| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Division of Endocrinology (X.B., A.C.K.), Department of Medicine, and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montréal, Québec, Canada H3T 1E2; Calcium Research Laboratory (D.M., J.L., D.G.), Department of Medicine, McGill University Health Center and McGill University, Montréal, Québec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Andrew C. Karaplis, Division of Endocrinology, Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste. Catherine Road, Montréal, Québec, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A central role for fibroblast growth factor 23 (FGF23) in the pathogenesis of these hypophosphatemic disorders has been proposed (5, 6, 7, 8), although other factors such as matrix extracellular phosphoglycoprotein (9) and secreted frizzled-related protein 4 (10) have also been implicated. Targeted disruption of Fgf23 leads to high serum phosphate, increased renal phosphate reabsorption and elevation in serum 1,25(OH)2D3 (11). Alternatively, increased circulating levels of FGF23 may lead to urinary phosphate wasting and altered vitamin D metabolism. Hence, impaired FGF23 degradation due to reduction or loss of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) protease activity has been suggested to underlie the XLH phenotype (12). Specific mutations of arginine residues at position 176 or 179 of FGF23 that disrupt the sequence motif recognized by furin proteases, cause ADHR presumably due to altered processing or decreased degradation of the protein (13, 14). Overproduction of FGF23 by tumors (14, 15) and osteogenic cells in fibrous dysplastic lesions (16) is likely responsible for the hypophosphatemia in TIO and fibrous dysplasia, respectively.
By implanting nude mice with tumors that oversecrete FGF23 into the circulation, others and we (5, 17) have shown that this model recapitulates the biochemical and histological alterations in bone associated with TIO. In addition, overexpression of the mutant FGF23 form identified in patients with ADHR FGF23 (R176Q) exerted derangements more profound than the wild-type protein, perhaps due to its prolonged biological half-life (17).
Despite significant advances in our understanding of these disorders, a number of important questions remain unanswered (18). For example, it is unclear whether circulating FGF23 levels are increased in XLH patients (14, 19, 20, 21), whether FGF23 is a direct PHEX substrate (22, 23, 24), and whether FGF23 is the direct mediator responsible for decreased transport phosphate by the proximal renal tubules and derangement in the regulation of vitamin D metabolism or whether another factor contributes to these effects (6, 18). To answer such questions, an animal model of FGF23 overexpression devoid of the potential biological pitfalls and short life span associated with tumor implantation would be desirable. Here we describe the generation of a murine model of FGF23 overexpression using a transgene encoding the secreted form of human FGF23 (R176Q) cDNA and the evolution of associated biochemical and bone histological changes at 1 and 2 months postnatally. We report that, contrary to previous reports, increased circulating levels of FGF23 alter parameters of phosphate and vitamin D as well as calcium homeostasis and that elevated PTH levels likely contribute to the renal phosphate wasting associated with these disease states.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
798 bp) epitope and ligated into the pLiv plasmid at the MfeI site. When expressed in cultured human hepatoma (HepG2) cells, this vector produced a band corresponding to an approximately 30-kDa protein on Western blot analysis using an anti-myc antibody (data not shown).
Generation of transgenic mice
All animal experiments were reviewed and approved by the institutional animal care committee. Transgenic mice designed to express human FGF23 (R179Q) in a liver-specific manner were generated using standard protocols. After establishing the correct orientation of the cDNA, the entire transgene was isolated from plasmid sequences by SacII/SpeI restriction endonuclease digestion. The linearized 6.6-kb fragment was gel purified using NucleoTrap nucleic acid purification kit (Clontech, Palo Alto, CA), microinjected into single-cell C57B/6J x CBA embryos, and implanted into pseudopregnant female mice.
Identification of transgenic mice and establishing transgenic lines
Mice were screened for integration of the transgene by Southern blot analysis of tail DNA. Offspring from pseudopregnant foster mothers were weaned at 3 wk of age, and DNA was prepared from a 1-cm portion of their tails. In brief, 10 µg of genomic DNA were digested with EcoRI, separated by agarose gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with a 753-bp [
-32P]dCTP-labeled hFGF23 cDNA as probe. Signals were detected by autoradiography. Mice carrying the transgene were mated with C57BL/6J mice to generate transgenic F1 progeny.
The mice were housed in a 12-h light, 12-h dark cycle maintained in cages with wooden shavings and had free access to water and normal calcium diet (0.95% calcium, 0.67% phosphorus, 4.5 IU/g vitamin D3; 0.21% Mg, 23.4% protein, 4.5% fat, 5.3% fiber, 6.9% ash; PMI Feeds, Inc., St. Louis, MO) in pelleted form. Weight of mice was measured every month.
Immunoprecipitation and Western blot analysis
Liver tissue (50 mg) from wild-type or transgenic mice was homogenized in 0.5 ml of radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycorticosterone, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 7.5)] supplied with a cocktail of protease inhibitors (Roche Diagnostics, Laval, Québec, Canada) and 1 mM phenylmethylsulfonyl fluoride. Extracts were clarified by centrifugation at 10,000 x g for 20 min at 4 C and 10 mg of liver tissue lysate were precleared by incubating with 50 µl protein G beads (Roche Diagnostics). The supernatants were then incubated with 2 µg anti-FGF23 antibody (Immutopics, Inc., San Clemente, CA) and 50 µl protein G beads with rotation overnight at 4 C, the beads were collected by centrifugation, washed twice in radioimmunoprecipitation assay buffer, dissolved in Laemmli buffer, and analyzed by SDS-PAGE (12%). The proteins were transferred to supported nitrocellulose membrane by electroblotting, followed by incubation with anti-myc monoclonal antibody (Roche Diagnostics). After the addition of a secondary horseradish peroxidase-conjugated goat antimouse IgG (Sigma, Oakville, Ontario, Canada), the membrane was subjected to enhanced chemiluminescence analysis.
Serum and urine biochemistry
Serum and urine concentrations of calcium, phosphorus, and creatinine and serum alkaline phosphatase activity were determined by routine methods using diagnostics reagents (Sigma). Tubular maximum reabsorption of phosphate per 100 ml glomerular filtrate TmPO4/G.F.R. was calculated using the nomogram of Walton and Bijvoet (25). Serum-intact PTH and human FGF23 were measured using an ELISA (Immutopics), whereas 1,25(OH)2D3 determinations were performed using a commercially available RIA kit (Immunodiagnostic Systems, Fountain Hills, AZ).
Skeletal radiography, microcomputed tomography (microCT), and bone mineral density
Femurs were removed and dissected free of soft tissue. Contact radiographs were obtained using a radiographic inspection system (model 805, Faxitron Contact, Faxitron, Germany) (22-kV voltage and 4-min exposure time). X-Omat TL film (Eastman Kodak, Rochester, NY) was used and processed routinely.
Tibiae obtained from 2-month-old mice were dissected free of soft tissue, fixed overnight in 70% ethanol, and analyzed with a microCT scanner and associated analysis software (model 1072, SkyScan, Antwerp, Belgium). Image acquisition was performed at 100 kV and 98 µA with a 0.9-degree rotation between frames. During scanning, the samples were enclosed in a tightly fitting rigid plastic rube to prevent movement. Thresholding was applied to the images to segment the bone from the background, and the same threshold setting was used for all the samples. Two-dimensional images were used to generate three-dimensional (3D) reconstructions using the 3D Creator software supplied with the instrument.
For measurement of tibial and femoral bone mineral density (BMD), a PIXImus densitometer (LUNAR Corp., Madison, WI) was used with precision of 1% coefficient of variation for skeletal BMD. The PIXImus software automatically calculated the BMD and recorded the data in Excel files (Microsoft, Redmond, CA).
Histology
Thyroparathyroidal tissue, femurs, and tibiae were removed and fixed in PLP fixative [2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate) overnight at 4 C and processed histologically, as previously described (26)]. The proximal ends of the tibiae were decalcified in EDTA glycerol solution for 5–7 d at 4 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 or histochemically for collagen, alkaline phosphatase (ALP), or tartrate resistant acid phosphatase (TRAP) activity as described below. Alternatively, undecalcified femora were embedded in LR White acrylic resin (London Resin Co. Ltd., London, UK), and 1-µm sections were cut on an ultramicrotome. These sections were stained for mineral either with the von Kossa staining procedure and counterstained with toluidine blue or subjected to the Goldner’s-Masson-trichrome stain to discriminate mineralized from unmineralized tissues.
Histochemical staining for collagen, ALP, and TRAP
Total collagen was detected in paraffin sections using a modification of Lopez-De Leon and Rojkind (27). Dewaxed sections were exposed to 1% sirius red in saturated picric acid for 1 h. After washing with distilled water, sections were dehydrated and mounted with Biomount medium.
Enzyme histochemistry for ALP activity was performed as previously described (28, 29). Briefly, after preincubation overnight in 1% magnesium chloride in 100 mM Tris-maleate buffer (pH 9.2), dewaxed sections were incubated for 2 h at room temperature in a 100 mM Tris-maleate buffer containing naphthol AS-MX phosphate (0.2 mg/ml, Sigma) dissolved in ethylene glycol monomethyl ether (Sigma) as substrate and fast red TR (0.4 mg/ml, Sigma) as a stain for the reaction product. After washing with distilled water, the sections were counterstained with Vector methyl green nuclear counterstain (Vector Laboratories, Burlington, Ontario, Canada), and mounted with Kaiser’s glycerol jelly.
Enzyme histochemistry for TRAP was performed using a modification of a previously described protocol (26). Dewaxed sections were preincubated for 20 min in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (pH 5.0). Sections were then incubated for 15 min at room temperature in the same buffer containing 2.5 mg/ml naphthol AS-MX phosphate (Sigma) in dimethylformamide as substrate and 0.5 mg/ml fast garnet GBC (Sigma) as a color indicator for the reaction product. After washing with distilled water, the sections were counterstained with methyl green and mounted in Kaiser’s glycerol jelly.
Immunohistochemistry
Paraffin sections were stained immunohistochemically for aggrecan, PHEX, sodium-phosphate transport protein 2 (NPT2), and CYP27B1 using the avidin-biotin-peroxidase complex technique. Briefly, primary antibody was applied to tissues overnight at room temperature. Rabbit antiserum to bovine aggrecan (R130, courtesy of A. R. Poole, Shriners Hospital, Montréal, Canada), rabbit antiserum against a synthetic peptide, ESEEKPKEK, corresponding to residues 606–614 of the carboxyl-terminal amino acid sequence of PHEX (30), affinity-purified rabbit serum against NPT2 (courtesy of M. Knepper, Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health, Bethesda, MD), and sheep antimurine CYP27B1 IGg fraction (The Binding Site Ltd., Birmingham, UK) 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 antigoat IgG, biotinylated goat antirabbit IgG). Sections were then washed and incubated with the Vectastain ABC-AP reagent or the Vectastain Elite ABC reagent (Vector Laboratories) for 45 min. After washing, red pigmentation to demarcate regions of immunostaining was produced by a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (containing 1 mM levamisole as endogenous alkaline phosphatase inhibitor; Sigma), or gray pigmentation was likewise produced using a Vector SG kit (Vector Laboratories). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly.
Computer-assisted image analysis
After histochemical staining of sections from six mice of each genotype, images of fields were photographed with a digital camera (Sony). Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed using Northern Eclipse image analysis software. For determining the trabecular bone volume relative to the total volume in collagen-stained sections, the mineralized area of hypertrophic zone, and osteoid volume relative to the BV in von Kossa-stained sections, ALP-positive area and intensity (summary total gray) in ALP histochemical-stained sections, and the number and size of osteoclasts in TRAP histochemical-stained sections, thresholds were set using green and red channels. The thresholds were determined as described previously (30, 31). The trabecular volume was measured in the metaphyseal region from below the distal (metaphyseal) side of the growth plate to 2.0 mm toward the diaphysis. ALP and TRAP parameters were measured in the fields of metaphyseal regions.
Northern blot analysis
cDNA fragments corresponding to nucleotides 535-1586 of mouse 25-hydroxyvitamin D3 24-hydroxylase (Cyp24; GenBank accession no. D49438) to nucleotides 421-1471 of mouse 25-hydroxyvitamin D3 1--hydroxylase (Cyp27b1; GenBank accession no. AB006034) were prepared by RT-PCR of mouse kidney RNA, subcloned, and verified by sequencing. DNA probes for Cyp24, Cyp27b1, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were prepared by a random primed DNA labeling kit (Roche Molecular Biosciences, Indianapolis, IN) and [-32P]dCTP (800 Ci/mmol; PerkinElmer Life Sciences, Norwalk, CT). Total RNA was isolated from kidney and bone 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 20x saline sodium citrate (SSC) overnight, and hybridized to the radiolabeled cDNA fragment (48% formamide, 10% dextran sulfate, 5x SSC, 1x Denhardt’s solution, and 100 µg/ml salmon sperm DNA) at 42 C overnight. The membranes were washed in 0.1% SDS plus 2x SSC for 15 min at room temperature and then in 0.1% SDS plus 0.1%x SSC for another 15 min at 60 C. The autoradiograms were prepared using BioMax film (Kodak) at –80 C with intensifying screens. Quantification of signal intensity on autoradiograms was performed by personal densitometer (Amersham Biosciences, Piscataway, NJ) using ImageQuant software.
Statistical analysis
Data from image analysis are presented as mean ± SEM. Statistical comparisons were made using a two-tailed unpaired t test, with P < 0.05 being considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). This approach was used previously to obtain mice with high levels of secreted osteoprotegerin, a protein involved in the regulation of bone density, in the systemic circulation (32, 33). Eleven mice harboring the transgene were identified by Southern blot analysis of tail DNA, and most exhibited significant growth retardation, a round back, with a short snout and tail, features that became readily apparent after weaning. Only two survived into adulthood and were able to mate and transmit the transgene (Fig. 1B). The two founders were shown to have a low- and high-copy number of the transgene inserted in the genome and expressed corresponding levels of transgene mRNA exclusively in the liver (Fig. 1C). FGF23 (R176Q) was readily detectable in liver lysates (Fig. 1D) and serum (Fig. 1E) of transgenic mice but not control mice, indicating that the transgene product was successfully expressed and delivered to the systemic circulation. For all subsequent studies, animals from both lines of transgenic mice were used.
|
). High circulating levels of FGF23 (R176Q) were associated with profound hypophosphatemia with decreased renal tubular reabsorption of phosphate, increased serum ALP activity, and low serum levels of 1,25(OH)2D3 that were inappropriate for the prevailing degree of hypophosphatemia. These biochemical alterations are characteristic of inherited and acquired renal phosphate-wasting states associated with elevated circulating FGF23 levels. However, in addition, unanticipated changes in parameters of calcium homeostasis were also apparent in the transgenic mice that became more pronounced with age. Serum calcium levels were slightly diminished, and this was reflected in increased circulating levels of PTH and serum ALP activity. Urinary calcium excretion was also decreased in the transgenic mice, consistent with increased renal tubular calcium reabsorption in response to circulating PTH. These findings, indicative of secondary hyperparathyroidism, were further substantiated histologically at the level of the parathyroids as the glands became progressively enlarged over time, likely a consequence of the persistent stimulus for PTH secretion arising in the setting of elevated FGF23 levels.
|
are radiographs of femurs from wild-type and transgenic mice at 1 and 2 months of age. The transgenic bones were proportionately smaller in size and more radiolucent than their wild-type counterparts. The frontal view of 3D reconstructed proximal end of tibia obtained using microCT showed the wider, unmineralized growth plate in the transgenic mice (Fig. 3B). The reduced skeletal mineralization was subsequently confirmed on histological sections of the epiphyseal region of femurs as shown in Fig. 3C. In comparison with wild-type animals, growth plates from transgenic mice expressing FGF23 (R176Q) were wider, more disorganized, and less well mineralized (Fig. 3, D and E), consistent with an advanced form of rickets. These rachitic changes were more apparent in transgenic animals at 2 months of age.
|
), mineralized trabeculae were much less numerous as demonstrated by 3D reconstructed longitudinal sections of long bones obtained from microCT scans (Fig. 4B) and histological assessment (Fig. 3C
). Profound osteomalacic changes were apparent in trabecular and cortical bone because increased amounts of unmineralized osteoid was present in specimens from animals expressing FGF23 (R176Q) (Fig. 4, C and D). In addition to the impairment in bone matrix mineralization, bone resorption was also diminished in the transgenic mice as indicated by the low osteoclast number (Fig. 4E) and the persistence of aggrecan expression and chondrocytes in the bone trabeculae (Fig. 4F). In contrast, ALP activity was increased in the transgenic bones (Fig. 4G). Interestingly, PHEX expression was enhanced in this pathophysiological state because intense protein immunoreactivity was observed in growth plate chondrocytes (not shown), osteoblasts, and osteocyte cell bodies and canalicular cellular processes, specifically in regions of unmineralized bone (Fig. 4H). BMD measurements and histomorphometric analysis further confirmed and quantified the reduced BMD and other skeletal alterations arising from FGF23 (R176Q) overexpression (Fig. 5, A–F).
|
|
). These changes are likely to account, therefore, for the impairment in tubular phosphate reabsorption and inappropriate excretion of urinary phosphate in these animals.
|
, B and C). On the other hand, mRNA levels for renal Cyp27b1 were increased (Fig. 7, A and B), but this failed to translate to increased protein immunoreactivity at the level of the renal proximal tubule (Fig. 7C), suggesting that a decrease in Cyp27b1 activity likely results from a defect in translational or posttranslational modification of the enzyme.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Whereas similar observations were recently published by others, significant differences are apparent between the two studies. Shimada et al. (34) reported that serum calcium and PTH levels in the transgenic mice were lower than those of normal mice. In contrast, we consistently observed a mild decrease in serum calcium accompanied by a corresponding rise in circulating PTH levels in transgenic animals, similar to what we previously reported after implantation of nude mice with tumors that oversecrete FGF23 into the circulation (17). These findings (high circulating PTH but low-normal serum calcium) were in part confirmed in a third study published while this work was under review (35). Presently it remains unclear as to the reason for the observed discrepancy in parameters of calcium homeostasis between the various transgenic animal models. It is possible these differences may arise from different promoter usage [ApoE3 vs. chicken ß-actin vs. 1(I) collagen], although this is unlikely. The phenotypic similarities in mice expressing FGF23 in hepatocytes (ApoE3) or osteoblasts [1(I) collagen] would indicate that the biological effects of FGF23 are likely endocrine rather than acting locally on bone cells. Alternatively, the form of the protein overexpressed by the transgene is more likely to impact on the findings because the FGF23 (R176Q) protein would exert derangements more profound than the wild-type form, perhaps due to its prolonged biological half-life (17).
Abnormalities in parameters of calcium homeostasis are not consistently observed in patients with renal phosphate-wasting disorders. Although serum calcium levels are usually normal, mild hypocalcemia has been described in patients with XLH and TIO (18). Moreover, a trend toward higher PTH levels has been reported in patients with ADHR (2), whereas apparent secondary and even tertiary hyperparathyroidism has been reported in XLH patients not treated with phosphate and 1,25(OH)2D3 (21, 36, 37, 38, 39). Secondary hyperparathyroidism with increased circulating levels of PTH has also been reported in Hyp and Gy mice, the murine homologs of XLH (40, 41). Serum levels of PTH have been variably reported as low as well as elevated but are most frequently normal in TIO (21, 42, 43). The reason for the discrepancy between these clinical observations and our findings can be explained, in part, by the fact that in our animal model, deregulated overexpression would tend to raise more profoundly the circulating levels of FGF23. Conceivably, in patients with renal phosphate-wasting disorders, potential feedback mechanisms are set in motion to restrain its excess production. In a recent study, FGF23 concentrations were 481 ± 528 RefU/ml in patients with suspected oncogenic osteomalacia and 353 ± 510 RefU/ml in XLH patients, approximately 6% of the serum FGF23 levels determined in our transgenic mice. Whereas the nature of such mechanisms remains unclear, the apparent reversal of the phenotype in a number of patients with ADHR (2) corroborates their existence.
Although the pathophysiology of hyperparathyroidism in hypophosphatemic rickets remains unclear at present (44), abnormal PTH regulation has been suggested (37). Given the high abundance of PHEX mRNA in parathyroid glands of patients with XLH (39), PHEX may play a role in the regulation of PTH, perhaps by altering its cleavage within or outside the parathyroid gland either directly (45) or indirectly (46, 47). Alternatively, elevated PTH levels could be readily attributed to the decreased levels of 1,25(OH)2D3 synthesis, thereby leading to hypocalcemia. PTH secretion appropriately ensues aimed toward maintaining calcium homeostasis by increasing bone turnover (increased serum ALP activity) and decreasing urinary calcium excretion, all consistent with secondary hyperparathyroidism. Additional confirmatory support for this argument is provided by the diffuse parathyroid hyperplasia observed histologically in transgenic animals that became progressively pronounced with age. Interestingly, it was suggested recently that elevated levels of circulating FGF23 also promote the development of secondary hyperparathyroidism in predialysis patients through suppression of CYP27B1 activity (48).
What then is the cause for the decrease in circulating levels of 1,25(OH)2D3? It is rather remarkable that when FGF23 is overexpressed, a rise in circulating PTH levels ensues, which fails to normalize 1,25(OH)2D3 serum concentrations. Normally the renal hydroxylases Cyp27b1 and Cyp24 are very tightly and reciprocally regulated by PTH, which induces Cyp27b1 while down-regulating Cyp24 activity by making the Cyp24 mRNA susceptible to degradation (49, 50). Here we show that at 1 month of age, in mice overexpressing FGF23, Cyp27b1 expression, at least at the mRNA level, is decreased, whereas concurrently, Cyp24 transcript levels are increased, compared with littermates devoid of the transgene. These are effects that are diametrically opposed to what one would anticipate in the presence of high circulating PTH levels and the concomitant hypophosphatemia. Yet, as these animals age and PTH levels become more pronounced, this is paralleled by a corresponding increase in both renal Cyp24 and Cyp27b1 mRNA expression, compared with controls. Yet these alterations were again not reflected in corresponding rises in 1,25(OH)2D3 circulating levels. Two possibilities could account for this discrepancy: first, the relentless metabolism of 1,25(OH)2D3 to inactive forms by the rising levels of Cyp24, and second, by the observation that the rise in Cyp27b1 mRNA levels is not reflected by similar alterations at the protein levels. In fact, in our mice renal tubular Cyp27b1 immunoreactivity is comparable with that in wild-type specimens. This dichotomy between transcript and protein levels for Cyp27b1 and the inability of PTH and hypophosphatemia to stimulate its enzymatic activity have also been described recently in Hyp mice (51). Therefore, the defect in Cyp27b1 activity does not result from aberrant transcriptional regulation but likely from a defect in translational or posttranslational modification.
Our findings here support the scheme that increased circulating levels of FGF23 not only decrease Npt2 expression and renal tubular phosphate reabsorption but also, either directly or indirectly, impair regulation of Cyp24 expression by PTH and alter Cyp27b1 mRNA translation or posttranslational modification of the protein. 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 low-normal levels of 1,25(OH)2D3 associated with these three disorders despite the prevailing hypophosphatemia, a finding that is rather unique to them because in other renal phosphate-wasting states, synthesis of 1,25(OH)2D3 is up-regulated. For example, 1,25(OH)2D3 synthesis is appropriately increased by hypophosphatemia in Npt2-null mice (52) and patients with hypophosphatemia associated with heterozygote missense mutations in the NPT2 gene (53). Further studies will be necessary to define the mechanisms that underlie these defects.
The question then arises as to whether the increase in circulating PTH contributes, at least in part, to the decrease in Npt2 action at the level of the renal proximal tubule and the inappropriate phosphaturia associated with this condition. Shimada et al. (34) concluded that the reduction in Npt2 expression and hypophosphatemia in their transgenic mice appear to be induced by a PTH-independent mechanism. In contrast, based on our animal model, it is becoming apparent that in the presence of high serum FGF23 levels, a dissociation of PTH actions at the level of the kidney occurs, whereby the effects of PTH on reabsorbing urinary calcium and perhaps promoting phosphaturia by reducing Npt2 expression are preserved, whereas Cyp27b1 activity and Cyp24 expression are refractory to its action. The concept that hyperparathyroidism can contribute to the phosphate loss independent of that arising from the PHEX mutation is also supported by the observation that parathyroidectomy in patients with XLH can lead to an increase in serum phosphate (39). Experiments are presently underway using mice carrying targeted disruption of the Pth gene (54) to investigate this possibility.
In summary, we have generated a murine model of FGF23 overexpression that has provided us with new insights into the complex cascade of factors that underlie the pathophysiology of renal phosphate-wasting disorders. This animal model will now serve to further clarify the role of PTH in the phosphaturic process and define the molecular mechanisms by which FGF23 alters renal vitamin D metabolism in these conditions.
|
Footnotes |
|---|
Abbreviations: ADHR, Autosomal dominant hypophosphatemic rickets; ALP, alkaline phosphatase; APOE3, apolipoprotein E3; BMD, bone mineral density; BV, bone volume; Cyp24, 25-hydroxyvitamin D3 24-hydroxylase; Cyp27b1, renal 25-hydroxyvitamin D3-1-hydroxylase; 3D, three-dimensional; FGF23, fibroblast growth factor 23; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; microCT, microcomputed tomography; Npt2, sodium-phosphate transport protein 2; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TIO, tumor-induced osteomalacia; TRAP, tartrate resistant acid phosphatase; XLH, X-linked hypophosphatemic rickets.
Received February 23, 2004.
Accepted for publication July 22, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis. Endocrinology 145:3087–3094[CrossRef][Medline]
-hydroxylase activity in X-linked hypophosphatemia: a translational or posttranslational defect. J Bone Miner Res 18:434–442[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  R. Samadfam, C. Richard, L. Nguyen-Yamamoto, I. Bolivar, and D. Goltzman Bone Formation Regulates Circulating Concentrations of Fibroblast Growth Factor 23 Endocrinology, November 1, 2009; 150(11): 4835 - 4845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. S. Levine, C. R. Kleeman, and A. J. Felsenfeld The Journey From Vitamin D-Resistant Rickets to the Regulation of Renal Phosphate Transport Clin. J. Am. Soc. Nephrol., November 1, 2009; 4(11): 1866 - 1877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Liu, X. Bai, H. Wang, A. Karaplis, D. Goltzman, and D. Miao Hypophosphatemia-mediated hypotension in transgenic mice overexpressing human FGF-23 Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1514 - H1520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Carrillo-Lopez, P. Roman-Garcia, A. Rodriguez-Rebollar, J. L. Fernandez-Martin, M. Naves-Diaz, and J. B. Cannata-Andia Indirect Regulation of PTH by Estrogens May Require FGF23 J. Am. Soc. Nephrol., September 1, 2009; 20(9): 2009 - 2017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gattineni, C. Bates, K. Twombley, V. Dwarakanath, M. L. Robinson, R. Goetz, M. Mohammadi, and M. Baum FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1 Am J Physiol Renal Physiol, August 1, 2009; 297(2): F282 - F291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Razzaque FGF23-mediated regulation of systemic phosphate homeostasis: is Klotho an essential player? Am J Physiol Renal Physiol, March 1, 2009; 296(3): F470 - F476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Berndt and R. Kumar Novel Mechanisms in the Regulation of Phosphorus Homeostasis Physiology, February 1, 2009; 24(1): 17 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nakatani, B. Sarraj, M. Ohnishi, M. J. Densmore, T. Taguchi, R. Goetz, M. Mohammadi, B. Lanske, and M. S. Razzaque In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis FASEB J, February 1, 2009; 23(2): 433 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Razzaque Does FGF23 toxicity influence the outcome of chronic kidney disease? Nephrol. Dial. Transplant., January 1, 2009; 24(1): 4 - 7. [Full Text] [PDF]
|
|
|
|
 |
|
 X. Bai, Q. Dinghong, D. Miao, D. Goltzman, and A. C. Karaplis Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype Am J Physiol Endocrinol Metab, January 1, 2009; 296(1): E79 - E88. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, L. Vierthaler, W. Tang, J. Zhou, and L. D. Quarles FGFR3 and FGFR4 Do not Mediate Renal Effects of FGF23 J. Am. Soc. Nephrol., December 1, 2008; 19(12): 2342 - 2350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, J. Zhou, W. Tang, R. Menard, J. Q. Feng, and L. D. Quarles Pathogenic role of Fgf23 in Dmp1-null mice Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E254 - E261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Brownstein, F. Adler, C. Nelson-Williams, J. Iijima, P. Li, A. Imura, Y.-i. Nabeshima, M. Reyes-Mugica, T. O. Carpenter, and R. P. Lifton A translocation causing increased {alpha}-Klotho level results in hypophosphatemic rickets and hyperparathyroidism PNAS, March 4, 2008; 105(9): 3455 - 3460. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Marsell, T. Krajisnik, H. Goransson, C. Ohlsson, O. Ljunggren, T. E. Larsson, and K. B. Jonsson Gene expression analysis of kidneys from transgenic mice expressing fibroblast growth factor-23 Nephrol. Dial. Transplant., March 1, 2008; 23(3): 827 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Marsell, E. Grundberg, T. Krajisnik, H. Mallmin, M. Karlsson, D. Mellstrom, E. Orwoll, C. Ohlsson, K. B Jonsson, O. Ljunggren, et al. Fibroblast growth factor-23 is associated with parathyroid hormone and renal function in a population-based cohort of elderly men Eur. J. Endocrinol., January 1, 2008; 158(1): 125 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, W. Tang, J. Zhou, L. Vierthaler, and L. D. Quarles Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1636 - E1644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Krajisnik, P. Bjorklund, R. Marsell, O. Ljunggren, G. Akerstrom, K. B Jonsson, G. Westin, and T. E Larsson Fibroblast growth factor-23 regulates parathyroid hormone and 1{alpha}-hydroxylase expression in cultured bovine parathyroid cells J. Endocrinol., October 1, 2007; 195(1): 125 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Bai, D. Miao, D. Goltzman, and A. C. Karaplis Early Lethality in Hyp Mice with Targeted Deletion of Pth Gene Endocrinology, October 1, 2007; 148(10): 4974 - 4983. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S Razzaque and B. Lanske The emerging role of the fibroblast growth factor-23-klotho axis in renal regulation of phosphate homeostasis J. Endocrinol., July 1, 2007; 194(1): 1 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Khosravi, C. M. Cutler, M. H. Kelly, R. Chang, R. E. Royal, R. M. Sherry, F. M. Wodajo, N. S. Fedarko, and M. T. Collins Determination of the Elimination Half-Life of Fibroblast Growth Factor-23 J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2374 - 2377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu and L. D. Quarles How Fibroblast Growth Factor 23 Works J. Am. Soc. Nephrol., June 1, 2007; 18(6): 1637 - 1647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Segawa, S. Yamanaka, Y. Ohno, A. Onitsuka, K. Shiozawa, F. Aranami, J. Furutani, Y. Tomoe, M. Ito, M. Kuwahata, et al. Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in klotho mice Am J Physiol Renal Physiol, February 1, 2007; 292(2): F769 - F779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, P. S N Rowe, L. Vierthaler, J. Zhou, and L D. Quarles Phosphorylated acidic serine-aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity J. Endocrinol., January 1, 2007; 192(1): 261 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Sitara, M. S. Razzaque, R. St-Arnaud, W. Huang, T. Taguchi, R. G. Erben, and B. Lanske Genetic Ablation of Vitamin D Activation Pathway Reverses Biochemical and Skeletal Anomalies in Fgf-23-Null Animals Am. J. Pathol., December 1, 2006; 169(6): 2161 - 2170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Antoniucci, T. Yamashita, and A. A. Portale Dietary Phosphorus Regulates Serum Fibroblast Growth Factor-23 Concentrations in Healthy Men J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3144 - 3149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, J. Zhou, W. Tang, X. Jiang, D. W. Rowe, and L. D. Quarles Pathogenic role of Fgf23 in Hyp mice Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E38 - E49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. White, T. E. Larsson, and M. J. Econs The Roles of Specific Genes Implicated as Circulating Factors Involved in Normal and Disordered Phosphate Homeostasis: Frizzled Related Protein-4, Matrix Extracellular Phosphoglycoprotein, and Fibroblast Growth Factor 23 Endocr. Rev., May 1, 2006; 27(3): 221 - 241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liu, W. Tang, J. Zhou, J. R. Stubbs, Q. Luo, M. Pi, and L. D. Quarles Fibroblast Growth Factor 23 Is a Counter-Regulatory Phosphaturic Hormone for Vitamin D J. Am. Soc. Nephrol., May 1, 2006; 17(5): 1305 - 1315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-i. Nabeshima Toward a better understanding of klotho. Sci. Aging Knowl. Environ., March 22, 2006; 2006(8): pe11 - pe11. [Abstract] [Full Text]
|
|
|
|
|
|
T. J. Berndt, S. Schiavi, and R. Kumar "Phosphatonins" and the regulation of phosphorus homeostasis Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1170 - F1182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Yu, O. A. Ibrahimi, R. Goetz, F. Zhang, S. I. Davis, H. J. Garringer, R. J. Linhardt, D. M. Ornitz, M. Mohammadi, and K. E. White Analysis of the Biochemical Mechanisms for the Endocrine Actions of Fibroblast Growth Factor-23 Endocrinology, November 1, 2005; 146(11): 4647 - 4656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shimada, Y. Yamazaki, M. Takahashi, H. Hasegawa, I. Urakawa, T. Oshima, K. Ono, M. Kakitani, K. Tomizuka, T. Fujita, et al. Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism Am J Physiol Renal Physiol, November 1, 2005; 289(5): F1088 - F1095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Razzaque, R. St-Arnaud, T. Taguchi, and B. Lanske FGF-23, vitamin D and calcification: the unholy triad Nephrol. Dial. Transplant., October 1, 2005; 20(10): 2032 - 2035. [Full Text] [PDF]
|
|
|
|
|
|
T. Larsson, S. I. Davis, H. J. Garringer, S. D. Mooney, M. S. Draman, M. J. Cullen, and K. E. White Fibroblast Growth Factor-23 Mutants Causing Familial Tumoral Calcinosis Are Differentially Processed Endocrinology, September 1, 2005; 146(9): 3883 - 3891. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Imel and M. J. Econs Fibroblast Growth Factor 23: Roles in Health and Disease J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2565 - 2575. [Full Text] [PDF]
|
|
|
|
|
|
O. Gutierrez, T. Isakova, E. Rhee, A. Shah, J. Holmes, G. Collerone, H. Juppner, and M. Wolf Fibroblast Growth Factor-23 Mitigates Hyperphosphatemia but Accentuates Calcitriol Deficiency in Chronic Kidney Disease J. Am. Soc. Nephrol., July 1, 2005; 16(7): 2205 - 2215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Larsson, X. Yu, S. I. Davis, M. S. Draman, S. D. Mooney, M. J. Cullen, and K. E. White A Novel Recessive Mutation in Fibroblast Growth Factor-23 Causes Familial Tumoral Calcinosis J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2424 - 2427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Jonsson The role of fibroblast growth factor 23 in renal disease Nephrol. Dial. Transplant., March 1, 2005; 20(3): 479 - 482. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
