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Endocrinology Vol. 140, No. 3 1192-1199
Copyright © 1999 by The Endocrine Society

ARTICLES

1,25-(OH)2D3 Down-Regulates Expression of Phex, a Marker of the Mature Osteoblast1

Brigitte Ecarot and Marguerite Desbarats

Genetics Unit, Shriners Hospital, Departments of Surgery and Human Genetics, McGill University, Montréal, Québec H3G 1A6, Canada

Address all correspondence and requests for reprints to: B. Ecarot, Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal, Québec H3G 1A6, Canada. E-mail: becarot{at}shriners.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations in the PHEX/Phex gene, which encodes for a protein with homology to neutral endopeptidases, are responsible for human and murine X-linked hypophosphatemia. The present study examined Phex messenger RNA (mRNA) and protein expression in cultured osteoblasts and its regulation by 1,25-(OH)2D3. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relatively to GAPDH mRNA levels. Immunoreactive Phex protein levels were evaluated by immunoprecipitation using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide.

ß-Glycerophosphate-induced matrix mineralization in primary osteoblast cultures was associated with significant increases in Phex mRNA and protein. Phex mRNA and protein levels were low or undetectable in proliferating preosteoblastic MC3T3-E1 cells and dramatically increased concomitantly with initiation of matrix mineralization. The pattern of Phex expression, however, was similar in nonmineralizing cultures grown in the absence of ß-glycerophosphate, indicating that the induction of Phex expression in MC3T3-E1 cells was related to cell differentiation rather than matrix mineralization. 1,25-(OH)2D3 inhibited mineral deposition and down-regulated Phex mRNA and protein expression in a time- and dose-dependent manner.

These results indicate that Phex is a marker of the fully differentiated osteoblast and that its expression is stimulated during ß-glycerophosphate-induced mineralization in primary osteoblast cultures and down-regulated by 1,25-(OH)2D3, an inhibitor of matrix mineralization. These findings add support for Phex having an important role in bone mineralization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
X-LINKED hypophosphatemia is associated with hypophosphatemia that results from impaired renal phosphate reabsorption, rickets, and osteomalacia that lead to stunted growth and skeletal deformities (1). Despite extensive investigations of the murine homologue, the Hyp mouse (2), the underlying mechanism for the bone and renal abnormalities is not understood. The defective renal phosphate transport in Hyp mice (3) has been attributed to decreased renal expression of the Na-Pi cotransporter gene and immunoreactive protein (4) and is thought to result from the effect of a humoral factor (5, 6). The failure of phosphate therapy and the need for concurrent administration of supraphysiological doses of 1,25-(OH)2D3 to correct the bone osteomalacic lesions in affected patients (7, 8) and mice (9, 10) have indicated that hypophosphatemia is not the sole cause of the defective bone mineralization. Evidence has been provided for an associated osteoblast dysfunction in this disorder. They include the presence of characteristic hypomineralized periosteocytic lesions in cortical bone of XLH patients (11) that do not completely disappear in treated patients despite normalization of the mineralization process (12) and the failure of Hyp bone cells to produce normal bone when transplanted into normal mice (13, 14). In contrast to pair-transplanted normal cells, Hyp bone cells also failed to produce normal bone upon normalization of serum phosphate in recipient Hyp mice by phosphate supplementation (15) or 1,25-(OH)2D3 treatment (16).

The recent identification of the gene responsible for XLH through positional cloning indicates that mutations in a gene first designated PEX (phosphate-regulating gene with homologies to endopeptidases, on the X chromosome), then PHEX, underlie the phenotypic features of XLH (17). A large number of additional PHEX mutations have since been reported (18, 19), and a large deletion in the 3' region of the Phex gene has been identified in Hyp mice (20, 21). The PHEX/Phex gene encodes for a protein with homology to members of the membrane-bound zinc metallopeptidase family including neutral endopeptidase-24.11 (NEP), endothelin-converting enzyme (ECE) and and the Kell blood protein (22). The mechanism by which mutations in the PHEX/Phex gene cause the bone and renal abnormalities in XLH and Hyp is not known. It has been demonstrated that NEP, also known as neprilysin and common acute lymphocytic leukemia antigen (CALLA), degrades and inactivates a variety of regulatory peptides (23) whereas ECE-1 and ECE-2 catalyze the conversion of biologically inactive big endothelin-1 into mature vasoactive endothelin-1 (24, 25). Based on the homology of PHEX/Phex to these endopeptidases, it is conceivable that the PHEX/Phex gene product activates or inactivates a factor involved in the regulation of bone mineralization and phosphate homeostasis.

We previously reported the cloning of the mouse Phex complementary DNA (cDNA) and showed that the cDNA sequence for the mouse Phex protein encodes a 749 amino acid protein that shares 70% amino acid sequence homology with mouse NEP and 67% homology with human ECE-1 (26). We also demonstrated by Northern blot analysis that Phex was expressed in bone and cultured osteoblasts from normal mice but was undetectable in bone from Hyp mice, confirming the hypothesis of an intrinsic osteoblast defect in Hyp. PHEX/Phex RNA expression has also been identified by Northern blot analysis in lung and by RT-PCR in several additional tissues including ovary, testis, muscle, and fetal liver (21, 27).

The present study was undertaken to investigate the potential role of Phex in bone mineralization. We examined Phex messenger RNA (mRNA) and protein expression in relation to osteoblast differentiation and matrix mineralization and in response to 1,25-(OH)2D3. Primary osteoblast cultures derived from newborn mouse calvaria and the murine osteoblastic MC3T3-E1 cell line were used.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Osteoblasts were isolated from periost-free calvaria of 6- to 7-day-old C57BL/6J mice by a nonenzymatic technique as previously reported (28). Cell nodules were cultured in DMEM (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% FCS, 50 µg/ml ascorbic acid, 100 U/ml penicillin, and 100 µg/ml streptomycin. Matrix mineralization was induced at day 6 of culture by supplementation of medium with 5 mM ß-glycerophosphate. Cells were also isolated from mutant Hyp mice obtained by mating homozygous Hyp/Hyp females with Hyp/Y males, which therefore exhibited full expression of the mutation, regardless of sex.

MC3T3-E1 cells were cultured in {alpha}MEM (Life Technologies) supplemented with 10% FBS, 20 mM HEPES, 50 µg/ml ascorbic acid, 5 mM ß-glycerophosphate, 100 U/ml penicillin, and 100 µg/ml streptomycin (supplemented medium). Cells were passaged every 3–4 days when reaching confluence in the same medium but not supplemented with ascorbate and ß-glycerophosphate (unsupplemented medium) and were not used beyond passage 15. Matrix mineralization was assessed at different time periods by incubating cells for 5 h in {alpha}MEM supplemented with 10% FBS and 1 µCi/ml of 45CaCl2. Cells were then incubated with unlabeled culture medium for 15 min and rinsed three times with 0.9% NaCl. Cell layers were solubilized in 12.5% TCA and aliquots counted for radioactivity.

To determine the effect of 1,25-(OH)2D3 on Phex expression, primary osteoblasts were incubated at day 6 of culture with increasing concentrations of 1,25-(OH)2D3 (a generous gift from Hoffmann-La Roche, Nutley, NJ). The final concentration of ethanol was 0.01%. Control cultures were supplemented with an equivalent amount of ethanol.

To assess the effect of 1,25-(OH)2D3 on ß-glycerophosphate-induced matrix mineralization, primary osteoblasts were incubated at day 6 of culture with 10-7 M 1,25-(OH)2D3 or the vehicle alone for 72 h. Cultures were supplemented with 5 mM ß-glycerophosphate and 1 µCi/ml of 45CaCl2 for the last 24 h of treatment. MC3T3-E1 cells grown in supplemented medium were incubated at day 6 with 10-7 M 1,25-(OH)2D3 or the vehicle alone for 72 h. Cultures were incubated with 45CaCl2 for the last 5 h of the treatment period. 45Ca accumulation in cell layers was quantitated as described above.

RNA preparation and Northern analysis
Cells were lysed using TRIzol reagent (Life Technologies). RNA samples were electrophoresed on a 1% agarose gel, transferred to Hybond-N filters (Amersham Canada Ltd., Oakville, Ontario, Canada) and hybridized with multiprime-labeled 32P cDNA probes. The probes used included the 1.3-kb murine Phex cDNA corresponding to nt 385-1696 (26) and the full-length murine osteopontin cDNA (29). Hybridizations were performed at 42 C in the presence of 50% formamide as described (26). More recently, hybridizations were carried out overnight at 65 C in 1% BSA, 1 mM EDTA, 0.5 M Na2HPO4, pH 7.2, and 7% SDS and blots were washed at 65 C in 0.5% BSA, 1 mM EDTA, 0.04 M Na2HPO4, pH 7.2, and 5% SDS. Blots were then exposed to Kodak x-ray film at -70 C for 4 to 7 days. Stripped filters were rehybridized with a human GAPDH probe (900 bp), as a control for loading and transfer of RNAs, and exposed for 2–4 h. The intensity of Phex and GAPDH RNA bands was quantitated by densitometric scanning of the autoradiographs using the NIH Image Software on a Macintosh computer.

Anti-Phex antiserum characterization
A polyclonal antiserum was raised in rabbits against a synthetic peptide, (CGG)PRNSTMNRGADS corresponding to residues 734–745 of the carboxy-terminal sequence of Phex (26). The antiserum was characterized by Western blot analysis of calvaria extracts. Calvaria were homogenized using a polytron in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 0.02 µg/ml trypsin inhibitor, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM PMSF) and centrifuged at 4,000 x g for 10 min. The resulting supernatant was centrifuged at 100,000 x g for 1 h. Membrane pellets were solubilized at 4 C for 1 h in the buffer described above supplemented with 1% Triton X-100 and centrifuged at 100,000 x g for 1 h. Proteins in the supernatants were measured by the protein assay kit (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada). Fifty micrograms of protein were run on a SDS-polyacylamide gel (8%) and transferred to a Hybond C nitrocellulose membrane (Amersham Canada, Ltd.). After blocking with 5% nonfat dry milk at 4 C for 16 h, membranes were exposed to a 1:1,000 dilution of polyclonal rabbit antiserum, followed by the addition of goat antirabbit IgG conjugated to horseradish peroxidase. Blots were washed and bands visualized using the ECL chemiluminescence detection method (Amersham Canada, Ltd). The blots were stripped according to the manufacturer’ s instructions and probed with a monoclonal antibody raised against rabbit NEP (kindly provided by Dr Philippe Crine, University of Montréal, Montréal, Québec, Canada).

Phex immunoprecipitation
Cultured cells were incubated in methionine- and cystine-free DMEM containing 0.5% FBS for 1 h, then labeled with [35S] methionine (100 µCi/ml) in the same medium for 4 h. Cells were washed three times with medium and lysed in RIPA buffer [50 mM Tris-HCl (pH 7.2), 0.15 M NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 3 µg/ml aprotinin, 5 µg/ml trypsin inhibitor, and 2 mM PMSF]. The lysates were sonicated and centrifuged at 100,000 x g for 1 h. Aliquots of supernatants containing similar amount of 10% TCA precipitable radioactivity were used for immunoprecipitation. Samples were first precleared with preimmune rabbit serum and protein A-Sepharose and precipitates discarded. Supernatants were immunoprecipitated using the rabbit polyclonal Phex antiserum. Immune complexes were precipitated with protein A-Sepharose (Pharmacia, Piscataway, NJ) and washed twice in RIPA buffer and once in 50 mM Tris-HCl, pH 7.0. Pellets were boiled in Laemmli buffer and proteins were resolved by electrophoresis on a 8% SDS polyacrylamide gel and visualized by autoradiography.

To determine the localization of the Phex protein, cytosolic and membrane fractions were prepared for immunoprecipitation. Briefly, MC3T3-E1 cells labeled with [35S] methionine were sonicated in 0.5 M sucrose, 5 mM Tris-HCl, pH 7.25, containing the protease inhibitors described above. After centrifugation at 1,000 x g for 5 min, the supernatant was collected and centrifuged at 100,000 x g for 1 h. The resulting supernatant was collected as the cytosolic fraction and mixed with 1 volume of RIPA buffer 2x. The pellet was resuspended in RIPA buffer and centrifuged at 100,000 x g for 1 h to obtain the soluble membrane fraction. Whole-cell lysates were prepared by directly solubilizing cell layers in RIPA buffer. The culture medium was concentrated by ethanol precipitation and solubilization of the precipitate in RIPA buffer. Aliquots of whole-cell lysates, cytosolic and membrane fractions, and culture medium corresponding to one P60 dish were immunoprecipitated using the anti-Phex antiserum.

Statistical analysis
Data are expressed as the mean ± SEM. Results were analyzed by one-way ANOVA or Student’s t test, where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of anti-Phex antiserum
To investigate Phex protein levels, an antiserum against a Phex carboxy-terminal peptide was raised in rabbits. Western immunoblotting of membrane extracts of normal mouse calvaria using this antiserum revealed a diffuse band of 97 kDa on reduced SDS gels which was absent in bone extracts from Hyp mice and in renal extracts from normal and Hyp mice (Fig. 1Go). Material of identical apparent Mr was detected in all extracts with an anti-NEP monoclonal antibody, demonstrating lack of cross-reactivity of the anti-Phex antiserum with the mouse NEP. The apparent Mr of Phex immunoreactive material was slightly increased to about 100 kDa in nonreduced SDS gels (data not shown). The anti-Phex antiserum also immunoprecipitated material of approximately 97 kDa from [35S] methionine labeled lysates of primary osteoblasts derived from normal mice but not derived from Hyp mice (Fig. 2Go). To determine the subcellular localization of the Phex protein, an immunoprecipitation assay was performed on total lysates, cytosolic and membrane fractions, and conditioned medium from MC3T3-E1 osteoblastic cells. As shown in Fig. 3Go, the immunoreactive Phex protein was detected almost exclusively in detergent extracts of membrane fractions. These observations indicate that Phex is a membrane-bound protein, in agreement with the identification of a transmembrane domain in Phex predicted amino acid sequence (26).



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  		Figure 1. Western immunoblots of bone and renal membrane extracts from normal and Hyp mice. Membrane extracts were prepared from mouse calvaria and kidney and equal amounts of proteins (50 µg) were separated by SDS-PAGE and transferred to nitrocellulose, as described in Materials and Methods. The blot was probed sequentially with a polyclonal antibody raised against a mouse Phex carboxy-terminal peptide and with an anti-NEP monoclonal antibody using a chemiluminescence detection system. The Mr of the immunoreactive bands is indicated.

 


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  		Figure 2. Immunoprecipitation of Phex protein from osteoblast lysates. Primary osteoblasts derived from normal (N) and Hyp (H) mice were incubated for 72 h in the presence of 10-7 M 1,25(OH)2D3 or ethanol vehicle only. Cultures were then labeled with 35S-methionine for 5 h and cell lysates were immunoprecipitated with an anti-Phex peptide antiserum, as described in Material and Methods. The Mr of the Phex protein band is shown.

 


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  		Figure 3. Membrane localization of the Phex protein. Total cell lysates, cytosolic and membrane fractions were prepared from 35S methionine-labeled MC3T3-E1 cell cultures, as described in Material and Methods. Aliquots of these preparations and of conditioned medium corresponding to one P60 dish were immunoprecipitated with an anti-Phex antiserum.

 
Effects of ß-glycerophosphate on Phex mRNA and protein production
Northern blot analysis of total RNA from primary osteoblast cultures revealed Phex transcripts of approximately 6.6 kb (26). The steady-state level of Phex mRNA was examined in primary osteoblast cultures supplemented with 5 mM ß-glycerophosphate by comparison with unsupplemented cultures. We have previously shown that addition of ß-glycerophosphate to primary osteoblast cultures at day 6–7 induced rapid matrix mineralization, which was visible after 24 h (28). The effect of ß-glycerophosphate on Phex mRNA levels were assessed in cultures incubated 72 h with ß-glycerophosphate. The steady-state Phex mRNA levels were increased by 2-fold in mineralizing cultures compared with control cultures incubated without ß-glycerophosphate (201 ± 7.4% of control value, n = 3). In a subsequent series of experiments, we investigated the time course of the effect of ß-glycerophosphate on Phex mRNA expression. Figure 4AGo shows that Phex mRNA levels were increased at 24 h (1.5-fold) and 48 h (1.9-fold) after addition of ß-glycerophosphate but not at 72 h. Similar results were obtained in a separate culture. These findings show that treatment of primary osteoblast cultures with ß-glycerophosphate caused significant elevations in Phex mRNA levels but that, depending on variables in the culture conditions that remain to be established, these increases occurred at different time points.



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  		Figure 4. Stimulation of Phex mRNA and protein production in primary osteoblast cultures by ß-glycerophosphate. Primary mouse osteoblasts were incubated in the absence (C) or in the presence of 5 mM ß-glycerophosphate (+ßGP) for the time periods indicated. A, Phex RNA levels were assessed by Northern blot analysis of total RNA (20 µg/lane) using 32P-labeled mouse Phex and human GAPDH cDNAs. B, Phex protein synthesis was measured by immunoprecipitation with the anti-Phex peptide antiserum using equal counts of 35S methionine-labeled cell lysates. PI, Samples immunoprecipitated with preimmune rabbit serum.

 
To determine whether the increase in the steady state amount of Phex mRNA correlated with an increase in Phex protein levels, an immunoprecipitation of osteoblast lysates was performed. Primary osteoblast cultures were supplemented with 5 mM ß-glycerophosphate for 24, 48, and 72 h and labeled with 35S-methionine for the last 4 h of the culture. Figure 4BGo shows that ß-glycerophosphate-induced mineralization was associated with a significant increase in the synthesis of Phex protein after 24 h and that this increase was persistent for up to 72 h. Nonspecific bands were seen below the immunoreactive Phex band in samples derived from ß-glycerophosphate-treated cultures. These results were observed in two independent experiments.

Phex mRNA expression was also investigated in the murine osteoblastic MC3T3-E1 cell line that is a good in vitro osteoblast differentiation and mineralization model (30, 31). MC3T3-E1 cells maintained in supplemented medium containing 5 mM ß-glycerophosphate initiate matrix mineralization as measured by 45Ca incorporation in cell layers (Fig. 5AGo). The mineralization lag time in MC3T3-E1 cell cultures was dependent on cell passage. The initiation of matrix mineralization occurred at day 14–15 for cells between passage 2 to 7 and at day 7–8 for cells between passages 8 to 15. Such enhancement of osteogenic potential has previously been reported for rat tibial cells upon extended propagation in unsupplemented medium (32). Although Phex mRNA and immunoreactive protein levels were low or undetectable before mineralization and dramatically increased during mineralization (Fig. 5Go, B and C), the induction of Phex expression was not necessarily related to matrix mineralization but could rather be related to cell differentiation. To determine whether the induction of Phex expression was related to matrix mineralization or cell differentiation, we evaluated Phex mRNA and protein expression in MC3T3-E1 cells grown in the absence of ß-glycerophosphate, which do not initiate mineral deposition (Fig. 5AGo). As shown in Fig. 5Go (B and C), the pattern of Phex expression in nonmineralizing cultures was similar to that observed in mineralizing cultures. No systematic differences between mineralizing and nonmineralizing cultures were seen of the Phex mRNA and protein levels in two independent experiments. These results indicate that the induction of Phex expression in MC3T3-E1 cells is linked with osteoblast differentiation rather than the mineralization process.



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  		Figure 5. Phex expression pattern in mineralizing and nonmineralizing MC3T3-E1 cell cultures. MC3T3-E1 cells at passage 9 were cultured in the presence (+ßGP) or absence (-ßGP) of 5 mM ß-glycerophosphate for 21 days. At different time periods A, 45Ca accumulation in cell layers was measured during the last 5 h of the culture; B, Phex RNA levels were assessed by Northern blot analysis of total RNA (20 µg/lane) using 32P-labeled Phex and GAPDH cDNAs; and C, Phex protein synthesis was measured by immunoprecipitation with the anti-Phex antiserum using equal counts of 35S methionine-labeled cell lysates. The mol wt marker (14C methylated phosphorylase-b, M) is shown on the left. Results shown in ABC are from the same experiment. Values depicted in A represent the mean ± SEM (for some data points too small to be seen) of three dishes. The experiment was conducted twice with similar results.

 
Effects of 1,25-(OH)2D3 on Phex expression
Treatment of nonmineralizing primary osteoblast cultures with 10-7 M 1,25-(OH)2D3 for 4–48 h revealed a time-dependent decrease in Phex mRNA levels (Fig. 6AGo). A significant decrease was observed after 4 h treatment and was maximal after 24 h with suppression of Phex mRNA expression. Similar results were obtained in mineralizing cultures grown in the presence of ß-glycerophosphate (data not shown). Figure 6BGo shows that the inhibitory effect of 1,25-(OH)2D3 was dose dependent in both mineralizing and nonmineralizing cultures. In mineralizing cultures, 10-11 M 1,25-(OH)2D3 had no significant effect on Phex mRNA levels after 24 h (91.2 ± 8.3% of controls, n = 3); 10-9 M 1,25-(OH)2D3 decreased Phex mRNA to 53.7 ± 7.0% of control levels (n = 3, P < 0.001), whereas 10-7 M 1,25-(OH)2D3 reduced Phex mRNA to 4.5 ± 2.3% (n = 5, P < 0.001). 1,25-(OH)2D3 also inhibited osteocalcin mRNA expression, as previously found in mouse bone cells (33, 34). The decrease in Phex mRNA was not due to a nonspecific effect of 1,25-(OH)2D3 treatment, as indicated by an increase in the levels of osteopontin mRNA. The inhibitory effect of 1,25-(OH)2D3 was reversible as Phex mRNA returned to control levels 48 h after removal of 1,25-(OH)2D3 (Fig. 5CGo). 1,25-(OH)2D3 also decreased Phex mRNA levels in mineralizing MC3T3-E1 cell cultures. The inhibitory effect was less dramatic than in primary osteoblasts with approximately 50% inhibition after 24 h exposure to 10-7 M 1,25-(OH)2D3 (data not shown).



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  		Figure 6. Inhibition of Phex mRNA expression in primary osteoblast cultures by 1,25(OH)2D3. In A, nonmineralizing cultures were treated with 10-7 M 1,25(OH)2D3 for different periods of time. In B, mineralizing (+ßGP) and nonmineralizing (-ßGP) cultures were exposed to 0–10-7 M 1,25(OH)2D3 for 24 h. The lower panel (C) shows reversibility of the effect in nonmineralizing cultures incubated for 24 h in the presence of 10-7 M 1,25(OH)2D3 and switched to 1,25(OH)2D3-free medium. Total RNA was extracted before (0 h) and at 24 and 48 h after incubation in 1,25(OH)2D3-free medium. Control cultures received ethanol vehicle only. Total RNA (20 µg/lane) was analyzed by Northern blot analysis using 32P-labeled Phex, osteopontin (OP), osteocalcin (OC), and GAPDH cDNAs.

 
To examine the effects of 1,25-(OH)2D3 on Phex protein production, an immunoprecipitation assay was performed on primary osteoblasts treated with varying concentrations of 1,25-(OH)2D3 for 72 h. The 1,25-(OH)2D3-mediated decreases in Phex mRNA levels were associated with corresponding decreases in Phex protein synthesis (Fig. 7Go). Phex protein synthesis was suppressed at 10-7 M 1,25-(OH)2D3, as shown in Figs. 2Go and 7Go.



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  		Figure 7. Inhibition of immunoreactive Phex production by 1,25(OH)2D3. Immunoprecipitation of equal counts of 35S methionine-labeled lysates of primary osteoblasts exposed to 0–10-7 M 1,25(OH)2D3 for 72 h was performed using the anti-Phex peptide antiserum as indicated in Materials and Methods. The results are representative of three independent experiments. The mol wt marker (14C methylated phosphorylase-b) is shown on the right.

 
To determine whether de novo protein synthesis was required to promote the inhibitory effect of 1,25-(OH)2D3 on Phex mRNA production, primary osteoblasts were treated with 1,25-(OH)2D3 for 24 h in the presence or absence of 2 µg/ml cycloheximide. At this concentration, cycloheximide inhibited about 90% of new protein synthesis (data not shown). Figure 8Go shows that the inhibitory effect of 1,25-(OH)2D3 on the expression of Phex mRNA was not blocked by cycloheximide. Cycloheximide treatment itself reduced the expression of control Phex mRNA, as revealed by a lower Phex/GAPDH mRNA ratio for cycloheximide-treated cultures relative to untreated cultures.



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  		Figure 8. Effect of cycloheximide on the inhibitory effect of 1,25(OH)2D3 on steady state Phex mRNA. Primary osteoblasts were treated for 24 h with 1,25(OH)2D3 at the concentrations indicated or with ethanol vehicle alone in the presence or absence of 2 µg/ml cycloheximide (CHX). Total RNA (20 µg/lane) was analyzed by Northern blot analysis using 32P-labeled Phex and GAPDH cDNAs.

 
To evaluate the effect of 1,25-(OH)2D3 on mineralization, cultures were incubated with 10-7 M 1,25-(OH)2D3 or vehicle alone and 45Ca accumulation in cell layers was determined after 72 h treatment. MC3T3-E1 cells were treated for 72 h with 1,25-(OH)2D3 after matrix mineralization was initiated (day 6 of culture), and 45Ca accumulation was measured for the last 5 h of treatment. 1,25-(OH)2D3 inhibited mineral deposition by about 40% (control: 1462858 ± 20058 cpm/dish vs. 1,25-(OH)2D3: 889209 ± 31071 cpm/dish, n = 5, P < 0.001). The effect of 1,25-(OH)2D3 on mineralization was also examined in primary osteoblast cultures. Cells were treated for 72 h with 10-7 M 1,25-(OH)2D3 at day 6 of culture and incubated in the presence of 5 mM ß-glycerophosphate and 45CaCl2 for the last 24 h of treatment. 1,25-(OH)2D3 inhibited the early mineralization of nodules by about 85% (control: 19113 ± 1504 cpm/nodule vs. 1,25-(OH)2D3: 2611 ± 731 cpm/nodule, n = 6, P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutations in the PHEX/Phex gene have been identified in patients with X-linked hypophosphatemia (17, 18, 19) and in the animal model for the disorder, the Hyp mouse (20, 21). Loss of function of the PHEX/Phex gene product appears therefore responsible for the phenotypic features of the disorder, i.e. defective bone mineralization and reduced renal reabsorption of phosphate. Consistent with a role for Phex in bone mineralization, we previously demonstrated by Northern blot analysis high levels of Phex expression in bone and cultured osteoblasts with undetectable levels of Phex transcript in bone from Hyp mice (26). The present study demonstrates for the first time that the Phex gene encodes for a 97-kDa protein present in membrane extracts from calvaria and cultured osteoblasts. Experiments using the MC3T3-E1 osteoblastic cell line indicate that the Phex protein is expressed coincidentally with mineral deposition and is a marker of the fully differentiated osteoblast. Finally, we found that Phex expression in primary osteoblasts is up-regulated in association with mineralization and is down-regulated by 1,25-(OH)2D3, an inhibitor of matrix mineralization.

Phex protein abundance in calvaria and cultured osteoblasts was determined by Western immunoblotting and immunoprecipitation respectively, using antibodies raised against a carboxy-terminal peptide of the mouse Phex protein (26). No immunoreactive material was detected in bone and cultured osteoblasts derived from Hyp mice and in kidney from normal and mutant mice. As the 3' end coding sequence of the Phex gene is deleted in the Hyp mouse (20, 21), the antibody used will not recognize a COOH-truncated Phex protein that might be translated from the Phex gene in the mutant strain. However, because Phex transcripts are undetectable on Northern blots of bone of the Hyp mouse, severe reduction in Phex protein production is expected from the mutant allele (26). To clarify this issue, an antibody directed against the N-terminal portion of the protein is needed.

To characterize the pattern of expression of Phex during osteoblast differentiation in vitro, we used the murine calvaria-derived MC3T3-E1 cell line. The MC3T3-E1 cells have the potential to proliferate and differentiate in long term cultures to form mineralized nodules when provided with ascorbate and ß-glycerophosphate (30, 31). A previous study evaluating Phex RNA expression in MC3T3-E1 cell cultures by RT-PCR showed up-regulation of Phex RNA in association with up-regulation of osteocalcin expression and alkaline phosphatase activity, suggesting developmental stage-specific expression of Phex (35). It remains unclear at which stage of osteoblast differentiation Phex is expressed and whether Phex expression is associated with mineralization. In the present study, we show that Phex expression in MC3T3-E1 cells was induced concomitantly with the initiation of mineralization and remained constant during matrix mineralization. These results suggested that Phex expression might be linked with matrix mineralization. However, the finding of a similar time-course pattern of Phex expression in cultures maintained in the absence of ß-glycerophosphate that do not undergo mineralization indicates that induction of Phex expression is associated with osteoblast differentiation. Thus, Phex appears to be a marker of the fully differentiated osteoblast, the cell that controls matrix mineralization.

Primary osteoblasts obtained from murine periostea-free calvaria by a nonenzymatic technique (28) were also used to examine Phex expression in relation to mineralization. We previously demonstrated that these cells retained their differentiated phenotype as they produce a mineralized matrix with ultrastructural properties similar to woven bone (36). Matrix mineralization in this system can occur spontaneously. However, addition of ß-glycerophosphate promotes rapid mineral deposition as cultures treated with ß-glycerophosphate at day 6–7 showed visible calcification 24 h after addition of ß-glycerophosphate (28). We found that the steady-state levels of Phex mRNA were increased significantly during ß-glycerophosphate-induced mineralization, and that these increases were associated with increases in Phex protein synthesis. Thus, in contrast to the observations in MC3T3-E1 cell cultures, mineralization appears to be associated with increased Phex expression in primary mouse osteoblast cultures. One possible explanation for the discrepancies could be differences in the cell populations present in freshly isolated bone cells compared with those present in the clonal MC3T3-E1 cells. Primary cultures may include cells at various stages of maturation that may exhibit selective responsiveness to the action of ß-glycerophosphate. Several studies show that ß-glycerophosphate can modulate the phenotypic expression of osteogenic cells. Using fetal rat calvaria cells grown continuously in the presence of ß-glycerophosphate, Lee et al. (37) found that ß-glycerophosphate-induced mineralization was associated with increases in alkaline phosphatase mRNA and activity but produced no significant effect on the expression of type I collagen, osteopontin, osteocalcin and bone sialoprotein. Gertensfeld et al. (38) and Aronow et al. (39) also reported significant changes in alkaline phosphatase activity in calvaria cell cultures continuously exposed to ß-glycerophosphate compared with untreated cultures. In the present study, we examined the response of mature osteoblasts, that expressed mRNA for osteopontin, osteocalcin and Phex, to 72-h treatment with ß-glycerophosphate. Data on the effects of short-term treatment with ß-glycerophosphate in mature cultures are not available. In view of the evidence of the present study that Phex is a marker of the differentiated osteoblast, it is conceivable that development of a more differentiated osteoblast phenotype for a fraction of the cell population accounts for the stimulatory effect of ß-glycerophosphate on Phex expression.

1,25-(OH)2D3 plays an essential role in bone formation and mineralization in vivo. However, the mechanism by which 1,25-(OH)2D3 promotes bone mineralization is not clearly understood. Weinstein et al. (40) showed that apparent normal bone mineralization occurs in vitamin D-deficient rats infused with adequate amounts of calcium and phosphorus, suggesting that 1,25-(OH)2D3 exerts its effect indirectly by increasing serum calcium and phosphorus levels. On the other hand, several studies have indicated that 1,25-(OH)2D3 has a direct action on osteoblasts. Administration of 1,25-(OH)2D3 to osteomalacic patients promotes mineralization only at osteoid surfaces covered by osteoblasts (41). Furthermore, in vitro studies have shown that 1,25-(OH)2D3 has numerous and diverse effects on the expression of several osteoblast phenotypic markers. 1,25-(OH)2D3 can exert both stimulatory and inhibitory effects on type I collagen synthesis (42, 43, 44, 45, 46), alkaline phosphatase activity (46, 47, 48, 49), osteocalcin (33, 34, 42, 49, 50, 51) and osteopontin (44, 51, 52) expression depending on the species, the cell culture conditions, the stage of osteoblast differentiation and the duration of hormone treatment. In the present study, we assessed whether Phex expression in osteoblasts is modulated by 1,25-(OH)2D3. The effect of 1,25-(OH)2D3 on Phex expression is of specific interest. Pharmacological doses of 1,25-(OH)2D3 are required in combination with phosphate supplementation to improve bone lesions in patients with X-linked hypophosphatemia (7, 8) and in Hyp mice (10). Treatment of Hyp mice with 1,25-(OH)2D3 for 14 days (16) had no effect on calvarial Phex transcript levels that remained undetectable by Northern blot analysis (B. Ecarot, unpublished data). The beneficial effect of 1,25-(OH)2D3 on bone mineralization in Hyp mice is therefore not consequent to a stimulatory effect on Phex expression. This result is not surprising because loss of function of the Phex gene product in Hyp mice has been ascribed to a large deletion in the Phex gene (20, 21). In fact, the present study demonstrates that 1,25-(OH)2D3 inhibits Phex expression in primary osteoblasts and MC3T3-E1 cells. The inhibitory effect of 1,25-(OH)2D3 in primary osteoblast cultures was time and dose dependent with suppression of Phex mRNA and protein at 10-7 M.

In parallel with inhibition of Phex expression, we show that 1,25-(OH)2D3 inhibited matrix mineralization in primary osteoblast and MC3T3-E1 cell cultures. 1,25-(OH)2D3 treatment of primary osteoblasts at day 6 of culture almost completely suppressed the early mineralization of nodules, whereas 1,25-(OH)2D3 treatment of mineralizing MC3T3-E1 cells resulted in about 40% inhibition of further mineral deposition. These observations are consistent with the data of Owen et al. (44) and Bellows et al. (53) who found that continuous or transient exposure of proliferating rat calvaria cells to 1,25-(OH)2D3 completely blocked the formation of mineralized nodules while treatment of differentiated cells in later cultures caused smaller but significant (about 25%) inhibition of mineral deposition. Similar results have been found in chicken calvaria cell cultures (51). Our results, however, are in contrast to the findings of Matsumoto et al. (54) who reported stimulation of mineral deposition by 1,25-(OH)2D3 in late cultures of MC3T3-E1 cells. The reason for the discrepancy seems not related to differences in 1,25-(OH)2D3 concentration. While the present study has been carried out at 10-7 M 1,25-(OH)2D3 to observe maximal effects, we found that 10-9 M 1,25-(OH)2D3, the highest dose used in the study by Matsumoto et al. inhibited mineral deposition in MC3T3-E1 cell cultures by about 30%. One possible explanation of these results may reside in the different culture conditions used in the two studies. In our experiments, cells were cultured in media containing 10% FCS unlike those of Matsumoto et al. that were maintained in the presence of 1% FCS. Different amounts of serum-derived factors may influence the effects of 1,25-(OH)2D3 on the cells.

The mechanism by which 1,25-(OH)2D3 inhibits mineral deposition in vitro is not clear. The inhibition of extracellular matrix formation and osteocalcin production observed when 1,25-(OH)2D3 treatment was initiated during the proliferative period likely contributes to the inhibition of mineral deposition (44, 51, 53). When given to cells expressing the mature osteoblast phenotype, the hormone also inhibited expression of several genes associated with the differentiated function (44, 51). In the present study, we found that 1,25-(OH)2D3 given to phenotypically mature primary cultures inhibited the expression of osteocalcin and Phex. Some studies have reported a correlation between the expression of osteocalcin mRNA or protein and mineralization (38, 44, 55). Thus, part of the inhibitory effect of 1,25-(OH)2D3 on matrix mineralization by mature mouse osteoblasts may occur through its ability to inhibit osteocalcin expression. The inhibition of Phex expression by 1,25-(OH)2D3 may also contribute to the observed inhibition of mineralization. In support of this idea is the defective bone mineralization present in the Phex-deficient (Hyp) mouse, which is not corrected upon normalization of serum phosphate levels (9, 10). The in vitro findings of an inhibitory effect of 1,25-(OH)2D3 on mineralization may bear relevance to the in vivo observations that pharmacological doses of the hormone resulted in impaired bone mineralization in young animals (56, 57, 58).

In summary, the present study demonstrates that Phex is expressed by fully differentiated osteoblasts. Its expression in primary osteoblast cultures is up-regulated in association with matrix mineralization and down-regulated by 1,25-(OH)2D3, in association with inhibition of mineral deposition. Such findings add support for a functional role of Phex in bone mineralization.


   Acknowledgments
 
We thank Janique Viel for technical assistance, Elisa de Miguel for peptide synthesis, and Jane Wishart for preparing the figures.


   Footnotes
 
1 This work was supported by the Shriners of North America. Back

Received March 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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