This study was presented in abstract form as an oral presentation at the 25th Annual Meeting of the American Society for Bone and Mineral Research held in Minneapolis, MN, September 19-23, 2003.
The authors have no conflict of interest.
XLH in humans and the Hyp phenotype in mice are caused by inactivating Phex mutations. Overexpression of human PHEX under the human β-actin promoter in Hyp mice rescued the bone phenotype almost completely, but did not affect phosphate homeostasis, suggesting that different, possibly independent, pathophysiological mechanisms contribute to hyperphosphaturia and bone abnormalities in XLH.
Introduction: Mutations in PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH) in humans, and its mouse homologs, Hyp, PhexHyp-2J, PhexHyp-Duk, Gy and Ska1. PHEX is thought to inactivate a phosphaturic factor, which may be fibroblast growth factor 23 (FGF)-23. Consistent with this hypothesis, FGF-23 levels were shown to be elevated in most patients with XLH and in Hyp mice. The aim of this study was, therefore, to examine whether transgenic overexpression of PHEX under the human β-actin promoter would rescue the Hyp phenotype.
Materials and Methods: We tested this hypothesis by generating two mouse lines expressing human PHEX under the control of a human β-actin promoter (PHEX-tg). With the exception of brain, RT-PCR analyses showed transgene expression in all tissues examined. PHEX protein, however, was only detected in bone, muscle, lung, skin, and heart. To assess the role of the mutant PHEX, we crossed female heterozygous Hyp mice with male heterozygous PHEX-tg mice to obtain wildtype (WT), PHEX-tg, Hyp, and Hyp/PHEX-tg offspring, which were examined at 3 months of age.
Results: PHEX-tg mice exhibited normal bone and mineral ion homeostasis. Hyp mice showed the known phenotype with reduced body weight, hypophosphatemia, hyperphosphaturia, and rickets. Hyp/PHEX-tg mice had almost normal body weight relative to WT controls, showed a dramatic improvement in femoral BMD, almost normal growth plate width, and, despite remaining disturbances in bone mineralization, almost normal bone architecture and pronounced improvements of osteoidosis and of halo formation compared with Hyp mice. However, Hyp and Hyp/PHEX-tg mice had comparable reductions in tubular reabsorption of phosphate and were hypophosphatemic relative to WT controls.
Conclusion: Our data suggest that different, possibly independent, pathophysiological mechanisms contribute to renal phosphate wasting and bone abnormalities in Hyp and XLH.
PHOSPHATE IS AN integral part of the bone matrix, and it is of major importance for numerous cellular functions. However, despite these essential biological functions, the regulation of mammalian phosphate homeostasis remains only incompletely understood.(1,2) Important new insights into the underlying regulatory mechanisms have emerged from studying inherited phosphate wasting disorders in humans and mice and defining their underlying genetic defect through positional cloning. Hence, efforts to define the molecular cause of X-linked hypophosphatemia (XLH) have led to the identification of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), an endopeptidase with limited homologies to neutral endopeptidase (NEP), endothelin-converting enzyme (ECE-1), and the Kell antigen. Phex is primarily expressed in osteoblasts, osteocytes, odontoblasts, lung, ovary, and muscle.(3–9) Similarly, large deletions affecting Phex, the murine homolog of PHEX, and adjacent genes were identified in the Hyp and the Gy mouse, two well characterized strains that represent the rodent equivalent of human XLH.(6,10) A point mutation in the splice donor site of Phex that caused skipping of exon 8 was found in Ska1 mice(11) and smaller intragenic deletions were recently identified in PhexHyp-2J and PhexHyp-Duk mice.(12) As a consequence of these deletions, all above-mentioned mouse strains develop renal phosphate wasting, hypophosphatemia, and rickets.(13)
Despite the resulting low blood phosphate concentration, Hyp, Gy, and Ska1 mice show an insufficient renal production of the vitamin D hormone, 1,25(OH)2D3, a finding that is similar to that observed in humans affected by XLH.(14–16) Further, parabiosis and cross-transplantation experiments strongly suggested that hypophosphatemia and rickets in Hyp mice are caused by a circulating factor, referred to as “phosphatonin,” and that the defect does not reside in the kidney.(17–20) Because osteoblasts from Hyp mice fail to form normal matrix in culture or when transplanted into wildtype (WT) mice,(21,22)PHEX (or Phex) seems to have an additional, intrinsic role in bone.
The molecular definition of another phosphate-wasting disorder, autosomal dominant hypophosphatemic rickets (ADHR),(23–25) resulted in the identification of mutations in fibroblast growth factor (FGF)-23, a novel member of the FGF family of proteins.(26) In ADHR patients, one of two conserved arginines in FGF-23 that both constitute a subtilisin-like proprotein convertase cleavage site are replaced by Gln and Trp or Gln, respectively.(26) This finding led to the hypothesis that FGF-23 is the long-sought phosphatonin(27) and that Phex inactivates FGF-23 by cleavage. In this model, ablation of Phex function or mutations in FGF-23 interfering with normal cleavage would result in the accumulation of this phosphaturic factor with all its metabolic and skeletal consequences. Consistent with this hypothesis, it has been shown that injection of WT FGF-23 into mice caused a significant increase in urinary phosphate excretion without affecting other tubular functions.(28) Furthermore, when Chinese hamster ovary (CHO) cells stably expressing WT or (ADHR type) mutant FGF-23 were subcutaneously implanted into nude mice, renal phosphate wasting occurred leading to the development of hypophosphatemia and rickets,(28) and more recent data indicate that the transgenic expression of FGF-23 under the control of the chicken β-actin promoter(29) or the collagen type Iα1 promoter(30) causes increased urinary phosphate excretion and hypophosphatemia. However, the underlying cellular mechanisms by which FGF-23, directly or indirectly, impairs renal tubular phosphate reabsorption remain to be defined.
At present, there is conflicting evidence regarding the role of PHEX in the regulation of the phosphaturic activity of FGF-23. Consistent with the hypothesis that PHEX is directly involved in the degradation of intact FGF-23, about two-thirds of patients with XLH revealed elevated circulating concentrations of FGF-23,(31,32) and Fgf-23 levels in Hyp mice seem to be even higher.(29) Furthermore, FGF-23 is expressed in osteoblasts in humans(33) and Hyp mice.(34) Initially, PHEX expressed by COS-7 cells was shown to cleave PTH(1-34),(35) and later on it was reported that a soluble form of PHEX degrades in vitro transcribed-translated WT FGF-23, but not FGF-23 with one of the known ADHR mutations.(27) However, more recent findings have shown that this endopeptidase degrades in vitro synthetic PTH-related peptide (PTHrP)(107-139),(36) but not full-length FGF-23 or its N- or C-terminal fragments.(34) Thus, it remains unclear whether FGF-23 is a substrate of PHEX.
In an attempt to clarify the role of PHEX expression in bone cells in the pathogenesis of hypophosphatemia and rickets in XLH and Hyp, two transgenic mouse models were developed that express PHEX under the control of two osteoblast-specific promoters, osteocalcin and type I collagen.(37,38) However, after crossing the transgene into Hyp mice, the hypophosphatemia present in Hyp mice remained unchanged, and the bone phenotype was only slightly improved, implying that Phex produced locally in osteoblastic cells is not involved in the regulation of the biological activity of systemic phosphaturic factors such as FGF-23, frizzled-related protein 4 (FRP4), or matrix extracellular phosphoglycoprotein (MEPE).(39–42) To explore the possibility that PHEX expressed in tissues other than bone plays a role in the degradation of phosphatonin, we generated transgenic mice expressing PHEX under the control of the human β-actin promoter, which allows broader expression. Here we show that overexpression of PHEX under this latter promoter was able to rescue the Hyp bone phenotype to a large extent, but not the renal phosphate wasting present in these mice.
MATERIALS AND METHODS
Generation of transgenic mice
The 4-kb fragment of the human β-actin promoter was excised from the plasmid phACT-FRT-neoFRT-lacZ (Genbank accession no. U46492), kindly provided by S Dymecki (Carnegie Institution of Washington, Baltimore, MD, USA)(43) by double digestion with HindIII/SalI. The fragment was cloned together with the full-length human PHEX cDNA into the pcDNAI plasmid (Invitrogen), placing the promoter fragment upstream of human PHEX. Restriction endonuclease digestions and nucleotide sequence analysis confirmed the correct orientation of the construct. The insert containing the 4-kb fragment of the human β-actin promoter, 2393 bp encoding human PHEX, and 1058 bp from the pcDNAI vector (which provides a splice sequence and the consensus polyadenylation signal absent in the cDNA encoding PHEX) were released from the modified vector by digestion with SalI and purified according to standard techniques. Microinjections into the pronucleus of fertilized oocytes from FVB/N strain mice were performed at the Massachusetts General Hospital Transgenic Facility (Boston, MA, USA). Two individual transgenic lines were established from the male founders 236 and 237 showing different insertion patterns of the transgene (Fig. 1) by mating the founders to female FVB/N wildtype mice. FVB/N PHEX transgenic (PHEX-tg) mice were backcrossed for one to two generations into C57/BL6 background. All animals analyzed for this study were offspring from matings of heterozygous Hyp females (background C57/BL6) with heterozygous PHEX-tg males of line 236 and 237 (background intercross FVB/N and C57/BL6).
All mice were housed at 24°C with a 12-h/12-h light/dark cycle and were allowed free access to tap water and a normal rodent chow (1314; Altromin, Lage, Germany) containing 0.9% calcium, 0.7% phosphorous, and 600 IU vitamin D/kg. Three-month-old (84-104 days) male mice were used for all studies. To examine alterations in renal excretion of minerals, the mice were kept individually in metabolic cages for a 15-h period overnight for urine collection. During that period, the mice had free access to tap water but were deprived of food. To prevent hypothermia in the metabolic cages, the room temperature was raised to 28°C. The next morning, 100 μl of blood was drawn from the retroorbital sinus into heparinized capillaries under ether anesthesia for the measurement of ionized calcium. Immediately thereafter, blood was drawn from the abdominal vena cava under anesthesia with ketamine/xylazine (70/7 mg/kg IP) for serum collection. All animals were subcutaneously injected with calcein (20 mg/kg; Sigma-Aldrich, Deisenhofen, Germany) on days 3 and 1 before necropsy. All animal procedures were approved by the local Ethical Committee and the government authorities.
Blood ionized calcium was measured with an AVL 9140 electrolyte analyzer (Roche Diagnostics, Mannheim, Germany). Serum and urinary calcium was measured with flame photometry (EFOX 5053; Eppendorf, Hamburg, Germany). Serum alkaline phosphatase activity, creatinine, and phosphorus, as well as urinary creatinine and phosphorus concentrations, were determined using a Hitachi 766 autoanalyzer (Boehringer Mannheim, Mannheim, Germany). The values for mouse serum creatinine (24.0-28.8 μM) obtained by the Hitachi 766 autoanalyzer were two to three times higher than the ones reported by Meyer et al. (8.7 μM).(44) Renal tubular reabsorption of phosphorus (TRP) and calcium (TRCa) were calculated according to the formulas: %TRP = [1 − (UrP × SeCrea)/(SeP × UrCrea)] × 100 and %TRCa = [1 − (UrCa × SeCrea)/ (SeCa × UrCrea)] × 100 (Ur, urinary; Se, serum; P, phosphorus; Ca, calcium; Crea, creatinine). Alternatively, %TRCa was calculated using blood ionized calcium instead of serum calcium. PTH concentrations were assessed using a two-sided enzyme-linked immunosorbent assay specific for intact mouse PTH (Immutopics, San Clemente, CA, USA). Serum concentrations of 1,25(OH)2D3 were measured using a radioreceptor assay (Immundiagnostik, Bensheim, Germany). Total deoxypyridinoline concentrations in urine were determined after acid hydrolysis using an ELISA technique (Metra Biosystems, Mountain View, CA, USA).
Genotyping by Southern blot analysis and PCR
Founders and the derived F1 generation were genotyped by Southern blot analysis. Genomic DNA purified from tail clips was digested with BamHI and hybridized to a specific DNA probe (XbaI/KpnI; 952 bp) derived from the pcDNA1 plasmid (Fig. 1). For subsequent genotyping by PCR, we used the forward primer 5′-TTCCACCACTGCTCCCATTCATC-3′ and the reverse primer 5′-GTTATGCTCATGTGAGGTGC-3′, yielding a 542-bp product in the transgenic, but none in the WT mice. Annealing temperature for the genotyping PCR (30 cycles) was 64°C.
For tissue distribution studies of the human PHEX transgene in transgenic mice with RT-PCR, sex-matched littermates were killed, and kidney, heart, lung, intestine, brain, spleen, skin, ovary, testis, muscle, pancreas, bone, and larynx including thyroid and parathyroid glands were isolated and snap frozen in liquid nitrogen. Total RNA was extracted from tissues using the RNeasy Mini Kit (Qiagen) and with Trizol (Gibco BRL) from bone. Two micrograms of DNase (Ambion, Austin, TX, USA)-treated total RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). In control samples, the reverse transcription step was omitted. The PCR protocol consisted of initial denaturing at 94°C for 3 minutes, and each of the 35 subsequent cycles consisted of denaturing at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute, and a final extension for 10 minutes. For expression analysis of the transgene, we used primers specific for human PHEX, forward 5′-CCTGGATCCTTGGTGGTCTACT-3′ and reverse 5′-GAGCTGGTTGTTGGTGAATGTG AT-3′, yielding a 265-bp product in tissues expressing the human PHEX. The primers used do not amplify the mRNA encoding murine Phex.
Fgf-23 mRNA expression by quantitative real-time PCR
Total RNA extracted from long bones was DNase-treated and reverse transcribed with random hexamer using the Taqman reverse transcription kit according to the manufacturer's protocol (Applied Biosystems, Branchburg, NJ, USA). PCR reactions contained 100 ng of template (RNA with or without reverse transcription), 300 nM each of Fgf-23 forward (5′-ACTTGTCGCAGAAGCATC-3′) and Fgf-23 reverse (5′-GTGGGCGAACAGTGTAGAA-3′) and GAPDH forward (5′-ACTGAGGACCAGGTTGTC-3′) and GAPDH reverse (5′-TGCTGTAGCCGTATTCATTG-3′) primer, and 1× SybrGreen PCR Master Mix (Applied Biosystems) in 50 μl. Samples were amplified for 40 cycles with an initial melt at 95°C for 10 minutes, followed by 95°C for 15 s and 60°C for 1 minute as reported previously.(34) Postamplification melting curves were generated to confirm that a single PCR product was produced in each reaction. Contamination of genomic DNA was determined by the threshold cycle (CT) given by the RNA template, which was usually <0.1%. Real-time PCR was always performed in duplicate. All the reactions were controlled by standards (nontemplate control and without adding reverse transcriptase). The quantity of mRNA was calculated by normalizing CT of FGF-23 to the CT of the housekeeping gene GAPDH of the same RNA sample.
Western blot analysis
Protein expression of the human PHEX transgene was determined by Western blot analysis as described previously.(45,46) Sex-matched littermates were killed, and bone, kidney, muscle, lung, intestine, skin, adrenal glands, heart, brain, and ovary were isolated and snap frozen in liquid nitrogen (N2). Femur midshafts were lyophilized for 1 week, crushed in liquid N2 with a mortar and pestle, and shaken in 0.05% Triton-PBS at 4°C for 3 days. Samples were centrifuged at 4°C for 30 minutes at 14,000g. Soft tissues were homogenized in Triton buffer (0.05 M Tris-HCl, pH 7.8, 0.14 M NaCl, 1% Triton X-100 containing protease inhibitor cocktail tablets; 1836170; Roche) at 4°C. Samples were centrifuged at 4°C for 10 minutes at 3000g. COS-7 cells were transfected with pcDNAI expression vector (Invitrogen) containing human PHEX cDNA or vector alone as positive and negative controls, respectively. Protein concentrations in supernatants were analyzed by the Bradford method (Pierce, IL, USA). Fifteen micrograms of protein was loaded per lane and fractionated on a 10% SDS-PAGE, transferred onto nitrocellulose filter for 2 h using standard methods. PHEX protein was detected by immunoblotting with anti-PHEX 13B12 monoclonal antibody (1/200 dilution, kindly provided by G Boileau).(46) This was followed by incubation with peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology, MA, USA) at 1/3000 dilution. An ECL detection kit (Amersham Biosciences, Westborough, MA, USA) was used to visualize the bands according to the manufacturer's instructions.
Immunohistochemical analysis of transgene expression was performed on sagittal, 5-μm-thick sections from hindlimbs of 3-day-old mice fixed in 4% paraformaldehyde (PFA) at 4°C overnight and embedded in paraffin. The 13B12 anti-PHEX monoclonal antibody was used as the primary antibody at a 1/100 dilution. After pretreatment with Proteinase K (10 μg/ml) for 5 minutes, the Histomouse-SP Kit (Zymed Laboratories, San Francisco, CA, USA), designed for examination of mouse tissues with mouse primary antibodies, was used as recommended by the manufacturer. Immunoreactivity was detected with the horseradish peroxidase-streptavidin-biotin amplification method, using the 3-amino-9-ethyl-carbazole (AEC) chromogen/substrate system. The sections were counterstained with hematoxylin, which was also furnished in the kit.
BMD of the left femur was measured by pQCT using a XCT Research M+ pQCT machine (Stratec Medizintechnik, Pforzheim, Germany). The measurements were made with a collimator opening of 0.2 mm on specimens stored in 70% ethanol. One slice in the mid-diaphysis of the femur and three slices in the distal femoral metaphysis located 1.5, 2, and 2.5 mm proximal to the articular surface of the knee joint were measured. All BMD values of the distal femoral metaphysis were calculated as the mean over three slices. A voxel size of 0.070 mm and a threshold of 600 mg/cm3 were used for calculation of cortical BMD.
Bone histology and histomorphometry
At necropsy, the right femurs were fixed in 4% PFA at 4°C overnight and were processed and embedded in methylmethacrylate as described.(47) Midsagittal sections of the distal femurs were prepared using a HM 360 microtome (Microm, Walldorf, Germany), and were stained with von Kossa/McNeal(48) or left unstained for evaluation of fluorochrome staining. Histomorphometric measurements in the distal femur were made on sections stained with von Kossa/McNeal using a semiautomatic system (Videoplan; Carl Zeiss) and a Zeiss Axioskop microscope with a drawing attachment. For the osteoid measurements, five fields (1.25 mm2) in the cancellous bone of the distal femoral metaphysis were evaluated in each section. The area within 0.20 mm from the growth plate was excluded from the measurements. Osteoid area (O.Ar./B.Ar) was measured at ×20 and was expressed as percentage of (mineralized and unmineralized) bone area. Osteoid width (O.Wi) was determined directly at ×400, sampling each osteoid seam every 50 μm. Growth plate width (GPl.Wi) was measured at ×10 in at least 10 sites distributed evenly over the whole growth plate in each animal. In addition, the mean area of at least 40 osteocyte lacunae (Ot.Lc.Ar) located in the anterior and posterior cortical bone was determined at ×40. Fluorochrome-based measurements could not be performed because of the lack of distinct calcein double labels in Hyp and in Hyp/PHEX-tg mice.
Statistics were computed using SPSS for Windows 11.0 (SPSS, Chicago, IL, USA). The data were initially analyzed using three-way factorial ANOVA. Three-way factorial ANOVA evaluated the effects of the line of transgenic animals (line 236 and 237), of the Hyp mutation (Hyp) and of the PHEX transgene (PHEX), and also determined whether there were two-way interactions between the individual factors (i.e., whether the different genetic factors mutually influenced each other in a nonadditive way). Subsequently, the combined data from both lines of animals were analyzed by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test. p values of <0.05 were considered significant for all statistical analyses. The data are presented as the mean ± SE.
Factorial ANOVA showed that the variable line did not have a significant effect on any of the parameters shown, showing that the results were not influenced by the insertion pattern of the transgene in the two different transgenic lines 236 and 237 (Table 1). In contrast, Hyp was associated with significant effects on body weight, blood ionized calcium, serum phosphate, percent tubular reabsorption of phosphate, urinary total DPD excretion, BMD of the femoral shaft and of the femoral metaphysis, and cortical thickness of the femoral shaft. With the exception of blood ionized calcium and phosphate homeostasis, the variable PHEX also had significant effects on these parameters. For all parameters showing a significant effect of PHEX, we found a positive two-way interaction between PHEX and Hyp, because PHEX had effects only in the presence but not in the absence of Hyp. The lack of a significant effect for line showed that it was legitimate to combine the data from both lines of PHEX-tg animals. Therefore, the data from both lines were combined for all subsequent analyses by one-way ANOVA.
Table Table 1.. Three-Way ANOVA Analysis of Selected Biochemical and Bone Parameters in 3-Month-Old Male WT, PHEX-tg, Hyp, and Hyp/PHEX-tg Mice
Expression of human PHEX in transgenic mice
With the exception of brain, RT-PCR analyses showed that the human PHEX transgene was expressed in all tissues examined (Fig. 2A). The expression pattern was identical in both lines of transgenic mice (data not shown). To exclude that the introduced human cDNA was a confounding factor in the expression analysis, PCR was performed on DNase-treated total RNA samples from transgenic mice, omitting the reverse transcription step. No PCR product was observed in these samples. Similarly, we found no PCR product in WT tissues known to express Phex such as lung and bone (data not shown), showing that the primers used were indeed specific for human PHEX. To confirm the expression pattern identified by RT-PCR, we performed Northern analyses using a human PHEX-specific 0.5-kb probe. However, we were unable to detect the presence of the transgene by Northern blot analysis in all tissues examined (data not shown). To evaluate the expression of PHEX protein in various tissues, we performed Western blot analyses (Fig. 2B). A 97-kDa band (fully glycosylated) was detected in bone and lung of WT animals, which was similar in size as that from COS-7 cells expressing human PHEX. Expression of PHEX protein in extracts from bone and lung of transgenic animals was distinctly higher, suggesting an active transgene. In addition, PHEX protein was detected in extracts from muscle, skin, and heart of transgenic, but not of WT, animals. Furthermore, some extracts showed lower molecular weight bands (87-83 kDa), which may represent deglycosylated forms or degradation products of the PHEX protein. The 13B12 antibody has been reported to detect slightly different sizes for recombinant PHEX and endogenous Phex in extracts of femurs. The difference results probably from specific glycosylation patterns. Similarly, two forms of PHEX have previously been shown using the same monoclonal antibody, which might be caused by incomplete deglycosylation.(46) We also detected a band in the brain extract of the transgenic animal. This band was, however, of different size, and because we did not detect a signal in brain by RT-PCR, we think that this band is unspecific.
To examine the localization of the transgene in bone, we performed immunohistochemistry on sagittal sections of limbs using the 13B12 anti-PHEX monoclonal antibody (Fig. 2C). Sections of WT and PHEX-tg animals showed intense staining of PHEX protein in osteoblasts and osteocytes, suggesting correct spatial distribution of transgene expression. Interestingly, other cells in bone or bone marrow displayed only weak or absent labeling in PHEX-tg mice. No positive signal could be detected in sections of Hyp animals or in sections in which the primary antibody was omitted (data not shown). In contrast, osteocytes and osteoblasts of Hyp/PHEX-tg animals showed clear expression of the PHEX transgene.
PHEX-tg mice do not have an abnormal phenotype
Body weight, BMD, and cortical thickness of the femoral shaft, as well as BMD of the distal femoral metaphysis, as measured by pQCT (Figs. 3A-3E), bone histology (Figs. 4A and 4B), bone mineralization (Fig. 4C), osteoid area and width (Figs. 5A and 5B), size of osteocytes lacunae (Fig. 5C), growth plate width (Fig. 5D), and mineral homeostasis (Figs. 6A-6F) in PHEX-tg mice were indistinguishable from those of WT littermates. These findings suggest that overexpression of PHEX under the human β-actin promoter is not associated with any detectable changes in transgenic mice.
PHEX transgene improves bone phenotype in Hyp mice, but does not affect hypophosphatemia and renal phosphate wasting
Figures 3, 4, 5, 6 show that Hyp mice showed the known phenotype consisting of reduced body weight, mild hypocalcemia, hypophosphatemia, reduced renal tubular reabsorption of phosphate, and rickets. The ricketic bone phenotype in Hyp mice was characterized by severe osteoidosis (Figs. 5A and 5B), halo formation around cortical bone osteocytes (Fig. 5C), and an almost 5-fold increase in growth plate width (Fig. 5D). Hyp mice carrying the PHEX transgene had almost normal body weight relative to WT controls and showed a dramatic, albeit incomplete, improvement in femoral BMD (Figs. 3A-3E). Compared with Hyp mice, total BMD of the femoral shaft increased by 29% in Hyp/PHEX-tg mice, and total BMD of the femoral metaphysis increased by 21%. Thus, the transgene compensated for 55% of the BMD deficit in Hyp mice in the femoral shaft and for 47% of the BMD deficit in the femoral metaphysis. Cortical thickness of the femoral shaft was completely normalized in Hyp mice by the PHEX transgene (Fig. 3C). Compared with Hyp mice, bone histology in Hyp/PHEX-tg mice revealed pronounced improvements in overall bone structure with almost normal bone architecture, a normal growth plate, a dramatic improvement of the osteoidosis, and almost complete disappearance of halos surrounding Hyp osteocytes (Fig. 4D). However, bone mineralization as visualized by fluorochrome labeling remained impaired, as indicated by the absence of distinct double labels (Fig. 4C). Histomorphometric analysis showed a 75% reduction in osteoid area (Fig. 5A), a 70% reduction in osteoid width (Fig. 5B), a 26% reduction in osteocyte lacunae area (Fig. 5C), and a 65% reduction in growth plate width (Fig. 5D) in Hyp/PHEX-tg versus Hyp mice, documenting the significant improvement of the ricketic Hyp bone phenotype induced by the transgene. However, osteoid area, osteoid width, and size of osteocytes lacunae were still significantly elevated in Hyp/PHEX-tg mice relative to WT controls.
In agreement with the pronounced improvement of the bone phenotype in Hyp/PHEX-tg mice, biochemical markers of bone turnover, serum alkaline phosphatase, and urinary excretion of DPD showed distinct reductions in Hyp/PHEX-tg mice relative to Hyp controls (Figs. 6A and 6B), but still remained slightly higher than those of WT controls. Interestingly, the PHEX transgene almost normalized blood ionized calcium in Hyp mice (Fig. 6C). However, despite the pronounced improvement in bone phenotype, Hyp and Hyp/PHEX-tg mice had comparable reductions in tubular reabsorption of phosphate (Fig. 6E) and remained hypophosphatemic (Fig. 6D) relative to WT controls. Serum PTH concentrations were higher in Hyp and Hyp/PHEX-tg mice compared with WT and PHEX-tg mice, but did not show statistically significant differences between Hyp and Hyp/PHEX-tg (Fig. 6F). However, we did not find differences in renal tubular reabsorption of calcium between any of the groups, regardless of whether blood ionized calcium or serum total calcium was used for the calculation of TRCa (data not shown). Serum 1,25(OH)2D3 was slightly lower in Hyp (33 ± 6 pg/ml; n = 15) compared with Hyp/PHEX-tg mice (46 ± 6 pg/ml; n = 15). However, this difference did not reach statistical significance. 1,25(OH)2D3 concentrations in WT mice and PHEX-tg mice were 41 ± 9 (n = 14) and 44 ± 9 pg/ml (n = 14), respectively.
To examine the expression of Fgf-23 in bones from 3-month-old Hyp and Hyp/PHEX-tg mice, we performed quantitative RT-PCR analysis on RNA extracted from bones of WT, PHEX-tg, Hyp, and Hyp/PHEX-tg littermates (N = 1). In agreement with earlier reports,(34)Fgf-23 mRNA expression was about 1.7-fold increased in the Hyp compared with the WT littermate. Interestingly, the transgene did not correct the increased Fgf-23 expression in the Hyp/PHEX-tg mouse (2.2-fold over control). This result suggests that the significant improvement of the Hyp bone phenotype induced by the PHEX transgene occurred in the presence of elevated Fgf-23 mRNA expression and further questions the idea that FGF-23 is a physiological substrate of PHEX.
Our data indicate that overexpression of PHEX under the control of a human β-actin promoter in transgenic mice is not associated with alterations in bone phenotype or mineral ion homeostasis per se. Similarly, mice showing targeted expressing of Phex in osteoblasts did not exhibit an abnormal phenotype relative to WT controls.(37,38) These findings are in agreement with the hypothesis that the biological role of Phex, an endopeptidase, is the activation or inactivation of its substrate(s), and that overexpression of the enzyme will not result in changes in mineral ion or bone homeostasis. Although we used the strong human β-actin promoter to ubiquitously overexpress human PHEX, expression of the transgene transcript was low, and PHEX protein could only be detected in bone, lung, muscle, skin, and heart. In addition, immunohistochemical analysis revealed that in bones the transgene was mainly expressed in osteoblasts and osteocytes, cells normally expressing Phex. The same promoter construct has yielded high expression of several other genes in transgenic mouse models.(43) We do not have a good explanation for this discrepancy. However, as-of-yet undefined regulatory sequences required for efficient transcription and/or translation of this relatively complicated gene may be lacking in our transgenic construct. In addition, it is important to consider that both the human and the murine PHEX/Phex genes do not contain classic Kozak sequences, suggesting that expression of these genes may indeed be regulated mainly by posttranscriptional mechanisms.(49,50) In line with our inability to detect Phex expression by Northern analysis in WT mice, expression levels of endogenous Phex in adult mice have been reported to be very low.(9,46)
Previous attempts to rescue the bone mineralization defect or the systemic hypophosphatemia in Hyp mice by targeted overexpression of Phex to osteoblasts using the osteocalcin promoter failed.(38) In comparison, overexpression of Phex in osteoblasts using the mouse proα1(I) collagen gene promoter partially improved the defective mineralization of bone and teeth but also failed to correct the hypophosphatemia.(37) In this study, we showed that overexpression of PHEX under the human β-actin promoter rescued the Hyp bone phenotype including the abnormal halo formation around osteocytes to a large extent, and also the hypocalcemia, but not renal phosphate wasting. It is likely that the impairment in bone mineralization that continued to be present in Hyp/PHEX-tg mice was caused entirely by the persistent hypophosphatemia in these animals and that the alterations in bone cells or bone matrix associated with the Hyp phenotype were fully rescued in Hyp/PHEX-tg mice, despite the fact that FGF-23 expression in bone was still elevated. In agreement with this notion, Hyp/PHEX-tg mice showed almost normal bone architecture and growth plate width, albeit with a remaining defect in bone mineralization.
Because XLH/Hyp is a disease that is genetically dominant, the incomplete rescue of the Hyp phenotype by the human PHEX transgene in our model may be explained (1) by insufficient activity of the PHEX transgene, (2) by the lack of ubiquitous PHEX expression, or (3) may be related to the possibility that human PHEX might be able to cleave the murine factor(s) that cause the bone lesions, but may not be able to fully inactivate the murine homolog of the circulating factor(s) that causes renal phosphate wasting. Nevertheless, these scenarios are difficult to reconcile with the idea that the bone abnormalities and the renal phosphate wasting are caused by a single factor originating from bone. Rather, our data show that different, possibly independent, pathophysiological mechanisms contribute to the hyperphosphaturia and the bone abnormalities in Hyp and XLH. This idea is corroborated further by reports of some females from kindreds with familial hypophosphatemia and vitamin D-resistant rickets showing renal phosphate wasting but normal bones.(51) In line with this argument, osteoblasts from Hyp mice show altered mineralization that is independent of the mineral ion environment.(22) Thus, Phex may have different substrates in the same or in different tissues.
An interesting finding in our study was that the PHEX transgene almost normalized blood ionized calcium in Hyp mice. It is thought that hypocalcemia occurring especially in young, fast growing Hyp mice is caused by impaired intestinal absorption of calcium because of inappropriately low serum 1,25(OH)2D3 concentrations.(52) In fact, intestinal calcium absorption in Hyp mice responds normally to external administration of 1,25(OH)2D3.(53) 1,25(OH)2D3 levels increased only slightly in Hyp/PHEX-tg mice, and renal tubular calcium reabsorption remained unchanged relative to Hyp controls. Therefore, the mechanism underlying the normalization of blood ionized calcium in the presence of the PHEX transgene remains unclear. Possible explanations for this finding may be (1) that the small, nonsignificant increase in 1,25(OH)2D3 concentration contributed to the increased blood calcium concentration, (2) that the endopeptidase Phex may also be, directly or indirectly, involved in the regulation of intestinal calcium absorption, and (3) that PTH-dependent osteoclastic bone resorption or calcium fluxes across resting bone surfaces may be more efficient for calcium homeostasis in Hyp/PHEX-tg compared with Hyp mice because of improved bone mineralization.
Despite normal blood ionized calcium, serum PTH levels remained elevated in Hyp/PHEX-tg mice. Therefore, disturbances in the regulation of PTH secretion in the parathyroid glands may contribute to the development of hyperphosphaturia in Hyp mice, and the persisting phosphate wasting in Hyp/PHEX-tg mice in our experiment may partially be attributable to the elevated PTH levels in these mice. Similarly, it has been reported that serum phosphate levels in Hyp/PTH−/− mice were increased in comparison with Hyp mice,(54) indicating a significant role of PTH in the renal phosphate wasting associated with the Hyp phenotype. Moreover, recent studies have shown that Fgf-23 (−/−) mice(55) display considerable elevations in serum phosphate, increased renal phosphate reabsorption, and elevated serum 1,25(OH)2D3 levels caused by enhanced expression of renal 25-hydroxyvitamin D-1α-hydroxylase. Vice versa, renal 1α-hydroxylase is suppressed after administration of FGF-23 to sham-operated or parathyroidectomized rats. Thus, the elevated serum levels of FGF-23 in Hyp and XLH may cause secondary hyperparathyroidism by suppression of renal 1α-hydroxylase and the accompanying, inadequate production of 1,25(OH)2D3. Secondary hyperparathyroidism may therefore be an integral component of the renal phosphate-wasting observed in Hyp/XLH.(55)
In conclusion, this study has shown that overexpression of PHEX under a human β-actin promoter greatly improves the ricketic bone phenotype and corrects hypocalcemia, but not renal phosphate wasting and secondary hyperparathyroidism in Hyp mice, suggesting that the pathophysiology of the ricketic bone phenotype and of the hypophosphatemia in Hyp mice are largely independent. Clearly, more experimentation is required to determine which tissue produces the circulating factor that causes renal phosphate wasting in Hyp mice and what role Phex has in regulation of calcium homeostasis. Further elucidation of the pathogenetic mechanisms responsible for the bone lesions and the renal phosphate wasting in Hyp mice may help to design more effective treatment strategies for XLH and related disorders.
This work was supported by grants from Roche Diagnostics and the Bavarian Ministry for Economic Affairs, Transport and Technology to BL and NIH Grant RO1 46718-10 to HJ. The authors thank S Lutz, C Bergow, and M Kohlross for help with the biochemical and pQCT analyses, K Begsteiger for help with the histological processing of the bone samples, and H-J Grön and E Bopp for their help with the 1,25(OH)2D3 measurements.