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Am J Physiol Endocrinol Metab 293: E1636-E1644, 2007. First published September 11, 2007; doi:10.1152/ajpendo.00396.2007
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Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice

Shiguang Liu, Wen Tang, Jianping Zhou, Luke Vierthaler, and L. Darryl Quarles

The Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas

Submitted 22 June 2007 ; accepted in final form 7 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice.

PHEX endopeptidase; X-linked hypophosphatemia; fibroblast growth factor 23; rickets; osteomalacia

X-LINKED HYPOPHOSPHATEMIA (XLH) is caused by inactivating mutations of the endopeptidase PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (32), a member of the M13 family of the type II cell-surface zinc-dependent proteases that is predominately expressed in bone (33). The mouse Phex cDNA sequence is highly homologous to that of humans, and a 3' deletion of the Phex gene in the Hyp mouse results in an animal model of XLH (4, 29). PHEX mutations result in impaired renal tubular reabsorption of phosphate and aberrant regulation of 1,25(OH)2D production, leading to hypophosphatemia and defective calcification of cartilage and bone, which result in rickets, osteomalacia, and growth retardation (32). Recent studies have indicated that the phosphaturia and impaired production of 1,25(OH)2D in XLH are due to increased production of fibroblast growth factor 23 (FGF23) by osteocytes in bone (15). The causative role of FGF23 in Hyp mice is supported by the findings that crossing FGF23-null mice onto the Hyp background reverses the hypophosphatemia (28) and that blocking antibodies to FGF23 ameliorates hypophosphatemia and rickets in Hyp mice (1). In addition, FGF23 is the key phosphaturic factor underlying hereditary hypophosphatemic disorders, including autosomal-dominant hypophosphatemic rickets (ADHR), autosomal-recessive hypophosphatemic rickets, and XLH. Mutations in FGF23 that prevent its cleavage by furin-like proprotein convertases and mutations in DMP1 that stimulate FGF23 gene transcription cause ADHR and autosomal-recessive hypophosphatemic rickets, respectively (8, 16, 27, 31, 36).

FGF23 is a member of the fibroblastic growth factor family and is a critical hormonal regulator of systemic phosphate homeostasis. Circulating FGF23 targets Klotho-FGFR complexes in the kidney to inhibit sodium-dependent phosphate reabsorption and 1{alpha}-hydroxylase in the proximal tubule (10, 34). FGF23's phosphaturic activity and its ability to suppress 1,25(OH)2D have been shown by administration of recombinant FGF23 to wild-type (WT) mice and overexpression of FGF23 in transgenic mice. Conversely, FGF23 deficiency (2, 3, 12, 24–26, 31, 37) or mutations increasing FGF23 degradation (11) result in hyperphosphatemia, increased serum 1,25(OH)2D3 levels, and soft tissue calcifications.

The exact mechanism whereby mutations of PHEX cause elevated FGF23 is not known. Despite earlier reports to the contrary (6), FGF23 does not appear to be a PHEX substrate (5, 13). Rather, increased serum FGF23 levels correspond to increased production of FGF23 by osteocytes in the Hyp mouse model of XLH (13), indicating that PHEX mutations somehow stimulate FGF23 gene transcription. This might result from a PHEX-dependent intrinsic abnormality in osteocytes or from aberrant production of a humoral factor due to altered metabolism of unknown PHEX substrates. These possibilities have been inadequately explored, and previous studies have produced conflicting data. With regard to systemic factors, PHEX could either proteolytically inactivate an FGF23 stimulatory factor or activate a FGF23 suppressive factor that would respectively accumulate or be deficient in the presence of inactivating PHEX mutations. Although no physiologically relevant substrates for PHEX have been identified to date, the highly variable serum FGF23 levels in both patients with XLH (39) and ADHR (9) implicate additional factors that regulate circulating FGF23 concentrations beyond the causative mutations in these hereditary disorders. On the other hand, factors intrinsic to inactivating PHEX mutations in the local osteocyte microenvironment might regulate FGF23 gene transcription. In this regard, although PHEX is expressed in cells within the osteoblast lineage, including osteoblasts and osteocytes, FGF23 is selectively upregulated in osteocytes embedded in bone, but not in surface-lining osteoblasts derived from Hyp mice. The fact that PHEX is necessary but not sufficient to upregulate FGF23 expression is consistent with the requirement for additional factors related to terminal differentiation into osteocytes and/or matrix-derived factors to upregulate FGF23 in the setting of PHEX deficiency (15).

The mechanisms whereby inactivating PHEX mutations lead to defective mineralization of bone and cartilage is also not clear. Evidence that FGF23 directly targets bone cells is presently lacking (34). Rather, the bone mineralization defect in XLH appears to be due either to hypophosphatemia and/or to a nascent defect in osteoblast function that impairs the mineralization process. Several initial observations provide compelling support for an intrinsic defect in mineralization of extracellular matrix in PHEX-deficient osteoblasts (7, 18, 21, 38), possibly due to the production of a putative mineralization inhibitory factor called minhibin (38). More recently, however, the nascent defect in osteoblast-mediated mineralization in PHEX deficiency was brought into question, based on the observation that correction of hypophosphatemia alone significantly improved rickets and osteomalacia in Hyp mice (20). In addition, PHEX is expressed in chondrocytes, and loss of PHEX in Hyp mice leads to a widened hypertrophic zone and impaired mineralization, retained cartilage remnants, and abnormal resorption of the subchondral primary spongiosa. These effects could also be due to hypophosphatemia or intrinsic abnormalities in PHEX-deficient chondrocytes (19).

Thus, although elevated FGF23 is a fundamental abnormality in XLH rickets, it remains unknown whether the increased production of FGF23 by osteocytes is the consequence of an intrinsic bone abnormality or aberrant production of another upstream factor resulting from inactivating PHEX mutations. In addition, it remains unclear to what extent the mineralization defect of bone and cartilage in XLH is due to local actions of PHEX or to systemic changes in phosphate and vitamin D metabolism induced by FGF23. In the present study, we developed a technique for bone explantation to comprehensively examine whether FGF23 regulation and bone mineralization result from an intrinsic or systemic effects of PHEX mutations. Assessment of FGF23 expression and bone mineralization in bone from WT mice engrafted into Hyp mice with an altered hormonal/metabolic milieu and in bone from mutant Hyp mice transplanted into a WT mouse with a normal hormonal/metabolic milieu permit examination of the relative importance of intrinsic and systemic effects of inactivating PHEX mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and genotyping. The FGF23-eGFP reporter mouse model was created by knocking in an enhanced green fluorescent protein (eGFP) reporter by replacing exon 1 of the Fgf23 gene with eGFP cDNA as described previously (15). The FGF23-eGFP reporter mice used in the embryo experiment and the bone explantation experiment in the present study were in a 129Sv/Ev and C57BL/6J mixed genetic background and a C57BL/6J genetic background, respectively. The mice were genotyped with the REDExtract-N-Amp tissue PCR kit (Sigma-Aldrich, St. Louis, MO) with the following primers: for FGF23, 5'-CTGACCTCTGATGGCACTGA-3' (forward) and 5'-GAAGATTGTTCGCACAGCAA-3' (reverse); for Neo, 5'-ATTCGGCAAGCAGGCATC-3' (forward) and 5'-CTGTTCTCCTCTTCCTCATCTC-3' (reverse) for FGF23-null mice; and for PHEX19, 5'-GCTTGGGCTAGTTTGCTATCT-3' (forward) and 5'-TGAGTTGGTGCTATACACGGAG-3' (reverse) for Hyp mice.

All mice were fed the Labdiet JL rat and mouse/auto 6F diet (Labdiet, Brentwood, MO) containing 1.15% calcium and 0.85% phosphorus and tap water. All mice were maintained and used in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, 1996), and experiments were reviewed and approved by the Institutional Animal Care and Use Committee (protocol number 2007-1632).

Intramuscular bone explantation. Intramuscular bone explantations were modified by using a previously reported method (30). To generate WT and Hyp newborn mice carrying the FGF23-eGFP reporter, male WT/FGF23-eGFP+/– and female Hyp/FGF23-eGFP+/– mice were mated. Newborn male mice at 4.5 days old were genotyped by PCR. Then, male WT and Hyp newborn mice carrying FGF23-eGFP reporter (WT/FGF23-eGFP+/– and Hyp/FGF23-eGFP+/–, respectively) were euthanized, and both tibias and femurs were isolated and explanted into the back muscles of 8- to 10-wk-old WT and Hyp male mice. The tibias or femurs from one side were explanted to WT mice, and the tibias or femurs from the other side were explanted to Hyp mice. Each host mouse received one explanted bone on each side of its back muscles. Three weeks after explantation, explanted bones were harvested to measure bone length and were examined by X-ray radiography, microcomputer tomography, and histological analysis. Serum samples from the host mice were also collected for serum biochemical assays.

In some studies, long bones from Hyp mice were first transplanted into WT and Hyp mice for a 3-wk period and then reexplanted into Hyp and WT mice, respectively, for an extra 2-wk period. The explanted bones were then harvested for histological eGFP assessment. To see the fluorescent labeling in bone, some mice explanted with femurs were prelabeled by intraperitoneal injection with alizarin complex one (10 mg/kg; Acros Organics, Fair Lawn, NJ) and calcein (5 mg/kg; Sigma-Aldrich) 4 and 1 days, respectively, before the mice were killed.

Embryo studies. To examine whether a circulating factor exists in Hyp mice to stimulate FGF23 promoter, we mated male WT/FGF23-eGFP+/– with female Hyp mice to obtain pregnant Hyp mice bearing both WT/FGF23-eGFP+/– and Hyp/FGF23-eGFP+/– embryos. We also mated male Hyp/FGF23-eGFP+/– with WT female mice to obtain pregnant WT mice bearing both WT/FGF23-eGFP+/– and Hyp/FGF23-eGFP+/– embryos. We isolated both Hyp and WT embryos carrying the FGF23-eGFP reporter gene at embryonic day 17.5 from both pregnant Hyp and pregnant WT mice. The femurs were dissected from the embryos and fixed in 4% paraformaldehyde for cryosection. The eGFP expression in osteocytes was compared among WT embryos in WT mothers, WT embryos in Hyp mothers, Hyp embryo in WT mothers, and Hyp embryos in Hyp mothers.

Histological analysis. eGFP fluorescent imaging in bone was performed with previously described methods (15). Briefly, mouse bones were quickly dissected and fixed in 4% paraformaldehyde in PBS (pH 7.4) and then embedded in frozen embedding medium. Cryosectioning was performed on a Leica CM1900 cryostat (D-69226; Leica, Nussloch, Germany) equipped with a CryoJane frozen sectioning kit (Instrumedics, Hackensack, NJ). Sections (5 µm) were obtained from embedded bone samples. eGFP was examined with a Leica DM IRB inverted microscope equipped with a Leica DM 500 digital camera. The frozen sections were also stained with Von Kossa's stain and analyzed under bright-field microscopy.

Explanted femurs prelabeled with alizarin complex one and calcein were collected and fixed in 70% ethanol and embedded in methyl methacrylate. Sections (5 µm) were stained with Goldner's stain and analyzed under microscope with transmitted light. Unstained sections (10 µm) were evaluated under UV light. Quantitative analysis of bone sections was performed with the OsteoMeasure bone histomorphometry system (OsteoMetrics, Atlanta, GA). The following static and dynamic parameters were measured: osteoid volume (%), osteoid surface (%), osteoid seam thickness (µm), mineralizing osteoid (%), bone formation rate (µm3·µm–2·day–1), bone formation rate/bone surface referant, and mineralization lag time (day) (22).

High-resolution radiography, bone densitometry, and microcomputed tomography analysis of femurs. Bone samples were fixed in 70% ethanol after they were harvested from the animals. The radiography of femurs was performed with the Faxitron specimen radiography system MX-20 (Faxitron X-Ray, Wheeling, IL). Bone mineral density (BMD) of femurs was measured with the use of a PIXImus bone densitometer (Lunar, Madison, WI). High-resolution microcomputed tomography (µCT40; Scanco Medical, Basserdorf, Switzerland) was used to scan and evaluate bone volume fraction and BMD in the femurs as previously described (15). Briefly, bone samples were scanned in a sample holder with 10.2 mm diameter at medium resolution. The three-dimensional images were generated with the built-in software using a threshold of 250. The mineralized tissue volume fractions were evaluated from the beginning of calcified tissue underneath the growth plate to the midpoint of the distal femur to adjust for the different bone lengths. The region of interest for quantitative analysis was manually drawn.

Serum biochemical measurements. Serum biochemical measurements were performed as previously described (15). Briefly, serum FGF23 levels were measured using an FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan), serum calcium was measured using a colorimetric assay (Stanbio Laboratories, Boerne, TX), and serum phosphorus was measured by the phosphomolybdate-ascorbic acid method.

Statistics. We evaluated differences between the WT and Hyp preexplantation and postexplantation by both two-way and factorial ANOVA. A Tukey's post hoc test was subsequently performed with the adjusted P values. For multiple group comparison in quantitative histomorphometric analysis of postexplanted bone sections, one-way ANOVA and Tukey's post hoc test were used to determine differences among groups. Results were considered to be significantly different at P < 0.05. All values are expressed as means ± SE. Computations were performed using either GraphPad Prism (GraphPad Software, San Diego, CA) or STATISTICA (StatSoft, Tulsa, OK).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of autologous bone explantation. Initially, we evaluated growth and mineralization characteristics of bone from 4.5-day-old WT and Hyp mice expressing eGFP under the control of the FGF23 promoter after explantation into mice of the same genetic background (e.g., WT into WT and Hyp into Hyp). The gross appearance and length of bones at the time of isolation were similar between WT and Hyp mice (Fig. 1A; Table 1), indicating that the full features of rickets and osteomalacia had not yet emerged. However, Faxitron radiography and microcomputed tomography identified increased bone volume in the metaphyseal region of Hyp mice (Fig. 1, B and C) that resulted in a measurable increase in BMD and in metaphyseal mineralized tissue volume (Table 1). Analysis of preexplant bone revealed the presence of normal bone architecture and bone histology in WT mice, as expected; however, rickets and osteomalacia were present in Hyp bone at 4.5 days of age. This was evidenced by the widened growth plate and excess unmineralized osteoid on bone surfaces (Fig. 2, A and B). Consistent with increased FGF23 production in osteocytes of Hyp mice, we observed eGFP expression in osteocytes of Hyp mice and no detectable eGFP in WT mice at 4.5 days of age (Fig. 3A).


Figure 1
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  		Fig. 1. Gross appearance and radiological analysis of femurs from wild-type (WT) and Hyp mice before and after explantation. Shown are gross appearance images (A), radiographs (B), and 3-dimensional microcomputed tomography images (C) of femurs obtained from WT/FGF23+/– and Hyp/FGF23+/– mice (where FGF23 is fibroblast growth factor 23) before explantation and 3 wk after explantation into WT and Hyp adult mice as indicated. Femurs of 4.5-day-old WT/FGF23+/– and Hyp/FGF23+/– reporter mice were explanted into the back muscles of WT and Hyp adult mice as described in MATERIALS AND METHODS.

 

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  		Table 1. Bone length and radiological analysis of femurs from WT and Hyp mice before and after explantation

 

Figure 2
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  		Fig. 2. Effects of bone cross-explantation on bone histology. A: von Kossa-stained sections from tibias of WT/FGF23+/– and Hyp/FGF23+/– mice at 4.5 days of age before and after explantation viewed under transmitted light (x25). Mineralized tissue stains black, and unmineralized osteoid stains red. B: Goldner-stained sections from distal femurs before and after explantation. Mineralized bone stains blue-green, and unmineralized osteoid stains orange-red. Shown are endochondral bone and adjacent trabecular structures from femurs obtained from the indicated genotypes before and after explantation (x200). C: micrographs viewed under fluorescent light showing alizarin red and calcein fluorescent labels in unstained sections of femurs after explantation (x200).

 

Figure 3
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  		Fig. 3. FGF23 expression before and after bone explantation. A: increased expression of FGF23 in osteocytes of Hyp mice. Images are of frozen sections of femurs obtained from WT/FGF23+/– and Hyp/FGF23+/– neonates at 4.5 days of age viewed under fluorescent light. FGF23 promoter activity is measured by enhanced green fluorescent protein (eGFP) expression and is present in osteocytes of Hyp mice, consistent with increased FGF23 production in association with inactivating PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) mutations. B: effect of cross-explantation on FGF23 expression. Images of frozen sections were obtained from WT bone explanted into either WT or Hyp mice (WT->WT and WT->Hyp) or Hyp bone explanted in WT or Hyp (Hyp->WT and Hyp->Hyp) (x200). C: reverse reexplantation of explanted bone. Hyp bone explanted into Hyp or WT mice was removed and reimplanted into WT or Hyp mice as described in MATERIALS AND METHODS. Return of Hyp bone to the WT milieu restored FGF23 expression in osteocytes. The micrographs represent frozen sections viewed under fluorescent light (x200).

 
We found that the bone explanted into muscles remained viable and increased in size over the 3-wk observation period (Fig. 1). Explantation of bone between donor and recipient of the same genotype produced bone morphogenic changes consistent with those observed during bone development in the intact animal. In this regard, WT bone explanted into WT recipients after 3 wk had a normal gross appearance (Fig. 1A), had increased in length from 5.5 ± 0.1 to 8.8 ± 0.1 mm (P < 0.001, n ≥ 6 in each group) (Table 1), and had no identifiable abnormalities detectable by radiography and microcomputed tomography (Fig. 1B and C). WT bone explanted into WT mice also retained a normal histological appearance of the growth plate and bone. Trabecular and cortical bone were characterized by narrow osteoid seams (Fig. 2B) and distinct fluorescent labels consistent with ongoing mineralization (Fig. 2C). No eGFP expression, a marker of FGF23 promoter activity, was detected in osteocytes from WT mice after explantation (Fig. 3B).

In contrast, Hyp bone explanted into Hyp mice displayed growth retardation and evidence of rickets and osteomalacia, as detected by radiography and microcomputed tomography (Fig. 1). The change in length of the Hyp femurs explanted into Hyp was small (from 5.2 ± 0.10 to 7.0 ± 0.12 mm; P > 0.05, n ≥ 6). Moreover, we observed evidence of widened growth plates and decreased BMD and mineralized tissue volume in explanted Hyp bone compared with WT bone explanted into WT mice (Fig. 1 and Table 1). Histological examination confirmed the widened growth plate and excess osteoid (Fig. 2, A and B). In addition, diffuse fluorescent labels were present in explanted Hyp bone, consistent with defective mineralization (Fig. 2C). Surprisingly, the increase in eGFP expression in osteocytes of Hyp mice preexplantation (Fig. 3A, right) was lost in Hyp bone explanted into Hyp mice (Fig. 3B), suggesting the presence of a circulating FGF23 suppressive factor (see below).

Effects of cross-explantation of bone between WT and Hyp mice. Next, we examined the effects of bone cross-explantation in WT and Hyp mice with donors and recipients of different genotypes. Three weeks after surgery, WT femurs explanted into Hyp mice demonstrated slower growth rates (from 5.5 ± 0.1 to 6.8 ± 0.2 mm; P < 0.001, n ≥ 6 in each group) and exhibited gross features of Hyp bone (Fig. 1 and Table 1). In this regard, WT bone explanted into Hyp mice acquired a widened metaphysisis, consistent with rickets (Fig. 1) and developed decreased BMD and mineralized tissue volume (Table 1). Histologically, WT bone in the Hyp mouse developed excess osteoid due to increased extent and width of osteoid seams and exhibited impaired mineralization, as evidenced by diffuse fluorescent labels and the complete absence of double labels (Fig. 1 and Table 2). Quantitative histological analysis of WT bone explanted into Hyp mice confirmed that exposure to the Hyp milieu resulted in increased relative osteoid volume, osteoid surface, and osteoid thickness compared with WT bone explanted into WT mice (Table 2). WT bone explanted into Hyp, however, failed to upregulate eGFP expression (Fig. 3B), suggesting that stimulation of FGF23 in Hyp is not due to a systemic factor but represents an intrinsic osteocyte defect.


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  		Table 2. Histomorphometric analysis of explanted bones

 
In contrast, Hyp bone explanted into WT mice largely rescued the ricketic appearance of bone (Fig. 1). However, additional abnormalities were present, consisting of osteosclerotic-like changes in subchondral bone (Fig. 1), leading to increases in BMD and mineralized tissue volume to values greater than WT bone explanted into WT mice (Table 1). By bone histological evaluation, we found, after explantation of Hyp bone into the WT milieu, near normalization of the growth plate width, although there was persistence of previously described Hyp-related changes in the subchondral bone (19). These subchondral abnormalities included a widened zone of primary spongiosa, which contained cartilage remnants and abnormal resorption of mineralized tissue in the metaphyseal region (Fig. 4), which explain the sclerotic-looking changes observed by radiography and microcomputed tomography (Fig. 1 and Table 1). In addition, examination of bone also revealed excessive amounts of unmineralized osteoid and narrow distances between the fluorescent labels, consistent with persistent impairment of mineralization (Fig. 2, B and C). The presence of osteomalacia was confirmed by quantitative histological analysis of the explanted bone, which showed increased relative osteoid volume, osteoid surface, and osteoid thickness and increased mineralization lag time (Table 2). Except for a measurable bone formation rate due to the presence of a few discernable double-labeled surfaces, the degree of osteomalacia in Hyp bone explanted into WT was similar to that of WT bone explanted into Hyp mice (Table 2). Because explantation of Hyp bone into WT mice did not alter serum phosphate (data not shown), these findings suggest that PHEX deficiency per se may be regulating bone mineralization. Moreover, FGF23 expression as assessed by eGFP remained elevated in Hyp bone transplanted into WT mice, consistent with PHEX mutations, leading to an intrinsic osteocyte defect in Hyp (Fig. 3B).


Figure 4
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  		Fig. 4. Effects of bone explantation on growth plate cartilage and primary spongiosa. A: growth plate of WT bone explanted into a WT mouse showing normal resting, proliferating, and hypertrophic zones of cartilage (top) and remodeling of the subchondral region (bottom) as evidenced by vascular channels, calcified cartilage, and areas of resorption. B: growth plate of Hyp bone explanted into a WT mouse showing near normalization of the growth plate architecture (top) but expansion of the primary spongiosa due to retained cartilage elements and abnormal subchondral remodeling (bottom). Micrographs represent Goldner-stained sections (x200).

 
Further evidence for an intrinsic defect in Hyp leading to elevated FGF23 expression. To further confirm that the elevated FGF23 expression in osteocytes in Hyp mice is not caused by a circulating stimulatory factor in Hyp mice, we compared FGF23 promoter activities in osteocytes in femurs from WT/FGF23+/– and Hyp/FGF23+/– embryos at day 17.5 from pregnant WT and Hyp mothers, respectively (Fig. 5). We found that eGFP expression was increased in osteocytes from PHEX-deficient day 17.5 Hyp embryos but not in osteocytes of WT littermates, regardless of the maternal genotype, indicating that increased FGF23 production by osteocytes is a nascent defect rather than being due to circulating maternal-derived factors.


Figure 5
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  		Fig. 5. Analysis of FGF23 promoter activities in osteocytes of femurs from day 17.5 WT/FGF23+/– and Hyp/FGF23+/– embryos derived from either WT or Hyp pregnant mothers. From left to right shows, respectively, frozen sections of femurs from WT/FGF23+/– and Hyp/FGF23+/– embryos of pregnant WT female mice and WT/FGF23+/– and Hyp/FGF23+/– embryos of pregnant Hyp female mice viewed under fluorescent light (x200).

 
Additional evidence for induction of an FGF23 suppressive factor with age in Hyp mice. To further explore whether the paradoxical suppression of FGF23 expression in Hyp bone transplanted into Hyp mice represents the presence of additional factors capable of suppressing FGF23 production, we removed the Hyp bone explanted into Hyp mice and reimplanted it into WT mice, as well as took Hyp bone explanted into WT mice that continued to express eGFP and reimplanted it into Hyp mice. Two weeks after reimplantation in WT mice, we found that the Hyp bone, which had lost eGFP expression in Hyp mice, regained eGFP expression after being reexplanted in WT mice. On the other hand, Hyp bone that initially retained eGFP expression in WT mice lost eGFP expression after being reexplanted in Hyp mice (Fig. 3C).

Because donor bone from Hyp mice is younger than that from recipient mice, we explored whether age-dependent suppression of FGF23 occurs in Hyp mice. For these studies, we compared eGFP expression as a measure of FGF23 promoter activity and circulating FGF23 levels in Hyp mice at ages ranging from 10 days to 12 wk (Fig. 6). We found that both FGF23 promoter activity in osteocytes in Hyp bones (Fig. 6A) and serum FGF23 levels (Fig. 6B) are highly elevated in Hyp mice up to 3 wk of age. Thereafter, both osteocyte expression and serum levels decline by 6 and 12 wk of age, indicating an age-dependent suppression in FGF23.


Figure 6
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  		Fig. 6. Age-dependent FGF23 promoter activities in osteocytes and corresponding serum FGF23 levels in Hyp mice. A: FGF23 promoter activity is indicated by eGFP expression using fluorescent microscopy (x200) in osteocytes of trabecular (Trab; top) and cortical (Cort; bottom) bone as a function of age in Hyp mice. FGF23 promoter activity decreases by 6 wk of age. B: serum FGF23 levels in 10-day and 3-, 6- and 12-wk-old mice. Data are means ± SE from 4–6 mice in each group. **Values significantly different from 10-day old mice (P < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PHEX is a cell surface endopeptidase expressed predominantly in bone. Inactivating PHEX mutations lead to inhibition of renal phosphate reabsorption and 1,25(OH)2D production in the kidney proximal tubule by increasing the production of FGF23 by osteocytes. How PHEX regulates FGF23 levels has not been established, and previous studies suggesting possible mechanisms have produced conflicting results. For example, after an initial study on purported PHEX-dependent cleavage of FGF23, subsequent studies found that FGF23 is not a substrate for PHEX (5, 13). Additional studies found that the production of FGF23 by osteocytes was increased in the setting of PHEX mutations (15). These findings suggested an alternative hypothesis that PHEX mutations may indirectly stimulate FGF23 production in osteocytes through the accumulation of yet to be identified extracellular PHEX substrates (14, 15). In the present study, we used bone cross-explantation between WT and Hyp mice to comprehensively examine the influence of local and systemic regulation of FGF23 by osteocytes in vivo. This experimental approach allowed us to examine the function of bone from Hyp mice in a normal hormonal/metabolic milieu and the function of normal bone in the abnormal metabolic environment of the Hyp mouse, thereby permitting additional insights into how PHEX mutations regulate FGF23 expression. We found that the bone explantation procedure does not alter growth or mineralization of the explanted bone. Indeed, both bone and growth plate development progressed normally in bone explanted into WT mice. The surgical procedure, therefore, did not confound interpretation of data from the cross-explantation studies.

In contrast to the possibility that accumulation of PHEX substrates stimulates FGF23 production, our observations support the alternative hypothesis that FGF23 production by osteocytes is an intrinsic defect derived from inactivating PHEX mutations in these cells. In this regard, inactivating mutations of PHEX in Hyp mice result in the increased production of FGF23 in osteocytes, and this increase in FGF23 expression in Hyp bone is retained when transferred to the normal hormonal/metabolic milieu of the WT mouse. In contrast, cross-explantation of WT bone into Hyp did not result in increased FGF23 expression in osteocytes, further indicating the absence of a humoral factor in Hyp capable of stimulating FGF23 production. The absence of a circulating FGF23 stimulatory factor was confirmed in studies of embryos in Hyp mothers, which also expressed FGF23 only in embryos with the inactivating PHEX mutation. Thus the increased expression of FGF23 in osteocytes, both in the cross-explantation and embryo models, corresponds to the bone genotype but not the hormonal/metabolic milieu. The proximate signal linking PHEX to FGF23 expression remains to be identified, but additional data suggest that factors in the bone extracellular matrix, such as dentin matrix protein 1, may participate in the upregulation of FGF23 (8, 15).

In contrast to this intrinsic regulation of FGF23, we also found evidence for a putative hormonal/metabolic change in older Hyp mice that suppresses FGF23 production, thereby attenuating the intrinsic defect leading to increased FGF23 in younger Hyp mice. In this regard, transplantation of Hyp bone into Hyp mice paradoxically resulted in loss of FGF23 expression in osteocytes. This loss of FGF23 is not due to gross abnormalities in bone development or osteocyte viability, since the bone grows in size and osteocytes are present. In addition, we confirmed that this was not due to the surgical intervention by reimplanting the explanted Hyp bone back into a WT mouse. The reimplanted Hyp bone regained its high level of FGF23 expression in osteocytes when returned to the WT environment. Conversely, reimplanting Hyp bone that had previously been cross-explanted into WT mice and that displayed high levels of FGF23 expression lost FGF23 expression when transferred to the Hyp mouse. To explore the potential for an age-dependent suppression of FGF23, since we implanted younger bone into older mice, we examined FGF23 expression in Hyp mice at different ages (Fig. 6). We found a concordant suppression of FGF23 circulating levels and bone expression in older mice. The loss of FGF23 production appears to be due to either the presence of an inhibitory factor or the loss or degradation of an FGF23 stimulatory factor. Moreover, other evidence does exist that supports the existence of yet to be identified humoral factors that regulate FGF23. For instance, recent studies of selective vitamin D receptor deletion in cartilage identified the production of putative factors by chondrocytes that regulate FGF23 expression in bone (17). In addition, clinical observations that FGF23 levels are highly variable in humans (such as in XLH and ADHR, which are known to be caused by FGF23) (9, 35) support the presence of factors that regulate FGF23 independent of PHEX. The existence of putative hormonal or metabolic inhibitors in older Hyp mice may also reflect an adaptive response of the Hyp mice to suppress the elevated FGF23 in the younger Hyp mice. Moreover, a suppressive factor for FGF23 may have important implications with regard to treatment of children with hypophosphatemic rickets and may explain why the disease appears to lessen in severity with age (23).

Impaired mineralization of bone is also observed in XLH or Hyp mice. Studies attempting to discern the possible direct role of PHEX from secondary effects of hypophosphatemia on extracellular matrix mineralization have also produced conflicting results. An intrinsic defect in osteoblasts or osteocytes caused by PHEX deficiency is supported by both in vivo and in vitro observations (7, 18, 38). In this regard, osteoblasts from Hyp mice, when transplanted into WT mice or grown in culture, produce abnormally mineralizing extracellular matrix, consistent with a functional effect of PHEX deficiency in bone-forming cells (7, 38). However, these data are difficult to reconcile with more recent studies showing thatHyp osteoblasts cultured in the presence of phosphate mineralize extracellular matrix to the same extent as WT osteoblasts and the finding that feeding Hyp mice a high-phosphorus diet results in almost complete rescue of the bone mineralization defect (20). These latter findings suggest that hypophosphatemia, rather than an intrinsic osteoblast defect, is primarily responsible for impaired mineralization of extracellular matrix (20).

Our study provides insights into the relative importance of hypophosphatemia and intrinsic osteoblast defect. On the one hand, the observations that WT bones explanted into Hyp mice for 3 wk developed defective mineralization, as evidenced by increased unmineralized osteoid and diffuse fluorescent labeling (Fig. 2), suggest that the impaired mineralization is largely a consequence of the hypophosphatemic Hyp milieu. On the other hand, Hyp bone explanted into WT mice also displayed impaired mineralization, as evidenced by the excess osteoid and impaired fluorescent labeling of bone. Moreover, the abnormalities of subchondral bone observed in juvenile Hyp mice before explantation and bone from Hyp mice explanted into the WT milieu reflect the previously reported abnormalities in primary spongiosa associated with PHEX mutations (19). Together, these findings suggest the presence of an intrinsic mineralization defect related to PHEX mutations in both osteoblasts and chondrocytes. An alternative possibility that FGF23 might also stimulate osteoblast-mediated matrix production is not supported by existing data, which indicate that osteoblasts are not targets for FGF23 actions (34).

In summary, the present findings provide unequivocal evidence that the mechanism whereby PHEX mutations lead to increased FGF23 expression in osteocytes is intrinsic to bone. In addition, we observed for the first time the possible presence of putative circulating factors that mitigate the increase in FGF23 production in older Hyp mice. Our studies also support the presence of an intrinsic mineralization defect related to PHEX mutation in osteoblasts/osteocytes that is modified by the humoral/metabolic milieu. A complete understanding of the molecular pathogenesis of XLH will require future studies to define the exact mechanisms for the stimulation of osteocyte production of FGF23 by PHEX-dependent changes in the bone microenvironment, to identity the putative circulating factor that reduces FGF23 expression in the adult Hyp mouse, and to characterize the local factors responsible for PHEX-dependent regulation bone extracellular matrix mineralization.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The project described was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant RO1 AR-45955 and National Center for Research Resources Grant P20 RR-17708.


   ACKNOWLEDGMENTS
 
We thank Steffani Webb for editorial assistance and Dr. Valentin David for assistance on statistical analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Darryl Quarles, 3901 Rainbow Blvd., MS 3018, Kansas City, KS 66160 (e-mail: dquarles{at}kumc.edu); or S. Liu, 3901 Rainbow Blvd., MS 3018, Kansas City, KS 66160 (e-mail: sliu{at}kumc.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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S. Liu, J. Zhou, W. Tang, R. Menard, J. Q. Feng, and L. D. Quarles
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