1,25-Dihydroxyvitamin D3
upregulates natriuretic peptide receptor-C expression in mouse
osteoblasts
Noriyuki
Yanaka,
Hiroyuki
Akatsuka,
Eri
Kawai, and
Kenji
Omori
Discovery Research Laboratory, Tanabe Seiyaku, Osaka 532-8505, Japan
 |
ABSTRACT |
1,25-Dihydroxyvitamin D3
[1,25(OH)2D3],
a key regulator of mineral metabolism, regulates expression of several
genes related to bone formation. The present study examined the
1,25(OH)2D3-mediated regulation of natriuretic peptide receptor-C (NPR-C) expression in
osteoblasts.
1,25(OH)2D3
treatment significantly increased NPR-C-dependent atrial natriuretic
peptide-binding activity and synthesis of the NPR-C protein in mouse
osteoblastic cells in a cell-specific manner. Western blot analysis
also demonstrated that
1,25(OH)2D3
upregulated expression of NPR-C protein in slow kinetics. Next,
Northern blot analysis revealed a significant increase in the
steady-state NPR-C mRNA level by
1,25(OH)2D3. Sequence analysis of the 9 kb of the 5'-flanking region of the mouse NPR-C gene revealed an absence of consensus vitamin D-response elements, and promoter analysis using osteoblastic cells stably transfected with mouse NPR-C promoter-reporter constructs showed a
slight increase of promoter activity with
1,25(OH)2D3
treatment. In addition, a nuclear run-on assay exhibited that the
transcriptional rate of the NPR-C gene was unchanged by
1,25(OH)2D3,
whereas that of the osteopontin gene was increased. Evaluation of NPR-C
mRNA half-life demonstrated that
1,25(OH)2D3
significantly increased the NPR-C mRNA stability in osteoblastic cells.
1,25(OH)2D3
attenuated intracellular cGMP production in osteoblastic cells
stimulated by C-type natriuretic peptide (CNP) without a significant
change of the natriuretic peptide receptor-B mRNA level, suggesting
enhancement of the clearance of exogenously added CNP via NPR-C.
Furthermore, NPR-C and osteopontin mRNAs in mouse calvariae were
significantly increased by administration of
1,25(OH)2D3,
and immunohistological analysis demonstrated that NPR-C is actually and
strongly expressed in mouse periosteal fibroblasts. These findings
suggest that
1,25(OH)2D3 can play a critical role for determination of the natriuretic peptide
availability in bones by regulation of NPR-C expression through
stabilizing its mRNA.
natriuretic peptide
 |
INTRODUCTION |
THE STEROID HORMONE 1,25-dihydroxyvitamin
D3
[1,25(OH)2D3]
is now recognized as a key regulator of both bone formation and resorption, with a principal role in calcium homeostasis and skeletal metabolism, and influences the expression of genes related to the
establishment and maintenance of the bone cell phenotype. Although the
overall function of
1,25(OH)2D3
in bones is to promote resorption, it is the osteoblasts, and not the
osteoclasts, that contain a nuclear receptor (vitamin D receptor, VDR)
that belongs to the steroid/retinoid/thyroid hormone receptor
superfamily and acts via binding to distinct vitamin D response
elements (VDRE). As a consequence, the expression of several genes in
osteoblasts is regulated by
1,25(OH)2D3.
For instance, the expression of the type I collagen gene and the bone
sialoprotein gene are downregulated by the hormone, whereas those of
the osteopontin and osteocalcin, noncollagenous proteins of the bone
extracellular matrix that regulates bone formation, are significantly
upregulated in humans and rats (18, 25, 26). To date, the role of
1,25(OH)2D3 in the regulation of osteocalcin gene transcription has been studied extensively. However, little is known regarding the effect of 1,25(OH)2D3
on gene expression through nongenomic mechanisms.
Natriuretic peptides (NPs) are known to play important roles in
cardiovascular homeostasis. Three isoforms, termed atrial natriuretic
peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic
peptide (CNP; see Refs. 7 and 31), constitute an NP family. ANP and BNP
have been considered to be responsible for systemic blood pressure
control and body fluid homeostasis (3, 11). These biological functions
of NPs were mediated with production of intracellular cGMP through the
guanylyl cyclase (GC)-coupled receptors, termed GC-A and GC-B. Recent
works have demonstrated that NPs were involved in the suppression of
cell proliferation and in phenotypic development of chondrocytes and osteoblasts (12, 27, 30). In addition, CNP secreted from cultured
osteoblastic cells has been shown to increase expression of alkaline
phosphatase and osteocalcin and to induce formation of mineralized
nodules in an autocrine manner (12). Natriuretic peptide receptor-C
(NPR-C) has a very short putative intracytoplasmic extension with no GC
activity (8, 10, 28). One of the possible roles of NPR-C is believed to
determine the biological availabilities of NPs by elimination from
circulation, therefore suggesting that the NPR-C expression is tightly
associated with the maintenance of the cardiovascular system and with
the regulation of bone formation. Recent work has shown that NPR-C
decreased in amount during phenotypic differentiation of osteoblastic
cells and chondrocytes (9, 14), suggesting that the NPR-C protein is a
marker molecule representing the developmental stages of these cells.
On the other hand, NPR-C has been reported to control adenylyl cyclase
activity via Gi protein,
phospholipid hydrolysis, thymidine kinase activity, and
mitogen-activated protein kinase activity in a variety of cells (1, 2,
4, 13, 29) without any cGMP response, suggesting it has a physiological
significance other than the clearance of ligands.
Here, we have demonstrated that
1,25(OH)2D3
treatment significantly increased NPR-C expression at protein and mRNA
levels in mouse osteoblastic cells in a cell-specific manner. We
recently reported highly specific antibody that recognizes the NPR-C
cytoplasmic domain (9), and the availability of this antibody enabled
us to investigate the NPR-C expression at total protein level and by
immunohistochemical analysis. We focused on the significant effect of
1,25(OH)2D3
on NPR-C mRNA expression and investigated the mechanisms underlying its
upregulation. The steady-state mRNA level can be affected by the rate
of gene transcription and by that of mRNA degradation. Our recent
studies have shown the structure of the 5'-flanking regulatory
regions of the mouse and human NPR-C genes and functional features
using serial 5'-deletions of the promoter regions (34, 35).
Further investigations were carried out to examine the
cis-acting sequences in the regulatory
region of the mouse NPR-C gene and its mRNA stability using a
transcription inhibitor, actinomycin D. Moreover, our findings in this
study were that the NPR-C mRNA was significantly increased in mouse calvariae by
1,25(OH)2D3
administration and that NPR-C was actually expressed in mouse
periosteal fibroblasts, suggesting that
1,25(OH)2D3 can modulate NPR-C expression with its physiological roles in vivo.
 |
MATERIALS AND METHODS |
Materials. Restriction endonucleases
and DNA-modifying enzymes were obtained from Takara Shuzo (Kyoto,
Japan). [
-32P]dCTP,
[
-32P]UTP, rat
125I-ANP, Hybond-N plus nylon
filter, and cGMP EIA system were from Amersham.
1,25(OH)2D3
was obtained from Biomol. CNP was a product of Peptide Institute
(Osaka, Japan). The ANP analog
des[Gln18,Ser19,Gly20,Leu21,Gly22]
ANP-(4
23)-NH2
[C-ANF-(4
23)] was purchased from Sigma.
-Modified Eagle's medium (
-MEM), Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), and G418 were obtained from GIBCO Biotech. The
vectors pGVP and pGVB containing a firefly luciferase gene were
obtained from Toyo Inki (Japan). The plasmid pMAM-neo was a product of Clontech.
Cell cultures. MC3T3-E1 cells, COS
cells, and BALB/3T3 clone A31 were obtained from Dainippon
Pharmaceutical (Osaka, Japan). C3H10T1/2 clone 8 (10T1/2) was from
RIKEN Cell Bank (Tsukuba, Japan). A population of osteoblastic cells
was isolated from calvariae of newborn ICR mice and Wistar
rats as previously reported (12). MC3T3-E1 cells, 10T1/2 cells, and
osteoblastic cells from calvariae were cultured in
-MEM supplemented
with 10% FCS. COS cells and BALB/3T3 cells were cultured in DMEM
supplemented with 10% FCS. These cells were passaged in a controlled
atmosphere of 5% CO2-95% air at
37°C.
Binding assay of NPR-C. Cells in
24-well dishes were washed two times with 1 ml of ice-cold
phosphate-buffered saline (PBS). Binding of 1 nM rat
125I-ANP was allowed to proceeded
for 30 min at 4°C to equilibrium in the presence or absence of 1 µM of unlabeled C-ANF-(4
23). After 30 min, cells were washed two
times with PBS and solubilized with 500 µl of 0.5 N NaOH. Aliquots
were assayed for radioactivity in a Cobra
-counter (Packard).
Western blot and immunohistological
analyses. The antiserum against the NPR-C protein used
was obtained according to our previously published procedures (9).
Confluent cells (1 × 107) were washed two times with
ice-cold PBS, harvested in the homogenizing buffer [10 mM
Tris · HCl (pH 7.5), 5 mM EDTA, 5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol], sonicated for 20 s, and centrifuged at 30,000 g for 30 min. The pellet was
rehomogenized in the homogenizing buffer. The solubilized membrane
proteins (~30 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon P filter
(Millipore). The blots were blocked for 18 h at 4°C by soaking in
5% nonfat dried milk in PBS and were incubated for 18 h at 4°C
with anti-NPR-C antiserum (diluted 1:1,000). Signals were detected
using horseradish peroxidase-conjugated anti-rabbit IgG and the
enhanced chemiluminescence systems (Amersham). Neonatal ICR mice were
used in the immunohistological experiment. Tissues were rapidly fixed
with paraformaldehyde, and 4-µm-thick sections were prepared on a
cryostat. Immunostaining was performed with a primary rabbit anti-NPR-C
antibody (diluted 1:1,000) with or without preabsorption by specific
antigen and by the avidin-biotin-peroxidase complex (ABC) kit (Vector
Laboratories, Burlingame, CA), as described in the manufacturer's directions.
Promoter analysis by stable transfection and reporter
assay. Nine kilobases of the 5'-flanking region
of the mouse NPR-C gene were subcloned into reporter plasmid pGVB to
yield pmNPRCluc1 (35). For stable transfection of MC3T3-E1 cells, the
cells were electroporated at 200 V and 960 µF using 1 µg of
pmNPRCluc1 or 1 µg of control plasmid pGVP with 1 µg of pMAM-neo.
After replating, the cells were treated with 500 µg/ml of G418 for 14 days. G418-resistant colonies were identified, and 10 independent
colonies were repropagated until they reached a higher density. After
18 h of incubation with or without 1 × 10
8 M
1,25(OH)2D3
treatment, the cells were washed two times with PBS, collected in a
microcentrifuge, and disrupted by a freeze-thaw cycle in 300 µl of
cell lysis solution (Promega). The supernatants obtained by
centrifugation for 5 min were pooled to measure firefly luciferase
activity. Luciferase activity was measured using TD20e (Turner) with 10 µl of cell extracts.
In vivo treatment with
1,25(OH)2D3.
Five-week-old ICR mice were injected intraperitoneally with 1 or 100 µg of
1,25(OH)2D3
or with vehicle and killed 18 h later. Total RNAs were isolated from
calvariae that were dissected out and cleaned of soft tissues.
RNA analysis by Northern blotting
hybridization. Total RNAs were isolated using ISOGEN
(Nippon gene, Toyama, Japan), and
poly(A)+ RNAs purified by an
oligo(dT)-cellulose column chromatography using an mRNA separator kit
(Clontech) were fractionated in a 1% agarose gel containing 0.66 M
formaldehyde and 0.02 M MOPS (pH 7.0). Fractionated RNAs were
transferred onto a nylon filter by capillary blotting and then
cross-linked by ultraviolet irradiation. 32P-labeled cDNA fragments
encoding mouse NPR-C (35), mouse osteopontin (5), rat
-actin, mouse
GC-B, rat
-actin, or human glyceraldehyde phosphate dehydrogenase
were used for Northern blotting hybridization as probes. Hybridization
was performed as described previously (35). Values were obtained from
densitometric scanning of hybridized signals using a Micro Computer
Imaging Device (MCID; Imaging Research).
Nuclear run-on transcription assay.
After incubation with or without 1 × 10
8 M
1,25(OH)2D3
treatment for 16 h, MC3T3-E1 cells were rinsed with ice-cold PBS two
times. Cultured cells were suspended in 400 µl of
buffer A [10 mM
Tris · HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5 % Nonidet
P-40] and chilled on ice for 15 min. After centrifugation at
3,000 g for 5 min, nuclear fractions
were resuspended in 400 µl of buffer
A. The nuclei were precipitated by centrifugation at
3,000 g for 5 min and suspended in 100 µl of buffer B [50 mM
Tris · HCl (pH 8.3), 0.1 mM EDTA, 5 mM
MgCl2, and 40% glycerol].
To label the nascent RNA transcripts, the nuclei were incubated in 200 µl of 2× reaction buffer (10 mM Tris · HCl, 5 mM MgCl2, and 300 mM KCl)
supplemented with 1 mM ATP, GTP, and CTP (Pharmacia Biotech), 250 µCi
of [
-32P]UTP (3,000 Ci/mmol), and 5 mM dithiothreitol for 30 min at 30°C with shaking.
The nuclear extracts were digested for 5 min with RNase-free DNase I
(GIBCO Biotech), and the reaction was terminated by the addition of SDS
buffer [83.3 mM EDTA, 330 mM Tris · HCl (pH
7.5), and 3.33% SDS] followed by treatment with proteinase K. 32P-labeled RNA transcripts were
purified by precipitation on Millipore type HA (0.45 µm) filters with
ice-cold 5% trichroloacetate, 30 mM sodium pyrophosphate, and ethanol
precipitations. Equal counts (~4 × 106 counts/min) of radiolabeled
RNA transcripts were produced from the control and
1,25(OH)2D3-treated
MC3T3-E1 cells. The radiolabeled RNA transcripts were hybridized for
two nights at 42°C with mouse NPR-C and mouse osteopontin cDNA
probes that had been denatured and immobilized on a nylon filter.
Filters were washed at room temperature in 2× saline sodium
citrate (SSC) with 0.1% SDS, followed by 0.5× SSC with 0.1% SDS
at 60°C for 15 min two times and subjected to autoradiography. The
plasmid pUC19 was used to determine nonspecific background
hybridization. Values were obtained from densitometric scanning of
hybridized signals using MCID.
Measurement of intracellular cGMP
accumulation. After 18 h incubation with or
without 1 × 10
8 M
1,25(OH)2D3
treatment, MC3T3-E1 cells were washed two times with
-MEM
supplemented with 10% FCS and then incubated with the indicated
concentration of CNP and 0.5 mM isobutyl methylxanthine (Sigma) in
-MEM supplemented with 10% FCS for 30 min at 37°C. After being
washed two times with PBS, cells were solubilized with 500 µl of
ice-cold 50% ethanol and extracted by evaporation. The concentrations
of cGMP were determined after acetylation using a cGMP EIA system (Amersham).
Statistical analysis. All values are
expressed as means ± SD. Statistical significance was determined by
the unpaired Student's t-test.
 |
RESULTS |
1,25(OH)2D3-induced
NPR-C expression in a cell-specific manner.
1,25(OH)2D3
treatment increased ANP-binding activity in MC3T3-E1 cells (Fig.
1). Binding analysis with rat
125I-ANP revealed that
C-ANF-(4
23), a specific NPR-C agonist, effectively competed for
increased ANP-binding sites with a high affinity (data not shown). To
directly estimate the NPR-C protein level, we performed Western blot
analysis using the antibody against the rat NPR-C cytoplasmic domain
(Fig.
2A). At
the reducing condition, the solubilized NPR-C protein was detected as a
single band of ~63 kDa. Strong signals corresponding to NPR-C were
seen in the lysates from
1,25(OH)2D3-treated
MC3T3-E1 cells compared with those from untreated cells, indicating
that augmentation of the NPR-C-dependent ANP-binding activity by
1,25(OH)2D3
was accompanied by upregulation of de novo synthesis of the NPR-C
protein. The relative amounts of NPR-C protein (487% increase) did not
appear to correlate with the percentage increase in ANP-binding
activity, suggesting that Western blot analysis could detect not only
NPR-C protein on the cellular surface but also a larger amount of
intracellular NPR-C protein. Next, we wanted to test whether
1,25(OH)2D3
could modulate the ANP-binding activity in a variety of cultured cells that express NPR-C protein abundantly. As shown in Fig. 1,
a significant increase in NPR-C-dependent ANP-binding activity was also
observed in 10T1/2 cells and in mouse cultured osteoblastic cells with 1,25(OH)2D3
treatment. However, NPR-C expression was not upregulated by
1,25(OH)2D3
in COS cells (Fig. 1A), HeLa
cells, and rat aortic smooth muscle cells (data not shown). As shown in
Fig. 2B, it is noteworthy that the
kinetics of the increase in the NPR-C protein level were slower than
that of the rapid increase of
25(OH)2D3 24-hydroxylase by
1,25(OH)2D3
treatment as previously reported (24).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of 1,25-dihydroxyvitamin D3
[1,25(OH)2D3]
on atrial natriuretic peptide (ANP)-binding activity. After treatment
with the indicated concentration of
1,25(OH)2D3
for 18 h, ANP-binding activity was determined as described in
MATERIALS AND METHODS. Natriuretic peptide receptor-C
(NPR-C)-dependent binding activity was determined by competitive
binding assay in the presence of 1 µM of unlabeled
des[Gln18,Ser19,Gly20,Leu21,Gly,22]
ANP-(4 23)-NH2
[C-ANF-(4 23)]. Error bars represent SD.
** P < 0.01 compared with
basal.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of
1,25(OH)2D3
on the NPR-C protein level by Western blot analysis.
A: after treatment with 1 × 10 8 M of
1,25(OH)2D3
for 18 h, cells (1 × 107)
were washed two times with ice-cold PBS and harvested in homogenizing
buffer. After centrifugation, the resulting pellets were homogenated
and subjected to 10% SDS-PAGE. Transferred blots were detected using
anti-NPR-C antiserum (diluted 1:1,000), horseradish
peroxidase-conjugated anti-rabbit IgG, and the enhanced
chemiluminescence systems as described in MATERIALS AND
METHODS. B: at the day of
confluence, MC3T3-E1 cells were exposed to 1 × 10 8 M of
1,25(OH)2D3
for the indicated time. Western blot analysis was performed as
described in MATERIALS AND METHODS.
|
|
Northern blot analysis revealed that
1,25(OH)2D3
treatment significantly increased the steady-state level of the NPR-C
mRNA in MC3T3-E1 cells (Fig. 3). The
existence of two discrete NPR-C mRNA species was shown in Northern
blotting analysis. We have previously isolated two different sized
cDNAs corresponding to mouse NPR-C mRNA (data not shown), revealing
that alternative polyadenylations in the 3'-untranslated region
lead to the presence of two different species of mouse NPR-C mRNA. In
the same experiment, we observed the significant increase in the
osteopontin mRNA level with
1,25(OH)2D3
treatment. Previous works have shown that an AP-1 sequence was located
in the 5'-flanking region of the mouse and chicken osteopontin
genes and that the osteopontin mRNA level was upregulated in phorbol
myristate acetate-treated MC3T3-E1 cells (5). Although our previous
work has revealed an AP-1 sequence in the 5'-flanking region of
the mouse NPR-C gene, no significant change of NPR-C mRNA level was
observed with phorbol myristate acetate treatment for 18 h.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
RNA analysis by Northern blotting hybridization. After treatment of
MC3T3-E1 cells with the indicated concentration of
1,25(OH)2D3
or with 50 ng/ml of phorbol myristate acetate (PMA) for 18 h,
poly(A)+ RNAs were purified as
described in MATERIALS AND METHODS.
Poly(A)+ RNA (5 µg) was
subjected to Northern blot analysis. Hybridization was performed using
32P-labeled mouse NPR-C, mouse
osteopontin, or human glyceraldehyde phosphate dehydrogenase (GAPDH)
cDNA as probes, as described in MATERIALS AND METHODS.
|
|
Mechanism of
1,25(OH)2D3-induced
increase in the NPR-C mRNA.
The
1,25(OH)2D3-induced
increase in steady-state NPR-C mRNA levels could be the result of
transcriptional effects, an effect on NPR-C mRNA stability, or a
combination of both. Our previous work revealed that no consensus VDRE
was identified in 2 kb of the 5'-flanking region of the mouse
NPR-C gene. We analyzed a further upstream region of the
5'-flanking region. (The nucleotide sequence has been submitted
to the GenBank/EMBL Data Bank with accession number AB007853.) However,
sequence analysis has shown that the 9-kb region upstream from an ATG
codon contained no candidate VDRE, based on a homology search using the
VDRE sequences of rat osteocalcin (21), rat calbindin-D9k (6), rat
25(OH)2D3 24-hydroxylase (24), and mouse osteopontin (23) genes. Additionally, we
investigated the effect of
1,25(OH)2D3
on the mouse NPR-C promoter activity in stably transfected MC3T3-E1
cells using a reporter construct, carrying the 9 kb of the
5'-flanking region (pmNPRCP1; Fig.
4).
1,25(OH)2D3
treatment slightly increased transcriptional activity only in stable
transformants carrying pmNPRCP1. Furthermore, we performed a nuclear
run-on assay to determine whether
1,25(OH)2D3 increased the transcription rate of the NPR-C gene. Nuclei from the
control or
1,25(OH)2D3-treated
cells were isolated, and nascent transcripts were hybridized to
filter-bound plasmid probes. Although 1,25(OH)2D3
significantly increased the transcriptional rate of the mouse
osteopontin gene, it had no effect on that of the NPR-C gene (Fig.
5).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Stable expression analysis of the mouse NPR-C promoter-luciferase (LUC)
reporter chimeric gene in MC3T3-E1 cells. Transcriptional activity was
measured by DNA transfection experiments in MC3T3-E1 cells.
A: mouse NPR-C gene was inserted
upstream of the firefly luciferase gene, and the nucleotide boundaries
were confirmed by nucleotide sequencing.
B: in each experiment, cells were
treated with or without 1 × 10 8 M of
1,25(OH)2D3
for 18 h. pGVP is a control plasmid carrying the luc gene
downstream of SV40 promoter. Values are relative degree of induction
compared with the activity obtained without
1,25(OH)2D3.
Results presented here are averages from 10 isolated independent
clones. Error bars represent SD.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
Transcription rate of the NPR-C gene in
1,25(OH)2D3-treated
MC3T3-E1 cells. A: nuclei were
isolated from confluent MC3T3-E1 cells untreated or treated with 1 × 10 8 M of
1,25(OH)2D3
for 12 h. Labeled RNAs were hybridized to nylon membrane containing 1 µg of linearized mouse NPR-C cDNA, mouse osteopontin cDNA, and pUC19
as a background control. Hybridization was carried out as described in
MATERIALS AND METHODS. B:
values shown are obtained from densitometric scanning of signals
(n = 3), as described in
MATERIALS AND METHODS. Error bars represent SD.
* P < 0.05 compared with
basal.
|
|
Next, we assessed whether
1,25(OH)2D3
treatment affects the half-life of the NPR-C mRNA. After incubation in
the presence or absence of
1,25(OH)2D3
for 16 h,
1,25(OH)2D3
was then removed, and parallel dishes were incubated in the presence of
the transcription inhibitor actinomycin D. We estimated the NPR-C mRNA
half-life in
1,25(OH)2D3-treated
or -untreated MC3T3-E1 cells. The half-life of the NPR-C
mRNA was significantly lengthened for 4.68 h by
1,25(OH)2D3 treatment in comparison with that in untreated cells (0.98 h). In the
same experiment, we observed that
1,25(OH)2D3
had no effect on the half-life of the osteopontin mRNA (data not
shown). Further experimentation was performed to test the effect of
1,25(OH)2D3 on the stability of the NPR-C mRNA. We first exposed MC3T3-E1 cells to
1,25(OH)2D3
for 16 h to increase NPR-C mRNA.
1,25(OH)2D3 was then completely removed, and parallel dishes were incubated in the
presence or absence of actinomycin D, plus or minus
1,25(OH)2D3 for 6 h. We observed, in the presence of actinomycin D, that the NPR-C
mRNA level without
1,25(OH)2D3
treatment was quite lower (63.5% decrease) than with its treatment
(Fig. 6,
B,
right). These experiments were
repeated independently to confirm the results described above. These
results strongly suggested that
1,25(OH)2D3 could have an effect on the stability of the NPR-C transcripts.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of
1,25(OH)2D3
on NPR-C mRNA half-life in MC3T3-E1 cells.
A: confluent MC3T3-E1 cells were
pretreated with or without 1 × 10 8 M of
1,25(OH)2D3
for 16 h. Medium was removed and incubated in the presence of 5 µg/ml
of actinomycin D for the indicated time.
Poly(A)+ RNA was purified as
described in MATERIALS AND METHODS.
Poly(A)+ RNA (10 µg) was
subjected to Northern blot analysis. Hybridization was performed using
32P-labeled mouse NPR-C cDNA
fragments as a probe. Values shown are obtained from densitometric
scanning of hybridized signals, as described in MATERIALS AND
METHODS. B: confluent MC3T3-E1
cells were pretreated with 1 × 10 8 M of
1,25(OH)2D3
for 16 h to increase the NPR-C mRNA. After the medium was removed, the
cells were incubated in the presence or absence of 5 µg/ml of
actinomycin D (AcD), with or without 1 × 10 8 M of
1,25(OH)2D3
for 6 h. Poly(A)+ RNA (10 µg)
was purified and subjected to Northern blot analysis. Hybridization was
carried out as described in MATERIALS AND METHODS.
|
|
Effect of
1,25(OH)2D3 on
cellular responsiveness to CNP.
To evaluate the functional significance of
1,25(OH)2D3-mediated
NPR-C upregulation, we examined the biological responsiveness to NPs.
After pretreatment of MC3T3-E1 cells with or without 1 × 10
8 M of
1,25(OH)2D3,
CNP (0.1 nM
1 µM) was then added to the medium. At the end
of incubation, the intracellular cGMP concentration was measured. The
CNP-induced cGMP accumulation was shown to be reduced by
1,25(OH)2D3
pretreatment (Fig.
7B).
cGMP production in MC3T3-E1 cells by CNP was shown to be mediated
through GC-B. Northern blot analysis has shown that GC-B mRNA level was
not significantly changed by
1,25(OH)2D3
treatment for 16 h (Fig. 7A). These
results suggested that the attenuation of cGMP accumulation in
1,25(OH)2D3-pretreated
cells is caused by the increased metabolic clearance of CNP in the
medium through the NPR-C upregulation.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of
1,25(OH)2D3
on guanylate cyclase (GC)-B mRNA level and cGMP production by CNP in
MC3T3-E1 cells. A: after pretreatment
with or without 1 × 10 8 M of
1,25(OH)2D3
for 16 h, poly(A)+ RNAs were
isolated as described in MATERIALS AND METHODS.
Poly(A)+ (5 µg) RNA was
subjected to Northern blot analysis. Hybridization was performed using
32P-labeled mouse GC-B or human
GAPDH cDNA as probes, as described in MATERIALS AND
METHODS. B: after pretreatment
with or without 1 × 10 8 M of
1,25(OH)2D3
for 16 h, MC3T3-E1 cells were incubated with the indicated
concentration of C-type natriuretic peptide (CNP) for 30 min.
Intracellular cGMP concentration was determined as described in
MATERIALS AND METHODS. Error bars represent SD.
* P < 0.05 and
** P < 0.01 compared with
basal.
|
|
Induction of NPR-C mRNA expression with
1,25(OH)2D3
treatment in vivo.
Recent work has demonstrated that expression of the osteocalcin gene is
significantly downregulated in mouse calvariae with intraperitoneal
injection of
1,25(OH)2D3
as well as in
1,25(OH)2D3-treated MC3T3-E1 cells (36). Further experiments were performed to test the
effect of
1,25(OH)2D3
on NPR-C expression in vivo.
1,25(OH)2D3 was injected intraperitoneally to mice, and, after 18 h,
poly(A)+ RNAs from calvariae were
isolated and subjected to Northern blot analysis. The steady-state
NPR-C mRNA level in calvariae was markedly increased by
1,25(OH)2D3
administration (Fig. 8). This induction was
also observed with 100 ng injection of
1,25(OH)2D3
(data not shown), whereas further investigations are needed to ensure
the hypercalcemia did not occur in the animals with a
1,25(OH)2D3 treatment. In these experiments, a significant increase in osteopontin gene expression was also seen in calvariae from
1,25(OH)2D3-injected mice. As shown in Fig.
9A,
immunohistochemical analysis by the ABC method using anti-NPR-C
antiserum demonstrated positive staining in periosteal fibroblasts in
1,25(OH)2D3-treated
mice. Preabsorption of anti-NPR-C antiserum by a specific antigen
resulted in complete loss of the staining (Fig.
9B).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 8.
RNA analysis by Northern blotting hybridization. Five-week-old ICR mice
were injected intraperitoneally with 1 or 100 µg of
1,25(OH)2D3
or with vehicle and killed 18 h later.
Poly(A)+ RNAs were isolated from
mouse calvariae as described in MATERIALS AND METHODS.
Poly(A)+ RNA (5 µg) was
subjected to Northern blot analysis. Hybridization was performed using
32P-labeled mouse NPR-C, mouse
osteopontin, or rat -actin cDNA fragments as probes, as described in
MATERIALS AND METHODS.
|
|

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 9.
NPR-C expression at the histological level. Immunostaining was
performed with a primary rabbit anti-NPR-C antiserum without
(A) or with
(B) preabsorption by specific
antigen and control staining with hematoxylin
(C), as described in MATERIALS
AND METHODS. Br and M indicate brain and bone matrix,
respectively. Arrows indicate positive-stained periosteal cells.
Original magnification is ×40.
|
|
 |
DISCUSSION |
We have investigated the regulation of the mouse NPR-C gene by
1,25(OH)2D3
and demonstrated that the expression is significantly upregulated by
this hormone.
1,25(OH)2D3
is known to regulate expression of several genes related to osteoblast
differentiation (28). The role of
1,25(OH)2D3
in the transcriptional regulation of the osteocalcin and osteopontin
genes, which are noncollagenous proteins of the bone extracellular
matrix, has been extensively studied. This regulation occurs at the
transcriptional level, that is, wherein the hormone-receptor complex
binds to a VDRE located in the 5'-flanking region of these genes.
We have previously demonstrated the structure of the 5'-flanking
regulatory region of the mouse and human NPR-C genes and functional
features using serial 5'-deletions of the promoter region (34,
35). In this study, we have cloned a further upstream sequence of the
5'-flanking region of the mouse NPR-C gene. However, sequence
analysis and promoter analysis using stable transfections in MC3T3-E1
cells indicated that a hormone response element for
1,25(OH)2D3
was not located in the mouse NPR-C promoter region. Although most of
the VDREs identified to date are located within 1 kb of the 5'-flanking regions of the
1,25(OH)2D3-inducible
genes, these observations do not exclude the possibility that VDRE is
located further upstream or downstream in the NPR-C gene locus, as has been demonstrated for the regulation of the HoxA cluster by retinoic acid (16). However, nuclear run-on assays have revealed that the
transcriptional rate of the NPR-C gene was not significantly affected
by
1,25(OH)2D3
treatment. Furthermore, we observed the augmentation of NPR-C mRNA
stability by
1,25(OH)2D3
according to experiments using a transcription inhibitor, actinomycin
D. Finally, we concluded that the
1,25(OH)2D3-mediated
effect is mainly dependent on stabilization of its mRNA. Although the
role of
1,25(OH)2D3
in the gene transcription has been well studied, little is known
regarding the effect of this hormone via the posttranscriptional pathway. Recent works have demonstrated that
1,25(OH)2D3
could increase aromatase cytochrome
P-450 mRNA in the presence of
actinomycin D in cultured human osteoblasts (32) and could stabilize
the VDR mRNA in human MG-63 osteosarcoma cells (22). On the other hand,
retinoic acid together with thyroid hormone is the main known regulator
of metabolism, differentiation, and development in vertebrates (20).
Gene regulation by retinoic acid follows binding to specific nuclear
receptors and formation of heterodimer with members of the RXR
proteins. However, several genes have been described as regulated
posttranscriptionally by retinoic acid. Retinoic acid has been shown to
prolong the half-life of the proteolipid protein mRNA in
C6 glioma cells (19) and that of
calbindin-D28k mRNA in the human medulloblastoma cell line D283 (33)
without enhancing the gene transcription.
Our other finding is that
1,25(OH)2D3
mediates a significant increase of NPR-C expression in a cell-specific
and a species-specific manner.
1,25(OH)2D3
was shown to be able to upregulate NPR-C-dependent ANP-binding activity
in MC3T3-E1, 10T1/2, and cultured osteoblastic cells from mouse
calvariae. The steady-state level of NPR-C mRNA in mouse calvariae was
also markedly increased with in vivo
1,25(OH)2D3 treatment. Although MC3T3-E1 is a fibroblastic cell line from calvariae
of C57BL/6 mice, 10T1/2 cells established from an early embryo have the
pluripotent activity to differentiate into osteoblastic cells by bone
morphogenetic protein-2 (15). Our immunohistological analysis has
demonstrated that NPR-C is actually and strongly expressed in
periosteal fibroblasts in
1,25(OH)2D3-treated
mice, suggesting that
1,25(OH)2D3
might upregulate NPR-C expression in fibroblasts, which could
differentiate into osteoblasts. Although we could not find positive
staining in control mouse calvariae, it is not fully understood whether
the upregulation of NPR-C mRNA in mouse calvariae with in vivo
1,25(OH)2D3
treatment could be a result of induced expression in undifferentiated
fibroblasts, in osteoblasts, or a combination of both. Moreover, a
significant upregulation of NPR-C expression was not observed in
cultured osteoblastic cells from rat calvariae. Although recent studies demonstrated the species-specific
1,25(OH)2D3
responsiveness for regulation of osteocalcin gene transcription (17,
36), little is known regarding a species-specific effect of this
hormone via the posttranscriptional pathway.
Recent work has demonstrated that an increase in intracellular cGMP by
CNP leads to expression of alkaline phosphatase and osteocalcin in
osteoblastic cells and formation of mineralized nodules (12). Current
study suggests that one of the functional significances of upregulation
of NPR-C by
1,25(OH)2D3
is to significantly attenuate biological responsiveness to NPs. To
date, NPR-C has been reported to directly control adenylyl cyclase
activity via Gi protein,
phospholipid hydrolysis, thymidine kinase activity, and
mitogen-activated protein kinase activity without any cGMP response.
1,25(OH)2D3
might modulate these biological activities of NPR-C in osteoblastic cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H. Hagiwara for kind instructions for preparing and
culturing osteoblastic cells, K. Fujishige for technical support, and
Drs. M. Sugiura and T. Ishizuka for continuous kind help.
 |
FOOTNOTES |
The nucleotide sequence reported in this paper has been submitted to
the GenBank/EMBL Data Bank with accession number AB007853.
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: K. Omori, Discovery Research Laboratory,
Tanabe Seiyaku Co., Ltd., 16-89 Kashima-3-chome, Yodogawa-ku,
Osaka 532-0085, Japan.
Received 15 May 1998; accepted in final form 6 August 1998.
 |
REFERENCES |
1.
Anand-Srivastava, M. B.,
M. R. Sairam,
and
M. Cantin.
Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system.
J. Biol. Chem.
265:
8566-8572,
1990[Abstract/Free Full Text].
2.
Anand-Srivastava, M. B.,
P. D. Sehl,
and
D. G. Lowe.
Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase.
J. Biol. Chem.
271:
19324-19329,
1996[Abstract/Free Full Text].
3.
Burnett, J. C., Jr.,
J. P. Cranger,
and
T. J. Opgenorth.
Effects of systemic atrial natriuretic factor on renal function and renin release.
Am. J. Physiol.
247 (Renal Fluid Electrolyte Physiol. 16):
F863-F866,
1984[Abstract/Free Full Text].
4.
Cahill, P. A.,
and
A. Hassid.
ANF-C-receptor-mediated inhibition of aortic smooth muscle cell proliferation and thymidine kinase activity.
Am. J. Physiol.
266 (Regulatory Integrative Comp. Physiol. 35):
R194-R203,
1994[Abstract/Free Full Text].
5.
Craig, A. M.,
J. H. Smith,
and
D. T. Denhardt.
Osteopontin, a transformation-associated cell adhesion phosphoprotein, is induced by 12-O-tetradecanoylphorbol 13-acetate in mouse epidermis.
J. Biol. Chem.
264:
9682-9689,
1989[Abstract/Free Full Text].
6.
Darwish, H. M.,
and
H. F. DeLuca.
Identification of a 1,25-dihydroxyvitamin D3-response element in the 5'-flanking region of the rat calbindin D-9k gene.
Proc. Natl. Acad. Sci. USA
89:
603-607,
1992[Abstract].
7.
deBold, A. J.,
H. B. Borenstein,
A. T. Veress,
and
H. Sonnenberg.
A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats.
Life Sci.
28:
89-94,
1981[Medline].
8.
Engel, A. M.,
J. R. Schoenfeld,
and
D. G. Lowe.
A single residue determines the distinct pharmacology of rat and human natriuretic peptide receptor-C.
J. Biol. Chem.
269:
17005-17008,
1994[Abstract/Free Full Text].
9.
Fujishige, K.,
N. Yanaka,
H. Akatsuka,
and
K. Omori.
Localization of clearance receptor in rat lung and trachea: association with chondrogenic differentiation.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L425-L431,
1998[Abstract/Free Full Text].
10.
Fuller, F.,
J. G. Porter,
A. E. Arfsten,
J. Miller,
J. W. Schilling,
R. M. Scarborough,
J. A. Lewicki,
and
D. B. Schenk.
Atrial natriuretic peptide clearance receptor.
J. Biol. Chem.
19:
9395-9401,
1988.
11.
Garcia, R.,
M. Cantin,
G. Thibault,
H. Ong,
and
J. Genest.
Relationship of specific granules to the natriuretic and diuretic activity of rat atria.
Experientia
38:
1071-1073,
1988.
12.
Hagiwara, H.,
A. Inoue,
A. Yamaguchi,
S. Yokose,
M. Furuya,
S. Tanaka,
and
S. Hirose.
cGMP produced in response to ANP and CNP regulates proliferation and differentiation of osteoblastic cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1311-C1318,
1996[Abstract/Free Full Text].
13.
Hirata, M.,
C.-H. Chang,
and
F. Murad.
Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells.
Biochim. Biophys. Acta
1010:
346-351,
1996.
14.
Inoue, A.,
Y. Himura,
S. Hirose,
A. Yamaguchi,
M. Furuya,
S. Tanaka,
and
H. Hagiwara.
Stimulation by C-type natriuretic peptide of the differentiation of clonal osteoblastic MC3T3-E1 cells.
Biochem. Biophys. Res. Commun.
221:
703-707,
1996[Medline].
15.
Katagiri, T.,
A. Yamaguchi,
T. Ikeda,
S. Yoshiki,
J. M. Wozney,
V. Rosen,
E. A. Wang,
H. Tanaka,
S. Omura,
and
T. Suda.
The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human morphogenetic protein-2.
Biochem. Biophys. Res. Commun.
172:
295-299,
1990[Medline].
16.
Langston, A. W.,
and
L. J. Gudas.
Identification of a retinoic acid responsive enhancer 3' of the murine homeobox gene Hox-1.6.
Mech. Dev.
38:
217-227,
1992[Medline].
17.
Lian, J. B.,
V. Shalhoub,
F. Aslam,
B. Frenkel,
J. Green,
M. Hamrah,
G. S. Stein,
and
J. L. Stein.
Species-specific glucocorticoid and 1,25-dihydroxyvitamin D3 responsiveness in mouse MC3T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression.
Endocrinology
138:
2117-2127,
1997[Abstract/Free Full Text].
18.
Lichtler, A.,
M. L. Stover,
J. Angilly,
B. Kream,
and
D. W. Rowe.
Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D3.
J. Biol. Chem.
264:
3072-3077,
1989[Abstract/Free Full Text].
19.
López-Barahona, M.,
M. Miñano,
E. Mira,
T. Iglesias,
H. G. Stunnenberg,
A. Rodríguez-Peña,
J. Bernal,
and
A. Muñoz.
Retinoic acid posttranscriptionally up-regulates proteolipid protein gene expression in C6 glioma cells.
J. Biol. Chem.
268:
25617-25623,
1993[Abstract/Free Full Text].
20.
Lotan, R.
Retinoids as modulators of tumor cells invasion and metastasis.
Semin Cancer Biol.
2:
197-208,
1991[Medline].
21.
MacDonald, P. N.,
C. A. Haussler,
C. M. Terpening,
M. A. Galligan,
M. C. Reeder,
G. K. Whitfield,
and
M. R. Haussler.
Baculovirus-mediated expression of the human vitamin D receptor. Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor.
J. Biol. Chem.
266:
18808-18813,
1991[Abstract/Free Full Text].
22.
Mahonen, A.,
and
P. H. Maenpaa.
Steroid hormone modulation of vitamin D receptor levels in human MG-63 osteosarcoma cells.
Biochem. Biophys. Res. Commun.
205:
1179-1186,
1994[Medline].
23.
Noda, M.,
R. L. Vogel,
A. M. Craig,
J. Prahl,
H. F. DeLuca,
and
D. T. Denhardt.
Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression.
Proc. Natl. Acad. Sci. USA
87:
9995-9999,
1990[Abstract].
24.
Ohyama, Y.,
K. Ozono,
M. Uchida,
T. Shinki,
S. Kato,
T. Suda,
O. Yamamoto,
M. Noshiro,
and
Y. Kato.
Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene.
J. Biol. Chem.
269:
10545-10550,
1994[Abstract/Free Full Text].
25.
Oldberg, A.,
B. Jirskog-Hed,
S. Axelsson,
and
D. Heinegard.
Regulation of bone sialoprotein mRNA by steroid hormones.
J. Cell Biol.
109:
3183-3186,
1989[Abstract].
26.
Owen, T. A.,
M. S. Aronow,
L. M. Barone,
B. Bettencourt,
G. S. Stein,
and
J. B. Lian.
Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures.
Endocrinology
128:
1496-1504,
1991[Abstract].
27.
Pfeifer, A.,
A. Aszodi,
U. Seidler,
P. Ruth,
F. Hofmann,
and
R. Fässler.
Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II.
Science
274:
2082-2086,
1996[Abstract/Free Full Text].
28.
Porter, J. G.,
A. Arfsten,
F. Fuller,
J. A. Miller,
L. C. Gregory,
and
J. A. Lewicki.
Isolation and functional expression of the human atrial natriuretic peptide clearance receptor cDNA.
Biochem. Biophys. Res. Commun.
171:
796-803,
1990[Medline].
29.
Prins, B. A.,
M. J. Weber,
R.-M. Hu,
A. Pedram,
M. Daniels,
and
E. R. Levin.
Atrial natriuretic peptide inhibits mitogen-activated protein kinase through the clearance receptor.
J. Biol. Chem.
271:
14156-14162,
1996[Abstract/Free Full Text].
30.
Suda, M.,
Y. Ogawa,
K. Tanaka,
N. Tamura,
A. Yasoda,
T. Takigawa,
M. Uehira,
H. Nishimoto,
H. Itoh,
Y. Saito,
K. Shiota,
and
K. Nakao.
Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide.
Proc. Natl. Acad. Sci. USA
95:
2337-2342,
1998[Abstract/Free Full Text].
31.
Sudoh, T.,
N. Minamino,
K. Kangawa,
and
H. Matsuo.
C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain.
Biochem. Biophys. Res. Commun.
168:
863-870,
1990[Medline].
32.
Tanaka, S.,
M. Haji,
R. Takayanagi,
S. Tanaka,
Y. Sugioka,
and
H. Nawata.
1,25-Dihydroxyvitamin D3 enhances the enzymatic activity and expression of the messenger ribonucleic acid for aromatase cytochrome P450 synergistically with dexamethasone depending on the vitamin D receptor level in cultured human osteoblasts.
Endocrinology
137:
1860-1869,
1996[Abstract].
33.
Wang, Y. Z.,
and
S. Christakos.
Retinoic acid regulates the expression of the calcium binding protein, calbindin-D28K.
Mol. Endocrinol.
9:
1510-1521,
1995[Abstract].
34.
Yanaka, N.,
J. Kotera,
and
K. Omori.
Isolation and characterization of the 5'-flanking regulatory region of the human natriuretic peptide receptor-C gene.
Endocrinology
139:
1389-1400,
1998[Abstract/Free Full Text].
35.
Yanaka, N.,
J. Kotera,
I. Taguchi,
M. Sugiura,
K. Kawashima,
and
K. Omori.
Structure of the 5'-flanking regulatory region of the mouse gene encoding the clearance receptor for atrial natriuretic peptide.
Eur. J. Biochem.
237:
25-34,
1996[Abstract].
36.
Zhang, R.,
P. Ducy,
and
G. Karsenty.
1,25-Dihydroxyvitamin D3 inhibits osteocalcin expression in mouse through an indirect mechnism.
J. Biol. Chem.
272:
110-116,
1997[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 275(6):E965-E973
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society