Intrinsic mineralization defect in Hyp mouse osteoblasts
Z. S. XIAO,1 M. CRENSHAW,2 R. GUO,1 T. NESBITT,1 M. K. DREZNER,1 AND L. D. QUARLES1
1Department of Medicine, Duke University Medical Center, Durham 27710; and 2Dental Research
Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Xiao, Z. S., M. Crenshaw, R. Guo, T. Nesbitt, M. K.
Drezner, and L. D. Quarles. Intrinsic mineralization defect
in Hyp mouse osteoblasts. Am. J. Physiol. 275 (Endocrinol.
Metab. 38): E700–E708, 1998.—X-linked hypophosphatemia
(XLH) is caused by inactivating mutations of PEX, an endo-
peptidase of uncertain function. This defect is shared by Hyp
mice, the murine homologue of the human disease, in which a
38 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 accumula-
tion into extracellular matrix (37 6 6 vs. 1,484 6 68
counts · min21 · µg protein21 ) and reduced formation of miner-
alization 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.
X-linked phosphaturia; osteomalacia; osteocalcin
X-LINKED HYPOPHOSPHATEMIA (XLH) is inherited as a
dominant disorder and is characterized by hypophos-
phatemia, growth retardation, and rickets/osteomala-
cia (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 associ-
ated with renal phosphate wasting and defects in
osteoblast-mediated mineralization (2, 31). This mu-
rine homologue provides a model to study the molecu-
lar 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 constella-
tion 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
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E700 0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
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Hyp mouse have produced inconsistent results (3, 6, 10,
13), carefully performed studies suggest that osteoblast
cultures derived from Hyp mice do display mineraliza-
tion 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 observa-
tions suggest that bone is a physiologically relevant site
of Pex expression and that a potential relationship
exists between mutations of Pex and aberrant osteo-
blast-mediated mineralization. Indeed, Pex may func-
tion in osteoblasts to metabolize endogenously or exog-
enously synthesized factors that regulate the process of
osteoblast-mediated mineralization. Accordingly, osteo-
blast 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 de-
rived from SV40 transgenic normal and Hyp mice, and
we confirmed that Pex abnormalities are associated
with osteoblast dysfunction and impaired mineraliza-
tion in vitro. Moreover, we found that osteoblast cul-
tures 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 endog-
enously synthesized substrates of the gene product lead
to impaired mineralization in XLH.
METHODS
Reagents. a-Minimum essential medium (a-MEM), DMEM/
F-12, penicillin-streptomycin-amphotericin (antibiotic-anti-
mycotic) 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 (Lo-
gan, UT). Pronase E, ascorbic acid, b-glycerophosphate, BSA,
p-nitrophenol, diethanolamine, and p-nitrophenolphosphate
used for alkaline phosphatase assay were purchased from
Sigma (St. Louis, MO). [3H]thymidine, 45CaCl2, and
[a-32P]dCTP were purchased from Du Pont-NEN (Boston,
MA). Bio-Rad reagent for protein assay was obtained from
Bio-Rad Laboratories (Hercules, CA).
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 recommen-
dations 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 Institu-
tional Animal Care and Use Committee of Duke University.
 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS
We established immortalized osteoblast cell lines from cal-
varia obtained from male normal and Hyp mice transgenic for
the large T antigen of simian virus 40 (SV40).
These transgenic animals were created by mating C57BL6J
males, heterozygous for the SV40 large T antigen, with
female Hyp mice, as previously described (21). Progeny
containing the SV40 transgene were identified by PCR am-
plification of a ,500-bp product of SV40 from individual
genomic DNA, using forward primer 58-CAGAGCAGAATT-
GTGGAGTGG-38 and reverse primer 58-GGACAAACCA-
CAACTAGAATGCAGTG-38. The normal and Hyp mice litter-
mates were distinguished among the SV40-positive mice on
the basis of serum phosphorus values that were measured by
colorimetric techniques on a Roche COBRAS MIRA-S.
We used a nonenzymatic method for obtaining the initial
osteoblast cell lines (11). A fragment of the frontal and/or
parietal bone from a single calvaria was aseptically removed
from a 6- to 7-day-old mouse. Suture lines and endosteum
were dissected away, and the bone fragment was placed in a
culture dish. One or two metal strips were positioned on the
endocranial surface and incubated for 3–4 days in DMEM/
F-12 media containing 10% (vol/vol) FBS and 1% (vol/vol)
antibiotic-antimycotic solution until the outgrowth of osteo-
blasts. The metal strips were removed, and the cells were
allowed to grow until ,60% confluent. The cells were scraped
from the dish, transferred to a flask, and propagated by
incubation in DMEM/F-12 containing 10% FBS and 50 µg/ml
ascorbic acid in 5% CO2 at 37°C. We designated these cell
lines as TMOb, for transgenic mouse osteoblasts. This tech-
nique produced both low and high alkaline phosphatase-
producing cell lines. We selected for detailed analysis TMOb
lines from normal and Hyp mice (TMOb-Nl and TMOb-Hyp,
respectively) that displayed an osteoblast maturational se-
quence as evidenced by comparable postconfluent levels of
alkaline phosphatase activity. These cell lines also main-
tained their osteoblast phenotype after repeated passages.
To control for potential cell heterogeneity of our immortal-
ized cell lines, we also generated clonal osteoblast cell lines to
create more homogeneous osteoblast cultures. For selection of
clonal normal and Hyp mouse osteoblasts, 100 cells from
either normal or Hyp TMObs were plated on a 100-mm tissue
culture dish and grown at low density to permit isolation of
individual colonies. After 10 days of culture, colonies were
isolated using trypsin-EDTA-saturated filter paper discs (2–3
mm) to lift individual colonies from the culture plates (8).
After ,3–5 min of trypsinization, the filter paper discs were
removed and placed directly into individual wells of a 24-well
culture dish and colonies were grown to near confluence.
Clonal cells were then transferred to T-75 flasks and ex-
panded for characterization.
We studied immortalized clonal osteoblasts rather than
primary osteoblasts because the phenotype of clonal immortal-
ized osteoblasts from Hyp mice is more likely to be due to
intrinsic abnormalities related to the Pex mutation than to
the Hyp mouse milieu, can be studied after multiple passages,
and is more uniform and reproducible.
All stock cultures of TMObs were grown in a-MEM [contain-
ing 10% (vol/vol) FBS, penicillin (100 U/ml), and streptomycin
(100 µg/ml)] in a humidified atmosphere of 10% CO2-90% air
at 37°C and were passed at a frequency sufficient to maintain
subconfluence. Until the time of study, cells were subcultured
every 3–5 days with 0.001% (wt /vol) pronase to achieve cell
detachment. For studies characterizing the temporal se-
quence of osteoblast maturation, we plated either 40,000 cells
into 35-mm-diameter multiwell dishes or 100,000 cells into
100-mm plates. TMOb-N1 and TMOb-Hyp cells were grown
for periods of up to 14 days in a-MEM containing 10% FBS
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E701
(vol/vol) supplemented with 5 mM b-glycerophosphate and 25
µg/ml of ascorbic acid, with media being replaced every 3
days.
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 har-
vested 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 phos-
phatase 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
45CaCl at the indicated times after seeding. Subsequently,
2
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 osteo-
blasts after 14 days of culture in differentiation medium
consisting of a-MEM containing 10% FBS, 25 µmol/ml ascor-
bate, and 5 mM b-glycerophosphate. Matrix was prepared by
lysing cells with deuterated H2O and rinsing in 0.5% Triton
X-100. After the ECM was extracted with 0.5 M EDTA and
washed with water, samples of each preparation were taken
for hydroxyproline (5, 27).
The formation of in vitro mineralization nodules was
determined by alizarin red-S histochemical staining (29).
Cells were fixed for 24 h in 1:1:1.5 solution of 10% Formalin,
methanol, and water; the fixative was removed; and the fixed
cells and matrices were stained for 15 min with a 2% (wt /vol)
solution of alizarin red-S at pH 4.0. The stained samples were
washed three times with water and then air dried.
RT-PCR analysis. We isolated total cellular RNA by a
single-step method using Trizol reagent, as previously de-
scribed (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 (58-TTCTGA-
TGGAAGCAGAAACAGGGA-38) and exon 8, M1930R
(58-GGGAATCATAGCGCTGAGTTCTGA-38) to amplify the
58 end of Pex; and exon 7, M1786F (58-TAATAGCTCTCGAGC-
TGAACATGA-38) and exon 20, M11983R (58-TATCCA-
TTTCCTGTAAGCCC-38) to amplify the 38 end of Pex. To
define the Pex deletion break point, we used reverse primers
to exon 15, M11619R (58-AAAGGCATTGACTGTTGTTG-38)
or to exon 16, M11680R (58-AAAGAAAGGCTTCTGCAGCT-
38) in combination with M1786F. 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 3 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 Technolo-
gies). 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 electro-
phoresis and stained with ethidium bromide. The 58 and 38
E702 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS

fragments were cloned into pCR 2.1 (Invitrogen) and con-
firmed as Pex by direct sequencing.
In addition, we performed RT-PCR to characterize osteo-
blast gene expression in clonal TMObs derived from Hyp and
normal mice. We used the following primer sets to amplify
osteopontin (mop-F 58-ACACTTTCACTCCAATCGTCC-38
and mop-R 58-TGCCCTTTCCGTTGTTGTCC-38), osteocalcin
(moc-F 58-CAAGTCCCACACAGCAGCTT-38 and moc-R
58-AAAGCCGAGCTGCCAGAGTT-38), and a1(I) procollagen
(m61-F 58-TCTCCACTCTTCTAGTTCCT-38 and m61-R
58-TTGGGTCATTTCCACATGC-38). We used mouse b-actin
primers (mActinF 58-GTGGGCCGCTCTAGG CAC CA-38,
mActinR 58-CGGTTGGCCTTA GGGTTCAGGGGG G-38) to
amplify a 245-bp fragment as a control for the amount and
integrity of RNA in the PCR reactions. Gel-separated prod-
ucts were blotted on Nytran membrane (Schleicher & Schuell,
Keene, NH) and immobilized on the membrane by ultraviolet
(UV) cross-linking with a Stratalinker (Stratagene, La Jolla,
CA). In some studies, the identity of the bands generated by
PCR was confirmed by hybridization with radiolabeled Pex
and b-actin cDNA probes.
Northern blot hybridizations. Northern analysis was car-
ried 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 hybrid-
ized overnight at 42°C in the prehybridization solution con-
taining 10% dextran sulfate and 2 3 106 counts · min21 · ml21
of the random-labeled mouse osteocalcin and 28S probe. The
blot was washed twice for 1 min at room temperature in a
solution containing 23 standard sodium citrate (SSC) and
0.1% SDS, followed by washing two additional times for 15
min at 50°C in a solution containing 0.13 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 electropho-
resed on 0.7% agarose gel and blotted to nylon membranes
(Schliecher & Schuell) by alkaline transfer. Hybridizations
were generally performed in a hybridization buffer containing
1.53 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

Fig. 1. Expression of a phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Pex) in
normal and Hyp mouse osteoblasts. A: Southern blot analysis of genomic DNA derived from normal (NL) and Hyp
mouse osteoblasts. Genomic DNA was digested by EcoR I and hybridized with 32P-labeled Pex cDNA probe, as
described in METHODS. A size calibration was obtained by simultaneous loading of l-DNA/Hind III fragments and a
1-kb DNA ladder. Bands ,4.0 kb [corresponding to exons 16–22 (40)] are absent in Hyp mouse, consistent with
deletion of 38 end of Pex gene. B: RT/PCR amplification of Pex transcripts using 58 and 38 specific primer pairs. To
amplify 58 and 38 ends of Pex, respective primer pairs M-5F/ M1930R and M1786/M11983R were used (see
METHODS ) with total RNA derived from normal and Hyp mouse osteoblasts cultured for 10 days. Amplified products
were transferred to nylon membranes and probed with a radiolabeled Pex cDNA probe. As a control, b-actin was
amplified and identified by hybridization. The 38 end of Pex could not be amplified from RNA derived from Hyp
mouse osteoblasts, and the 58 end was present in low abundance. Both 58 and 38 ends of Pex could be amplified in
normal osteoblasts. C: RT-PCR detection of break points in Hyp mutant Pex transcripts. Using the forward primer
M1786F in combination with exon 15 primer M11619R and exon 16 primer M11680R and RNA from normal and
Hyp mouse osteoblasts, we found evidence for a deletion beyond exon 15.

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 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS
labeled by random hexamer priming, and washing was done
in 0.13 SSC, 0.1% SDS at 65°C for 15 min.
Coculture experiments. Coculture experiments were per-
formed using a 6-well culture plate (Becton-Dickinson, Frank-
lin Lakes, NJ) that contained a 10-cm2 lower plate well size
and a 4.2-cm2 upper well insert that incorporated polyethyl-
ene 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 b-glycerophos-
phate 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 6 SE. All computations were performed using the
Statgraphic statistical graphics system (STSC, Rockville,
MD).
RESULTS
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
38 deletion of the Pex gene beyond exon 15 (31).
Accordingly, in TMOb-Hyp cells, RT-PCR amplification
of the 38 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 58 end of
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E703
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 58
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.
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 replica-
tion and subsequent postmitotic expression of osteoblas-
tic 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.
During the period of rapid cell growth, both TMOb-Nl
and TMOb-Hyp cells expressed low levels of alkaline
phosphatase (Fig. 2D), consistent with their immature,
preosteoblastic state. As anticipated, however, down-
regulation of replication was associated with a signifi-
Fig. 2. Temporal changes in osteoblast
phenotype during culture of normal
and Hyp mouse-derived osteoblasts.
Cell no. (A), protein content (B), DNA
synthesis (C), and alkaline phospha-
tase (ALP) activities (D) were assessed
at different time points, as described in
METHODS. Cell nos. increased rapidly
and progressively during initial period
of culture, corresponding to a high rate
of DNA synthesis. By day 10, replica-
tion rate diminished and DNA synthe-
sis decreased, consistent with near
growth arrest. Protein concentration
also increased as a function of culture
duration and showed a disproportion-
ate increase between days 10 and 14,
consistent with formation of extracellu-
lar matrix (ECM). In addition, there is
a time-dependent upregulation of ALP
activity, consistent with expression of
the osteoblast phenotype in postmitotic
mature osteoblasts. Data are means 6
SE of 3 separate experiments. Values
sharing the same letter superscript are
not significantly different at P 5 0.001.
E704 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS

cant increase in the expression of alkaline phosphatase

activity in both TMOb-Nl and TMOb-Hyp cells (Fig.
2D), although by 14 days of culture, activity was
slightly greater in normal cells. Similarly, the process
of osteoblast maturation in the immortalized cells was
marked by the absence of osteocalcin transcripts in
4-day-old cultures but with high levels of osteocalcin in
14-day-old cultures (data not shown). In concert, we
found that Pex expression increased in TMOb- Nl and
TMOb-Hyp cells as a function of culture duration, a
temporal increase corresponding to the osteoblast matu-
rational stage (Fig. 3). Thus both immortalized osteo-
blast cell lines retain their capacity in vitro to undergo
a normal temporal upregulation of osteoblast-related
gene expression. Collectively, these observations indi-
cate that the immortalized cells represent an excellent
in vitro model system in which to study the bone
mineralization defect in XLH.
Impaired mineralization in TMOb-Hyp osteoblast
cultures. In ensuing experiments, we assessed mineral-
ization 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 incor-
poration (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 im-
paired mineralization. The impaired mineralization
was not related to differences in the amount of collagen
produced in the normal and Hyp mice osteoblast cul-
Fig. 4. Mineralization defect in Hyp mouse osteoblasts. A: 45Ca
incorporation. Mineralization was quantified in normal and Hyp
osteoblasts by radioactive calcium accumulation within cell layer and
ECM. Corresponding to developmental upregulation of osteoblast
phenotype, there was an increase in ECM mineralization by day 14 of
culture in TMOb-Nl but not in TMOb-Hyp. Values are means 6 SE of
3 separate determinations. Values sharing the same letter super-
script are not significantly different at P 5 0.0001. B: in vitro
mineralization nodule formation. Histochemical staining with aliza-
rin red-S was performed to assess mineralized nodule formation, as
described in METHODS. Panel I depicts representative 100-mm plates
derived from 14-day-old normal (NL) and Hyp osteoblasts stained for
mineralized nodules with alizarin red-S. Panel II is a high-powered
(3100) magnification, showing well-defined mineralization nodules
in TMOb-Nl and indiscrete foci of stain in TMOb-Hyp osteoblasts.

tures. In this regard, hydroxyproline content was simi-

Fig. 3. Effect of osteoblast development on Pex expression in normal
and Hyp mice osteoblasts. Total RNA was isolated at different time
points, and Pex transcripts were identified by RT-PCR with M-5F and
M1930R to amplify a region extending from exon 1 to exon 8. b-Actin
was amplified as a control. Transcripts were transferred to Nytran
membrane, blotted with specific radiolabeled probes, and identified
by autoradiography. Both transgenic mouse osteoblasts (TMOb)-Nl
and TMOb-Hyp exhibit a developmental stage-dependent upregula-
tion of Pex transcripts. As in Fig. 1, Pex transcripts were lower in Hyp
than in normal mice osteoblasts.
lar between TMOb-Nl and TMOb-Hyp cell culture-
derived matrix (0.125 6 0.001 vs. 0.126 6 0.001 mg/mg
dry wt).
Persistence of defective mineralization in clonal osteo-
blasts derived from TMOb-Hyp cell cultures. In addi-
tion, 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 miner-
alization (Fig. 5, A and B) in association with incre-
ments in alkaline phosphatase activity (Fig. 5C) and
normal Pex expression (Fig. 5D). In contrast, clonal
osteoblasts obtained from TMOb-Hyp cultures mani-
fest impaired mineralization (Fig. 5, A and B) in
association with the 38 Pex deletion (Fig. 5D) and
significantly greater alkaline phosphatase activity com-

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 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS E705

Fig. 5. Characterization of clonal osteoblast cell lines derived from TMOb cultures. Osteoblasts were subcloned
from parent TMObs, as described in METHODS. A: histochemical staining of mineralization nodules. Normal and Hyp
clonal osteoblast cell lines were cultured for 14 days and stained with alizarin red-S. Similar to corresponding
parent cell lines (Fig. 4), clonal Hyp osteoblasts failed to form mineralization nodules, whereas clonal osteoblasts
from normal TMObs formed abundant mineralized nodules. B: quantification of mineralization. Alizarin red-S stain
was extracted with 10% cetylpyridinium chloride and quantified as described in METHODS. Clonal Hyp osteoblasts
had significantly lower alizarin red-S accumulation at day 14 of culture compared with clonal normal osteoblasts. C:
ALP activity. Clonal Hyp osteoblasts displayed degrees of osteoblast maturation similar to normal osteoblasts. Both
Hyp and normal cell lines displayed a culture duration-dependent increase in ALP activity. D: mRNA phenotype
analysis of clonal osteoblasts. Mouse specific primers were used to RT-PCR amplify osteopontin (OP), osteocalcin
(OC), and a1(I) procollagen from clonal osteoblasts derived from TMOb-Nl and TMOb-Hyp parent cell lines.
Predicted-size products for OP (239 bp), OC (370 bp), and a1(I) procollagen (268 bp) were expressed at similar
abundance in normal and Hyp clonal osteoblasts, whereas the 38 end of Pex was found only in normal osteoblasts,
consistent with our findings in parent cell lines (Fig. 1). b-Actin served as a control for relative mRNA abundance.
Numeric values represent means 6 SE of 3 separate determinations. Values sharing the same letter superscript are
not significantly different at P , 0.01.

pared with normal clonal osteoblasts (Fig. 5C). More-
over, we could identify no differences in osteopontin,
osteocalcin, and type I collagen mRNA expression
between clonal osteoblasts derived from Hyp and nor-
mal mice (Fig. 5D). These findings suggest that a
nascent defect in osteoblast-mediated mineralization is
a characteristic of osteoblasts with the Pex deletion.
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 miner-
alization, 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 signifi-
cant reductions in alizarin red-S staining (Fig. 6B).
Identical results were obtained in three replicative
studies, consistent with the production of factors ca-
pable of inhibiting normal mineralization by TMOb-
Hyp cells.
DISCUSSION
The bone mineralization defect in XLH may be due to
inadequate circulating levels of mineral and/or hor-
monal/metabolic factors that influence osteoblast func-
tion 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 associ-
ated with abnormal Pex function. In this regard, we
found that TMOb-Hyp cells manifest a 38 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 osteo-
blasts (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

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E706 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS
Fig. 6. Coculture experiments comparing normal and Hyp mouse
osteoblasts. TMOb-Nl and TMOb-Hyp osteoblasts separated by a
semipermeable membrane were cocultured for 14 days. A: histochemi-
cal staining of mineralization nodules with alizarin red-S. Normal
osteoblasts cocultured with normal osteoblasts formed mineralized
nodules, and normal osteoblasts cocultured with Hyp mouse osteo-
blasts did not mineralize. B: measurement of alizarin red-S staining,
which was extracted with 10% cetylpyridinium chloride and quanti-
fied as described in METHODS. TMOb-Hyp osteoblasts had lower
alizarin red-S accumulation than TMOb-Nl, consistent with impaired
mineralization. TMOb-Hyp, however, inhibited mineralization of
cocultured normal osteoblasts, consistent with the production/
accumulation of a soluble inhibitor of mineralization. Values are
means 6 SE of 3 separate determinations. Values sharing the same
letter superscript are not significantly different at P , 0.01.
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 expres-
sion 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 mineraliza-
tion-promoting factor, is inconsistent with our cocul-
ture experiments in which the Hyp phenotype predomi-
nates (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 investiga-
tions, efforts to discriminate whether the mineraliza-
tion inhibitor represents phosphatonin (17) or an addi-
tional putative PEX substrate will be essential.
Although Pex substrates appear to be present in
osteoblasts expressing the 38 Pex deletion, the mecha-
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nism whereby the accumulated Pex substrate causes
the mineralization defect remains unknown. The im-
paired 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 mineraliza-
tion inhibitor. In this regard, provided that Pex in the
normal cells is not saturated and is located extracellu-
larly (issues that require confirmation), the Pex endo-
peptidase in the normal cocultured cells should de-
grade any diffusible substrates, precluding a negative
effect on mineralization. Given the results of our cocul-
ture experiments (Fig. 6), it is more likely that im-
paired mineralization results from a downstream ki-
nase 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 osteo-
blasts (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 local-
ization. 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 38 deletion of Pex (Fig. 1).
Similar to prior Southern analysis of genomic DNA
(31), we identified the absence of bands corresponding
to the 38 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 extracel-
lular 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 38 end of the Pex tran-
script 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 condi-
tions, lower abundance of the truncated message, and/or
differences related to amplification from contaminating
genomic DNA. Regardless, we found that Pex expressed
a truncated 58 transcript, albeit at lower levels com-
pared with normal TMOb cells (Fig. 1B). Lower levels
of Pex expression in Hyp mice osteoblasts suggest that
the 38 deletion may result in additional abnormalities
of message stability. The possibility that message insta-
bility may also be clinically relevant is supported by the
recent identification in certain families with XLH of
mutations in the 58- and 38-untranslated regions of
PEX that may be important in stabilizing messenger
RNA (7).
 COEXPRESSION OF PEX AND ITS SUBSTRATES IN OSTEOBLASTS
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 support-
ing the concept that osteoblastic cells are a physiologi-
cally relevant site of Pex expression and have signifi-
cant 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.
We thank Suzanne Ellett for secretarial assistance in the prepara-
tion of this manuscript.
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).
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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