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J. Biol. Chem., Vol. 275, Issue 26, 20197-20203, June 30, 2000
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From the ¶ Department of Bioengineering, University
of Washington, Seattle, Washington 98195 and the
Received for publication, November 16, 1999, and in revised form, March 15, 2000
Osteopontin (OPN) is a non-collagenous,
glycosylated phosphoprotein associated with biomineralization in
osseous tissues, as well as ectopic calcification. We previously
reported that osteopontin was co-localized with calcified deposits in
atherosclerotic lesions, and that osteopontin potently inhibits calcium
deposition in a human smooth muscle cell (HSMC) culture model of
vascular calcification. In this report, the role of phosphorylation in osteopontin's mineralization inhibitory function was examined. The
ability of OPN to inhibit calcification completely depended on
post-translational modifications, since bacteria-derived recombinant OPN did not inhibit HSMC mineralization. Following casein kinase II
treatment, phosphorylated OPN (P-OPN) dose-dependently
inhibited calcification of HSMC cultured in vitro about as
effectively as native OPN. The inhibitory effect of osteopontin
depended on the extent of phosphorylation. To determine the specific
structural domains of OPN important for inhibition of calcification, we
compared OPN fragments (N-terminal, C-terminal, and full-length), and
compared the inhibitory effect of both phosphorylated and
non-phosphorylated fragments. While none of the non-phosphorylated OPN
fragments effected calcification, P-OPN caused dose dependent
inhibition of HSMC calcification. P-OPN was treated with alkaline
phosphatase to create dephosphorylated OPN. Dephosphorylated OPN did
not have an inhibitory effect on calcification. The expression of OPN
mRNA and P-OPN secretion by HSMC were decreased in a
time-dependent manner during culture calcification. These
results indicate that phosphorylation is required for the inhibitory
effect of OPN on HSMC calcification, and that regulation of OPN
phosphorylation represents one way in which mineralization may be
controlled by cells.
Vascular calcification is often encountered in the development of
atherosclerotic intimal lesions and is a common consequence of aging
(1). In diabetic patients and individuals with renal failure, vascular
calcification contributes to both the morbidity and mortality
associated with these diseases (2). For example, vascular calcification
is positively correlated with increased risk of myocardial infarction
and increased risk of dissection following angioplasty (3). Moreover,
calcification is a major cause of failure for both native and tissue
prosthetic heart valves, affecting 1-2% of the aging population (4).
Until recently, vascular calcification was considered to be a passive,
degenerative, and end-stage process of vascular disease. However, bone
morphogenetic proteins including bone morphogenetic proteins-2, and
noncollagenous bone matrix proteins such as osteopontin, osteonectin,
osteocalcin, and matrix Gla protein have been demonstrated in calcified
vascular tissues (5-8). In addition, vascular cell calcification
in vitro was regulated by calcitropic hormones such as
parathyroid hormone-related peptide (9) and vitamin D (10), as well as
lipid oxidation products (11). These findings suggest that the process
of vascular calcification may share some mechanisms with mineralization
seen in bone, cartilage, and teeth, and that vascular calcification is
in fact an actively regulated process.
OPN1 is a secreted,
glycosylated phosphoprotein found normally in mineralized tissues such
as bones and teeth, in addition to kidney, urine, and epithelial lining
cells of numerous organs. OPN is associated with calcified deposits in
soft tissues, such as Monckeberg's sclerosis, aortic stenosis,
prosthetic valves, renal stones, and tumor-associated calcifications.
We and others have reported that OPN is abundant at sites of
calcification in atherosclerotic plaques and in calcified aortic valves
(7, 12). OPN is a multifunctional protein that promotes cell adhesion and migration (13), inhibits hydroxyapatite formation (14), and binds
Ca2+ (15). OPN can exist in multiple forms depending on the
extent of post-translational modification. In addition to sulfation
(16), glycosylation (17), and transglutamination (18), osteopontin can
undergo extensive phosphorylation. A highly phosphorylated form of OPN
can be isolated from the mineralized extracellular matrix of bone
tissue (19) and is synthesized by osteoblasts (20, 21). Breast milk has
also been shown to contain highly phosphorylated OPN (22). In some
cells, OPN phosphorylation is highly regulated. For example, normal rat
kidney cells as well as smooth muscle cells secrete both phosphorylated
and non-phosphorylated OPN (23, 24). Likewise, JB6 epidermal cells
treated with phorbol esters secrete phosphorylated OPN while JB6 cells
treated with vitamin D3 secrete non-phosphorylated OPN
(25). While an extensive tissue survey has yet to be performed, it is
likely that tissue-specific expression of OPN differs not only in
protein levels but phosphorylation state. Such differences in the
extent of phosphorylation of OPN may be important in OPN's
physiological function, in particular, in the formation of mineralized tissues.
Previously we reported that native smooth muscle derived-OPN inhibited
calcium deposition in a bovine smooth muscle cell calcification system,
and that OPN was localized to the surface of calcified deposits (26).
In this study, we investigated the role of phosphorylation in OPN's
ability to inhibit calcification in vitro. We found that bacterial-derived recombinant OPN (reOPN) containing no
post-translational modifications did not inhibit HSMC mineralization,
while native OPN derived from rat neonatal smooth muscle cells
inhibited HFSMC culture calcification. The ability of OPN to inhibit
mineralization could be restored to reOPN using casein kinase II (CKII)
to generate phosphorylated OPN (P-OPN). P-OPN dose dependently
inhibited calcification and was about as effective as native OPN. The
inhibitory effect of osteopontin on HSMC culture calcification was
strictly dependent on the number of phosphorylated sites. Moreover,
phosphorylated OPN treated with alkaline phosphatase to generate
dephosphorylated OPN did not inhibit HSMC culture calcification.
Finally, both the expression of endogenous OPN mRNA and
phosphorylated OPN secretion decreased in a time-dependent
manner during HSMC culture calcification. These results indicated that
phosphorylation of OPN is required for its inhibitory effects on HSMC
biomineralization, and that this is an actively regulated process in
HSMC probably contributing to the propensity of the cultures to calcify.
Reagents--
Dulbecco's modified Eagle's medium (high
glucose, 4.5 g/liter of glucose) (DMEM) and fetal bovine serum (FBS)
were purchased from Life Technologies, Inc. (Grand Island, NY). Casein
kinase II was purchased from Calbiochem (LA Jolla, CA).
H3[32P]O4,
[ Native Osteopontin and Neutralizing Antibody--
Native OPN was
purified from the conditioned medium of rat neonatal smooth muscle cell
cultures as described previously (29). This preparation was judged to
be >95% pure, on the basis of Coomassie staining and N-terminal
sequence analysis. Goat anti-rat osteopontin antibody OP-199 and
non-immune goat serum were prepared, and IgG fractions were purified as
described previously (27).
Generation of ReOPN Fragments--
Full-length human reOPN was
generated as described previously (27). An expression plasmid
containing histidine-tagged protein was generated by cloning a
polymerase chain reaction fragment containing the full-length splice
variant of human OPN (OPN10), amino acid residues 1-317, into the
BamHI site of vector pQE30 (Qiagen, Chatsworth, CA).
Escherichia coli transformed with the His-OPN plasmid was
grown in LB with 100 µg/ml ampicillin and induced with
isopropyl-1-thio-
Osteopontin N- and C-terminal proteins were generated by thrombin
cleavage of bacterially expressed GST-OPN fusion proteins. Expression
plasmids containing GST-OPN were generated by cloning polymerase chain
reaction-amplified N-terminal (amino acid residues 17-169) and
C-terminal (amino acid residues 170-317) osteopontin fragments into
BamHI/EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). The N-terminal 10N and 30N fragments were amplified from
cDNAs encoding two different splice forms of OPN, OP10, and OP30,
respectively. The 30N fragment is identical to the 10N fragment except
that it includes the alternate splice exon 5 (amino acid residues
59-72). The C-terminal 10C fragment was amplified from OP10. The
plasmid OP10 was provided by Dr. Larry Fisher (28). OP30 was obtained
from ATCC (29). The GST-OPN fusion constructs were DNA sequence
verified. E. coli JM109 cells transformed with these GST-OPN
plasmids were grown in LB with 150 µg/ml ampicillin and then induced
with 0.1 mM
isopropyl-1-thio- Cell Culture--
HSMC were obtained by enzymatic digestion as
described previously (30). Briefly, medial tissues were separated from
segments of human fetal aorta obtained at autopsy. Small pieces of
tissue (1 to 2 mm3) were digested overnight in DMEM
supplemented with 165 units/ml collagenase type I, 15 units/ml elastase
type III, and 0.375 mg/ml soybean trypsin inhibitor at 37 °C. Single
cell suspensions were placed in 6-well plates and cultured for several
weeks in DMEM supplemented with 20% FCS at 37 °C in a humidified
atmosphere containing 5% CO2. Cultures which formed
colonies were collected at confluence and maintained in growth medium
(DMEM containing 15% FBS and 1 mM sodium pyruvate
supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin;
final inorganic phosphate concentration = 1.4 mM).
Purity of cultures was assessed by positive immunostaining for Induction of Calcification--
HSMCs were routinely subcultured
in growth medium. At confluence, the cells were switched to
calcification medium (DMEM containing 15% FBS in the presence of 2 mM inorganic phosphate (unless otherwise stated)
supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin)
for up to 14 days. The medium was replaced with fresh medium every 2 days. For time course experiments, the first day of culture in
calcification medium was defined as day 0.
Quantification of Calcium Deposition--
Cells were decalcified
with 0.6 N HCl for 24 h. The calcium content of HCl
supernatants was determined colorimetrically by the
o-cresolphthalein complexone method (Calcium Kit; Sigma) as described previously (9). After decalcification, the cells were washed
three times with phosphate-buffered saline and solubilized with 0.1 N NaOH, 0.1% sodium dodecyl sulfate (SDS). The protein content was measured with a BCA protein assay kit (Pierce, Rockford, IL). The calcium content of the cell layer was normalized to protein content.
Preparation of P-OPN--
The reOPNs (10 µg) were
phosphorylated in the presence of 0.3 mM ATP with or
without [ RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from HSMCs by extraction with Trizol as suggested by the
manufacturer (Life Technologies, Inc.). Twenty micrograms of total RNA
were electrophoresed on 1% agarose gels containing formaldehyde, and
transferred to a nylon filter (Zeta-Probe GT, Bio-Rad). Blots were
prehybridized at 42 °C for 1 h in a buffer containing 50%
formamide, 0.75 M NaCl, 50 mM Tris-HCl (pH
7.5), 1% SDS, 10% dextran sulfate, 20 µg/ml denatured salmon sperm
DNA, and 1× Denhardt's solution and then hybridized at 42 °C for
24 h with cDNA probe for human OPN which was labeled with
[ Preparation of Dephosphorylated OPN--
5 µg of P-OPN was
dephosphorylated in the presence of 2 units of alkaline phosphatase in
50 mM HEPES (pH 10), 1 mM MgCl2 for up to 24 h at 37 °C. The samples were analyzed by SDS-PAGE on 10% gels, and radiolabeled proteins were detected by autoradiography.
Metabolic Labeling of HSMCs and Immunoprecipitation of
OPN--
HSMCs were cultured in DMEM supplemented with 15% FBS until
confluent and then switched to calcification medium containing 2 mM phosphate. HSMC were grown in the calcification medium
for 10 days to promote mineralization. To detect phosphorylated OPN, HSMC were incubated in phosphate-free DMEM for 30 min, followed by
incubation in phosphate-free DMEM (1 ml/dish) containing
[32P]orthophosphate (1 mCi of 32P/dish) for
6 h. After 6 h incubation, the medium was carefully collected. The supernatants were immunoprecipitated with anti-OPN antibody (OP-199) or a goat IgG as a negative control at 4 °C. Immune complexes were recovered by binding to protein A-Sepharose and
washing five times with IP wash buffer (50 mM Hepes, pH
7.4, 50 mM NaCl, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 5 mM EDTA, 1% Nonidet
P-40, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 200 µM phenylmethylsulfonyl fluoride). The immunoprecipitated proteins were suspended in 20 µl of sample buffer (0.07 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, and 0.01%
bromphenol blue). The samples were analyzed by SDS-PAGE on 10% gels,
and radiolabeled proteins were detected by autoradiography. Western
blot confirmed that the isolated protein was OPN.
Statistics--
Data were analyzed for statistical significance
by ANOVA with post-hoc Scheffe's F analysis, unless otherwise stated.
These analyses were performed with the assistance of a computer program (StatView version 4.11, Abacus Concepts, Berkeley, CA).
We have developed an in vitro model for human vascular
calcification. In this system elevating inorganic phosphate to the hyperphosphatemic range (2 mM) induced matrix
calcification. We first examined the effect of native smooth muscle
cell-derived OPN (native OPN) on HSMC calcification. Native OPN was
previously shown to be both phosphorylated and glycosylated (24).
Native OPN inhibited HSMC calcification in a dose-dependent
manner (calcified control (vehicle-treated cells) versus 15 nM native OPN-treated cells: 153.4 ± 27.1 versus 62.7 ± 4.8 (µg/mg protein), mean ± S.D.
(n = 3)) (Fig.
1A). This finding was
consistent with previous studies showing that native OPN inhibited
bovine smooth muscle cell calcification (26). We next examined the
effect of bacterial-derived rat and human reOPNs on HSMC calcification.
In contrast to native OPN, both rat and human reOPNs dose dependently
promoted calcification (calcified control (vehicle-treated cells)
versus 15 nM rat reOPN-treated cells: 153.4 ± 27.1 versus 244.6 ± 31.5 (µg/mg of protein),
mean ± S.D. (n = 3)) (calcified control
(vehicle-treated cells) versus 15 nM human
reOPN-treated cells: 153.4 ± 27.1 versus 254.4 ± 14.9 (µg/mg protein), mean ± S.D. (n = 3))
(Fig. 1, B and C). Since the bacterial products
contain neither phosphorylation nor glycosylation, these data suggested
that the ability of OPN to inhibit calcification was dependent on
post-translational modification.
In order to compare the bioactivity of phosphorylated and
non-phosphorylated OPN, human reOPN was phosphorylated with CKII. CKII
phosphorylated OPN in a time-dependent manner for up to 90 min (Fig. 2). A mean molar ratio of
phosphate:OPN of approximately 20 was achieved (Table
I). This is in good agreement with the number of putative CKII phosphorylation sites found in the human OPN
sequence (23).
Phosphorylation of Osteopontin Is Required for Inhibition of
Vascular Smooth Muscle Cell Calcification*
§,
Second
Department of Internal Medicine, Osaka City University Medical School,
Osaka 545, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, and [
-32P]dCTP were
obtained from NEN Life Science Products Inc. (Boston, MA). Unless
otherwise mentioned, all other reagents were obtained from Sigma.
-D-galactopyranoside at 37 °C to express the histidine-tagged protein. The reOPN was purified from bacterial cells according to the manufacturer's instructions (QIA expression kit, Qiagen), chromatographed on
Ni2+-nitrilotriaacetic acid resin, and eluted with 0.2 M imidazole. The purified reOPN was analyzed by
SDS-PAGE.
-D-galactopyranoside for 2 h at
37 °C to express the fusion proteins. The GST-OPN fusion proteins
were purified basically according to the manufacturer's instructions
(GST gene fusion system, Amersham Pharmacia Biotech) with
glutathione-Sepharose beads. The OPN N- or C-terminal fragments were
separated from GST-bound beads by treating with 0.1 unit of
biotinylated thrombin/µg of GST-OPN (Novagen, Madison, WI) for 2 h. The cleavage reaction was stopped with biotinylated-PPACK (400 ng/unit of biotinylated thrombin). Supernatants were collected and
biotinylated thrombin and PPACK were removed by incubation with
streptavidin-agarose beads (Pierce) and separation of beads from supernatant.
-SM
actin and calponin, and absence of von Willebrand factor staining as
described previously (30). HSMC up to passage 8 were used for these experiments.
-32P]ATP (specific activity 1 µCi/mmol) and
100 ng of CKII in 100 µl of assay buffer (20 mM HEPES, pH
7.5, 15 mM NaCl, 12 mM MgCl2). At
various times during the reaction, incorporation of
[-32P]ATP into proteins was monitored by spotting 1 µg of proteins on glasswool, followed by washing with 5%
trichloroacetic acid to remove unincorporated
[-32P]ATP and counting incorporated 32P in
5 ml of liquid scintillant. Incorporation of 32P into
proteins was evaluated by SDS-PAGE on 10% gels, and radiolabeled proteins were detected by autoradiography. Western blot confirmed that
the isolated protein was OPN.
-32P]dCTP (3000 Ci/ml; NEN Life Science Products
Inc., Boston, MA) by use of a random priming method (Megaprime cDNA
labeling system, Amersham Pharmacia Biotech). Blots were washed and
autoradiographed with x-ray film at 
70 °C. The amounts of RNA were
quantified by densitometric scanning and normalized by comparison with GAPDH.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Effect of native OPN and reOPN on HSMC
calcification. HSMCs were cultured in growth medium or
calcification medium for 4 days in the presence of indicated
concentration of: A, native OPN or its vehicle alone;
B, rat reOPN or its vehicle alone; or C, human
reOPN or its vehicle alone. The calcium contents were measured by the
o-cresolphthalein complexone method, normalized by cellular
protein content, and are presented as mean ± S.D.
(n = 3). The differences compared with calcified
control were statistically significant (*, p < 0.01, Scheffe's test). The + and indicate the presence and absence
of calcification medium, respectively.
View larger version (38K):
[in a new window]
Fig. 2.
Time course of OPN phosphorylation by
CKII. A, phosphorylation was performed by incubation of
15 nM OPN with CKII for the indicated times as described
under "Materials and Methods." Incorporation of 32P
into proteins was evaluated by trichloroacetic acid precipitation and
SDS-PAGE on 10% gels. A, time course of trichloroacetic
acid precipitable counts/min in OPN following incubation with CKII.
Data represent mean ± S.D. (n = 3). B,
radiolabeled P-OPN was electrophoresed, transferred to membrane, and
detected by autoradiography (C). Immunoblot was performed
with anti-rat OPN polyclonal antibody. The position of migration of
molecular weight markers (kDa) is indicated on the left
side.
Incorporated phosphate per mole of OPN
-32P]ATP and counting incorporated 32P in 5 ml of
liquid scintillant. The data indicate mean molar ratio of
phosphate:OPN and are presented as mean ± S.D., n = 3.
We next examined the effect of P-OPN on HSMC calcification. P-OPN
inhibited calcification in a dose-dependent manner, and at
75 nM P-OPN, calcium deposition decreased to 31% of
control cultures (calcified control (vehicle-treated cells)
versus 75 nM phosphorylated OPN-treated cells:
142.2 ± 2.5 versus 42.8 ± 5.0 µg/mg of
protein, mean ± S.D. (n = 3)) (Fig.
3A). To determine whether the
extent of OPN phosphorylation effected its inhibitory potential, we
prepared differentially phosphorylated OPNs by incubating reOPN with
CKII for limiting periods of times. OPN was phosphorylated with CKII
for times ranging from 5 to 90 min. As shown in Fig. 3B,
P-OPN inhibited calcification in proportion to the extent of
phosphorylation (calcified control vehicle-treated cells
versus 15 nM OPN phosphorylated with CK II for
90 min-treated cells: 169.6 ± 1.5 versus 73.8 ± 7.9 µg/mg of protein, mean ± S.D. (n = 3))
(Fig. 3B).
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To identify the specific phosphorylated domain of OPN important for
inhibition of calcification, we made use of reOPN N- and C-terminal
fragments. These fragments represent the fragments that result from
thrombin cleavage of osteopontin at serine 169. The N-terminal fragment
contains amino acids 1-169 and the C-terminal fragment contains amino
acids 170-317 of human OPN. In addition, two different N-terminal
fragments were prepared, 10N and 30N, representing two alternative
splice variants described for human OPN (28, 29). The 10N fragment
differs from the 30N fragment by deletion of exon 5. This exon encodes
14 amino acids including potential sites for phosphorylation (28, 29).
Each fragment was phosphorylated by incubating with CKII for 90 min. As
shown in Table I, mean molar ratios of phosphate:30N-terminal,
10N-terminal, 10C-terminal OPN fragments of 12.1, 9.7, and 8.6, respectively, were achieved. The slightly lower level of
phosphorylation of the 10N fragment compared with the 30N fragment is
likely due to the extra phosphorylation sites present in the 30N
fragment that contains exon 5 (29). The effect of N-terminal and
C-terminal P-OPN fragments on HSMC calcification was then investigated.
Whereas nonphosphorylated OPN fragments did not significantly decrease calcification, all phosphorylated OPN fragments potently inhibited calcification (calcified control (vehicle-treated cells)
versus 15 nM phosphorylated OPN (full-length)
versus 15 nM phosphorylated 30N-OPN
versus 15 nM phosphorylated 10N-OPN
versus 15 nM phosphorylated 10C-OPN-treated
cells: 145.0 ± 10.2 versus 27.3 ± 3.1 versus 25.8 ± 0.6 versus 27.6 ± 3.5 versus 20.6 ± 0.6 µg/mg protein, mean ± S.D.
(n = 3)) (Fig.
4A). These data suggested that
the organization of phosphate groups guided by OPN primary structure in
both the N- and C-terminal fragments were most critical for
anticalcification properties of OPN. Furthermore, these data indicate
that anticalcification properties of OPN are RGD-independent in this
in vitro model system. This is consistent with previous
observations that OPN's ability to bind and block hydroxyapatite
crystal growth most likely explains its ability to inhibit
biomineralization in vitro (26, 31).
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We next examined the effect of alkaline phosphatase on OPN's ability
to inhibit HSMC calcification. P-OPN was dephosphorylated by alkaline
phosphatase treatment as confirmed by 10% SDS-PAGE (Fig.
5A) with no loss of
osteopontin protein (Fig. 5B). Although P-OPN inhibited
calcification, after treatment with alkaline phosphatase, calcium
deposition was restored (calcified control (vehicle-treated cells)
versus recombinant OPN versus phosphorylated OPN
versus dephosphorylated OPN-treated cells: 147.3 ± 9.6 versus 172.5 ± 5.8 versus 44.6 ± 5.2 versus 162.4 ± 10.4 µg/mg of protein, mean ± S.D. (n = 3)) (Fig. 5C). These data
suggested that alkaline phosphatase could be a physiological regulator
of OPN's anticalcification activity.
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Finally, we examined the expression and phosphorylation state of
endogenous OPN during in vitro calcification of HSMC
cultures by Northern blot analysis. A 1.6-kilobase OPN mRNA was
detected in both calcified and non-calcified HSMC. The expression of
OPN mRNA was clearly decreased during the calcification process
(Fig. 6). To determine the
phosphorylation state of OPN during culture calcification, HSMC were
metabolically labeled by the addition of
[32P]orthophosphate to the culture medium. The labeled
osteopontin in the medium was immunoprecipitated and separated by 10%
SDS-PAGE and visualized by autoradiography. While a strong band
corresponding to phosphorylated OPN was visualized in non-calcifying
HSMC, no phosphorylated OPN was detected calcifying HSMC (Fig.
7A). This was not due to the
inability of the antibody to detect non-phosphorylated OPN since
Western blot analysis indicated that OPN protein was present in both
noncalcified and calcified HSMC culture supernatants (Fig.
7B). Consistent with the mRNA data, somewhat less OPN
was detected in calcified versus noncalcified cultures.
These data suggest that decreased synthesis and secretion of
phosphorylated OPN may contribute to calcification of HSMC under the
conditions used in this study.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we have demonstrated that the ability of OPN to inhibit calcification of HSMC cultures is dependent on post-translational modification. Although bacteria-derived reOPN did not inhibit HSMC culture mineralization, rat native OPN showed strong anticalcification activity. We found that reOPN phosphorylated by CKII dose dependently inhibited calcification. Inhibition of calcification was proportional to the number of phosphorylated sites in OPN. While nonphosphorylated N-terminal and C-terminal reOPN fragments did not effect HSMC culture calcification, phosphorylated versions of these fragments strongly inhibited HSMC calcification. Furthermore, OPN dephosphorylated with alkaline phosphatase did not have an inhibitory effect on HSMC culture calcification. Finally, the expression of OPN mRNA, secretion of protein, and fraction of phosphorylated osteopontin decreased during calcification. These results indicate that phosphorylation of OPN is required for its inhibitory effect on HSMC culture calcification.
We previously found that the major mineral deposited in bovine smooth muscle cell cultures was hydroxyapatite (HA) (26). OPN has a high affinity for HA (31), and was previously shown to inhibit de novo HA formation in both metastable calcium phosphate solutions, and steady state agarose (32) and gelatin (33) gels. In contrast, OPN showed no ability to nucleate HA (32). Consistent with these findings, native OPN inhibited calcification of both human (present study) and bovine smooth muscle cell cultures (26), and was shown by immunogold electron microscopy to bind to growing hydroxyapatite crystals within the extracellular matrix (26). Thus, it is likely that the ability of OPN to bind to HA and block crystal formation underlies its potent effect on vascular smooth muscle calcification in vitro.
OPN is highly anionic due to its elevated content of the acidic amino acid, aspartate, and its high degree of phosphorylation. The primary structure of OPN contains over 20 potential phosphorylation sites for various protein kinases (28, 29). Not all of these sites appear to be utilized, however, since it has been reported that rat bone OPN contains 12 phosphoserines and 1 phosphothreonine (19), bovine milk OPN contains 27 phosphoserines and 1 phosphothreonine (17), and chicken osteoblast OPN contained 7 phosphoserine and 1 phosphothreonine (33). The majority of the phosphorylations occur on serines within consensus phosphorylation motifs for casein kinases such as mammary gland Golgi casein kinase and CKII (34). Indeed, in purified systems, CKII was the predominant enzyme capable of phosphorylating chicken OPN (35), and Golgi kinase had strong activity toward rat recombinant OPN (36). Consistent with those findings, human recombinant OPN was phosphorylated with CKII and a mean molar ratio of phosphate:OPN of 20 was achieved in the present studies. Which of these enzymes phosphorylates OPN in vivo, however, is still controversial.
Our studies indicate that the presence of phosphorylated residues is particularly important for OPN's anticalcification effects in HSMC cultures. We found that bacterial-derived recombinant OPN, devoid of any post-translational modification, did not inhibit HSMC culture calcification, and on the contrary, showed a slight stimulatory effect. However, following phosphorylation with CKII, bacterial OPN was as potent as native OPN in inhibiting HSMC calcification. Furthermore, the anticalcific potency of OPN depended on the extent of phosphorylation, with minimal inhibition occurring unless >9 mol of phosphate were incorporated per mole of osteopontin. While the precise sites of phosphorylation in our CKII-treated bacterial OPN have not yet been identified, these data suggest that either specific phosphorylated sequences or arrangement of phosphorylated sequences is required for OPN function in anticalcification. In addition, dephosphorylation of P-reOPN with alkaline phosphatase completely inhibited calcification inhibitory activity. These studies are consistent with previous observations in cell-free systems, showing that treatment of OPN with alkaline phosphatase removed 84% of the covalently bound phosphate and reduced HA inhibiting activity by more than 40-fold (37). Phosphorylation has also been suggested to regulate the cell binding activity of OPN. In one study, partial dephosphorylation of bovine OPN by tartrate-resistant acid phosphatase resulted in decreased osteoclast binding (38). On the other hand, CKII treatment of recombinant rat OPN enhanced osteoclast adhesion, even though only low mean molar ratio of phosphate:OPN of approximately 1.5 was achieved (39).
The present studies are the first to use defined OPN peptide fragments to examine sequences important for OPN's calcification inhibitory activity. The data indicate that OPN's inhibitory activity on HSMC calcification is independent of the RGD sequence and polyaspartic acid domain since a fragment lacking both the RGD and polyaspartate sequences (10C) exhibited inhibitory potency equivalent to fragments which contained both domains (30N and 10N). This was somewhat unexpected, since previous studies in a cell-free system showed that poly-L-aspartic acid was nearly as potent as bone-derived OPN in inhibiting HA formation (37). One explanation of this discrepancy is that the calcium binding properties of OPN may be more important in inhibiting HA formation in cell-free systems than in our cell culture system, since the polyaspartic acid sequence and both phosphorylated and nonphosphorylated forms of OPN have been shown to bind calcium with specificity (40).
Finally, to determine whether regulation of OPN phosphorylation might occur during the development of HSMC culture mineralization, we examined endogenous OPN mRNA, OPN protein, and phosphorylated OPN levels with time in mineralizing cultures. Our data indicate that OPN mRNA levels and total as well as phosphorylated OPN protein levels decline as HSMC cultures calcify. Thus OPN synthesis as well as phosphorylation are inversely correlated with tissue culture mineralization.
Our findings suggest that regulation of phosphorylation state may be a
common mechanism controlling OPN's functional activities. Several
recent studies support this notion. Normal rat kidney cells secrete
both the phosphorylated (pp69) and non-phosphorylated (np69) form of
OPN. pp69 is cell surface-associated, whereas np69 is not. On the other
hand, np69 can form a heat-dissociable complex with fibronectin, while
pp69 cannot (23). Furthermore, phorbol ester stimulation of P-OPN in
JB6 epidermal cells was correlated with tumorigenic morphological
changes and anchorage independent growth. On the other hand, calcitriol
stimulated synthesis and secretion of nonphosphorylated OPN in JB6
cells, and these transformed cells lacked the tumorigenic properties
observed in phorbol ester-treated cells (25). These observations,
combined with our studies, suggest that phosphorylated and
nonphosphorylated forms of OPN have different functional properties.
Identification of mechanisms controlling OPN phosphorylation state is
thus of paramount interest in future studies.
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FOOTNOTES |
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* This work was supported in part by Grant R01 Hl62329-01 and National Science Foundation Grant EEC9529161.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. Section 1734 solely to indicate this fact.
§ Recipient of a grant from the Lilly Fellowship Program for Bone and Mineral Research (Ely Lilly Japan, KK, and the Japan Osteoporosis Foundation).
Established Investigator of the American Heart Association. To
whom all correspondence and reprint requests should be addressed: Bioengineering Dept., Box 351720, University of Washington, Bagley Hall
Rm. 479, Seattle, WA 98195. Tel.: 206-543-0205; Fax: 206-616-9763; E-mail: ceci@u.washington.edu.
Published, JBC Papers in Press, April 13, 2000, DOI 10.1074/jbc.M909174199
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ABBREVIATIONS |
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The abbreviations used are: OPN, osteopontin; DMEM, Dulbecco's modified Eagle's medium; HSMC, human smooth muscle cell; P-OPN, phosphorylated OPN; reOPN, recombinant OPN; CKII, casein kinase II; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hydroxyapatite; PPACK, thrombin-[D-phenylalanyl-N-[4-(aminoiminomethyl)amino]-1-[(chloroacetyl)butyl]-L-prolinamide.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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