Division of Endocrinology (X.B., S.G., A.C.K.), Department of Medicine, and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montréal, Quebec, Canada H3T 1E2; Calcium Research Laboratory (D.M., D.P., D.G.), Department of Medicine, McGill University Health Centre and McGill University, Montréal, Quebec, Canada H3A 1A1; and Faculty of Dentistry (M.D.M.), and Department of Anatomy and Cell Biology, McGill University, Montréal, Quebec, Canada H3A 2B2
Address all correspondence and requests for reprints to: Andrew C. Karaplis, Lady Davis Institute for Medical Research, 3755 Cote Sainte Catherine Road, Montréal, Québec, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca.
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ABSTRACT |
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INTRODUCTION |
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Much of our understanding of XLH has been facilitated by the availability of the murine homolog, the Hyp mouse, which exhibits the skeletal and biochemical features of XLH (3). Considerable effort has been placed in elucidating the molecular basis of the renal defect in Pi transport and altered vitamin D metabolism in this animal model. In vitro (4, 5, 6), parabiosis (7), and cross-transplantation studies (8) indicate that Hyp is not a primary renal defect. Rather, the Hyp gene product is likely involved, either directly or indirectly, in the elaboration of a humoral factor that regulates phosphate uptake and vitamin D metabolism in the renal proximal tubule.
As with human studies, work with the Hyp mouse has also pointed to a primary osteoblast metabolic defect in this disorder. Osteoblasts isolated from Hyp mice do not produce adequate amounts of mineralized matrix when transplanted in a normal environment (9, 10, 11) or when cultured in vitro (12). This intrinsic cellular defect in Hyp mouse osteoblasts is believed to be responsible for the altered release of and/or the modification of a factor that can block mineralization of extracellular matrix (12) and, also independently, reach the circulation and modify Pi reabsorption (6).
Through positional cloning, a gene that spans the deleted region Xp22.1 in XLH patients, or is mutated in nondeletion patients with the disorder, has been identified (designated PEX and subsequently PHEX, for phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (13). The predicted human PHEX gene product, as well as its murine homolog (14), exhibits structural similarity to a family of neutral endopeptidases involved in either activation or degradation of peptide hormones. Therefore, PHEX likely functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism (15, 16, 17). Extensive mutation analysis of XLH families has revealed a range of defects in the PHEX gene, all of which appear to be loss-of-function mutations (18). In the Hyp mouse, the gene has been shown to harbor a deletion of its 3'-end (19, 20). Taken together, these data provide unequivocal evidence that XLH and the homologous disease in mice (Hyp) arise from loss of PHEX/Phex function.
PHEX/Phex expression has been observed primarily in human and mouse fetal bone, specifically in cells of the osteoblast lineage (14, 15, 19, 21). In the mouse, ontogeny studies for Phex expression by in situ hybridization have shown the presence of Phex mRNA in osteoblasts and odontoblasts, suggesting that the protein product is involved in the development of bone and teeth (22). Gene expression was detectable on d 15 of embryonic development, which coincides with the beginning of matrix deposition in bone. The abundance of the transcript, however, decreases in bones as early as 3 d post partum and in the adult skeleton. Phex expression in the adult murine bone is localized primarily in osteocytes (23), thereby providing a link between the hypomineralized periosteocytic lesions, which are a hallmark of XLH/Hyp and Phex expression. Together, these findings indicate that bone and teeth are physiologically relevant sites of PHEX expression and that PHEX plays an active role in osteoblast/odontoblast-mediated matrix mineralization.
These observations notwithstanding, the physiological function of the PHEX gene product and the mechanisms that lead to the biochemical and skeletal abnormalities of XLH remain to be defined. One approach to elucidate the role of PHEX is to reintroduce wild-type Phex gene expression in the Hyp genetic background, thereby rescuing the Hyp phenotype. Here, we have used transgenic technology to target PHEX expression to osteoblasts in the Hyp background. In such a genetically modified mouse strain, PHEX expression will be restricted exclusively to osteoblasts, while other cells, normally expressing the protein would remain devoid of it. In this report, we demonstrate that targeted PHEX expression in osteoblasts/odontoblasts in Hyp mice partially reverses the bone and tooth abnormalities while failing to correct the biochemical derangements associated with the disorder.
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RESULTS |
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A 2.3-kb proximal promoter of the mouse pro-1(I) collagen gene, previously shown to be highly active in these cells with only faint expression in skin and tendons and no activity in other tissues, was used to drive human PHEX cDNA expression in mice (Fig. 1A
and Ref. 24). Southern blot analysis of tail genomic DNA showed that the two male founders obtained had low (35) and high (810) copy numbers of the transgene inserted in the genome. For all subsequent studies, unless otherwise indicated, only results obtained from the high-copy number founder mouse and mouse line will be presented and discussed (Fig. 1B
). The transgene was subsequently transferred into the Hyp genetic background (PHEX/XHyp), anticipating that PHEX transgene expression would correct the biochemical and morphological abnormalities associated with deletion of the endogenous Phex gene (Fig. 1C
).
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Amelioration of Osteomalacic Lesions
In Hyp mice, generalized impairment of mineral deposition (osteomalacia) and, particularly, hypomineralized lesions surrounding osteocytes (periosteocytic lesions) are the major morphological features of the skeleton (Fig. 4). In sharp contrast to the impaired response of biochemical parameters, targeting PHEX expression in cells of the osteogenic lineage led to a marked improvement in the mineralization defects in trabecular as well as cortical bone (Fig. 4
). Quantification of the intensity of von Kossa staining in bone sections demonstrated that the Hyp genotype was associated with an 83% and 51% decrease in mineralization of trabecular and cortical bone, respectively, compared with wild-type littermates and wild-type C57BL/6J inbred mice. On the other hand, in Hyp mice carrying the PHEX transgene, this decrease was reduced to 64% and 32%, an improvement of 20% in the mineralization defect at both skeletal sites. Interestingly, this amelioration included the periosteocytic lesions that are unique to this disorder. The improved mineralization seen histologically following PHEX gene transfer, was further substantiated from radiographs of long bones obtained from wild type, Hyp, and PHEX/XHyp transgenic animals. Radiological analysis showed that PHEX/XHyp long bones were markedly more radiopaque than those derived from Hyp mice due to improved mineralization of the cortices and metaphyseal trabeculation (Fig. 4
). Lastly, as expected, no improvement was noted histologically in the mineralization defect of the hypertrophic zone of the growth plate, since PHEX transgene expression was excluded from chondrocytes (Fig. 5
).
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Partial Rescue of Osteogenic Cell Differentiation and Mineralization in ex Vivo Cultures
Given that PHEX gene transfer to Hyp osteoblasts in vivo partially improved the abnormalities in bone without altering the hypophosphatemic environment, we postulated that PHEX expression likely alters the intrinsic mineralization defect proposed to exist in these cells. To investigate this further, bone marrow cells (BMCs) from the three mice groups (wild type, Hyp, and PHEX/XHyp), were isolated in culture and induced to undergo osteogenic differentiation. Osteogenic colonies derived from wild-type and PHEX/XHyp mice expressed PHEX, whereas colonies from Hyp mice did not display PHEX immunoreactivity, as anticipated (Fig. 6). After culture, the cells were examined for proliferation, and colony formation, and for markers of osteogenic differentiation/function such as alkaline phosphatase (ALP) activity, total collagen content, and mineralization of extracellular matrix. All three groups of cultured cells displayed similar proliferative capacity, as indicated by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) (data not shown). However, bone marrow cultures derived from Hyp mice, compared with cultures from wild-type littermates, exhibited a marked decrease in the differentiation and mineralization parameters examined, indicating the presence of an intrinsic defect in osteogenic differentiation and mineralization that arises in this genetic background (Fig. 7
). In contrast, parameters of osteogenic cell differentiation and mineralization were markedly improved, although not completely normalized, in bone marrow cultures derived from PHEX/XHyp animals, suggesting that a partial rescue of the intrinsic osteoblast defect had occurred consequently to PHEX gene transfer.
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DISCUSSION |
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The reason that the osteoblast-specific PHEX gene transfer did not ameliorate the deranged Pi handling and vitamin D metabolism in Hyp mice is not entirely clear. One potential explanation is that the human PHEX protein is not completely functional in the mouse. This is unlikely, however, given that the gene is highly conserved between man and mouse, with the predicted murine Phex cDNA showing 91% identity at the DNA level and 96% identity at the protein level to the human form (19, 20). This high degree of homology increases greatly the expectation that the human transgene would be totally functional in the mouse. Alternatively, PHEX expression controlled by the proximal mouse pro-1(I) collagen gene promoter may not be adequate to totally correct the metabolic abnormalities arising from the absence of PHEX function. Hyp is a truly dominant condition with similar levels of Pi handling abnormalities seen in individuals heterozygous and homozygous for the defective allele. Hence, the protein may have to attain a threshold level for normal function, and that threshold may not have been reached with our transgene. However, this explanation is also unlikely, given that the levels of PHEX expression (shown by RT-PCR, immunohistochemistry, and Western blot analysis) and endopeptidase activity after gene transfer in Hyp osteoblasts are comparable to those seen in wild-type cells. It could also be argued that transgene expression did not parallel temporally that of the endogenous Phex gene. PHEX expression is absent from immature osteoblasts but increases as the osteoblast differentiation process progresses (21). Interestingly, the 2.3-kb murine pro-
1(I) collagen gene promoter, which has been extensively characterized in transgenic animals as well as in vitro, has been detected in osteoblasts at a time when BSP begins to be expressed and mineralization ensues, as well as in mature osteoblasts and osteocytes (28). This pattern temporally parallels that of endogenous PHEX. A more plausible explanation for the inability of the transgene to correct the hypophosphatemia and low vitamin D levels would therefore appear to be that PHEX expression is required in cells other than osteoblasts. Miyamura et al. (29), using syngeneic bone marrow transplantation, were able to partly reverse the biochemical abnormalities in Hyp mice but showed that engraftment was not restricted to cells of the osteoblast lineage but also occurred in the bone marrow, thymus, and spleen. Engraftment of donor cells to tissues other than osteoblasts may therefore have been critical for correcting the associated biochemical abnormalities. In addition to its expression in developing osteoblasts, osteocytes, and odontoblasts, PHEX transcripts have been detected in other fetal and adult tissues such as brain, kidney, thymus, heart, liver, muscle, ovaries, and lung (19, 30, 31, 32). Expression of PHEX in tissues such as lung may be important in regulating circulating levels of humoral factors involved in the renal handling of Pi and vitamin D metabolism. In fact, recent studies have identified a member of the fibroblast growth factor family, FGF23, as one such factor implicated in these processes (26, 33, 34, 35). This protein has been reported to serve as substrate for PHEX endopeptidase activity (17), yet it is not expressed in osteoblasts (Ref. 36 and results herein). Although the product of the MEPE gene has also been proposed as a candidate for this role, our data demonstrate that despite correction of Mepe transcript levels in osteoblasts by PHEX gene transfer, hypophosphatemia due to Pi wasting persisted. Failure of MEPE overexpression to cause Pi wasting in vivo has also been recently reported (26). These findings would indicate that MEPE plays no obvious role in the regulation of renal Pi handling and vitamin D metabolism but more likely partakes in extracellular matrix mineralization (see below).
Our results are also consistent with the clinical features of XLH that display an unusual organ-specific gene dosage (1). Thus, the defects in bone and teeth in XLH reveal a direct correlation with the dosage of the PHEX gene, with heterozygous alterations distributed in severity between those in mutant hemizygotes and normal homozygotes. On the other hand, serum Pi concentrations do not show such a relationship as phosphate values are similar in mutant hemizygotes, mutant homozygotes, and heterozygotes. The role of gene dosage on vitamin D metabolism has not been examined, but, based on the findings of this study, it is likely that, here again, correlation with the dosage of the PHEX gene is not applicable and likely related to factors underlying the pathophysiology of abnormal Pi homeostasis.
We and others (12, 23) have previously reported that osteoblast cultures derived from Hyp mice display an impaired capacity to form mineralized matrix and inhibit normal mineralization in coculture experiments with wild-type cells. This finding suggests that Hyp cells serve as source for a diffusable factor inhibiting mineralization (12) and is consistent with the notion that loss of Phex enzymatic function in Hyp osteoblasts leads to the accumulation of an osteoblast-derived substrate resulting in the defective mineralization. Perhaps one such protein is MEPE/Mepe (27). This protein has major similarities to a group of secreted bone-tooth mineral matrix phosphoglycoproteins such as osteopontin, dentin sialo-phosphoprotein, dentin matrix protein-1, and BSP which contain Arg-Gly-Asp (RGD) sequence motifs essential for integrin-receptor interactions. MEPE, like PHEX, is expressed in osteoblasts but in contrast to PHEX, its expression is markedly increased during osteoblast-mediated matrix mineralization. Moreover, increased Mepe mRNA expression was observed by others (37) and us in osteoblasts from Hyp mice, suggesting that Phex may be responsible for processing Mepe (or a derived fragment) and, for the first time, we show here that transcript levels normalize after PHEX gene transfer. Nevertheless, processing of Mepe by Phex, and a potential role for Mepe in the mineralization of extracellular matrix by osteoblasts, remains to be demonstrated. Furthermore, whether it is the Phex substrate itself that acts as the mineralization inhibitor, or pathways downstream from it, remains to be clarified.
The aforementioned studies suggested that a cause-and-effect relationship exists between Phex expression and a defective osteoblast phenotype in vivo. Here, we have used a genetic approach to demonstrate that indeed it is the loss of the Phex protein that leads in part to the mineralization defect, as PHEX gene transfer to Hyp osteoblasts partially corrects this hypomineralization. The present work also indicates that the expression of at least one protein related to Mepe, BSP, is altered by the absence of Phex. This protein binds tightly to hydroxylapatite and appears to form an integral part of the mineralized matrix. Moreover, it is important for cell-matrix interaction by promoting RGD-dependent extracellular matrix-mediated cell adhesion. The decreased levels of vitronectin, an Vß3 integrin ligand containing the RGD sequence, would add to this impairment in cell attachment as well as affect migration, cytoskeletal organization, cell proliferation, survival, and differentiation. Transfer of PHEX cDNA into osteoblasts partly corrects these abnormalities, resulting in improved extracellular matrix mineralization.
Recently, Liu et al. (36) also reported on the capacity of Phex gene transfer to osteoblasts to correct the Hyp phenotype. In this study and in our hands, there was no improvement in the hypophosphatemia and abnormal vitamin D metabolism. Therefore, despite restoration of Phex function in osteoblasts, persistent Pi wasting and low-normal 1,25-(OH)2D3 levels impaired complete healing of bones. In our studies, we saw definite improvement in mineralization in bone and regard this as a partial rescue of the phenotype because the Pi level was not corrected. Moreover, we saw healing of the periosteocytic hypomineralization that is characteristic of XLH and observed a major improvement in dentin mineralization. Interestingly, these authors also showed a significant increase in bone mineral density and in dry ashed weight in the rescued Hyp animals but chose to interpret this as inconsistent with even partial rescue of the Hyp phenotype. Perhaps, in our studies, the improvement in mineralization is quantitatively larger, although it is difficult to say without side-by-side comparison. Part of the differences between the two studies may reflect different promoter usage, the pro-1(I) collagen gene promoter in contrast to the osteocalcin promoter, to drive Phex expression to osteoblasts.
In conclusion, the present study provides direct genetic evidence that PHEX gene transfer in cells of the Hyp osteogenic lineage that restored Phex mRNA levels, protein expression, and endopeptidase activity only partly rescued proper osteoblast/odontoblast-mediated matrix mineralization in vivo and failed to correct the hypophosphatemia and associated vitamin D abnormalities. Therefore, other signals related to the enzymatic action of PHEX, and perhaps distant from those in bone, are likely to contribute to the regulation of Pi handling and vitamin D metabolism.
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MATERIALS AND METHODS |
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Transgene Plasmid
A 3.6-kb BglII-DraIII fragment containing the previously cloned human PHEX cDNA (15) and a 231-bp sequence encompassing the bovine GH polyadenylation signal positioned 3' to the PHEX cDNA to enhance mRNA stability was inserted into BamHI-SphI restriction enzyme sites of the pJ251 plasmid (24), downstream to the 2.3-kb fragment of the murine pro-1(I) collagen gene promoter (kindly provided by Dr. B. de Crombrugghe, M.D. Anderson Cancer Center, University of Texas, Houston, TX). The choice for the human transgene was based on sequence differences that would allow distinction between transgene and endogenous Phex expression.
Generation of Transgenic Mice
Transgenic mice designed to express PHEX in an osteo/odontoblast-specific manner were generated using standard protocols. The entire transgene [pro-1(I) collagen gene promoter, PHEX cDNA, and 3'-polyadenylation signal] were released from the vector by NotI/ClaI restriction endonuclease digestion, separated from the vector by agarose gel electrophoresis, recovered by electroelution, and diluted in Tris-EDTA buffer at a concentration of 2 ng/µl. Pronuclei of mouse fertilized oocytes (B6 x D2F2) were microinjected with the purified linearized DNA using standard procedures, and injected eggs were transferred into pseudopregnant CD1 foster mothers.
Identification of Transgenic Mice and Establishing Transgenic Lines
Mice were screened for integration of the transgene by Southern blot analysis of tail DNA. In brief, 10 µg of genomic DNA was digested with BamHI, separated by agarose gel-electrophoresis, transferred to a nitrocellulose membrane, and hybridized with an 5.9-kb [32P]dCTP-labeled transgene, as probe. Signals were detected by autoradiography. Two founder male mice were generated carrying the transgene.
After 11 backcrosses into the C57BL/6J inbred strain, the transgene was transferred into the Hyp genetic background by crossing the transgenic animals with C57BL/6J XHyp mice. Male transgenic mice were crossed with heterozygous female Hyp mice to obtain male Hyp mice carrying the PHEX transgene (PHEX/XHyp). Assuming integration of the transgene in an autosomal chromosome, then one of eight offspring from such matings would have the desired genotype.
To identify mice carrying the Hyp mutation, a set of oligonucleotide primers was designed (GenBank accession no. U73915: forward, 256 bp 5'-CAGGAGCATCTACAGTCAGTAAGC-3' 279 bp; and reverse, 453 bp 5'-ATAGAGAAGGTTTCCAAGCAAATG-3' 430 bp) to amplify a 197-bp fragment from intron 20 of the murine Phex gene, reported to be deleted in Hyp mice. The PCR product was sequenced using an automated ABI 310 sequencer for verification and then used as probe in Southern blot analysis of tail-tip genomic DNA digested with EcoRI.
Transgene Expression
For transgene expression, total RNA was extracted from 7-wk-old murine bones, after the marrow was flushed, using TRIzoL (Life Technologies, Inc., Gaithersburg, MD), and 3 µg were used for reverse transcription reaction. Reverse transcriptase products were then amplified using oligonucleotide primers specific for human PHEX cDNA (GenBank accession no. U75645: forward, 2097 bp 5'-CAGGCATCACATTCACCAAC-3' 2119 bp; and reverse, 2299 bp 5'-TCTGTTCATCGTGGAATTGG-3' 2280 bp), and primers specific for 5'- and 3'-regions of the murine Phex gene (38).
PHEX/Phex endopeptidase activity was assessed in homogenates (100 µg) prepared from calvaria of 6-wk-old wild-type, Hyp, and PHEX/XHyp mice using minor modifications of methods described previously (15, 36). In some studies, membranes were preincubated for 5 min with 10 µM thiorphan (Sigma-Aldrich Corp.) before the addition of PTH 134 (Peninsula Laboratories, Inc., Belmont, CA) (39).
Blood and Urine Biochemistry
Serum calcium and serum and urine levels of Pi were measured colorimetrically (Sigma, St. Louis, MO). Serum 1,25-(OH)2D3 was determined using an assay kit from Immunodiagnostic System, Ltd. (Boldon, UK).
Histology and Radiography
All animal and tissue processing protocols used in this study were approved by the Institutional Review Board of McGill University. Mice were killed by cervical dislocation, and the distal ends of femurs and the mandibles with their dentition were rapidly removed, dissected free of soft tissue, and fixed by immersion in PLP (2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate solution) fixative for 24 h at 5 C. The specimens were then decalcified in a 10% EDTA-glycerol solution for 57 d at 5 C. Tissue samples were dehydrated in a series of graded ethanols and embedded in low-melting-point paraffin, and 5-µm sections were cut on a rotary microtome. Undecalcified distal ends of femurs and mandibular segments containing the molar teeth were also embedded in LR-White acrylic resin, 1-µm sections were cut on an ultramicrotome, and mineral was visualized with von Kossa stain. Mineralization of bone was determined by measuring the intensity of von Kossa staining in the positively stained areas of trabecular and cortical bone (referred to as summary average gray) using digital image capture and image analysis (Northern Eclipse version 6.0; Empix Imaging, Inc., Mississauga, Ontario, Canada). Summary average gray correlates with mineralization in the extracellular matrix, as determined by electron microscopy (40). The mineralized percentage of trabecular and cortical bone was calculated by the following formula: mineralization (% of bone) = von Kossa-positive area/von Kossa-positive area + von Kossa-negative area of bone x 100%.
For radiography, long bones were removed and dissected free of soft tissue, and x-ray images were taken with a Faxitron (model 805), under constant conditions (22 kV, 4 min exposure), using X-Omat TL film (Eastman Kodak Co., Rochester, NY).
BMCs
Tibias and femurs of wild-type, Hyp, and PHEX/XHyp mice were removed under aseptic conditions, and BMCs were flushed out with DMEM containing 15% fetal calf serum, 5 µg/ml ascorbic acid, 10 mM ß-glycerophosphate, and 10-8 dexamethasone (differentiation medium). Cells were dispersed by repeated pipetting and a single-cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle. BMCs from five mice were pooled and 106 cells were cultured in 36-cm2 petri dishes in 5 ml differentiation medium that was changed every 4 d. Cultures were maintained for 18 d. At the end of the culture period, cells were washed with PBS, fixed with PLP fixative, and stained, as described below.
Methylene Blue Stain for Total Colonies
Total colonies were estimated by staining with methylene blue. Cells were first washed in borate buffer (10 mM, pH 8.8) and then stained with 1% methylene blue (wt/vol) in borate buffer for 30 min at room temperature. Cells were then washed three times with borate buffer and left to dry before the number of colonies was counted by image analysis, as described below.
Cytochemical Staining for ALP Activity
Cultured cells were incubated for 15 min at room temperature in 100 mM Tris-maleate buffer containing naphthol AS-MX phosphate (0.2 mg/ml, Sigma) dissolved in ethylene glycol monomethyl ether (Sigma) as substrate, and fast red TR (0.4 mg/ml, Sigma) as stain for the reaction product. After washing with distilled water and drying in air, ALP-positive colonies were counted by image analysis.
Picrosirius Red Staining for Collagen
Total collagen was measured in the fixed cell layers using methodology previously described, with minor modifications (41). Cells were exposed to 1% sirius red in saturated picric acid for 18 h. In the presence of saturated picric acid, the negative sulfonic groups of the dye reversibly bind with the positive charged groups on the proteins. Stained cell layers were washed in water thoroughly to remove any nonspecific staining and dried before images were taken. Colonies were destained in 0.1 M NaOH/methanol (50:50, vol:vol).
Alizarin Red S Staining for Calcium
Cell cultures were exposed to a solution of Alizarin Red at pH 6.2 (1 mg/ml) for 30 min at RT, after which they were gently washed under running water and left to dry. Images of stained dishes were taken as described below. The colonies were destained by briefly washing with 5% perchloric acid.
Image Analysis
After each staining, plates were photographed over a light box with a Sony charge-coupled device camera. Images were analyzed using Northern Eclipse image analysis software. The data were imported to a spreadsheet program and processed (23).
Immunocyto/histochemistry
Cultured cells or paraffin sections were stained immunocyto/histochemically for PHEX protein using the ABC immunoperoxidase technique. The cells or dewaxed sections were incubated overnight at room temperature with the primary antibodies diluted in 0.1% BSA in Tris-buffered saline (TBS: 50 mM Tris-HCl; 150 mM NaCl; 0.01% Tween 20, pH 7.6) containing 5% normal serum from the same species as the origin of the secondary antibodies. As a negative control, the normal serum or TBS was substituted for the primary antibody. After washing with high-salt buffer (50 mM Tris-HCl, 2.5% NaCl, 0.01% Tween 20, pH 7.6) for 10 min at room temperature followed by 2 x 10-min washes with TBS, the cells were incubated with secondary antibody, washed as before, and incubated with Vectastain ABC-AP kit (Vector Laboratories, Inc., Peterborough, UK) for 30 min at room temperature. After washing, red pigmentation was produced by a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma, containing 1 mM levamisole as endogenous ALP inhibitor). After washing with distilled water, the sections were counterstained with methyl green and mounted under coverslips with Kaisers glycerol jelly.
Evaluation of BMC Proliferation by Immunocytochemical Staining for Proliferating Cell Nuclear Antigen (PCNA)
Cells cultured in petri dishes for 8 d were stained immunocytochemically for PCNA by the ABC immunoperoxidase technique. Cells were counterstained with methyl green. PCNA-positive and total BMCs were counted by image analysis.
Double Staining for Collagen Type I and Mineral
Cells cultured in petri dishes for 18 d were stained immunocytochemically for type I collagen by the ABC immunoperoxidase technique and cytochemically for calcium by the von Kossa staining method.
Western Blot Analysis
Proteins were extracted from 18-d total BMC cultures, and aliquots (100 µg) were fractionated by SDS-PAGE and were transferred to polyvinylidenedifluoride membranes. Immunoblotting was carried out using the antibodies described in Materials and Methods.
Northern Blot Analysis
Osteoblasts were isolated from calvaria of 1-wk-old wild-type, Hyp, and PHEX/XHyp mice by sequential digestion using collagenase (1 mg/ml) and trypsin (0.05%) and were cultured in MEM (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 50 µg/ml ascorbic acid. When osteoblasts were fully differentiated (
d 18 of culture), total RNA was extracted and examined using Northern blot analysis. Mepe cDNA probe was prepared from calvarial RNA by RT-PCR using mouse-specific primers F290/R800 (38). Quantification of signal intensity on autoradiograms was performed by a Personal Densitometer using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical Analysis
Differences between groups were analyzed by Students t test and two-way ANOVA using the software program Prism, version 3.0 (GraphPad Software, Inc., San Diego, CA). All data are presented as mean ± SE. The value of P < 0.05 was considered significant.
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FOOTNOTES |
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This work was supported by the Canadian Institutes of Health Research (CIHR) and the Canadian Arthritis Network. M.D.M. is a Scholar of the Fonds de la recherche en santé du Québec (FRSQ). D.M. and A.C.K. are recipients of CIHR Fellowship and Scientist Awards, respectively.
Abbreviations: ABC, Avidin-biotin-peroxidase complex; ALP, alkaline phosphatase; BMC, bone marrow cell; BSP, bone sialoprotein; Fgf23, fibroblast growth factor 23; Mepe, matrix extracellular phosphoglycoprotein; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PCNA, proliferating cell nuclear antigen; Pi, inorganic phosphate; RGD, Arg-Gly-Asp sequence mofif; TBS, Tris-buffered saline; XLH, X-linked hypophosphatemic rickets.
Received for publication March 20, 2002. Accepted for publication August 22, 2002.
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REFERENCES |
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