Osteopontin inhibits inducible nitric oxide synthase
activity in rat vascular tissue
JEREMY A. SCOTT,1,3,4 M. LYNN WEIR,2,5 SYLVIA M. WILSON,2,5
JIM W. XUAN,2,5 ANN F. CHAMBERS,2,5 AND DAVID G. MCCORMACK1,3,4
1A. C. Burton Vascular Biology Laboratory, 2London Regional Cancer Centre,
London Health Sciences Centre, and Departments of 3Medicine, 4Pharmacology and Toxicology,
and 5Oncology, University of Western Ontario, London, Ontario, Canada N6A 4G5
lipopolysaccharide; sepsis; endotoxin
OSTEOPONTIN (OPN) is an integrin- and calcium-binding
phosphoprotein produced by a limited set of normal
cells, including cells of mineralized tissue, epithelial
cells, and activated cells of the immune system (4, 31).
OPN production has been reported to be increased in a
number of pathological conditions, including inflammation, atherosclerosis, nephritis, and malignancy, as well
as normal situations of morphogenesis, such as bone
remodeling (10, 31, 39). OPN is expressed in various
tissues of the vascular system in response to injury and
appears to play a role in the maintenance of normal
vascular development and function (9, 10, 12, 30). The
function of OPN in the normal and pathological contexts in which it is expressed remains poorly understood. However, many of its effects appear to be mediated by interaction of OPN, via its conserved GRGDS
(glycine-arginine-glycine-aspartic acid-serine) amino
acid sequence, with integrin molecules (especially avb3 )
(21, 41, 42), and perhaps other receptors, on the
surfaces of target cells.
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
H2258
OPN has been shown to inhibit the induction of the
inducible nitric oxide synthase (iNOS) expression and
function in kidney epithelial cells in culture (13),
suggesting that OPN may play a role in regulating the
nitric oxide (NO) synthetic pathway in the kidney. OPN
has also been demonstrated to inhibit induced NO
production and reduce cytotoxic activity in macrophages (35, 36). An OPN fragment has also been shown
to modulate iNOS mRNA levels in microvascular endothelial cells from the heart (40). On the basis of these
findings, coupled with the demonstration that OPN
produced by tumor cells can contribute functionally to
their malignancy (1, 8, 28) and that NO mediates the
cytotoxic activity of macrophages (11), it has been
hypothesized that OPN produced by tumor cells could
function to protect them from NO-mediated cytotoxic
attack by host vascular and immune tissues (3, 7).
We tested the hypothesis that OPN inhibits the
induction of iNOS in vascular tissue. Rat thoracic
aortas were treated with lipopolysaccharide (LPS) to
induce iNOS activity, and the effect of added recombinant mouse OPN on iNOS and constitutive NO synthase (cNOS) activities was determined. OPN proteins
lacking the RGD sequence (and, therefore, unable to
bind to integrins) or with a substituted RGE (arginineglycine-glutamic acid) sequence, which binds poorly to
integrins, were also used to address the requirement
for the RGD sequence in OPN inhibition of iNOS
activity. Furthermore, Western blotting and RT-PCR
were used to assess iNOS protein and mRNA levels,
respectively, in the vascular tissue. These investigations provide evidence that OPN and NOS expression
may be functionally linked in vascular tissues.
METHODS
Preparation of Recombinant OPN
Recombinant mouse OPN was expressed in Escherichia
coli as a glutathione S-transferase (GST)-fusion protein and
purified as described previously (42, 43). Mutated OPN
proteins in which the RGD sequence was deleted (and thus
unable to bind to integrins) or substituted with RGE (and
thus poorly bound to integrins) were produced by sitedirected mutagenesis, as described previously (43). GST
protein was used as a control.
Incubation of Thoracic Aorta
Male Sprague-Dawley rats (280–350 g) were anesthetized
with pentobarbital sodium (50 mg/kg ip) and killed by cervical dislocation and exsanguination. Thoracic aortas were
flushed with Krebs solution (pH 7.4; in mM: 118 NaCl, 4.7
KCl, 2.5 CaCl2, 1.2 MgSO4 · 7H2O, 1.2 KH2PO4, 11 dextrose, 22
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
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Scott, Jeremy A., M. Lynn Weir, Sylvia M. Wilson, Jim
W. Xuan, Ann F. Chambers, and David G. McCormack.
Osteopontin inhibits inducible nitric oxide synthase activity
in rat vascular tissue. Am. J. Physiol. 275 (Heart Circ.
Physiol. 44): H2258–H2265, 1998.—We tested the hypothesis
that osteopontin (OPN) can inhibit the induction of inducible
nitric oxide synthase (iNOS) in vascular tissue. iNOS activity
was induced in rat thoracic aortas by incubation of the tissue
with lipopolysaccharide (LPS) and measured by conversion of
L-[3H]arginine to L-[3H]citrulline. Addition of $1 nM recombinant OPN protein significantly reduced the LPS-induced
increase in iNOS activity. Western blotting and the RT-PCR
were used to determine the effect of LPS with and without
OPN on tissue levels of iNOS protein and RNA, respectively.
LPS resulted in an increase in iNOS protein and RNA,
whereas OPN dose-dependently reduced tissue levels of iNOS
activity, protein, and RNA. Mutated OPN proteins, in which
the integrin-binding RGD amino acid sequence was deleted or
mutated to RGE, resulted in complete and partial loss,
respectively, of the ability of OPN to inhibit LPS-induced
iNOS activity, implicating integrin binding in the effect.
These results indicate that OPN can prevent induction of
iNOS in vascular tissue.
OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
NOS Assay
After homogenization in ice-cold homogenization buffer,
NOS activity was quantitated in thoracic aortas as the
conversion of L-[3H]arginine to L-[3H]citrulline, as previously
described (38). The protein concentrations of rat aortic homogenates were determined by the Bradford method (2), with
BSA as the standard and homogenization buffer as the blank.
Calcium-dependent (constitutive) NOS activity was calculated as the difference between a sample containing calcium
and calmodulin (cNOS 1 iNOS activities) and one containing
EDTA/EGTA (iNOS activity only). Nonspecific radioactivity
and/or metabolism of L-arginine was accounted for by incubating homogenization buffer or tissue homogenate with 10 µM
NG-nitro-L-arginine methyl ester (an inhibitor of the NOSmediated conversion of L-arginine), respectively, in the incubation buffer containing EDTA/EGTA, as described previously
(38). Resultant enzyme activities were expressed as picomoles of L-[3H]citrulline produced per minute per milligram
of protein.
Cell Culture
RAW 264.7 mouse macrophage cells (American Type Culture Collection, Rockville, MD) were grown at 37°C in 5% CO2
in DMEM with 10% fetal bovine serum (GIBCO BRL, Burlington, ON, Canada). Cells grown to confluence were untreated
(control) or treated with 20 ng/ml LPS plus 50 U/ml murine
interferon-g (IFN-g) for 6 h to induce iNOS.
Rat aortic smooth muscle cell cultures were grown at 37°C
in 5% CO2 in DMEM with 10% calf serum and 5% fetal bovine
serum. Cells grown to confluence were untreated (control) or
treated with 250 µg/ml LPS plus 1,000 U/ml murine IFN-g for
10 h to induce iNOS.
Western Blots
Rat thoracic aortas were homogenized in five volumes of
cold homogenization buffer (pH 7.4; 10 mM Tris, 1 mM EDTA,
2 µg/ml leupeptin, 1 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride). An aliquot of 20% SDS was added to give
1% final concentration, and samples were boiled for 5 min.
Protein extracts were prepared from cell cultures (RAW 264.7
and rat aortic smooth muscle cells) by washing each culture
dish twice with PBS (pH 7.5; in mM: 137 NaCl, 2.7 KCl, 8.15
Na2HPO4, 1.5 KH2PO4 ) and adding boiling buffer (pH 7.4; 10
mM Tris, 1% SDS, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 0.5
mM phenylmethylsulfonyl fluoride). Each cell lysate was
scraped from the dish, boiled 5 min, and passed through a
26-gauge needle. All samples were centrifuged at 16,000 g for
5 min to precipitate insoluble material, each supernatant was
removed, and its protein concentration was determined by
Peterson’s modification (32) of the Lowry method, with BSA
as the standard.
Proteins (5 µg each for RAW 264.7 macrophages and rat
aortic smooth muscle cells; 25 µg for rat thoracic aortic
homogenate) were separated by SDS-PAGE under reducing
conditions on a 6% separating gel and electrophoretically
transferred to Immobilon-P (Millipore, Bedford, MA). Blots
were blocked with 4% casein in Tris-buffered saline (TBS; pH
7.4; 20 mM Tris, 150 mM NaCl) for 1 h at 37°C. Blots were
then incubated overnight at room temperature in 4% casein
in TBS with a mouse iNOS-specific monoclonal antibody
(1:1,250), washed, and incubated for 3 h at room temperature
in 4% casein in TBS, with an anti-mouse Ig secondary
antibody linked to horseradish peroxidase (1:3,000). The
iNOS band was detected by enhanced chemiluminescence
protocol with Hyperfilm-ECL (Amersham, Oakville, ON,
Canada). Results were quantitated by densitometry using a
PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).
Primer Design
The rat iNOS cDNA sequence was obtained from GenBank
(accession no. X76881): 58-TCGAGCCCTGGAAGACCCACATCTG-38 (forward primer, located at nt 1654–1678) and
58-GTTGTTCCTCTTCCAAGGTGTTTGCCTTAT-38 (reverse
primer, located at nt 1901–1930). This primer set is perfectly
complementary to the rat iNOS and not complementary to
other NOS isoforms or unrelated cDNAs. Amplification of
cDNA synthesized from mRNA produces a 250-bp PCR band.
The mouse glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) sequence was obtained from GenBank (accession
no. M32599): 58-TATTGGGCGCCTGGTCACCA-38 (forward
primer, located at nt 79–98) and 58-CCACCTTCTTGATGTCATCA-38 (reverse primer, located at nt 806–825). This
primer set is perfectly matched to the rat and mouse GAPDH
cDNA. A 752-bp PCR fragment will be synthesized. Oligonucleotide primers for iNOS and GAPDH were synthesized
by the Medical Research Council Molecular Biology Core
Facility at London Regional Cancer Centre.
RT-PCR Analysis
Reverse transcription was performed using the reverse
oligonucleotide primers for rat iNOS and mouse GAPDH (as
an internal standard). One microgram of total cytoplasmic
RNA isolated from each tissue using TRIzol reagent (GIBCO
BRL) was reverse transcribed in a 20-µl reaction mixture
according to the recommended conditions in the Moloneymurine leukemia virus RT kit (GIBCO BRL). A negative
control of sterile water, in place of RNA, ensured no contamination of reaction materials.
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NaHCO3 ), removed, and cleared of surrounding connective
tissue. Each vessel was dissected into four equal segments,
such that each rat contributed a segment of thoracic aorta to
each of four groups for incubation. In each experimental
protocol, vessels were incubated for 5 h at 37°C and aerated
with 95% O2-5% CO2 in Krebs solution with or without LPS
and/or various OPN proteins, as described below. After the
incubation period the vessels were flash frozen in liquid
nitrogen and maintained at 280°C until the time of assay.
Pilot experiments documented no effect of the GST protein (to
which the recombinant OPN is linked) on the LPS induction
of iNOS activity.
To evaluate the concentration-dependent effect of OPN on
induction of iNOS activity and expression, rat thoracic aortas
were incubated with a previously determined concentration
of LPS (1 µg/ml) in Krebs solution (38). The tissues were
coincubated with and without OPN (0.1–100 nM).
The effect of OPN on cNOS and iNOS activities and
expression of iNOS in control and LPS-treated vascular
tissues were assessed. Rat thoracic aortas were incubated
with and without LPS (1 µg/ml) and with and without OPN
(10 nM). This supramaximal concentration of OPN was
selected on the basis of the results of the initial concentrationdetermination study.
To examine the role of integrin binding in the effect of OPN
on LPS-induced iNOS activity and expression in stimulated
rat vascular tissue, alternative forms of OPN, which had been
mutated at the GRGDS sequence, were used. Rat aortas were
incubated with LPS (1 µg/ml) under control conditions (no
OPN) or with the intact recombinant OPN, the RGD-deleted
form of OPN, or the RGE-mutated form of OPN (10 nM each).
H2259
H2260
OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
Chemicals
Cytoscint environmentally safe scintillation fluid and Coomassie brilliant blue G-250 were purchased from ICN Biomedical (Mississauga, ON, Canada). Orthophosphoric acid
was purchased from BDH (Toronto, ON, Canada). Murine
monoclonal iNOS-specific antibody was purchased from Transduction Laboratories (Lexington, KY). TRIzol reagent, SDS,
calf serum, DMEM, and recombinant murine IFN-g were
purchased from GIBCO BRL. Anti-mouse IgG antibody linked
to horseradish peroxidase and L-[3H]arginine were purchased
from Amersham. FCS was purchased from Whittaker Bioproducts (Walkersville, MD). E. coli LPS (serotype 026:B6) and all
other reagents were purchased from Sigma Chemical (Mississauga, ON, Canada).
Statistical Analysis
All enzyme activity and densitometry results are expressed
as means 6 SE and compared using factorial ANOVA with
Fisher’s post hoc test. P , 0.05 was considered significant. All
statistical tests were calculated using the StatView 14.5
program on a Macintosh computer.
RESULTS
NOS Activity of Rat Thoracic Aorta
Effect of OPN on control and LPS-stimulated iNOS
and cNOS activities. To determine whether OPN affected iNOS and cNOS activities in LPS-stimulated
and control tissues, vessels were incubated for 5 h with
and without LPS (1 µg/ml) in the presence of OPN
(0–10 nM). Tissues incubated with LPS alone exhibited
an elevated iNOS activity compared with control incubated tissues (Fig. 1A). Incubation of aortas with LPS
and OPN (0.1–10 nM) resulted in a progressive attenu-
Fig. 1. Effect of osteopontin (OPN) on lipopolysaccharide (LPS)mediated alteration of inducible (iNOS, A) and constitutive nitric
oxide synthase (cNOS, B) activities (pmol L-citrulline · min21 · mg
protein21 ) in rat thoracic aortas. Rat thoracic aortas incubated with
and without LPS (1 µg/ml) were coincubated with 0–10 nM OPN.
Values are means 6 SE, with number of observations in parentheses
within bars. * P , 0.05 compared with LPS only; # P , 0.05 compared
with untreated control.
ation of the LPS-induced iNOS activity, producing a
significant attenuation at $1 nM OPN.
A concomitant decrease in the cNOS activity of the
LPS-incubated tissues was demonstrated relative to
control tissues (Fig. 1B). cNOS activity in the vessels
incubated with LPS and $1.0 nM OPN was similar to
control and thus significantly greater than in vessels
incubated with LPS alone. Incubation of control vessels
with OPN (10 nM) alone did not result in any significant alteration of cNOS or iNOS activities compared
with controls. Incubation of control and LPS-stimulated tissues with the GST protein had no significant
effect on NOS activities (data not shown). We further
confirmed that OPN had no effect on induced iNOS
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The cDNAs (5 µl) produced by the RT reaction were then
amplified by the PCR by using the forward oligonucleotide
primers for iNOS and GAPDH. The PCR amplification was
performed according to the manufacturer’s protocol (GIBCO
BRL). RT-PCR conditions were optimized to ensure that the
procedure was performed in the linear portion of the reaction.
The RT-PCR products (5 µl) were identified by size on an
ethidium bromide-stained 1.5% agarose gel. The gel was then
denatured and neutralized, and the DNA capillary was
transferred onto GeneScreen Plus (NEN/DuPont, Mississauga, ON, Canada) in the presence of 103 saline-sodium
citrate. The blot was probed sequentially with a 58-end
[32P]ATP-labeled iNOS oligonucleotide probe complementary
to an internal rat iNOS-specific sequence (58-TCAGTAGCAAAGAGGACTGTGGCTCTGACG-38, nt 1785–1814) and a
denatured, oligo-labeled (deoxy-[32P]CTP) GAPDH cDNA
probe (oligo-labeling kit, Pharmacia Biotech, Baie d’Urfe, PQ,
Canada). The RT-PCR products were quantified by densitometry (PhosphorImager SI), and expression levels were calculated relative to GAPDH.
To confirm that the amplified PCR products represented
iNOS mRNA, PCR cDNA products were directly ligated into
the PCR II vector and transformed into bacteria (competent
INVaF8 cells) by the TA cloning kit (Invitrogen, San Diego,
CA). Selection was by blue-white screening and analysis by
Miniprep to verify the presence of cloned PCR products. The
products were then expanded and purified by plasmid preparation (Prep-a-Gene Kit, Bio-Rad, Mississauga, ON, Canada)
and confirmed by direct sequencing using the T7 sequencing
kit (Pharmacia Biotech).
OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
iNOS Protein Levels in Rat Thoracic Aorta
As OPN blocked the induction of iNOS activity by
LPS (Fig. 1) but was unable to directly inhibit previously induced iNOS activity, Western blotting was
performed using an antibody specific for mouse macrophage iNOS, to examine whether the OPN effect was
due to an inhibition of the upregulation of iNOS protein
levels (Fig. 3). To verify the specificity of this antibody,
iNOS protein amounts were examined in a mouse
macrophage cell line (RAW 264.7) and in cultures of rat
aortic smooth muscle cells, both of which were stimulated with LPS and IFN-g to induce iNOS. Western
blotting showed that this antibody detected an ,123kDa iNOS protein in both types of stimulated cells that
was not present in unstimulated cells (Fig. 3A). Hence,
this antibody recognized mouse macrophage iNOS and
rat vascular smooth muscle iNOS.
Analysis of rat aortic homogenates by Western blotting with this antibody showed an iNOS protein band
that comigrated with those seen in stimulated RAW
264.7 cells and rat aortic smooth muscle cells, although
the intensity of the band from the cell lines was
considerably greater than that from the tissue homogenates. A nonspecific binding band (,133 kDa) was
detected in all aortic tissue homogenate samples, even
when the iNOS antibody was omitted and only the
secondary antibody was used with aortic homogenate;
this nonspecific band was not detected in the RAW
264.7 cell line.
iNOS protein levels in each sample were determined
by densitometry and expressed relative to the LPSstimulated maximum (Fig. 3B). iNOS protein levels in
naive control and untreated aorta (control) were low
and comparable to those seen in vessels exposed to
OPN alone (NOS activities of these 2 groups were not
statistically significant, data not shown). A slight elevation of iNOS protein in tissues incubated under control
conditions (compared with naive control tissues) was
likely because of low levels of LPS normally found on
the glassware during the incubation (34). Addition of
LPS (1 µg/ml) to the incubation medium resulted in a
significant increase in aortic iNOS protein levels compared with control incubated tissues. The presence of
LPS plus increasing doses of OPN slightly, although not
significantly, attenuated iNOS protein levels relative to
LPS treatment alone, but not to the same extent as
with the iNOS enzyme activities. Coincubation of the
tissues with LPS and the RGD-deleted or RGEmutated OPN did not affect the iNOS protein levels
(not significantly different from LPS treatment alone,
n 5 3 for each group; data not shown).
iNOS RNA Levels in Rat Thoracic Aorta
To determine whether the OPN-mediated inhibition
of the LPS-induced iNOS activity was due to transcriptional regulation, iNOS RNA levels were examined by
RT-PCR followed by Southern blotting and probing
with an internal iNOS-specific oligonucleotide probe.
Analysis of the RT-PCR products from naive control
and LPS-stimulated tissues indicated significant induction of a 250-bp band that corresponded to the band
induced in rat aortic smooth muscle cells stimulated
with LPS and IFN-g (Fig. 4). Consistent with the
Western blot data (Fig. 3), this 250-bp iNOS band was
progressively decreased (again, not significantly) in
tissues coincubated with LPS and OPN (0.1–10 nM)
and, again, not to the same extent observed with the
iNOS activities.
DISCUSSION
Fig. 2. Effect of RGD-deleted and RGE-mutated OPN (10 nM each)
on LPS induction/depression of iNOS and cNOS activity (pmol
L-citrulline · min21 · mg protein21 ) in rat thoracic aortas. Values are
means 6 SE of 5 assays. * P , 0.05 compared with LPS only; # P ,
0.05 compared with untreated control (Fig. 1).
The experiments presented here indicate that OPN
can block the LPS-induced increase in iNOS activity
and reduce the expression of iNOS observed in rat
thoracic aorta and that this blockade is dependent on
the concentration of OPN used. This effect was not due
to direct inhibitory activity toward the iNOS enzyme,
as the addition of OPN to previously LPS-stimulated
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activity in tissues previously incubated for 5 h with LPS
only (18.1 6 2.7 vs. 22.1 6 1.0 pmol L-citrulline ·
min21 · mg21 for LPS-stimulated tissues with and without addition of 10 nM OPN at time of enzyme activity
determination, respectively; n 5 2 assays each).
Effect of RGD-mutated OPN on LPS-stimulated tissues. To determine whether the RGD amino acid sequence of OPN was required for the OPN blockade of
LPS-induced changes in iNOS and cNOS activities,
vessels were incubated with the RGD-deleted and
RGE-mutated forms of OPN. In contrast to the effect
seen with nonmutated OPN (Fig. 1), coincubation of
thoracic aortas with LPS and the RGD-deleted form of
OPN (10 nM) did not significantly alter the iNOS and
cNOS activities compared with LPS-incubated tissues
(n 5 5 assays; Fig. 2). Coincubation of thoracic aortas
with LPS and the RGE-mutated form of OPN (10 nM)
significantly attenuated the enhanced iNOS activity
observed with LPS-treated tissues (n 5 5 assays).
cNOS activity was also increased in the tissues incubated with LPS and RGE-mutated OPN compared with
LPS-incubated tissues (n 5 5 assays), although not to
the level of cNOS activity observed in control tissues.
H2261
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OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
tissues had no effect on iNOS activity. Furthermore,
this investigation demonstrates for the first time that
OPN blocks the LPS-induced depression of cNOS activity in the vasculature. Recombinant OPN protein from
which the RGD region had been deleted to produce a
protein that is unable to bind to cell surface integrins
was ineffective at blocking the LPS-induced increase in
iNOS. Mutated OPN in which the RGD sequence had
been mutated to RGE and, therefore, binds poorly to
integrins was only partially effective at blocking the
LPS-induced elevation of iNOS activity. Thus the effect
of OPN on the induction of iNOS in this tissue depends
on an intact integrin-binding RGD amino acid sequence
in the protein, consistent with an integrin-mediated
signal transduction process. Our study demonstrates
that OPN can modulate LPS-mediated alterations of
iNOS and cNOS in vascular tissue.
We have previously demonstrated that stimulation of
vascular tissues with LPS results in an increase in
iNOS activity, along with a concomitant decrease in
cNOS activity (38). In the present study we confirmed
this finding and found, for the first time, that OPN
coincubated with LPS was able to completely block the
LPS-mediated effects on iNOS and cNOS activities.
The decrease in cNOS activity, coordinated with an
increase in iNOS activity, has been hypothesized to be
mediated at the mRNA level through a reduction in the
stability of the cNOS mRNA (22, 44). Although this
study did not directly examine the mechanism of these
observed changes in cNOS activity, our data would be
consistent with this hypothesis and further suggest
that OPN interferes with this induction/downregulation signal transduction pathway.
In the present study, we used Western blotting and
RT-PCR to assess levels of iNOS protein and RNA,
respectively, in LPS-stimulated vascular tissue, with
and without OPN. We demonstrated that LPS increased tissue levels of iNOS protein and RNA, consistent with the observed increase in iNOS activity. Concomitant with the decrease in iNOS activity observed
in tissues coincubated with LPS and OPN, iNOS protein and RNA levels were attenuated compared with
tissue incubated with LPS alone, although not to
control levels. These results are consistent with previ-
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Fig. 3. Effect of OPN on LPS induction
of iNOS protein levels in rat thoracic
aortas. A: stimulated (1) and unstimulated (2) RAW 264.7 mouse macrophages (Mf) and rat aortic smooth
muscle cells (RASMC) and thoracic aortic homogenates from naive control (i.e.,
no incubation period), 5-h control incubated (Ctrl), and LPS-incubated tissues
with 0–10 nM OPN were probed using
an iNOS-specific primary antibody. Protein extracts from RAW 264.7 macrophages and aortic homogenates probed
with secondary antibody (2° Ab) alone
indicated a nonspecific binding (NSB)
of antibody in aortic homogenates. B:
quantification of iNOS protein from control and LPS-incubated rat thoracic aortas, normalized to LPS-stimulated
maximum, with 0–10 nM OPN. iNOS
expression is shown in stimulated (1)
and unstimulated (2) RASMC. Values
are means 6 SE, with number of observations in parentheses above bars. * P
, 0.05 compared with control or unstimulated tissues; # P , 0.05 compared
with LPS-stimulated tissues. (RASMC
were incubated with LPS and interferon-g, as described in METHODS ).
OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
H2263
ously reported findings for kidney proximal tubule
epithelial cells in culture (13), in which OPN reduced
the cytokine-induced increase in cellular levels of iNOS
protein, and with previously reported findings for cultured cardiac myocytes and endothelial cells, where
exogenous application of an OPN fragment concomitantly reduced iNOS RNA expression and protein levels
(40). However, the finding that the degree of inhibition
of induction of iNOS protein and mRNA did not precisely correlate with the observed restoration to control
levels of iNOS activity suggests that additional mechanisms of iNOS regulation exist. Recently, Eissa et al. (6)
reported that in human epithelial cells an iNOS splice
variant may be produced, which is unable to form the
active iNOS dimer. Hence, some of the increased iNOS
RNA and protein observed in our LPS-treated tissues
may produce nonfunctional iNOS protein, and the
observed reduction of RNA and protein might be sufficient
to account for the more significant attenuation of iNOS
activity. Thus we have demonstrated that OPN produces a
unique inhibition of the LPS-stimulated induction of iNOS
activity in the vasculature, which is mediated, at least in
part, by regulation of iNOS RNA and protein levels.
We have demonstrated that the presence of the RGD
amino acid sequence is essential for the OPN-mediated
blockade of the LPS-induced elevation of iNOS activity.
Although the exact mechanism of this inhibition remains unknown, the inability of the RGD-deleted OPN
to prevent the LPS-mediated induction of iNOS activity
and of the concomitant reduction in cNOS activity
(demonstrated with normal OPN) implicates integrin
binding, perhaps to avb3, and the resultant signal
transduction pathway in this response. Furthermore,
what appears to be a partial blockade of this iNOS
effect with the RGE-mutated OPN suggests a partial
agonist activity of this mutant form. This observation is
consistent with the previous finding of Hwang et al.
(13), who found that OPN was unable to block the
induction of nitrite production in stimulated kidney
epithelial tubule cells coincubated with GRGDS peptide fragments. Together, these investigations suggest
that an integrin binding-mediated event stimulates the
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Fig. 4. Effect of OPN on LPS induction of iNOS RNA in
rat thoracic aortas. A: Southern blot of iNOS and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
RT-PCR products from stimulated (1) and unstimulated (2) RASMC and rat thoracic aortas from naive
rats and vessels incubated with LPS (1 µg/ml) and 0–10
nM OPN from a representative incubation experiment,
for which tissue was obtained for Western and Southern
blotting and for NOS activity determination. B: quantification of iNOS RNA levels obtained from Southern
blotting of RT-PCR products, normalized to RNA levels
in LPS-stimulated tissues. Results are from quantification of Southern blot by PhosphorImager SI expressed
as ratio of 250-bp iNOS band to 750-bp GAPDH band to
control for variability. Values are means 6 SE, with
number of observations in parentheses above bars. * P ,
0.05 compared with control or unstimulated tissues;
# P , 0.05 compared with LPS-stimulated tissues.
H2264
OSTEOPONTIN AND INDUCIBLE NO SYNTHASE
The authors thank Dr. S. C. Pang and Judy Van Horne (Dept. of
Anatomy and Cell Biology, Queen’s University, Kingston, ON, Canada)
for providing the rat aortic smooth muscle cell culture.
This study was supported by the Ontario Thoracic Society (D. G.
McCormack and A. F. Chambers), the National Cancer Institute of
Canada Grant 6015 to A. F. Chambers, and the Medical Research
Council of Canada.
Present address of M. L. Weir: Dept. of Cell Biology, Hospital for
Sick Children, Toronto, ON, Canada M5G 1X8.
Address for reprint requests: D. G. McCormack, Div. of Respiratory Medicine, London Health Sciences Centre, Victoria Campus, 375
South St., London, ON, Canada N6A 4G5.
Received 7 July 1998; accepted in final form 28 July 1998.
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signal transduction pathway (14, 15) necessary to
produce the inhibitory effects of OPN on the LPSstimulated alteration of iNOS and cNOS.
Both of the major cellular components of vascular
tissue, namely, smooth muscle and endothelial cells,
have been shown to be capable of producing and
responding to OPN. Giachelli and co-workers (9, 10, 20,
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and repair, as well as in proliferating smooth muscle
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Uninjured rat endothelium expresses low levels of OPN
and one of its integrin receptors (avb3 ), and expression
of OPN and avb3-integrin transiently increases during
repair of vascular injury (21). Thus OPN can be produced by smooth muscle and endothelial cells under
situations of repair and morphogenesis (9). Clearly, the
interaction of these two cell types and their products
(i.e., NO and OPN) will contribute to the functional
response of vascular tissue to activating agents such as
LPS.
Excess production of NO through the action of iNOS
may be an important mechanism that contributes to
the hypotension (25) and abnormal vascular contractility characteristic of sepsis (38). Furthermore, NO has
significant effects on leukocyte adhesion and a variety
of other physiological processes that may be important
in conditions such as sepsis or inflammation. Corticosteroids have been demonstrated to inhibit the induction of iNOS that occurs in response to LPS (17, 33),
and recently, Singh et al. (40) reported that this effect of
corticosteroids in cardiac myocytes and endothelial
cells may be mediated through OPN. These data, along
with the results of the present investigation, suggest
that OPN may be important in the regulation of iNOS
activity in other tissues in conditions such as sepsis.
The results presented here are the first to demonstrate that OPN is capable of preventing the inducible
increase in iNOS and decrease in cNOS activity in
vascular tissue, suggesting that OPN plays a role in
regulating NOS activity in the vasculature. Because
NO is a highly reactive and potentially toxic molecule,
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tightly regulated (24, 26, 37). Our findings with RGDmutated OPN proteins implicate integrin-mediated
binding and likely signal transduction in the blockade
of LPS-induced iNOS activity. One potential mechanism of the OPN blockade of the iNOS activity may be
inhibition of the induction of iNOS mRNA and protein
in a subset of vascular cells (i.e., endothelial and
vascular smooth muscle cells). The results presented
here suggest that OPN may be an important mediator
of the physiological and pathophysiological regulation
of iNOS in vascular tissue in health and disease.
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