Here, we undertook to explore the biological actions of FGF23 proteins
in a relevant setting (i.e. the intact organism), by overexpressing the R176Q (mFGF23) and wild type forms of FGF23 cDNA
in the tumor-bearing nude mouse system. We show that the R176Q
substitution is a gain-of-function mutation that is resistant to
cleavage and in vivo is able to promote the phenotypic
alterations associated with the disorder. In addition, we provide an
explanation for the associated peculiarities in vitamin D metabolism by
describing, for the first time, a role for FGF23 in dissociating the
mineral and vitamin D actions of parathyroid hormone (PTH) at the level of the kidney.
Cloning Human FGF23--
Human FGF23 cDNA was cloned by PCR
using oligonucleotide primers derived from the published sequence
(GenBankTM accession number AF263537). The forward primer
was 5'-CCGACAGGAGTGTCAGGTTT-3' (93-112 bp), and the reverse primer
encompassed sequences encoding the c-myc epitope
(underlined) followed by FGF23 sequences (901-886 bp),
5'-CTACTAGTTGTTCAGGTCCTCTTCGCTAATCAGCTTTTGTTCCATAGAGATGAACTTGGCGA-3'. The PCR was conducted using as template aliquots of a cDNA
expression library generated from a human mesenchymal tumor
(hemangiopericytoma) associated with oncogenous osteomalacia (11). The
reaction was heated at 94 °C for 2 min and then cycled at 94 °C
for 10 s, 60 °C for 30 s, and 72 °C for 1 min for 35 cycles with a final extension at 72 °C for 10 min. The PCR product
(~0.9 kb) was first subcloned into the pGEM-T Easy vector (Promega,
WI), restricted with EcoRI, and ligated into the
EcoRI site of the polylinker region of the mammalian
expression vector pcDNA3 (Invitrogen). Its authenticity was
confirmed by direct DNA sequencing. The wild type cDNA was then
used as template to introduce the point mutation at nucleotide 527 (g Cell Culture--
Chinese hamster ovary (CHO-K1) cells were
purchased from ATCC (Manassas, VA) and were maintained on Ham's F-12
medium with 10% fetal calf serum. Cells were stably transfected with
empty pcDNA3 vector or vector expressing either wild type or mutant FGF23-myc cDNAs using LipofectAMINE 2000 Reagent
(Invitrogen). Following selection with G418 (400 µg/ml), cells were
trypsinized, the suspension was diluted with culture medium to
approximately one cell per 25 µl, and aliquots were plated in 96-well
plates. When colonies had formed, conditioned media were treated with 5 volumes of acetone, cooled on ice for 1 h, and centrifuged, and
the protein sediment was resuspended in lysis buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 EGTA, 10 mM
Animal Experimental Design--
All animal experiments were
reviewed and approved by the institutional animal care committee. Male
CD-1 nu/nu mice were purchased from Charles River and kept
in cages eating commercial rodent diet and drinking sterilized tap
water ad libitum. Groups of six mice were implanted on both
sides of the back with CHO cells transfected with pcDNA3 or CHO
cells expressing either the wild type or R176Q mutant form of
FGF23-myc (1 × 107 cells/0.1 ml of
phosphate-buffered saline). The experiment was performed twice.
Serum and Urine Biochemistry--
Serum and urine concentrations
of calcium, phosphorus, and creatinine and serum alkaline phosphatase
activity were determined by routine methods using Sigma Diagnostics
reagents (Sigma). Tubular maximum reabsorption of phosphate per 100 ml
of glomerular filtrate was calculated using the nomogram of Walton and
Bijvoet (12). Serum-intact PTH and human FGF23 were measured using an
enzyme-linked immunosorbent assay (Immutopics, Inc., San Clemente, CA),
whereas 1,25(OH)2D3 determinations were
performed using a commercially available radioimmunoassay kit
(Immunodiagnostic Systems).
Ribonuclease Protection Assay (RPA)--
cDNA fragment
corresponding to nucleotides 1177-1515 of mouse sodium-phosphate
cotransporter Npt2 (accession number L33878) (13) was
prepared by reverse transcription-PCR from mouse kidney RNA, subcloned,
and verified by sequencing. Riboprobes for Npt2 and
Northern Blot Analysis--
A cDNA fragment corresponding to
nucleotides 535-1586 of mouse 25-hydroxyvitamin D3 24-hydroxylase
(Cyp24; accession number D49438) (14) was prepared by
reverse transcription-PCR of mouse kidney RNA, subcloned, and
sequenced. DNA probes for Cyp24 and
glyceraldehyde-3-phosphate dehydrogenase were prepared by a random
primed DNA labeling kit (Roche Molecular Biosciences) and
[ Skeletal Radiographs--
The distal end of femur was removed
and dissected free of soft tissue, and radiographs were taken using a
Faxitron model 805 radiographic inspection system (22-kV voltage and
4-min exposure time). Eastman Kodak Co. X-Omat TL film was employed and
processed routinely.
Histology--
Thyroparathyroidal tissue, kidney, femurs, and
tibiae were removed and fixed in PLP fixative (2% paraformaldehyde
containing 0.075 M lysine and 0.01 M sodium
periodate solution) overnight at 5 °C and processed histologically,
as previously described (15). The proximal end of the left tibiae was
decalcified in EDTA glycerol solution for 5-7 days at 5 °C.
Decalcified tibiae and other tissues were dehydrated and embedded in
paraffin, after which 5-µm sections were cut on a rotary microtome.
The sections were stained with hematoxylin and eosin and immunostained,
as described below. Undecalcified, the proximal ends of left tibiae were embedded in LR White acrylic resin (London Resin Company Ltd.,
London, UK). 1-µm sections were cut on an ultramicrotome. These
sections were stained for mineral with the von Kossa staining procedure
and counterstained with toluidine blue.
Immunohistochemistry--
Paraffin sections were stained
immunohistochemically for PTH, Npt2, and Cyp40 using the
avidin-biotin-peroxidase complex technique. Briefly, primary antibody
was applied to tissues overnight at room temperature. Goat serum
against PTH (16), affinity-purified rabbit serum against NPT2 (courtesy
of M. Knepper, Laboratory of Kidney and Electrolyte Metabolism,
National Institutes of Health, Bethesda, MD), and affinity-purified
rabbit serum against Cyp40 (provided by G. Hendy, McGill University,
Montréal, Canada) were employed. As negative control, preimmune
serum or Tris-buffered saline was substituted for the primary antibody.
After washing, tissues were incubated with secondary antibody
(biotinylated rabbit anti-goat IgG, biotinylated goat anti-rabbit IgG).
Sections were then washed and incubated with the Vectastain ABC-AP
reagent (Vector Laboratories, Ontario, Canada) for 45 min. After
washing, red pigmentation to demarcate regions of immunostaining was
produced by a 10-15-min treatment with Fast Red TR/Naphthol AS-MX
phosphate (containing 1 mM levamisole as endogenous
alkaline phosphatase inhibitor; Sigma). After washing with
distilled water, the sections were counterstained with methyl green and
mounted with Kaiser's glycerol jelly.
Computer-assisted Image Analysis--
Computer-assisted image
analysis was performed, as previously described (15). The positive and
negative areas of von Kossa staining in trabecular and cortical bone
were measured by digital image capture and image analysis using
Northern Eclipse version 6.0 (Empix Imaging Inc., Mississauga, Canada)
image software. The mineralization percentage of trabecular and
cortical bone was calculated using the following formula: mineralized
bone (%) = von Kossa positive area/von Kossa positive area + von
Kossa negative area of bone × 100%.
Statistical Analysis--
Data from image analysis are presented
as mean ± S.E. (n = 6). Statistical comparisons
were made using a two-way analysis of variance, with p < 0.05 being considered significant.
A 0.9-kb fragment corresponding to the FGF23 transcript
was amplified from several pools of a cDNA expression library
generated from a hemangiopericytoma associated with TIO (Fig.
1A) (11). The addition of the
c-myc epitope at the carboxyl terminus of the protein was
employed for its subsequent identification and visualization. The R176Q
mutation, initially described in a family with ADHR (5), was also
introduced in the FGF23 cDNA using site-directed
mutagenesis. When transiently transfected into COS-1, OK, or CHO cells,
the wild type and mutant forms of FGF23 (mFGF23) were processed, as
previously described (results not shown) (8).
-hydroxylase
(Cyp40) to promote its synthesis and by decreasing that of
25(OH)D3-24-hydroxylase (Cyp24) to prevent its
catabolism. The findings provide direct in vivo evidence
that missense mutations from ADHR kindreds are gain-of-function
mutations that retain and increase the protein's biological potency.
Moreover, for the first time, they define a potential role for FGF23 in
dissociating parathyroid hormone actions on mineral fluxes and on
vitamin D metabolism at the level of the kidney.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
a; R176Q) by site-directed mutagenesis using the Transformer kit
(Clontech, Palo Alto, CA). The fidelity of clones
was verified by direct sequencing.
-mercaptoethanol, and a mixture of protease inhibitors (1 µg/ml
leupeptin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl
fluoride) in 0.5% Triton X-100) and examined for FGF23 content by
SDS-PAGE (15% gel) and Western blot analysis using an
anti-myc monoclonal antibody.
-actin (250-bp KpnI-XbaI fragment of the
mouse
-actin gene) were prepared by transcription of the
subcloned cDNA fragments using T7 RNA polymerase and
[
-32P]UTP (800 Ci/mmol; PerkinElmer Life
Sciences), and RPA was performed, as per the instructions of the
manufacturer (Ambion). Total RNA (10 µg) isolated from kidney with
Tripure Isolation Reagent (Roche Molecular Biosciences) was hybridized
with the appropriate labeled riboprobes (8 × 104 cpm)
at 42 °C overnight and treated with ribonuclease A/T1 mix (1:100)
for 30 min at 37 °C. The protected fragments were precipitated, heat-denatured, and electrophoresed on 5% denaturing polyacrylamide gel. The gel was exposed to Kodak BioMax film at
80 °C for the appropriate time, with intensifying screens.
-32P]dCTP (800 Ci/mmol; PerkinElmer Life
Sciences). Total RNA was isolated from kidney with Tripure
Isolation Reagent (Roche Molecular Biosciences), and 20-µg aliquots
were fractionated by electrophoresis on a 1% formaldehyde agarose gel,
transferred to nitrocellulose membranes by upward capillary transfer in
20× SSC overnight, and hybridized to the radiolabeled cDNA
fragment (48% formamide, 10% dextran sulfate, 5× SSC, 1×
Denhardt's solution, and 100 µg/ml salmon sperm DNA) at 42 °C
overnight. The membranes were washed in 0.1% SDS plus 2× SSC for 15 min at room temperature with rotation and then in 0.1% SDS plus 0.1×
SSC for another 15 min at 60 °C. The autoradiograms were prepared
using Kodak BioMax film at
80 °C with intensifying screens.
Quantification of signal intensity on autoradiograms was performed by
Amersham Biosciences personal densitometer using ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning of human FGF23
cDNA and its expression in CHO cells. A,
aliquots (one represented per lane) from a cDNA
expression library prepared from a hemangiopericytoma associated with
TIO were used as template for PCR amplification of FGF23 cDNA. The
amplified bands corresponding to ~0.9 kb were present in 6 of the 12 aliquots examined. Lane 1, molecular weight
marker. B, Western blot analysis of
FGF23-myc-tagged protein secreted in the conditioned medium
from clones of CHO cells stably transfected with empty vector, wild
type, and R176Q mutant (mFGF23) cDNAs using an anti-myc
monoclonal antibody. A filled arrowhead
designates the mature form, whereas an open
arrowhead indicates the carboxyl-terminal fragment of FGF23.
C, serum concentration of human FGF23, as measured by a
commercial assay that does not cross-react with the murine form of the
protein. Note logarithmic scale on the y axis. ***,
p < 0.001 versus serum from mice implanted
with empty vector-transfected CHO cells.
To investigate the in vivo biological properties of mFGF23, CHO cells were stably transfected with pcDNA3 mammalian expression vector alone or vector expressing either the myc-tagged wild type or the myc-tagged R176Q form of the protein. Following G418 selection, neomycin-resistant clones were further processed by limited dilution to identify high expressor cells. Shown in Fig. 1B is Western blot analysis of conditioned media from single isolated clones. Abundant secretion of mature FGF23 (32-kDa form) was apparent in both wild type and mutant form-expressing cells, whereas vector-transfected CHO cells did not express the protein. Whereas the wild type form was primarily processed to an amino-terminal fragment (not detected) and a 12-kDa carboxyl-terminal fragment, the mature mutant protein failed to be cleaved and remained intact in the conditioned medium.
The biological activity of the two FGF23 forms was then assessed
following subcutaneous implantation into nude mice of CHO cell clones
expressing either FGF23, mFGF23, or CHO cells transfected with vector
alone as control (each at a total of 1 × 107 cells).
Using an enzyme-linked immunosorbent assay that detects epitopes within
the carboxyl-terminal portion of human but not murine FGF23,
circulating serum levels of the protein were shown to be equivalent in
both groups of mice expressing FGF23, whereas none was detectable in
the control animals (Fig. 1C). The mice were followed for 45 days, and urine and blood samples were obtained periodically for
analyses. Tumor-bearing animals secreting FGF23 and mFGF23 developed
profound hypophosphatemia as early as 10 days following implantation
(the first time blood samples were obtained) that progressively became
even more profound, although at a slower rate, over the next 35 days
(Fig. 2A). This was
accompanied by inappropriate excretion of urinary phosphate (Fig.
2B), as indicated by the decrease in tubular phosphate
reabsorption (12). In addition, as observed in patients with TIO and
ADHR, FGF23-overexpressing animals developed reduced serum levels of
1,25(OH)2D3 that were inappropriate for the
prevailing degree of hypophosphatemia (Fig. 2C).
Interestingly, these biochemical alterations were somewhat more
pronounced in the mice implanted with CHO cells expressing mFGF23 than
in those secreting the wild type form of the protein. However, both
were significantly different from those from animals implanted with CHO
cells carrying the empty vector.
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Based on these observations, we next examined parameters of calcium
homeostasis. In all three groups of mice, serum calcium concentration
was maintained within the normal range (Fig.
3A). However, distinct
differences in the levels of circulating PTH were evident among the
three groups of animals, being markedly increased in mice
overexpressing the two forms of FGF23 (Fig. 3B). This
alteration was accompanied by concomitant decreases in urinary calcium
excretion (Fig. 3C) and increases in serum (Fig.
3D) and bone (Fig. 3E) alkaline phosphatase
activity, changes consistent with secondary hyperparathyroidism. This
was further confirmed by histological analysis of thyroparathyroidal
tissue from these animals (Fig. 3F). Predictably,
parathyroid glands from mice expressing FGF23 forms were enlarged, as
determined by immunoreactive staining for PTH, compared with glands
from animals transplanted with CHO cells carrying the empty vector.
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The abnormalities in phosphate, vitamin D, and calcium homeostasis were
studied further by first assessing the expression of renal sodium
phosphate transport protein 2 (Npt2) in the proximal renal tubule 45 days following tumor implantation. Fig.
4A shows that immunoreactivity
for the protein was markedly diminished in the presence of circulating
FGF23 forms, with a somewhat more pronounced decrease arising from
expression of the mutant protein. Moreover, RPA analysis for renal
Npt2 mRNA demonstrated that the FGF23-induced reduction
in Npt2 was in part due to a decrease in transcript levels, an effect
that was again more evident with the mutant FGF23 form. These changes
are likely to account, therefore, for the diminished tubular phosphate
reabsorption and inappropriate excretion of urinary phosphate in these
animals. Nevertheless, despite the marked hypophosphatemia, expected
alterations in the expression of enzymes aimed at increasing
circulating levels of 1,25(OH)2D3 failed to
take place. In fact, contrary to its anticipated up-regulation,
immunoreactivity for 25(OH)D3-1-hydroxylase
(Cyp40) in the proximal renal tubule was reduced (Fig.
4B), whereas transcript levels of
25(OH)D3-24-hydroxylase (Cyp24) were increased
(Fig. 4C) instead of diminished. Again, these inappropriate
renal adaptations in Cyp40 and Cyp24 expression were more
apparent in mice implanted with CHO-mFGF23 cells, as compared with the
wild type FGF23 form, suggestive of the increased biological potency of
the mutant protein.
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We next examined the long bones of these animals. Histological sections
of the epiphyseal region of tibiae are shown in Fig. 5. In comparison with the growth plate of
the control animals, the growth plate of mice implanted with CHO cells
expressing wild-type FGF23 was wider, more disorganized, and less well
mineralized. Once more, these rachitic changes were more apparent in
the animals implanted with CHO cells expressing the mutant form of
FGF23. Osteomalacic changes were also noted in the metaphyses of these bones (Fig. 6A). Increased
unmineralized osteoid was present in specimens from animals expressing
FGF23 and was even more abundant in bones from mFGF23-expressing mice.
This alteration was evident in both trabecular as well as cortical bone
(Fig. 6, B-D). The reduced skeletal mineralization arising
from expression of the FGF23 forms was further confirmed from
radiographic studies (Fig. 6E).
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DISCUSSION |
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FGF23 is mutated in the hereditary renal phosphate wasting disorder ADHR (5). Missense mutations described in the patients with ADHR have been postulated to stabilize the protein by impairing its cleavage and thereby potentially elevating circulating concentrations of FGF23, leading to phosphate wasting (6, 8). Here we show using an in vivo overexpression system that the R176Q form of FGF23 that is resistant to proteolytic cleavage has an equivalent and probably higher capacity than wild type FGF23 to recapitulate the biochemical and skeletal abnormalities associated with ADHR. Similar findings have recently been reported by other investigators (17). Although it is rather difficult to make direct comparisons on the in vivo potency of the two FGF23 forms, their nearly equivalent circulating concentrations that we describe in our experimental model would tend to add further credence to our conclusion. Hence, the mutant protein has profound effects on phosphate homeostasis and vitamin D metabolism, leading to the concomitant development of renal phosphate wasting, rickets, and osteomalacia.
The phenotypic similarities between ADHR, TIO, and XLH would suggest that deregulation of the same phosphate-regulating pathway, namely FGF23, is involved in the pathogenesis of all three renal phosphate-wasting disorders. Thus, as indicated by our studies here and by others (6), overexpression of wild type FGF23 in TIO is likely to overwhelm the subtilisin-like proprotein convertase processing of the protein, leading to increased circulating levels of the unprocessed 32-kDa FGF23 form with the ensuing biochemical and skeletal alterations. Similarly, it is reasonable to postulate that circulating FGF23 also serves as one of the humoral factor(s) that underlie the etiology of XLH. In this model, FGF23, secreted by one or more tissues, would serve as substrate processed by PHEX enzymatic activity. Consequently, inactivating mutations in PHEX endopeptidase would lead to impaired processing of FGF23 and persistent biological activity that would adversely affect renal phosphate handling, calcitriol synthesis, and bone metabolism. Therefore, overproduction of FGF23 by tumors (as in TIO), mutations that prevent its cleavage (as in ADHR), or mutations that inactivate PHEX (as in XLH) would all increase the level of active circulating FGF23, leading to renal phosphate wasting, hypophosphatemia, rickets, and osteomalacia.
The molecular and physiological mechanisms by which FGF23 and its naturally occurring mutant forms associated with ADHR cause derangement in renal phosphate handling, however, are currently unknown. One possibility is that FGF23 acts directly on the kidney to alter phosphate transport and renal parameters of vitamin D metabolism. Studies addressing this scenario, however, tend to be conflicting, since both confirmatory and negative findings have been reported (6, 10). The difficulty in clearly demonstrating a direct effect of FGF23 on phosphate transport in vitro suggests that perhaps a more complex mechanistic model exists. For example, FGF23 may act as an intermediary in causing phosphaturia, by altering either the expression of PHEX or the expression of its putative substrate or by mobilizing another factor whose nature at present remains unknown.
A somewhat unexpected finding in our study, not described by others (17), was the marked increase in circulating PTH levels in animals bearing FGF23- and mFGF23-secreting tumors. This change was attributed to the observed decrease in calcitriol synthesis, leading to hypocalcemia. The ensuing appropriate rise in PTH secretion would then be aimed toward maintaining calcium homeostasis by increasing bone turnover (increased alkaline phosphatase activity in bone and serum) and decreasing urinary calcium excretion, consistent with secondary hyperparathyroidism. Additional confirmatory support for this argument is provided by the diffuse parathyroid hyperplasia observed histologically in these two groups of animals. Increased circulating levels of PTH have been reported in some patients with TIO (18, 19) and in Hyp and Gy mice, the murine homologs of XLH (20). Although not described in ADHR, a trend toward higher PTH levels has been observed in patients with this disorder (1). The reason for the discrepancy between this observation and our findings can be explained, in part, by the fact that in our animal model deregulated overexpression would tend to raise the circulating levels of the FGF23 forms more profoundly. Conceivably, in patients with ADHR, potential feedback mechanisms are likely to be set in motion so as to restrain excess production of FGF23. Whereas the nature of such mechanisms remains unclear, their existence has been substantiated by the apparent reversal of the phenotype in a number of patients with ADHR (2).
The question then arises as to whether the concomitant increase in circulating PTH levels contributes, at least in part, to the decrease in Npt2 expression at the level of the renal proximal tubule and the inappropriate phosphaturia associated with this condition. Confirmation of this would have to await the completion of similar experiments that are presently under way using nude mice carrying targeted disruption of the Pth gene. Nevertheless, it is rather remarkable that when FGF23 is overexpressed, high circulating PTH levels do not normalize 1,25(OH)2D3 serum concentration. Normally, the enzymes Cyp40 and Cyp24 are very tightly and reciprocally regulated by 1,25(OH)2D3 and PTH (21). 1,25(OH)2D3 activates its own breakdown by strongly inducing Cyp24 while at the same time down-regulating Cyp40 (22). On the other hand, PTH induces Cyp40 while down-regulating Cyp24 expression (23, 24). Here we show that, following FGF23 overexpression, Cyp40 expression remains decreased while, concurrently, Cyp24 expression is increased, effects that are diametrically opposed to those one would anticipate from the presence of increased PTH and decreased 1,25(OH)2D3 circulating levels. Thus, an apparent dissociation of PTH actions at the level of the kidney occurs, whereby the effects of PTH on reabsorbing urinary calcium and perhaps promoting phosphaturia are preserved, whereas Cyp40 and Cyp24 expression are refractory to its action. FGF23, therefore, appears to be directly or indirectly responsible for the inappropriate alterations in the activity of the renal vitamin D-metabolizing hydroxylases observed both in our mice and likely in patients with ADHR, TIO, and XLH. This may also explain, in part, the inappropriately normal levels of 1,25(OH)2D3 associated with these three disorders despite the prevailing hypophosphatemia, a finding that is unique to them, since in other renal phosphate wasting states, 1,25(OH)2D3 synthesis is up-regulated. For example, 1,25(OH)2D3 synthesis is appropriately increased by hypophosphatemia in Npt2-null mice (25) and in patients with hypophosphatemia associated with heterozygote missense mutations in the NPT2 gene (26).
In summary, our findings demonstrate the enhanced in vivo
biological potency of the R176Q mutant form of FGF23 associated with
ADHR compared with the wild type protein and provide a novel perspective on the molecular intricacies that underlie the
pathophysiology of hereditary and acquired disorders of renal phosphate wasting.
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FOOTNOTES |
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* This work was supported by the Canadian Institutes of Health Research (CIHR) and the Canadian Arthritis Network.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.
§ Both authors contributed equally to this work.
Recipient of a CIHR fellowship award.
** Recipient of a CIHR scientist award.
To whom correspondence should be addressed: Dept. of Medicine
and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte Ste. Catherine Rd., Montréal, Québec H3T 1E2, Canada. Tel.: 514-340-8222 (ext.
4907); Fax: 514-340-7573; E-mail: akarapli@ldi.jgh.mcgill.ca.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M210490200
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ABBREVIATIONS |
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The abbreviations used are: XLH, X-linked hypophosphatemic rickets; ADHR, autosomal dominant hypophosphatemic rickets; FGF23, fibroblast growth factor 23; mFGF23, mutant FGF23; PTH, parathyroid hormone; CHO, Chinese hamster ovary; RPA, ribonuclease protection assay; 1, 25(OH)2D3, 1,25-dihydroxyvitamin D3.
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