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1The Kidney Institute, The University of Kansas Medical Center, Kansas City, Kansas; and 2Department of Genetics and Development Biology, University of Connecticut Health Center, Farmington, Connecticut
Submitted 6 January 2006 ; accepted in final form 26 February 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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X-linked hypophosphatemia; hypophosphatemia; osteomalacia; fibroblastic growth factor-23
The mechanisms whereby inactivation of PHEX leads to renal phosphate wasting, abnormal vitamin D metabolism, and rickets/osteomalacia are not clear. PHEX is predominately expressed in osteoblasts and osteocytes in bone (4, 6, 10). Several observations suggest the existence of putative PHEX substrates that regulate systemic phosphate homeostasis, called phosphatonins (20), and the mineralization process, called minhibins (36). Both cross-transplant and parabiosis studies in mice support the presence of a phosphaturic factor in Hyp mice that stimulates renal phosphate wasting and impairs production of 1,25(OH)2D3 (20). The evidence for minhibin is derived from both in vivo and in vitro studies demonstrating that osteoblasts and bone marrow stromal cell (BMSC) cultures from Hyp mice have an intrinsic defect in the mineralization of extracellular matrix due to the production of a mineralization inhibitory factor (36), although recent findings suggest that hypophosphatemia and alterations in the physiochemical milieu may be sufficient to account for the defective mineralization (19). Regardless, the presence of PHEX in bone, the importance of phosphate in regulating the mineralization process, and the presence of a circulating phosphaturic factor in PHEX-deficient states have given rise to the possibility of a bone-kidney axis involving PHEX and the production of systemic and local factors by bone that regulate phosphate homeostasis as well as mineralization of extracellular matrix (21).
Fibroblastic growth factor-23 (FGF23) is a leading candidate for phosphatonin (8). In this regard, FGF23 has phosphaturic activity in vivo (25), is also predominantly expressed in bone, and has been shown to be coexpressed with PHEX in osteoblast cultures (15). Moreover, the essential role of FGF23 in regulating serum phosphate levels is supported by the fact that overexpression of FGF23 in mice (2, 13, 26) or mutations that prevent its degradation (1, 3, 35) cause hypophosphatemia and suppress 1,25(OH)2D3 production by the kidney, whereas FGF23 deficiency (3, 24) or mutations increasing FGF23 degradation (12) result in hyperphosphatemia, increased serum 1,25(OH)2D3 levels, and soft tissue calcifications. In addition, serum levels of FGF23 are elevated in XLH (37) and Hyp mice (15), and the renal and skeletal phenotypes of Hyp mice resemble those of mice with excess Fgf23. Finally, recent reports indicate that crossing Fgf23 null mice onto the Hyp background reverses the hypophosphatemia (27), and blocking antibodies for Fgf23 ameliorates hypophosphatemia and rickets in Hyp mice (1a).
However, several questions remain about the regulation of Fgf23 by Phex and the role of Fgf23 in mediating the phenotype of Hyp mice. For example, the mechanism whereby inactivation of Phex results in increased Fgf23 levels is not clear. Although initial studies suggested that FGF23 might be a substrate for PHEX (28), other studies have been unable to confirm that FGF23 is metabolized by PHEX (5, 15). Furthermore, Fgf23 transcripts are increased in bone and osteoblasts derived from Hyp mice, suggesting that inactivation of Phex results in increased synthesis of Fgf23. In addition, the role that Fgf23 plays in the skeletal abnormalities in Hyp mice has not been determined. Fgf23 excess might directly contribute to the apparent intrinsic mineralization defect observed in Hyp-derived osteoblasts (7, 36), or, alternatively, Phex may have other substrates or actions in osteoblasts that impact upon bone independently of Fgf23.
In the present investigations, we examined the mechanism whereby Fgf23 is regulated by Phex and evaluated the contribution of elevated Fgf23 levels to the systemic and skeletal abnormalities observed in Hyp mice, namely hypophosphatemia, abnormal 1,25(OH)2D3 production, and rickets/osteomalacia. To accomplish this, we created a reporter mouse line where enhanced green fluorescent protein (eGFP) was inserted into the Fgf23 locus to replace the first exon of the Fgf23 gene with eGFP sequence, thereby creating a null allele called Fgf23+/– that was crossed onto the Hyp background to create combined Fgf23 and Phex-deficient mice. We found that Fgf23 was expressed predominately in osteocytes, but also identified a novel location in endothelial cells in the venous sinusoids of the bone marrow and thymus. Inactivating mutations of Phex resulted in increased expression of Fgf23 at these sites, presumably from the production of intrinsic or extrinsic factors stimulating Fgf23 gene transcription. In addition, we found that Fgf23–/– mice, despite profound hyperphosphatemia, lacked active mineralization, as assessed by fluorescent labeling of bone. Moreover, the Fgf23 null phenotype was dominant to that of Hyp, resulting in conversion of hypophosphatemia and inappropriately low 1,25(OH)2D3 levels in Hyp to hyperphosphatemia and elevated 1,25(OH)2D3 levels. With regard to the skeleton, superimposed Fgf23 deficiency corrected the rickets but not the defective mineralization in Hyp mice. Rather, there was a conversion of osteomalacia to a complete absence of mineralization in combined Fgf23–/–/Hyp mice. In addition, the presence of accentuated metaphyseal sclerosis in combined Fgf23–/–/Hyp compared with Fgf23–/– mice also suggests an independent effect of Phex on bone.
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METHODS |
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Transfer of Fgf23 deficiency onto Phex-deficient Hyp background. We crossed male heterozygous Fgf23-deficient mice (Fgf23+/–/XY) with Hyp mice to generate female heterozygous Fgf23 deficient Hyp mice (Fgf23+/–/HypX). We then crossed Fgf23+/–/XY with Fgf23+/–/HypX to generated Fgf23 and Phex-double-deficient mice. Cross between Fgf23+/–/XY with Fgf23+/–/HypX generated 12 genotypes at the predicted frequency. Wild-type (WT; Fgf23+/+), heterozygous (Fgf23+/–), and homozygous (Fgf23–/–) Fgf23-deficient mice were genotyped by PCR (see below) and were found to be born at the expected Mendelian frequency (Fig. 1B). Because of the inability to genotype female Hyp mice, we examined only male mice in these studies.
Genotyping. Genomic DNA tissue was extracted and purified from the tail of each mouse by use of a Qiagen DNeasy Tissue kit (Qiagen, Valencia, CA). Genotypes were determined by PCR using the following primers: Fgf23F (5'-CTGACCTCTGATGGCACTGA-3'), Fgf23R (5'-GAAGATTGTTCGCACAGCAA-3'), NeoF (5'-ATTCGGCAAGCAGGCATC-3'), and NeoR (5'-CTGTTCTCCTCTTCCTCATCTC-3') for FGF23 null mice; and Phex19F 5'-GCTTGGGCTAGTTTGCTATCT-3' and Phex19R 5'-TGAGTTGGTGCTATACACGGAG-3' for Hyp mice (14).
Serum biochemical measurements. Serum calcium was measured using a Calcium kit (Sigma-Aldrich, St. Louis, MO), and serum phosphorus was measured by the phosphomolybdate-ascorbic acid method, as previously described (16). Serum parathyroid hormone (PTH) levels were measured by a mouse intact PTH ELISA kit (Immutopics, Carlsbad, CA). Serum 1,25-(OH)2D3 levels were measured using a Gamma-B 1,25-Dihydroxy Vitamin D Kit 4. Serum FGF23 levels were measured by using an FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan), following the manufacturer’s protocol.
Bone marrow harvest and stromal cell culture.
BMSCs from long bones isolated from 6-wk-old male animals were cultured as previously described (14). Adherent cells were grown in the differentiation medium (
-MEM containing 10% FBS supplemented with 5 mmol/l 
-glycerophosphate and 25 µg/ml ascorbic acid) to induce osteoblastic differentiation. During the culture period, we checked the cells under an inverted microscope with both bright field and fluorescent every day. On day 14, the cells’ pictures were taken under bright field and fluorescent light microscopy and then fixed in 4% paraformaldehyde dissolved in PBS-4% formaldehyde for immunohistochemical analysis.
Fluorescent imaging and immunohistochemical analysis. eGFP fluorescent imaging in tissues was performed using modifications of previously described methods (11). Briefly, mouse bones were quickly dissected and fixed in 4% paraformaldehyde dissolved in PBS (pH 7.4). The fixed samples were then embedded in frozen embedding medium (Thermo Shandon). Cryosectioning was performed on a Leica CM1900 Cryostat (D-69226; Leica, Nussloch, Germany) equipped with a CryoJane frozen sectioning kit (Instrumedics, Hackensack, NJ). Five-micrometer sections were obtained from the femurs and the other organs. eGFP was examined with a Zeiss Axioplan 200 inverted microscope equipped with epifluoresence and a Zeiss AxioCam color digital camera. The filter set 51019 with wavelength of excitation 475/25, dichroic 495, and emission 525/45 was used to observe eGFP. To see the tissue structure of the slide, hematoxylin staining was used in the serial sections next to the section for eGFP image.
For immunohistochemical analysis of frozen section, 5-µm slides were rinsed with PBS and incubated for 30 min at room temperature with 3% hydrogen peroxide in distilled water to block endogenous peroxidase activity. After being washed in PBS, the slides were blocked for 20 min in 1x Universal Blocking Reagent (BioGenex, San Ramon, CA) and rinsed with PBS. The frozen sections were incubated overnight at 4°C with a 1:100 dilution of purified rat anti-mouse CD31 monoclonal antibody (BD Biosciences, San Diego, CA). After being washed in PBS, the sections were incubated with 1:200 dilution of biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) for 1 h at room temperature and then washed and incubated for 30 min at room temperature with Vector ABC reagent. The slides were rewashed and developed with a DAB kit (Vector Laboratories). To further characterize the eGFP-positive cells in bone marrow, 15-µm sections were used for fluorescent labeling and confocal microscopy analysis. Alexa 594-conjugated goat anti-rat IgG (1:200; Molecular Probes, Eugene, OR) was used to achieve fluorescent labeling, and a ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole special packaging (Invitrogen, Carlsbad, CA) was used to stain the nuclei. Photomicrographs were taken with a Zeiss LSM 510 confocal microscope using Zeiss LSM 510 software (Zeiss, Thornwood, NY). Immunofluorescence staining for CD31 in BMSCs was performed similarly to the above protocol for tissues. The primary rat anti-mouse CD31 monoclonal antibody (1:100) and the Alexa 594-conjugated secondary antibody (1:200) were used in the staining. Endothelial cells were identified by red fluorescence under fluorescent microscope with the filter set 51019.
High-resolution radiography of femurs, bone densitometry, and three-dimensional analysis of the femurs by microcomputed tomography. The femurs from 6-wk-old mice were collected and fixed in 70% ethanol. The radiography of femurs was performed using a Faxitron MX-20 Specimen Radiography system with a digital camera attached (Faxitron X-Ray, Buffalo Grove, IL). Bone mineral density (BMD) of femurs was measured using a PIXImus bone densitometer (Lunar, Madison, WI). High-resolution microcomputed tomography (µCT) was used to evaluate trabecular volume fraction and microarchitecture in the distal femur [microcomputed tomography (µCT40); Scanco Medical, Basserdorf, Switzerland] (14). The three-dimensional (3-D)structure was constructed and 3-D morphometric analysis conducted with the built-in software of the µCT system.
Histomorphometric analysis of nondecalcified bone. Skeletons of mice were prelabeled on day –6 with alizarin complexone (Acros Organics, Fair Lawn, NJ) and on day –1 with calcein (Sigma-Aldrich) by intraperitoneal injection before collection of bone samples. Bones were fixed in 70% ethanol and embedded in methyl methacrylate. Five-micrometer sections were stained with Goldner’s stain and analyzed under transmitted light, and 20-µm unstained sections were evaluated under fluorescent light.
Statistics. We evaluated differences between groups by one-way ANOVA. All values are expressed as means ± SE. A P value of <0.05 was considered statistically significant. All computations were performed using the Statgraphics statistical graphic system (STSC, Rockville, MD).
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RESULTS |
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50% in Fgf23–/– mice, consistent with the increase in 1,25(OH)2D3 or possible effects Fgf23 on the parathyroid gland (see below). Heterozygous Fgf23+/– mice, consistent with their normal levels of Fgf23, had serum phosphate, calcium, 1,25(OH)2D3, and PTH levels that did not differ from WT mice (Table. 2).
In Hyp mice, the marked elevations in circulating Fgf23 levels were associated with a 40% reduction in mean serumphosphate concentrations and an 63% reduction in 1,25(OH)2D3 levels compared with WT mice. Serum calcium levels were not different from WT controls, but serum PTH levels were significantly increased in Hyp mice (Table 2). In combined homozygous Fgf23–/–/Hyp mice, the serum phosphate levels were increased to levels indistinguishable from values in Fgf23–/– mice, and the 1,25(OH)2D3 concentrations were markedly elevated. Serum calcium and PTH levels in combined Fgf23–/–/Hyp mice were also identical to values found in Fgf23 null mice (Table 2). Indeed, the 10-fold reduction in PTH values in Hyp by superimposed Fgf23 deficiency could be due to increased 1,25(OH)2D3 or direct effects of Fgf23 on the parathyroid gland. Combined heterozygous Fgf23+/–/Hyp mice, which had an 40% reduction in Fgf23 levels, remained hypophosphatemic with serum phosphate levels not significantly different from Hyp mice, but 1,25(OH)2D3 levels in 6-wk-old combined heterozygous Fgf23+/–/Hyp mice increased significantly from the suppressed 1,25(OH)2D3 levels in Hyp alone (Table 2), and this nearly threefold increase was not associated with a reduction in PTH.
Expression of eGFP in vivo under the control of the endogenous Fgf23 promoter. Next, we examined the cell distribution and expression levels of eGFP driven by endogenous Fgf23 promoter in heterozygous Fgf23+/– (1 copy of eGFP) and Fgf23–/– (2 copies of eGFP) mice and the effect of superimposed Phex deficiency in combined heterozygous and homozygous Fgf23-deficient Hyp mice of eGFP expression. We found evidence for eGFP driven by the endogenous Fgf23 promoter in bone cortex, venous sinusoids in bone marrow, and thymus as well as in ventrolateral thalamus. In addition to cortical bone, eGFP expression was also found in calvaria, trabecular bone in long bone, and vertebrae. Interestingly, eGFP was not observed in growth plate cartilage (data not shown), where Phex has been shown to be expressed in proliferating and hypertrophic chondrocytes (17). The presence of two copies of eGFP resulted in higher levels of eGFP expression in tissues of homozygous Fgf23–/– compared with heterozygous Fgf23+/– mice (Fig. 2B), indicating an eGFP gene dosage effect. We found no eGFP expression in heart, spleen, kidney, or liver (Fig. 2A). In particular, although eGFP was expressed in the venous sinusoids of the central bone marrow (Figs. 2, A and B, and 3B) and in venules of the thymus, it was not present in vascular beds of other organs (Fig. 2A and data not shown).
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To define the cell types that express eGFP in the venules of bone marrow and thymus, we performed immunohistochemical staining with an antibody to the endothelial marker CD31. These studies colocalized eGFP and CD31 immunoreactivity in apparent endothelial cells in the venous sinusoids of both bone marrow (Fig. 3C) and thymus (data not shown). Confocal microscroscopic analysis confirmed the coexpression of eGFP and CD31 in the venous sinusoids (Fig. 3C, inset).
Expression of eGFP in bone marrow stromal cultures under control of the endogenous Fgf23 promoter. To further characterize the cell type expression of eGFP and its regulation by Phex and osteoblast development, we performed cultures of BMSCs derived from combined homozygous Fgf23–/–/Hyp for 14 days under conditions favoring the differentiation into osteoblasts. Similar to the in vivo findings, we observed eGFP expression in two cell populations (Fig. 4A). In early cultures, before the formation of mineralization nodules, we observed low levels of eGFP in CD31-positive fibroblast-like cells (data not shown). After the development of mineralization nodules in 14-day-old confluent cultures, we observed high levels of eGFP expression limited to cells within the mineralization nodules that were negative for CD31 and had dendritic processes resembling osteocytes. Fibroblast-like CD31-positive cells that were present in earlier cultures persisted in these late cultures as spindle-shaped cells interspersed with mesenchymal cells surrounding the mineralization nodules (Fig. 4A). In addition, we compared eGFP expression in BMSCs from Fgf23+/– and Fgf23+/–/Hyp mice to determine whether Phex deficiency regulates Fgf23 promoter activity in these different cell populations ex vivo. We found that the CD31-positive fibroblast-like cells located outside of the mineralization nodules continued to express low levels of eGFP (Fig. 4B, a and c) in the setting of superimposed Phex deficiency, whereas the osteocyte-like cells embedded in the mineralization nodules markedly upreglated eGFP expression (Fig. 4B, b and d) in the absence of Phex. Both the number of cells in mineralization nodules expressing eGFP and the intensity of eGFP expression per cell were increased in Hyp mice (Fig. 4B, d).
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20% reduction in BMD (Fig. 5C). Histologically, the growth plate of 6-wk-old Fgf23–/– mice demonstrated an increase in the hypertrophic zone relative to the proliferative zone without appreciable widening of the growth plate, consistent with a dyschondroplasia, leading to impaired long bone growth (Fig. 6A). Compared with WT mice, Fgf23–/– mice also displayed increased osteoid volume and impaired mineralization, as evidenced by the reduced intensity and the lack of double-fluorescent labeling of bone (Fig. 6B), as previously reported (24).
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The radiographic appearance of bone in heterozygous Fgf23+/–/Hyp mice did not differ from Hyp mice (Fig. 5A), although, similarly to the slight increase in bone length (Table 1), these animals also had a small but significant increase in BMD (Fig. 5C). In contrast, combined Fgf23–/–/Hyp mice showed correction of both the radiographic and histological manifestations of rickets, which were replaced with growth plate abnormalities and overall bone appearance resembling those of Fgf23–/– mice (Figs. 5, A and B, and 6A). In addition, combined Fgf23–/–/Hyp mice developed a more pronounced increased density of subchondral bone in the distal metaphyseal bone just beneath the growth plate (Fig. 5, A and B), suggesting that Phex deficiency results in changes distinct from FGF23 deficiency in this segment of bone. Overall, the BMD also increased in combined homozygous Fgf23–/–/Hyp mice to levels not significantly different from Fgf23 null mice but remained significantly lower than WT littermates (Fig. 5C). On histological examination, osteomalacia persisted in combined Fgf23–/–/Hyp mice but now resembled Fgf23–/– mice, resulting from a reduction in both the reduced amount of osteoid and diminished intensity of fluorescent labeling compared with Hyp mice (Fig. 5C).
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DISCUSSION |
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Our investigation of Fgf23-deficient mice expressing eGFP under the control of the endogenous Fgf23 promoter provides new insights into the cell type expression of Fgf23 in bone and clarifies the role of this hormone in the pathogenesis of renal and bone abnormalities in the Hyp homolog of XLH. The present studies show for the first time that Fgf23 is expressed in two distinct cell populations in the bone, namely osteocytes embedded in both cortical and trabecular bone and in cells lining the venous sinusoids in bone marrow. Only the Fgf23 expression in osteocyte appears to be upregulated by Phex deficiency. In contrast, Fgf23 was expressed at similar levels in venous sinusoids in both combined Fgf23-deficient/Hyp and Fgf23-deficient mice. The site-selective upregulation of Fgf23 by Phex deficiency in osteocytes, but not venous sinusoids, parallels our prior observations that increased Fgf23 transcript in Hyp mice was limited to bone, because no increases of Fgf23 were observed in bone marrow by real-time PCR (15).
In addition, the present results further clarify the spatial-cell type relationship between Phex and Fgf23 expression. On the basis of prior studies showing that Phex and Fgf23 are coexpressed in differentiated osteoblasts (15, 23, 36) and the inability to demonstrate that Fgf23 is a substrate for Phex (5, 15), we proposed that inactivating Phex mutations increase the biosynthesis of Fgf23 in osteoblasts, possibly as a result of direct consequences of Phex deficiency or through autocrine/paracrine actions of putative Phex substrates, accumulating in the bone milieu as a result of inactivating Phex mutations (21). The present findings, however, show that the cell type expression of Fgf23 in bone is more limited than Phex. Whereas the expression of Phex has been shown to be limited to osteoblasts and osteocytes by in situ and immunohistochemical analysis (17, 22, 23), we show that Fgf23 is selectively upregulated in osteocytes embedded in bone, but not in surface lining osteoblasts, in response to inactivating Phex mutations (Fig. 3A). Additional studies of BMSCs derived from Fgf23-deficient eGFP reporter mice confirm that Fgf23 gene transcription occurs when the cells within the osteoblast lineage become embedded in the extracellular matrix (Fig. 4). Thus additional factors related to terminal differentiation into osteocytes and/or matrix-derived factors are required to upregulate Fgf23 in the setting of Phex deficiency. The related observation that Phex immunostaining is pronounced in the cellular processes of osteocytes that transverse matrix favors indirect stimulation of Fgf23 resulting from altered Phex-dependent metabolism of extracellular matrix proteins. To date, only dietary phosphate, but not hyperphosphatemia per se, and 1,25(OH)2D3 have been shown to regulate FGF23 gene transcription, neither of which could account for the increased Fgf23 expression in Hyp. Additional information is needed to identify physiologically relevant Phex substrates and to define their role in the regulation of Fgf23 expression. Nevertheless, the present findings indicate that, although Phex deficiency is necessary, it is not sufficient to increase Fgf23 production in cells within the osteoblast lineage. Also, the upregulation of Fgf23 by inactivating Phex mutations in bone places Fgf23 downstream of Phex in what is likely to be a hormonal cascade involving yet to be discovered Phex substrates that are the proximate stimuli for increased Fgf23 expression.
The finding of Fgf23 in venous sinusoids of bone marrow is novel. Colocalized Fgf23 with CD31, a marker of endothelial cells in bone marrow, and the presence of eGFP and CD31-positive cells in BMSC cultures suggest that Fgf23 is produced by vascular endothelial cells, or possibly pericytes, that are in close proximity to vascular endothelial cells in venous sinusoids. The presence of Fgf23 in the bone marrow venules might explain the ability of bone marrow transplantation to partially rescue the hypophosphatemia in Hyp mice (18). The localization of Fgf23 to the venous circulation also raises interesting possibilities regarding the endocrine functions of Fgf23 as well as potential local function to regulate bone marrow hematopoietic lineages. A local role is suggested by the related finding that Fgf23 is also localized to venous sinusoids of the thymus, which undergoes involution in Fgf23 null mice (24). Expression of Fgf23 in the sinusoid does not appear to be regulated by Phex deficiency, making it less likely that Fgf23 production at this site is important in regulating systemic phosphate homeostasis or mineralization.
These results also provide insights into the biological effects of Fgf23 in Hyp. With regard to the kidney, the present studies are consistent with Fgf23 mediating the hypophosphatemia and abnormal vitamin D metabolism in Hyp. The absence of circulating Fgf23 levels results in increased serum phosphate and 1,25(OH)2D3 levels but fully corrects the hypophosphatemia and low 1,25(OH)2D3 levels in Hyp mice to levels identical to Fgf23 null mice, thereby showing the dominant role of Fgf23 in the regulating phosphate and 1,25(OH)2D3 levels in Hyp mice. If inactivating mutations of Phex were to result in the increased production of another phosphaturic factor, we might expect that the serum phosphate levels would remain lower in Fgf23–/– compared with combined Fgf23–/–/Hyp mice. Reduction of Fgf23 levels by 50% in combined heterozygous Fgf23+/–/Hyp mice, however, failed to significantly increase serum phosphate or correct the skeletal abnormalities but did normalize 1,25(OH)2D3 levels, suggesting that the regulation of sodium phosphate transport and 1-hydroxylase activity by Fgf23 in the kidney may have different sensitivities.
The potential interactions between Fgf23 and Phex on bone growth and mineralization remain to be fully elucidated. The presence of osteomalacia in Fgf23 null mice (i.e., excess osteoid and impaired fluorescent bone labeling), despite hyperphosphatemia, which should stimulate mineralization of extracellular matrix (19), the presence of growth plate abnormalities leading to growth retardation in Fgf23 null mice, and the expression of potential Fgf23 receptors in bone and cartilage (38) indicate a possible direct effect of Fgf23 on the skeleton. Preliminary reports (33) show that adenovirus-mediated overexpression of Fgf23 acts as a negative regulator of osteoblast development and matrix mineralization in fetal rat calvarial cultures. However, such actions would not account for the impaired mineralization observed in Fgf23-deficient mice. From our studies, it is difficult to distinguish possible direct osseous effects of Fgf23 from potential indirect effects resulting from hyperphosphatemia and elevated 1,25(OH)2D3. With regard to Phex, its inactivation causes both rickets and osteomalacia. The osteomalacia in Hyp differs from that observed in Fgf23 null mice in that Hyp has a greater degree of excess osteoid and greater accumulation of fluorescent labeling. Phex deficiency has also been purported to have indirect effects on bone mineralization through putative mineralization inhibitors, called minhibin, that mediate an intrinsic defect in mineralization, although recent data indicate that phosphate loading can correct the defective mineralization in Hyp mice (19). The greater complexity is indicated by the finding that superimposing Fgf23 deficiency onto Hyp reversed the bone abnormalities typically associated with Phex deficiency, namely rickets, and replaced the Hyp bone phenotype with that of Fgf23 null mice, except for the selective increase in subchondral sclerosis in distal metaphysical bone in Phex-deficient Hyp mice lacking Fgf23. The marked degree of subchondral sclerosis did not occur in Fgf23 null mice expressing wild-type Phex despite identical degrees of hyperphosphatemia and increased 1,25(OH)2D3. The presence of this Phex-dependent bone phenotype supports the presence of other Phex-dependent processes regulating mineralization.
On the basis of these studies, we conclude that inactivating mutations of Phex selectively increase Fgf23 transcription in osteocytes in bone, revealing that Phex deficiency is necessary, but not sufficient, for increased Fgf23 gene transcription in osteocytes. It is likely that the presence of other factors related to osteocyte differentiation and/or the presence of extracellular matrix factors are necessary for the selective upregulation of Fgf23 expression in Phex-deficient osteocytes. Insights into these additional factors and identification of physiologically relevant Phex substrates will be necessary to fill in the gap in our understanding of how inactivating Phex mutations increase Fgf23 production. Regardless, superimposing Fgf23 deficiency onto Hyp mice corrects the abnormalities in serum phosphate and 1,25(OH)2D3 associated with inactivation of Phex, thereby establishing Fgf23 as phosphatonin and placing this phosphaturic factor downstream of Phex. Finally, the presence of bone abnormalities in Phex-deficient mice not attributable to Fgf23 leaves open the possibility of additional Phex functions and/or substrates in bone that regulate mineralization.
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FOOTNOTES |
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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.
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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]
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G. J. Strewler Bone Talk: Klotho and FGF23 Signaling IBMS BoneKEy, December 1, 2006; 3(12): 36 - 40. [Full Text] [PDF]
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E. Schipani, S. Ferrari, N. S. Datta, L. K. McCauley, A. Vignery, T. Bellido, G. J. Strewler, C. H. Turner, Y. Jiang, and E. Seeman Meeting Report from the 28th Annual Meeting of the American Society for Bone and Mineral Research IBMS BoneKEy, November 1, 2006; 3(11): 14 - 50. [Full Text] [PDF]
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L. F. Bonewald Mechanosensation and Transduction in Osteocytes. IBMS BoneKEy, October 1, 2006; 3(10): 7 - 15. [Abstract] [Full Text] [PDF]
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G. J. Strewler Disordered Secretion of FGF23 Links Three Disorders of Phosphate Transport IBMS BoneKEy, August 1, 2006; 3(8): 14 - 17. [Full Text] [PDF]
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