Am J Physiol Cell Physiol 283: C1414–C1421, 2002;
10.1152/ajpcell.00135.2002.
Role of abnormal neutral endopeptidase-like activities
in Hyp mouse bone cells in renal phosphate transport
STÉPHANIE G. DUBOIS,1 ANDRÉA FROTA RUCHON,2 ALINE DELALANDRE,1
GUY BOILEAU,2 AND DANIEL LAJEUNESSE1
1
Unité de recherche en Arthrose, Centre de Recherche du Centre Hospitalier de l’Université
de Montréal, Hôpital Notre-Dame, Montreal H2L 4M1; and 2Département de Biochimie,
Université de Montréal, Montreal, Quebec, Canada H3C 3J7
Received 22 March 2002; accepted in final form 3 July 2002
X-linked hypophosphatemia; Phex; putative inhibitor of renal
phosphate uptake
in human X-linked hypophosphatemia (XLH) is attributable to a specific reduction
of phosphate (Pi) reabsorption in the proximal tubule
(reviewed in Ref. 37). Phenotypic characteristics of
XLH involve paradoxically low 1,25-dihydroxyvitamin
D3 [1,25(OH)2D3] levels in response to hypophosphatemia, osteomalacia, rickets, and lower limb deformities that sometimes require surgery. The murine
Hyp model reproduces these characteristics (15, 18, 48,
49) and has been extensively used to study the cause of
hypophosphatemia. In recent years it has become evident that the observed hypophosphatemia is not due to
an intrinsic renal cell defect but results from humoral
RENAL PHOSPHATE WASTING
Address for reprint requests and other correspondence: D. Lajeunesse, Unité de recherche en Arthrose, Centre Hospitalier de
l’Université de Montréal, Hôpital Notre-Dame, Pavillon J. A. DeSève
Y-2605, 1560, rue Sherbrooke Est, Montreal, Quebec, Canada H2L
4M1 (E-mail: [email protected]).
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mediation (25). This hypothesis was first proposed by
Meyer et al. (30) following the observation that parabiosis between a normal and a Hyp mouse results in
progressive hypophosphatemia in the normal animal
in association with a diminished renal reabsorption of
phosphate. Kidney transplantation from normal to
Hyp mice and from Hyp to normal mice and measurements of Pi uptake by immortalized cell cultures from
the renal proximal tubule of normal and Hyp mice also
argued in favor of a humoral mediation (32, 33). More
recently, we demonstrated the direct intervention of a
humoral factor, present in Hyp mice, on Pi uptake
inhibition by primary mouse proximal tubule cells in
vitro, and we also showed that Hyp osteoblasts could
produce and/or modify this factor (25).
The PHEX/Phex (human/mouse) gene (phosphateregulating gene with homologies to endopeptidases on
the X chromosome) has been shown to be mutated in
XLH patients (16) and in Hyp mice (9). Because PHEX/
Phex codes for a putative neutral endopeptidase, it was
suggested that the enzyme could either inactivate an
inhibitor of phosphate reabsorption or hydrolyze a
propeptide that stimulates phosphate reabsorption in
the kidney. The putative in vivo substrate(s) of PHEX/
Phex remains unknown, but it was recently demonstrated that parathyroid hormone (PTH)-related peptide107–139 (PTHrP) was an in vitro substrate of soluble
PHEX (3), suggesting that, in addition to this role in
modulating the activity of a phosphaturic peptide,
Phex also appears to be involved in bone cell differentiation and/or mineralization. Furthermore, increasing
evidence indicates that a primary bone cell defect is
present in XLH and in the Hyp mouse. Indeed, although high Pi diet and pharmacological doses of calcitriol improve phosphorus balance (4, 5, 8, 41, 48),
rickets never completely heal even in the face of active
mineralization (4, 5, 19). Reduction, but not normalization, of the osteoid thickness and volume, indexes of
abnormal mineralization, are observed following bone
cell transplants from Hyp mice into normal recipients,
whereas abnormal bone formation is observed when
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0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society
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Dubois, Stéphanie G., Andréa Frota Ruchon, Aline
Delalandre, Guy Boileau, and Daniel Lajeunesse. Role
of abnormal neutral endopeptidase-like activities in Hyp
mouse bone cells in renal phosphate transport. Am J Physiol
Cell Physiol 283: C1414–C1421, 2002; 10.1152/ajpcell.00135.
2002.—We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on
the X chromosome) in the Hyp mouse affects the expression
and activity of neprilysin (NEP) and of endothelin-converting
enzyme-like endopeptidase (ECEL1/DINE) in bone marrow
stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like
activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal.
Conditioned media (CM) from Hyp BMSC and Ob inhibited
inorganic phosphate (Pi) uptake by mouse proximal tubule
cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal Pi uptake. A
linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of Pi uptake.
NEP and ECEL1/DINE mRNA levels were higher in Hyp
cells than in normal cells, and in situ hybridization of
ECEL1/DINE confirmed higher levels of expression in the
Hyp mouse than in normal cells. In conclusion, we observed
a correlation between the inhibition of Pi uptake by CM from
Hyp cells and elevated NEP-like activities.
NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
METHODS
Animals
⫹/Y
Hyp/Y
Both normal (C57BL/6J ) and Hyp (C57BL/6J
) 6- to
8-wk-old male mice were obtained from our own breeding
colony. Hyp mice were identified by their shorter body length,
shorter tail length, lower weight, and reduced serum phosphorus levels (Table 1).
Preparation of Primary BMSC Cultures, Ob, and Mouse
Proximal Tubule Cells
Total bone marrow was recuperated by flushing posterior
limbs twice with 5 ml of Ham’s F-12/DMEM (Sigma, St.
Louis, MO) containing 5% penicillin-streptomycin (PenStrep; GIBCO BRL, Grand-Island, NY), cutting bones longitudinally, and vortexing twice for 30 s in the same medium
by the method of Levite et al. (27). The cell suspensions were
pooled and centrifuged at 1,200 g for 2 min at room temperature three times with Ham’s F-12/DMEM containing 5%
Pen-Strep. The last pellet was resuspended in Biggers, Gwatkin, and Judah medium (BGJb; Sigma) containing 10% fetal
bovine serum (FBS; BioWhittaker, Walkersville, MD). Total
bone marrow cells were plated at an initial density of 2 ⫻ 106
cells/cm2 in T25 flasks (Starsted). After 3 days of culture,
adhering BMSC were washed twice with Ham’s F-12/DMEM
containing 5% Pen-Strep. The cells were then fed with BGJb
medium containing 10% FBS and changed every 2 days
thereafter. At day 10, BMSC were split once with 0.025%
trypsin and 0.05% EDTA (Sigma), and cells distributed were
in 24-well plates. Primary Ob cultures and mouse proximal
tubule cell (MPTC) cultures were prepared following our
previously published protocols (24, 25).
Preparation of Conditioned Media From BMSC and Ob
After BMSC and Ob were plated at 50,000 cells/cm2 in
24-well plates, they were fed with BGJb medium containing
10% FBS for 4 days with one change of medium at day 2. At
day 4, cells were washed once with Hams’ F-12/DMEM (1:1)
without FBS and then fed with Ham’s F-12/DMEM containing 10% FBS for conditioning. The conditioned media (CM)
were recuperated and used to treat MPTC cultures for 48 h
before Pi uptake, as previously described (25). BMSC and Ob
were used to determine alkaline phosphatase and NEP-like
activities, protein content and/or cell count, or to extract
RNA. No differences were noted in protein content or cell
number between normal and Hyp cells under these conditions. CM in the absence of cells or conditioned in the presence of Chinese hamster ovary (CHO) cells were prepared in
parallel to BMSC-CM and Ob-CM to determine the specificity of the conditioning (negative control).
Alkaline Phosphatase and Neutral Endopeptidase Activities
of BMSC and Ob
Alkaline phosphatase was determined by the hydrolysis of
p-nitrophenylphosphate (12.5 mM), as previously described
(23, 24). Total NEP-like activity of BMSC and Ob was evaluated following a two-step enzymatic analysis, as described
Table 1. Physiological parameters for normal and Hyp mice
Genotype
Body Weight, g
Serum Phosphorus, mM
Total BMC,
106 cells/g body wt
BMSC,
106 cells/dish
Normal
Hyp
n
P
22.43 ⫾ 0.43
16.69 ⫾ 0.38
10
⬍0.001
2.99 ⫾ 0.22
1.56 ⫾ 0.1
10
⬍0.001
5.38 ⫾ 0.41
3.47 ⫾ 0.22
10
⬍0.005
2.78 ⫾ 0.29
1.92 ⫾ 0.16
5
⬍0.05
Values are presented as means ⫾ SE. Mice were sacrificed, and their posterior long bones were removed and cleaned of all surrounding
tissues. After both ends of the femur and tibia were out, they were flushed with Ham’s F-12/DMEM and total bone marrow cells (BMC) were
evaluated. Total BMC plated at 2 ⫻ 106 cells/cm2 in T25 flasks were cultured for 3 days in BGjb medium containing 10% FBS, the medium
was removed, and adhering cells were washed 3 times with Ham’s F-12/DMEM alone containing 5% penicillin/streptomycin. They were then
fed every 2 days with BGJb medium containing 10% FBS until day 10. These bone marrow stromal cells (BMSC) were split with trypsin and
counted. Statistical analysis was performed by using the 2-tailed Mann Whitney U-test; a difference of 0.05 was considered significant.
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normal bone cells are transplanted into mutant mice
(13, 14). This role of Phex is further supported by the
observations that the inability of Hyp osteoblasts to
produce normal bone is not due to prior exposure to an
abnormal low-phosphate environment (12) and that
primary cell cultures of Hyp osteoblasts show abnormal phenotypic expression compared with normal
mouse osteoblasts (7, 25).
PHEX/Phex is a member of a family of structurally
related zinc-containing endopeptidases that includes,
among others, neprilysin (NEP) and the endothelinconverting enzymes (ECEs) (50). Besides PHEX/Phex,
two more enzymes of the family, NEP and endothelinlike converting enzyme 1/damage-induced neuronal
endopeptidase (ECEL1/DINE ), have been shown to be
present in bone (10, 40, 43, 52, 53), suggesting an
important role for these enzymes in bone metabolism.
The relation between PHEX/Phex expression and expression of other members of the family has never been
investigated. In the present paper, we used specific
inhibitors to probe the role of NEP on the elaboration of
the inhibitor of phosphate uptake by Hyp mouse bone
marrow stromal cells (BMSC) and osteoblasts (Ob).
Finally, because Phex is absent from Hyp BMSC and
Ob, we evaluated whether this absence had an effect on
the total NEP-like activity of these cells and on the
expression of NEP and ECEL1/DINE. Our results
show that total NEP-like activity was higher in Ob
than in BMSC, regardless of genotype, and that Hyp
BMSC and Ob showed higher NEP-like activities than
normal cells. We also showed that the expression of
both NEP and ECEL1/DINE is increased in BMSC and
Ob from Hyp mice compared with normal mice and
that these enzymes may be involved in the elaboration
of the putative inhibitor of phosphate uptake by Hyp
BMSC and Ob.
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NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
by Shipp et al. (45), in the presence or absence of 10 ␮M
phosphoramidon or thiorphan, using the synthetic peptide
Glu-Ala-Ala-Phe-4-methoxy-2-naphthylamide (Sigma).
Phosphate Uptake by MPTC
Transport experiments with MPTC were performed at
37°C in the incubation buffer composed of 140 mM NaCl, 5
mM KCl, 1 mM MgCl2, and 10 mM HEPES-Tris buffer at pH
7.4, containing 0.1 mM 32P (NEN-DuPont, Mississauga, ON,
Canada) at 2 ␮Ci/ml, as previously described (25). After three
washings with stop solution, cells were covered with 500 ␮l of
0.5 N NaOH and agitated for 60 min at room temperature on
a rotating platform at 100 rpm. Aliquots were counted, after
neutralization with 0.5 N HCl, in a beta counter, and protein
content was determined by the BCA method (46).
Detection by RT-PCR. Total RNA from normal and Hyp
cells was extracted by the method of Chomczynski and Sacchi
(6), and 1 ␮g of RNA was used for RT-PCR amplification.
Reverse transcription reaction was performed by using
pd(N)6 random hexamers as recommended by the supplier
(Pharmacia Biotech, Baie d’Urfé, QC, Canada). Half of the
single-stranded cDNA obtained was used to generate, via
PCR, specific cDNAs of 670, 732, and 450 bp for ECEL1/
DINE, NEP, and Phex, respectively. The second half was
used to amplify a cDNA of 983 bp for GAPDH via PCR.
PCR reactions used 20 pmol of each primer (sense) 5⬘TCAAAAAGATTCGACAGGAGGTGG-3⬘ and (antisense)
5⬘-AGAGCACTTATGGACGGGGTTC-3⬘ for ECEL1/DINE,
(sense) 5⬘-AGATGAGTGGATAAGTGGAGCAGC-3⬘ and
(antisense) 5⬘-GTTTCTCTGAGGGAAGAGTTCCTG-3⬘ for
NEP, (sense) 5⬘-AAAATGGAAACCTTGATCCGTGG-3⬘
and (antisense) 5⬘-ATCTGCTCCTCTGTTCATAGTGG-3⬘
for Phex, and (sense) 5⬘-TGAAGGTCGGTGTGAACGGATTTGGC-3⬘ and (antisense) 5⬘-CATGTAGGCCATGAGGTCCACCAC-3⬘ for GAPDH. Thirty-five cycles (linear portion of the amplification curve) were needed for Phex and
NEP, and forty cycles were requested for ECEL1/DINE,
whereas twenty-eight cycles were enough for the amplification of GAPDH. Amplified DNA fragments were then visualized on 1.2% agarose gel.
Detection by Western blot analysis. Extracts of normal and
Hyp cells were prepared and analyzed by Western blotting
essentially as previously described with specific monoclonal
antibodies for NEP (40). Protein was detected by ECL-PLUS
chemiluminescence kit with Lumigen-PS3 (Amersham Pharmacia Biotech) and visualized with Kodak X-Omat films, and
scanning densitometry was performed directly on these films.
Detection by in situ hybridization. In situ hybridization
(ISH) was performed essentially as previously described (39),
except for the RNase A concentration, which was 20 mg/ml
instead of 200 mg/ml. Sense and antisense riboprobes labeled
with 35S-uridine triphosphate (UTP; 1,250 Ci/mmol; Amersham, Arlington Heights, IL) were prepared by in vitro transcription of appropriate linearized plasmids. Plasmids used
were, for ECEL1/DINE, pSPORT1 plasmid (catalog no.
1058129; American Type Culture Collection, Rockville, MD)
containing a 458-bp mouse ECEL1/DINE cDNA insert corresponding to nucleotides 2075–2533 of the nucleotide sequence (GenBank accession no. XM002262), and for PTH/
PTHrP receptor, pBluescript containing a 528-bp cDNA
insert corresponding to nucleotides 193–721 of PTH/PTHrP
receptor nucleotide sequence (GenBank accession no.
X78936).
AJP-Cell Physiol • VOL
Values represent means ⫾ SE for n individual culture
preparations. Transport experiments are performed in quadruplicate dishes per experiment per conditions tested,
whereas all other determinations were performed on individual samples in duplicate. Two-tailed Mann-Whitney U-tests
were performed, and a level of 0.05 was considered to be
significant.
RESULTS
Phenotypic Characterization of BMSC and Ob
BMSC are the reservoir of the osteoblastic cell lineage. The suggestion that normal mouse bone marrow
transplanted into an Hyp mouse can correct the Hyp
phenotype (31) could indicate that Hyp BMSC already
bear a cell defect leading to abnormal bone metabolism. We therefore investigated whether Hyp BMSC
are different from normal cells. Hyp mice showed a
significant reduction in total bone marrow cells and in
available BMSC compared with normal (Table 1).
Moreover, when normal and Hyp BMSC were plated at
high initial density at first passage, to eliminate the
possibility that growth characteristics may influence
enzymatic profiles, alkaline phosphatase activities in
confluent cell cultures were always significantly higher
in Hyp mouse BMSC compared with normal. Although
an increase in alkaline phosphatase activity was also
noted in Hyp Ob compared with normal, it never
reached statistical significance (Fig. 1).
Neutral Endopeptidase Activity in BMSC and Ob
It has been reported that NEP-like activity may vary
with bone cell maturation (1). Therefore, we questioned
whether an NEP-like activity could be affected in bone
lineage cells of the Hyp genotype. Regardless of genotype, NEP-like activity of Ob was much higher than for
BMSC (2- to 3-fold, Fig. 2). This activity was also
higher in Hyp BMSC compared with normal BMSC
Fig. 1. Alkaline phosphatase activity of bone marrow stromal cells
(BMSC) and osteoblasts (Ob) from normal and Hyp mice. Alkaline
phosphatase was determined by the hydrolysis of p-nitrophenylphosphate as described in METHODS. Values are means ⫾ SE of 6 experiments, and values are expressed as nmol 䡠 mg protein⫺1 䡠 30 min⫺1.
*P ⬍ 0.025 between normal and Hyp BMSC; NS, not significant.
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Detection of Phex, NEP, and ECEL1/DINE
in Cell Preparations
Statistical Analysis
NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
(89.9 ⫾ 13.5% increase, P ⬍ 0.001) and in Hyp Ob
compared with normal Ob (157.4 ⫾ 49.9% increase,
P ⬍ 0.02) (Fig. 2). Thiorphan (10 ␮M) and phosphoramidon (10 ␮M), both inhibitors of NEP-like activity,
significantly inhibited the NEP-like activity in normal
and Hyp cells (Fig. 2). Treatment of cells with 10 ␮M
phosphoramidon failed to significantly alter cell
growth characteristics in normal and Hyp BMSC and
Ob (data not shown). However, the same treatment
reduced alkaline phosphatase activity in Hyp BMSC
(24.4 ⫾ 8.8% decrease, P ⬍ 0.025) and Ob (30.7 ⫾ 7.9%
decrease, P ⬍ 0.025) but failed to influence this parameter in normal BMSC (3.7 ⫾ 2% decrease, not significant) and Ob (1.3 ⫾ 0.3% increase, not significant).
Fig. 3. Effect of conditioned medium (CM) from normal and Hyp
mouse BMSC or Ob (see Fig. 2 legend) on renal phosphate uptake by
mouse proximal tubule cells (MPTC). CM were prepared for the last
48 h of culture of cells before being added to MPTC for 48 h.
Phosphate uptake was then measured with 0.1 mM 32Pi for 5 min.
Values are means ⫾ SE of 4–10 experiments run in quadruplicate
dishes per condition. *P ⬍ 0.001; **P ⬍ 0.005 vs. medium alone.
CHO, Chinese hamster ovary cells.
4.3%, P ⬍ 0.001). Likewise, normal Ob-CM partly inhibited Pi uptake (P ⬍ 0.005), whereas Hyp Ob-CM
inhibited this uptake much more than normal Ob (P ⬍
0.001). Because the capacity to inhibit Pi uptake in
MPTC varies between Hyp BMSC and Ob and normal
BMSC and Ob, we evaluated whether a relationship
exists between NEP-like activity and Pi uptake. A
linear relationship was observed between the NEP-like
activity of normal and Hyp BMSC and Ob and the
inhibition of Pi uptake by MPTC (Fig. 4). The involvement of the NEP-like activity in the production of the
phosphaturic factor by Ob from Hyp animals is further
supported by the addition of phosphoramidon in culture medium. Phosphoramidon inhibited NEP-like activity in both normal and Hyp Ob, and the same treatment during the conditioning of Hyp mouse Ob
Effect of NEP-Like Activity Inhibition on Production
of the Putative Phosphate Uptake Inhibitor
To determine whether this NEP-like activity is involved in the production of the phosphaturic factor, we
measured phosphate uptake by MPTC in the presence
of CM from normal or Hyp BMSC and Ob. This biological assay provided a sensitive method to monitor the
production of the phosphaturic factor, because we successfully used MPTC to evaluate the time and dose
dependence of Hyp mouse serum and CM of Hyp Ob on
Pi uptake (25) and to monitor the purification of the
elusive Hyp humoral phosphaturic factor(s) (Loignon
M, Dubois SG, and Lajeunesse D, unpublished observations). Phosphate uptake by MPTC in response to
CHO-CM and normal BMSC-CM is not significantly
modified compared with cells treated with medium
alone (Fig. 3). In contrast, Hyp BMSC-CM produced a
significant inhibition of Pi uptake by MPTC (⫺36.1 ⫾
AJP-Cell Physiol • VOL
Fig. 4. Relationship between total neutral endopeptidase activity
and phosphate uptake inhibition capacity of both normal and Hyp Ob
and BMSC (see Fig. 2 legend). Total neutral endopeptidase activity
was measured as described in Fig. 2 and Pi uptake as described in
Fig. 3. Values are means ⫾ SE of 4–10 experiments per condition run
in quadruplicate dishes per condition.
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Fig. 2. Neprilysin (NEP)-like activity of normal and Hyp mice BMSC
and Ob treated with or without 10 mM phosphoramidon (Ph) or 10
mM thiorphan (Th). NEP-like activity of BMSC from normal (13
preparations) and Hyp (8 preparations) mice and primary Ob from
normal (10 preparations) and Hyp (8 preparations) was measured.
Values are means ⫾ SE and are expressed as nmol 䡠 mg
protein⫺1 䡠 h⫺1. *P ⬍ 0.001 between normal and Hyp BMSC. **P ⬍
0.005 between Hyp BMSC and Ob. ***P ⬍ 0.02 between normal and
Hyp Ob. ****P ⬍ 0.05 between normal BMSC and Ob.
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NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
prevented its inhibitory effect on Pi uptake by MPTC
(Fig. 5), whereas no clear effects could be observed with
normal mouse CM under these conditions.
Levels of Expression of NEP and ECEL1/DINE
In addition to Phex, two members of the NEP family
have been reported in bone: NEP and ECEL1/DINE.
Both these enzymes have been shown to be sensitive to
thiorphan and phosphoramidon (22, 38, 50, 51). To test
the possibility that expression of these enzymes is
increased and thus contributes to the higher level in
NEP-like activity observed in Hyp BMSC and Ob, we
determined the level of NEP and ECEL1/DINE mRNA
and protein. Specific oligonucleotides were used in a
RT-PCR strategy to amplify DNA fragments for
ECEL1/DINE, NEP, and Phex from total RNA isolated
from normal or Hyp BMSC and Ob (Fig. 6). In the
conditions used, ECEL1/DINE could not be detected in
normal mouse BMSC but was readily detectable in
normal Ob and in both Hyp BMSC and Ob, with higher
levels in Ob than BMSC. The ratio of ECEL1/DINE to
GAPDH mRNA levels measured by RT-PCR was
higher in Hyp Ob compared with normal Ob (1.317 ⫾
DISCUSSION
BMSC are the precursor cells of osteoblast lineage
cells (17, 35) and differentiate into Ob (2, 26). Several
studies have shown that BMSC can form a mineralized
collagen matrix and calcified nodules as found in bone
(29, 42, 44, 47). The first conclusion of the present
study is that BMSC from the Hyp mouse are less
numerous, grow at a slower rate, have higher alkaline
phosphatase activity, and produce more phosphate uptake inhibitor than cells from a normal mouse. Similar
features were previously reported for Hyp Ob (25).
Elevated alkaline phosphatase in Hyp BMSC may suggest that these cells are kept at an earlier stage of
development, because this activity should decrease as
Fig. 6. Representative expression levels of ECEL1/
DINE (endothelin-like converting enzyme 1/damageinduced neuronal endopeptidase), NEP, and Phex
(phosphate-regulating gene with homologies to endopeptidases on the X chromosome) by Ob and BMSC
(see Fig. 2 legend) from normal and Hyp mice. Cells
were grown to confluence in T25 flasks in the presence
of Ham’s F-12/DMEM containing 10% FBS. RT-PCR
analysis of ECEL1/DINE, NEP, and Phex was performed by using specific primers in the presence of 1 ␮g
of total RNA. Equivalent loading was established by the
parallel detection of GAPDH in the same samples.
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Fig. 5. Effect of inhibition of NEP-like activity by phosphoramidon
on production of the putative inhibitor of renal phosphate uptake by
Hyp Ob (see Fig. 2 legend). CM were prepared in the presence or
absence of 10 ␮M phosphoramidon for 48 h of conditioning before
being added to MPTC for 48 h. Phosphate uptake was then measured
with 0.1 mM 32Pi for 5 min. Values are means ⫾ SE of 4 determinations for phosphate uptake. **P ⬍ 0.025 vs. Hyp Ob with no treatment.
0.100 vs. 0.849 ⫾ 0.040, respectively, P ⬍ 0.025, n ⫽ 3).
NEP mRNA levels were also found to be slightly higher
in Hyp Ob vs. normal Ob, although this difference did
not reach significance (1.056 ⫾ 0.061 vs. 1.019 ⫾ 0.048
NEP-to-GAPDH ratio, respectively, n ⫽ 3), and levels
were significantly more elevated in Hyp than in normal
BMSC (0.994 ⫾ 0.021 vs. 0.841 ⫾ 0.034 NEP-toGAPDH ratio, respectively, P ⬍ 0.025, n ⫽ 3). As
expected, Phex expression measured by RT-PCR was
absent from Hyp cells. Interestingly, we observed
higher levels of Phex in normal BMSC than in Ob
(4.3-fold increase; 1.531 ⫾ 0.125 vs. 0.393 ⫾ 0.072
Phex-to-GAPDH ratio, respectively, P ⬍ 0.01, n ⫽ 4).
To determine whether changes in ECEL1/DINE expression observed in primary cultures of Ob and BMSC
cells of Hyp mouse are also present in whole bones, we
performed ISH on sections of 4-day-old (P4) normal
and Hyp animals using an ECEL1/DINE-specific
probe. Figure 7 shows an increased expression of
ECEL1/DINE in the calvarium of the Hyp mouse compared with normal mouse (Fig. 7, A and B for normal
and Hyp, respectively). As a control, the expression of
PTH/PTHrP receptor was measured by ISH in parallel
in these samples (Fig. 7, D and E for normal and Hyp,
respectively). No significant differences were noted for
PTH/PTHrP receptor expression between genotypes as
opposed to ECEL1/DINE. Similar results were observed in vertebrae (results not shown).
We next used specific antibodies to determine the
level of NEP in normal and Hyp cells (Fig. 8). In
contrast to mRNA levels, which were only slightly
higher in Hyp cells, larger amounts of NEP protein
were observed in Hyp than normal cells (Fig. 8).
NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
C1419
the cells progress into the osteoblast lineage (36), as
illustrated here by the decrease in alkaline phosphatase activity in both normal and Hyp Ob compared
with their respective BMSC. However, Hyp cells always showed higher alkaline phosphatase activities
than normal cells. These observations suggest that the
lack of Phex could result in an intrinsic defect in these
progenitor cells that did not disappear as the cells
progressed toward mature Ob.
The precise role of Phex is still obscure. Some have
proposed that mineralization by osteoblasts may be
linked to Phex expression (20, 54), whereas others have
suggested that Phex may be involved in osteoblast
differentiation, hence possibly leading to abnormal
mineralization (11). Recently, Liu et al. (28) demonstrated that in vivo and in vitro expression of Phex in
Hyp osteoblasts is not sufficient to fully rescue the Hyp
phenotype, hence indicating that additional factors are
likely to be important in the pathogenesis of XLH.
Indeed, because Phex may be linked to osteoblast maturation, its absence could modify the expression of
other genes in BMSC and/or Ob from Hyp animals. The
observation that NEP-like activity and expression levels of two NEP-like enzymes are increased in BMSC
and Ob of Hyp animals is the second important finding
of our study. mRNA levels of Phex in normal BMSC
compared with Ob varied oppositely to those of NEP
and ECEL1/DINE. Consistent with this observation,
Fig. 8. Protein levels of NEP in normal and Hyp BMSC and Ob (see
Fig. 2 legend). Total protein (50 ␮g) was separated by SDS-PAGE,
transferred to a polyvinylidene difluoride membrane, and detected
by Western blot analysis using monoclonal antibodies against NEP
that do not cross-react with other neutral endopeptidases.
AJP-Cell Physiol • VOL
the absence of Phex in Hyp cells resulted in increased
expression of NEP and ECEL1/DINE. Our results suggest a regulation, direct or indirect, of both NEP and
ECEL1/DINE expression in bone cells by the Phex gene
product. However, because this increase in NEP expression was not observed in bone extracts of 4-day-old
mice (40), it is suggested that the role of Phex in the
regulation of NEP expression is more important at an
earlier stage of growth.
It has been shown previously that NEP-like activity
increases during the differentiation of BMSC (1). Because NEP has been detected in growing bone plates
(10, 40) and NEP-like activity is inversely correlated
with bone cell growth (1), the present increase in NEPlike activity may explain the reduction in the reservoir
of BMSC of the Hyp mouse. However, because NEP
and ECEL1/DINE cannot be truly distinguished on the
basis of inhibitors used in our study, it is not possible
at the present time to determine which of these activities is more important for bone cell growth, differentiation, and/or mineralization. However, the partial
normalization of alkaline phosphatase activity in Hyp
BMSC and Ob following the inhibition of endopeptidase activities with 10 ␮M phosphoramidon (not
shown), which was not affected in normal cells, suggests that abnormally high NEP-like activity in Hyp
cells could contribute to the abnormal cell behavior of
these cells in vitro as well as in vivo. It is interesting to
note that increases in NEP protein in both Hyp BMSC
and Ob are much higher than the increase in mRNA
level. This finding could suggest the removal of a posttranscriptional inhibition on NEP synthesis in the Hyp
mouse. Other investigators (21) recently indicated that
another peptidase is elevated in Hyp mouse bone tissues, namely, cathepsin D, offering another link to
abnormal bone mineralization/turnover in these animals.
Our study is the first demonstration of Phex expression
in BMSC suggesting that Phex can be involved in prolif-
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Fig. 7. In situ hybridization (ISH) comparison of
ECEL1/DINE mRNA levels in normal (A) and Hyp (B)
mice and of parathyroid hormone(PTH)/PTH-related
peptide (PTHrP) receptor mRNA levels in normal (D)
and Hyp (E) mice. The ISH patterns are presented at an
atomic resolution in a sagittal plane on X-ray film after
hybridization with 35S-labeled antisense riboprobes.
Silver labeling is shown as dark shading. The arrow in
B indicates positive mRNA signal in Hyp mouse calvaria for ECEL1/DINE. Only background level is revealed in calvaria of its normal littermate (thin arrows
in A). In contrast, similar positive mRNA signals are
observed for PTH/PTHrP receptor in both normal (D)
and Hyp (E) mouse calvaria. C and F are negative
controls for ECEL1/DINE and PTH/PTHrP receptor
experiments, respectively. Scale bar, 1 cm.
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NEUTRAL ENDOPEPTIDASE ACTIVITY AND HYP MOUSE BONE CELLS
This study was supported by Canadian Institute of Health Research Grant MT-14666 (to D. Lajeunesse). S. G. Dubois is supported
by a studentship from the Canadian Institute of Health Research. D.
Lajeunesse is a senior scholar from the Fonds de la Recherche en
Santé du Québec.
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