1 Department of Medicine, X-linked hypophosphatemia (XLH) is caused by
inactivating mutations of PEX, an endopeptidase of uncertain function.
This defect is shared by Hyp mice, the
murine homologue of the human disease, in which a 3'
Pex deletion has been documented. In
the present study, we report that immortalized osteoblasts derived from
the simian virus 40 (SV40) transgenic
Hyp mouse
(TMOb-Hyp) have an impaired capacity
to mineralize extracellular matrix in vitro. Compared with immortalized
osteoblasts from the SV40 transgenic normal mouse (TMOb-Nl), osteoblast
cultures from the SV40 Hyp mouse
exhibit diminished 45Ca
accumulation into extracellular matrix (37 ± 6 vs. 1,484 ± 68 counts · min
X-linked phosphaturia; osteomalacia; osteocalcin
X-LINKED HYPOPHOSPHATEMIA (XLH) is inherited as a
dominant disorder and is characterized by hypophosphatemia, growth
retardation, and rickets/osteomalacia (1, 16). The genetic defect
underlying XLH rickets has been identified as mutations in the PEX gene
product, or the phosphate-regulating gene
Pex with homologies to
endopeptidases on the X chromosome (1, 12, 14, 15, 33, 35). The Hyp mouse, a murine homologue of XLH,
also has a loss of function Pex
deletion associated with renal phosphate wasting and defects in
osteoblast-mediated mineralization (2, 31). This murine homologue
provides a model to study the molecular and biochemical events linking
Pex mutations to phosphaturia and
impaired mineralization (16, 19, 31).
The physiological function of PEX is unknown. The presence of renal
phosphate wasting secondary to a mutation of this gene suggests that
this endopeptidase degrades a novel phosphaturic hormone
[referred to as phosphatonin (1, 17)] or inactivates a
phosphate-conserving factor (18-20). Given the broad constellation of phenotypic findings characteristic of XLH, it is also possible that
PEX has other actions that are independent of its effects on renal
phosphate transport, including regulation of bone mineralization. Although studies of primary osteoblast cultures derived from the Hyp mouse have produced inconsistent
results (3, 6, 10, 13), carefully performed studies suggest that
osteoblast cultures derived from Hyp
mice do display mineralization abnormalities when transplanted into
normal mice (10) and have alterations in osteoblast gene expression
that are independent of hypophosphatemia (6, 13, 25, 32, 34). Moreover,
Pex is expressed at high levels in
osteoblasts, and its expression is temporally associated with the
formation of mineralized extracellular matrix (ECM) in cultured
osteoblasts (2, 9, 14). These observations suggest that bone is a
physiologically relevant site of Pex
expression and that a potential relationship exists between mutations
of Pex and aberrant
osteoblast-mediated mineralization. Indeed,
Pex may function in osteoblasts to
metabolize endogenously or exogenously synthesized factors that
regulate the process of osteoblast-mediated mineralization.
Accordingly, osteoblast cell lines derived from Hyp mice should display a nascent
defect in osteoblast-mediated mineralization, if
Pex plays a role in mineralization
that is independent of hypophosphatemia.
In the present investigation, we characterized the maturational profile
of immortalized osteoblasts derived from SV40 transgenic normal and
Hyp mice, and we confirmed that
Pex abnormalities are associated with
osteoblast dysfunction and impaired mineralization in vitro. Moreover,
we found that osteoblast cultures derived from the
Hyp mice produce a diffusible factor
that inhibits normal mineralization in coculture experiments. Our
studies support the hypothesis that abnormalities of
Pex function in
Hyp mouse osteoblasts and that
attendant accumulation of putative endogenously synthesized substrates
of the gene product lead to impaired mineralization in XLH.
Reagents.
Isolation and culture of immortalized osteoblasts and clonal
osteoblast cell lines from normal and Hyp mouse calvaria.
Mice were maintained and used in accordance with recommendations in the
Guide for the Care and Use of Laboratory Animals, prepared by the
Institute on Laboratory Animal Resources, National Research Council
(DHHS Publ. NIH 86-23, 1985), and by guidelines established by the
Institutional Animal Care and Use Committee of Duke University. We
established immortalized osteoblast cell lines from calvaria obtained
from male normal and Hyp mice
transgenic for the large T antigen of simian virus 40 (SV40).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 · µg
protein
1) and reduced
formation of mineralization nodules. Moreover, in coculture
experiments, we found evidence that osteoblasts from the SV40
Hyp mouse produce a diffusible factor
that blocks mineralization of extracellular matrix in normal
osteoblasts. Our findings indicate that abnormal PEX in osteoblasts is
associated with the accumulation of a factor(s) that inhibits
mineralization of extracellular matrix in vitro.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-Minimum essential medium (
-MEM), DMEM/F-12,
penicillin-streptomycin-amphotericin (antibiotic-antimycotic) solution,
Hanks' balanced salt solution (HBSS), and Trizol Reagent for
single-step isolation of total RNA from cells were obtained from GIBCO
(Grand Island, NY). Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT). Pronase E, ascorbic acid,
-glycerophosphate, BSA,
p-nitrophenol, diethanolamine, and
p-nitrophenolphosphate used for
alkaline phosphatase assay were purchased from Sigma (St. Louis, MO).
[3H]thymidine,
45CaCl2,
and [
-32P]dCTP were
purchased from Du Pont-NEN (Boston, MA). Bio-Rad reagent for protein
assay was obtained from Bio-Rad Laboratories (Hercules, CA).
Assay of cell replication. We determined cell number at the various time points by direct counting with a hemocytometer. At the completion of the incubation period, cells were harvested by removing the media, washing twice with HBSS, and treating for 5 min with 0.25% trypsin-1 mM EDTA to achieve cell detachment. DNA synthesis was measured by determining TCA-precipitable radioactivity after a 3-h pulse with [3H]thymidine (1.5 µCi/ml), as previously described (24).
Alkaline phosphatase activity. We analyzed alkaline phosphatase in cell layers by colorimetric assay of enzyme activity with the substrate p-nitrophenolphosphate, as previously reported (24).
Mineralization assays. The time course of mineralization was measured by radioactive calcium accumulation within the cell layer and matrix, as previously described (4, 24). Cells were incubated for 48 h in medium containing 0.5 µCi/ml of 45CaCl2 at the indicated times after seeding. Subsequently, the cell layers were harvested and digested in 0.1 N NaOH, and aliquots were counted by liquid scintillation spectroscopy or analyzed for total protein by the Bio-Rad protein assay (24).
ECM was isolated from TMOb-Nl and TMOb-Hyp osteoblasts after 14 days of culture in differentiation medium consisting ofRT-PCR analysis. We isolated total cellular RNA by a single-step method using Trizol reagent, as previously described (23). RNA samples were pretreated with DNase to remove any contaminating DNA and were quantified by absorbance at 260 nm. To identify Pex expression in TMObs, we performed RT-PCR using RNA derived from each cell line with the following primers: exon 1, M-5F (5'-TTCTGATGGAAGCAGAAACAGGGA-3') and exon 8, M+930R (5'-GGGAATCATAGCGCTGAGTTCTGA-3') to amplify the 5' end of Pex; and exon 7, M+786F (5'-TAATAGCTCTCGAGCTGAACATGA-3') and exon 20, M+1983R (5'-TATCCATTTCCTGTAAGCCC-3') to amplify the 3' end of Pex. To define the Pex deletion break point, we used reverse primers to exon 15, M+1619R (5'-AAAGGCATTGACTGTTGTTG-3') or to exon 16, M+1680R (5'-AAAGAAAGGCTTCTGCAGCT-3') in combination with M+786F. One microgram of total RNA was reverse-transcribed into cDNA using the reverse primer. The RT reaction was incubated at 42°C for 1 h in 20 µl of 5 mM MgCl2, 1 × PCR buffer (Life Technologies, New York), 1 mM dNTP, 0.75 µM reverse primer, 20 units of RNase inhibitor, and 50 units of reverse transcriptase (Life Technologies). The conditions of PCR were 2 min at 94°C, followed by 38 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1-2 min, and 72°C for 10 min for final extension. Samples without reverse transcriptase treatment were analyzed as controls. All predicted products were separated by agarose gel electrophoresis and stained with ethidium bromide. The 5' and 3' fragments were cloned into pCR 2.1 (Invitrogen) and confirmed as Pex by direct sequencing.
In addition, we performed RT-PCR to characterize osteoblast gene expression in clonal TMObs derived from Hyp and normal mice. We used the following primer sets to amplify osteopontin (mop-F 5'-ACACTTTCACTCCAATCGTCC-3' and mop-R 5'-TGCCCTTTCCGTTGTTGTCC-3'), osteocalcin (moc-F 5'-CAAGTCCCACACAGCAGCTT-3' and moc-R 5'-AAAGCCGAGCTGCCAGAGTT-3'), andNorthern blot hybridizations.
Northern analysis was carried out as described (14). Briefly, 20 µg
of total RNA were electrophoresed on a 1.2% formaldehyde agarose gel
and transferred to Nytran membrane (Schleicher & Schuell), and the RNA
was immobilized on the membrane by UV cross-linking with a Stratalinker
(Stratagene). The blot was hybridized overnight at 42°C in the
prehybridization solution containing 10% dextran sulfate and 2 × 106
counts · min
1 · ml
1
of the random-labeled mouse osteocalcin and 28S probe. The blot was
washed twice for 1 min at room temperature in a solution containing 2× standard sodium citrate (SSC) and 0.1% SDS, followed by
washing two additional times for 15 min at 50°C in a solution
containing 0.1× SSC and 0.1% SDS. The blot was air dried, and
the bands were visualized by autoradiography.
Southern blotting. For Southern blot analysis, genomic DNA (~10 µg) from normal and Hyp mouse osteoblasts was digested with EcoR I. The digested DNAs were electrophoresed on 0.7% agarose gel and blotted to nylon membranes (Schliecher & Schuell) by alkaline transfer. Hybridizations were generally performed in a hybridization buffer containing 1.5× SSPE (15 mM Na H2PO4, pH 7.4, 225 mM NaCl, and 1.5 mM EDTA), 1% SDS, and 10% dextran sulfate at 65°C overnight. A probe containing mouse Pex exons 7-22 was labeled by random hexamer priming, and washing was done in 0.1× SSC, 0.1% SDS at 65°C for 15 min.
Coculture experiments.
Coculture experiments were performed using a 6-well culture plate
(Becton-Dickinson, Franklin Lakes, NJ) that contained a 10-cm2 lower plate well size and a
4.2-cm2 upper well insert that
incorporated polyethylene terephthalate track-etched membrane (pore
size 3 µm) to permit diffusion of soluble factors into a lower well.
We plated TMOb-Nl and TMOb-Hyp cells
in either the lower or upper well at an initial density of 40,000 cells
per well to achieve coculture. Controls consisted of coculture of
TMOb-Nl with TMOb-Nl and TMOb-Hyp with
TMOb-Hyp cells. After 14 days in
medium containing ascorbic acid and -glycerophosphate as described
above, mineralization was assessed by alizarin red-S staining and
quantified by modification of previously described methods (30).
Briefly, the stained matrix was washed with water and PBS, the dye was
diluted with 10% (wt/vol) cetylpyridinium chloride, and the
alizarin red-S was quantified at 562 nm.
Statistics. We evaluated differences between groups by one-way analysis of variance (29). All values are expressed as means ± SE. All computations were performed using the Statgraphic statistical graphics system (STSC, Rockville, MD).
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RESULTS |
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Characterization of the Pex mutation in Hyp mouse osteoblasts. To confirm the presence of a Pex mutation in Hyp mice, we performed Southern blot analysis of genomic DNA using a Pex cDNA probe (Fig. 1A). Consistent with previous observations, we identified a 3' deletion of the Pex gene beyond exon 15 (31). Accordingly, in TMOb-Hyp cells, RT-PCR amplification of the 3' end of Pex, with primers designed to amplify the gene segment extending to exon 19, failed to produce an RNA product, whereas this region was amplified in TMOb-Nl cells (Fig. 1B, top). In contrast, using primers designed to amplify exons in the 5' end of Pex, we identified the predicted-size band from normal as well as from Hyp mouse osteoblasts. However, in four separate experiments, Pex was in lower abundance in the mutant cells (Fig. 1B, middle). Additional RT-PCR studies with reverse primers to sequences in exons 15 and 16, in combination with an upstream 5' primer (Fig. 1C), further defined the deletion break point. Consistent with a deletion break point between exons 15 and 16, primer pairs, including exon 15, amplified the predicted Pex transcript, whereas no product was obtained using exon 16 primers in Hyp mouse osteoblasts.
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Phenotype characteristics of immortalized osteoblast cultures. In subsequent studies, we examined whether the immortalized cells exhibited a temporal sequence of maturation characterized by an initial period of replication and subsequent postmitotic expression of osteoblastic characteristics. Similar to primary cultures (22) and other established cell lines (24), both normal and Hyp mouse osteoblasts underwent an initial period of rapid cell proliferation that was characterized by increments in cell number (Fig. 2A) and high levels of DNA synthesis (Fig. 2C). Additionally, in both cell lines we observed a disproportionate increase in protein content relative to cell number after day 10 of culture (Fig. 2B), which corresponded to confluence of the cultures, a concordant decrement in the growth rate, and the formation of collagenous ECM (24). However, there was no significant difference between normal and Hyp mouse osteoblasts with regard to parameters of cell growth and protein content.
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Impaired mineralization in TMOb-Hyp osteoblast cultures. In ensuing experiments, we assessed mineralization in normal and Hyp mouse osteoblasts by use of 45Ca incorporation and alizarin red-S histochemical staining. In immature TMOb-Nl cells, we observed the absence of mineralization (data not shown), whereas marked increments in 45Ca incorporation (Fig. 4A) that corresponded to the presence of alizarin red-S-stained mineralization nodules were observed in these cells by day 14 of culture (Fig. 4B). In contrast, mature Hyp mouse osteoblasts exhibited significantly less 45Ca incorporation (Fig. 4A) after 14 days of culture. Moreover, alizarin red-S staining revealed only ill-defined patches with limited dye uptake and the absence of discrete mineralization nodules (Fig. 4B), consistent with impaired mineralization. The impaired mineralization was not related to differences in the amount of collagen produced in the normal and Hyp mice osteoblast cultures. In this regard, hydroxyproline content was similar between TMOb-Nl and TMOb-Hyp cell culture-derived matrix (0.125 ± 0.001 vs. 0.126 ± 0.001 mg/mg dry wt).
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Persistence of defective mineralization in clonal osteoblasts derived from TMOb-Hyp cell cultures. In addition, we showed that the impaired mineralization in Hyp mouse-derived osteoblasts was not attributable to differences in cellular composition of the cultures, because clonal cell lines obtained from the parent TMOb cultures displayed identical results (Fig. 5). In this regard, clonal osteoblasts obtained from normal TMOb cultures exhibited maturation-dependent mineralization (Fig. 5, A and B) in association with increments in alkaline phosphatase activity (Fig. 5C) and normal Pex expression (Fig. 5D). In contrast, clonal osteoblasts obtained from TMOb-Hyp cultures manifest impaired mineralization (Fig. 5, A and B) in association with the 3' Pex deletion (Fig. 5D) and significantly greater alkaline phosphatase activity compared with normal clonal osteoblasts (Fig. 5C). Moreover, we could identify no differences in osteopontin, osteocalcin, and type I collagen mRNA expression between clonal osteoblasts derived from Hyp and normal mice (Fig. 5D). These findings suggest that a nascent defect in osteoblast-mediated mineralization is a characteristic of osteoblasts with the Pex deletion.
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Transfer of the Hyp mouse phenotype in coculture experiments between TMOb-Nl and TMOb-Hyp. To examine whether the abnormal mineralization in TMOb-Hyp cells is due to production of a factor(s) that inhibits mineralization, we cocultured TMOb-Hyp and TMOb-Nl cell lines separated by a semipermeable membrane. TMOb-Hyp cells displayed abnormal mineralization, whether cocultured with TMOb-Nl or TMOb-Hyp cells (Fig. 6B). In contrast, coculture of TMOb-Hyp with TMOb-Nl cells inhibited the mineralization of the normal osteoblasts, as evidenced by a failure to form discrete mineralization nodules (Fig. 6A) and significant reductions in alizarin red-S staining (Fig. 6B). Identical results were obtained in three replicative studies, consistent with the production of factors capable of inhibiting normal mineralization by TMOb-Hyp cells.
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DISCUSSION |
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The bone mineralization defect in XLH may be due to inadequate circulating levels of mineral and/or hormonal/metabolic factors that influence osteoblast function or to nascent defects in osteoblast function that impair the mineralization process. Our studies indicate that the abnormal mineralization in Hyp mice is due, at least in part, to an intrinsic osteoblastic defect associated with abnormal Pex function. In this regard, we found that TMOb-Hyp cells manifest a 3' Pex deletion (Fig. 1) and, in a setting remote from the in vivo Hyp mouse environment, fail to mineralize under culture conditions supporting mineralization in normal osteoblasts (Figs. 4 and 5). More importantly, we found that the Hyp mouse osteoblasts produce a factor(s) that is capable of regulating the mineralization of ECM. To this end, the mineralization defect observed in TMOb-Hyp cell lines is transferable to normal osteoblasts in coculture experiments (Fig. 6). Such production of a mineralization inhibitor clearly represents a nascent defect in the osteoblasts from Hyp mice.
Because a physiologically relevant site of PEX expression is the osteoblast, it appears likely that production of this mineralization inhibitor is the result of the primary genetic abnormality underlying XLH, namely inactivating mutations of PEX. Indeed, dysfunction of the gene product may result in failure to degrade an endogenously synthesized but undefined inhibitor of mineralization that is a substrate of Pex. The alternate possibility, that Pex fails to activate a novel mineralization-promoting factor, is inconsistent with our coculture experiments in which the Hyp phenotype predominates (Fig. 6). In any case, further studies are necessary to identify the putative Pex substrates produced by osteoblasts and to determine their relationship to the osteoblast-synthesized factor(s). In these investigations, efforts to discriminate whether the mineralization inhibitor represents phosphatonin (17) or an additional putative PEX substrate will be essential.
Although Pex substrates appear to be present in osteoblasts expressing the 3' Pex deletion, the mechanism whereby the accumulated Pex substrate causes the mineralization defect remains unknown. The impaired mineralization might be a direct consequence of a Pex substrate or might result from a multistepped cascade linking the Pex mutation and the accumulation of its substrate with impaired mineralization. Several observations suggest that a downstream event, rather than the putative Pex substrate, may be the mineralization inhibitor. In this regard, provided that Pex in the normal cells is not saturated and is located extracellularly (issues that require confirmation), the Pex endopeptidase in the normal cocultured cells should degrade any diffusible substrates, precluding a negative effect on mineralization. Given the results of our coculture experiments (Fig. 6), it is more likely that impaired mineralization results from a downstream kinase cascade that is regulated by the Pex substrate. Consistent with this possibility, additional studies have identified reductions in casein kinase and decreased phosphorylation of matrix proteins in Hyp mouse osteoblasts (16, 25).
The possible coproduction of Pex and its substrate in osteoblasts is supported by several studies in which an endopeptidase and its substrate are found in the same cell (35). However, when the identity of the substrate is determined, in situ and immunohistochemical studies will be necessary to establish its precise cellular localization. In any event, our study establishes that Pex effects on bone are likely mediated by its metabolism of local factors derived from cells that are within the osteoblast lineage or coisolated with osteoblasts from calvaria.
The current investigations also clarify the nature of the Pex mutation in Hyp mice. We found that TMObs derived from Hyp mice have a 3' deletion of Pex (Fig. 1). Similar to prior Southern analysis of genomic DNA (31), we identified the absence of bands corresponding to the 3' end of the Pex gene in Hyp mice (Fig. 1A) and localized the site of the deletion between exons 15 and 16 by RT-PCR (Fig. 1C). This deletion predicts the production of a protein lacking a portion of the extracellular domain containing the putative catalytic sites; consequently, this is likely to result in loss of Pex function. We were unable to identify the putative intronic sequence or retained 3' end of the Pex transcript in TMOb-Hyp cells, as reported by Beck et al. (2). The reason for this apparent discrepancy is not clear but could be due to differences related to PCR conditions, lower abundance of the truncated message, and/or differences related to amplification from contaminating genomic DNA. Regardless, we found that Pex expressed a truncated 5' transcript, albeit at lower levels compared with normal TMOb cells (Fig. 1B). Lower levels of Pex expression in Hyp mice osteoblasts suggest that the 3' deletion may result in additional abnormalities of message stability. The possibility that message instability may also be clinically relevant is supported by the recent identification in certain families with XLH of mutations in the 5'- and 3'-untranslated regions of PEX that may be important in stabilizing messenger RNA (7).
Many questions remain regarding the pathogenesis of XLH, despite the identification of the PEX/Pex gene. Our results add to the growing body of evidence supporting the concept that osteoblastic cells are a physiologically relevant site of Pex expression and have significant implications regarding our understanding of the pathogenesis of the mineralization defect in XLH and Hyp mice. Further studies will be needed to determine the specific molecular abnormalities of ECM that are responsible for the impaired mineralization and whether these abnormalities are due to the accumulation of a Pex substrate itself or the downstream consequence of the Pex substrate. Our cell culture system also will permit molecular targeting and direct manipulation of Pex expression to prove a cause-and-effect relationship between Pex and the osteoblast phenotype. In turn, unraveling the pathogenesis of XLH and the function of Pex in osteoblasts may provide insights into novel factors that regulate bone mineralization.
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ACKNOWLEDGEMENTS |
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We thank Suzanne Ellett for secretarial assistance in the preparation of this manuscript.
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FOOTNOTES |
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This work was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants RO1-AR-37308 and RO1-AR-43468 (to L. D. Quarles) and R01-AR-27032 (to M. K. Drezner).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: L. D. Quarles, Dept. of Medicine, PO Box 3036 DUMC, Durham, NC 27710.
Received 8 April 1998; accepted in final form 19 June 1998.
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