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Department of Surgery, Georgetown University Hospital, Washington, D.C. 20007
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1
. In the presence of GRGDSP, a hexapeptide that blocks
binding of RGD-containing proteins to cell surface integrins, NO
production is significantly increased in the presence of LPS
stimulation. These data suggest a unique
trans-regulatory mechanism in which LPS-induced NO
synthesis feedback regulates itself through up-regulation of OPN
promoter activity and gene transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent transcriptional and
posttranslational control of iNOS has been described (1).
Substrate and cofactor availability can also modulate iNOS activity
(1). Lowenstein and colleagues have demonstrated that both
kalirin and NAP110 inhibit iNOS activity by preventing iNOS homodimer
formation (2, 3). These mechanisms do not appear to be NO
dependent. In contrast, a particularly unique feedback regulatory
mechanism is NO-mediated S-nitrosation of NF-
B, a key
transcription factor for iNOS gene transcription, with accompanying
inhibition of NF-B DNA binding and down-regulation of iNOS gene
transcription. Given the ubiquity of negative feedback regulation as a
mechanism for modulation of protein activity, NO-dependent inhibitory
mechanisms for iNOS may also exist. One approach to understanding regulatory mechanisms is the identification of patterns of gene expression associated with varying physiological states. A technique called suppression subtractive hybridization (SSH) has recently been described, which is based on technology similar to representational difference display but with modifications to normalize for mRNA abundance (4, 5, 6). In ANA-1 murine macrophages, we hypothesized that endotoxin (LPS)-mediated NO production induces a specific set of genetic programs that may serve to alter cellular NO metabolism. To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as a "tester" and RNA from LPS plus NG-nitro-L-arginine methyl ester (L-NAME)-treated cells was used as a "driver." Individual cDNA clones generated by SSH were used as probes in Northern blot analysis to identify differentially expressed genes.
Using SSH, osteopontin (OPN) was found to be specifically induced in
the presence of LPS-induced NO synthesis. This observation was
confirmed in both ANA-1 and RAW 264.7 murine macrophages and also in
the context of IFN- and IL-1 stimulation. OPN is a secreted,
acidic phosphoprotein that binds to an RGD integrin-binding motif; it
is produced by cells of mineralized tissue and activated cells of the
immune system (7). OPN production is increased in
inflammatory states, atherosclerosis, nephritis, malignancy, and bone
remodeling. Several investigators have demonstrated that OPN synthesis
inhibits iNOS expression and/or NO synthesis in kidney epithelium,
heart microvascular endothelium, macrophages, and rat thoracic aorta
(7, 8, 9, 10, 11, 12, 13). The molecular regulation of this functional
linkage has not been previously characterized. In this study, we
demonstrate that OPN gene transcription and promoter activity is
up-regulated by NO in endotoxin- and cytokine-stimulated murine
macrophages. Our results suggest that OPN-mediated inhibition of iNOS
expression and NO production is an NO-dependent negative feedback
regulatory loop.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse OPN promoter that included the 5' sequence from nt -777 to nt +79 cloned into pXP2 plasmid encoding luciferase was kindly provided by Dr. David T. Denhardt (Rutgers University, New Brunswick, NJ). ANA-1 macrophages were a gift from Dr. George Cox (Uniformed Services University of the Health Sciences, Bethesda, MD).
Induction of NO synthesis in ANA-1 and RAW 264.7 macrophages
ANA-1 and RAW264.7 macrophages were maintained in DMEM with 10%
heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml
streptomycin. LPS (100 ng/ml), IFN- (100 u/ml), or IL-1 (20
ng/ml) were used to induce NO synthesis. In selected instances, the
competitive substrate inhibitor of NO synthase, L-NAME (40
µM), or the NO donor,
S-nitroso-N-acetylpenicillamine (SNAP) (100
µM), or a combination of these compounds, was added. After incubation
for 12 h at 37°C in 5% CO2, the
supernatants and cells were harvested for assays.
Assay of NO production
NO released from cells in culture was quantified by measurement of the NO metabolite, nitrite (14). After stimulation, 50 µl of culture medium was mixed with 50 µl sulfanilamide (1%) in 0.5 N HCl. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-naphthyl)-ethylenediamine was added. Following incubation for 10 min at room temperature, the absorbance of samples at 540 nm was compared with that of a NaNO2.
RNA preparation and Northern blot analysis
Total RNA was isolated from ANA-1 and RAW 264.7 macrophages using TRIzol reagent (Life Technologies, Rockville, MD). The RNA samples (10 µg/lane) were fractionated by electrophoresis on a 1% agarose formaldehyde gel and transferred to Hybond-C nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization using [32P]dATP-labeled probes was performed at 42°C for 18 h in ULTRA hybridization buffer (Ambion, Austin, TX). Following hybridization, filters were washed twice and subjected to autoradiography. cDNA probes were prepared by random primer labeling, followed by purification using a Sephadex G-50 mini-column (BioMax, Odenton, MD).
Differential screening of the subtracted cDNA library
SSH was performed as previously described (4, 5, 6). To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as a "tester" and RNA from LPS plus L-NAME-treated cells was used as a "driver." Differentially expressed sequences in subtracted cDNA were amplified by PCR to amplify only cDNA with different adaptors at both ends. Further enrichment was performed by a second PCR amplification with nested primers. The differentially expressed sequences were inserted into a T/A vector, pT-Adv cloning vector (Clontech, Palo Alto, CA). After a blue/white visual assay, PCR was used to rapidly amplify cDNA inserts. PCR products were blotted on nylon membrane (Hybond N+; Amersham Pharmacia Biotech). Following hybridization, positive clones were sequenced with the ABI PRISM 377 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Resulting sequences were compared with the GenBank database.
Immunoblot analysis
Protein was extracted from both cells and media. To determine the amount of secreted protein, media were centrifuged and concentrated using Microcon YM-10 (Amicon, Beverly, MA). To determine cellular expression of protein, the cells were detached using PBS, centrifuged, and lysed in buffer (0.8% NaCl, 0.02 KCl, 1% SDS, 10% Triton X-100, 0.5% sodium deoxycholic acid, 0.144% Na2HPO4, and 0.024% KH2PO4, pH 7.4); the lysate was centrifuged at 12,000 x g for 10 min at 4°C. Protein concentration was determined by absorbance at 560 nm using protein assay reagent (Bio-Rad, Richmond, CA). Ten micrograms of protein in each lane was separated by SDS-12% PAGE, and then the products were electrotransferred to polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) for 60 min at 100 V. The membrane was blocked with 5% skim milk in PBS-0.05% Tween for 1 h at room temperature. After being washed three times, blocked membranes were incubated with the goat anti-mouse OPN Ab (R&D Systems, Minneapolis, MN) or rabbit anti-mouse iNOS Ab (Transduction Laboratories, Lexington, KY) for 1 h at room temperature, then washed three times in PBS-0.05% Tween and incubated with HRP-conjugated anti-goat-IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After an additional three washes, bound peroxidase activity was detected by the ECL detection system (Amersham Pharmacia Biotech).
Nuclear run-on assays
Macrophage nuclei were prepared in lysis buffer (10 mM Tris-HCl,
pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet
P-40) and pelletted at 500 x g. The nuclei (2 x
107) were resuspended in 100 µl glycerol
buffer, then 150 µCi of [
-32P]UTP (800
Ci/mmol) in 100 µl of 10 mM Tris-HCl (pH 8.0), 5 mM DTT, 5 mM
MgCl2, 300 mM KCl, and 1 mM (each) ATP, CTP, and
GTP were added for 30 min at 30°C with shaking. Labeled RNA was
treated with 10 U RNase-free DNase I (Life Technologies) for 5 min at
30°C and extracted with phenol:chloroform (24:1) once and chloroform
once. Before ethanol precipitation, 10 µg yeast tRNA was added, and
labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution
was neutralized by the addition of HEPES (acid free) to a final
concentration of 0.24 M. After ethanol precipitation, the RNA pellet
was resuspended in 10 mM
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH
7.4, 0.2% SDS, and 10 mM EDTA. Target DNA was spotted onto nylon
membranes with a slot blot apparatus; -actin and pT-Adv vector
served as positive and negative controls, respectively. Hybridization
was performed at 42°C for 48 h with 5 x
106 cpm of labeled RNA in hybridization buffer
(50% formamide, 4x SSC, 0.1% SDS, 5x Denhardt’s solution, 0.1 M
sodium phosphate, pH 7.2, and 100 µg/ml salmon sperm DNA). After
hybridization, the membranes were washed twice at room temperature in
2x SSC and 0.1% SDS and three times at 56°C in 0.1x SSC and 0.1%
SDS. The membranes were then exposed to x-ray film with an intensifying
screen.
Transient transfection reporter assay
ANA-1 macrophage cells were transfected with the pXP2-OPN
promoter by the calcium phosphate-DNA coprecipitation method. ANA-1
cells were plated onto 60-mm dishes at a density 6 x
105 cell/dish; after 24 h, cells were
transfected with the plasmid containing the pXP2-OPN promoter and
pCMV.SPORT-gal. The final amount of DNA added per dish was 8 µg of
the OPN promoter reporter construct and 2 µg pCMV.SPOT-gal. ANA-1
cells were incubated with LPS with or without L-NAME and/or
SNAP for 12 h. The cells were harvested and lysed by scraping in a
solution consisting of 25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM
1,2-diaminocycloheane-N,N,N,N-tetracetic
acid, 10% glycerol, and 1% Triton X-100. Luciferase activity was
assayed using the Luciferase assay system kit (Promega, Madison, WI);
-galactosidase expression was determined using the -galactosidase
enzyme assay kit (Promega) at 420 nm with a MAXLINE microplate reader.
Luciferase activity was normalized for transfection efficiency using
-galactosidase expression.
Statistical analysis
All data are presented as mean ± SEM of three or four experiments. Analysis was performed using a Students t test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ANA-1 macrophage production of NO in response to a 12-h incubation
with LPS (0–10 µg/ml) was determined in the presence and absence of
the competitive substrate inhibitor, L-NAME (40 µM) (Fig. 1
). Nitrite levels in unstimulated
control cells were 23.1 ± 3.3 nmol/mg. There was a significant
concentration-dependent increase in media levels of nitrite, the NO
metabolite, in response to LPS stimulation (ANOVA p =
0.001). In the presence of [LPS] = 100 ng/ml, nitrite production was
74 ± 5.6 nmol/mg. LPS plus L-NAME-treated
cells exhibited levels of NO production that were not significantly
from that of controls for all concentrations of LPS used. Nitrite
levels from cells treated with L-NAME alone did
not differ from that of unstimulated controls cells, 19.3 ± 2.9
nmol/mg vs 23.1 ± 3.3 nmol/mg (p = NS).
In a similar fashion, RAW 264.7 macrophages also exhibited a
significant concentration-dependent increase in nitrite following LPS
stimulation (ANOVA p = 0.001). In the presence of
[LPS] = 100 ng/ml, nitrite production in RAW 264.7 cells was 54
± 6.7 nm/mg. Nitrite levels in unstimulated control cells were
15.1 ± 1.7 nmol/mg. LPS plus L-NAME-treated
cells’ level of NO production was not significantly different from
that of controls for all concentrations of LPS used. In subsequent
assays, an LPS concentration of 100 ng/ml was used for both ANA-1 and
RAW 264.7 cells, unless otherwise stated.
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Using SSH and Northern blot screening in ANA-1 cells, genes
exhibiting greater than a 5-fold increase in expression in the presence
of both LPS and NO were completely sequenced and queried against the
National Center for Biotechnology Information GenBank database. One of
these genes was found to be OPN. Northern blot analysis was performed
to confirm that steady-state OPN mRNA expression was up-regulated in
ANA-1 and RAW 264.7 cells in the setting of LPS-induced NO synthesis
(Fig. 2). Baseline expression of OPN mRNA
was found in unstimulated control cells. In the presence of LPS, OPN
mRNA expression (1.5-kb band), normalized to that of -actin, was
increased 
10-fold. Inhibition of NO production by addition of
L-NAME to LPS-treated cells significantly decreased OPN
mRNA expression to a level equivalent to that of controls.
L-NAME alone did not alter OPN mRNA levels. When an
exogenous source of NO, SNAP (50 µM), was then added with LPS plus
L-NAME, normalized OPN mRNA expression was restored to a
level not statistically different from that of LPS-treated cells. When
NO alone, in the form of SNAP (50 µM), was delivered in the absence
of LPS, OPN mRNA expression was not different from that of controls for
both ANA-1 and RAW 264.7 macrophages (data not shown). These data
indicate that LPS-mediated NO production is associated with
significantly increased OPN mRNA expression in both ANA-1 and RAW 264.7
macrophages.
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Immunoblot analysis was used to determine OPN protein expression
in LPS-treated cells (Fig. 3). After
12 h of incubation, no immunoreactive OPN protein was found in
cell lysates from any of the ANA-1 or RAW 264.7 treatment groups. In
contrast, OPN protein (45 kDa) was detected in the cell culture
medium of LPS-treated cells. Inhibition of NO synthesis by addition of
L-NAME with LPS resulted in ablation of the secreted OPN
protein. Restoration of NO levels by treatment of cells with LPS plus
L-NAME plus SNAP resulted in detectable secreted OPN
protein. In the ANA-1 cells treated with LPS plus L-NAME
plus SNAP, the level of protein expression was only 60% of that
noted in LPS-treated cells. In contrast, LPS plus L-NAME
plus SNAP treatment normalized OPN protein expression in RAW 264.7
cells. Again, addition of SNAP alone did generate OPN protein secretion
in either ANA-1 or RAW 264.7 cells. These data suggest that
LPS-mediated NO production is associated with significantly increased
OPN protein secretion in both ANA-1 and RAW 264.7
macrophages.
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SNAP and OPN expression in ANA-1 and RAW 264.7 cells
To determine the effect of exogenous NO in the absence of an iNOS induction agent, such as LPS, SNAP (50 µM) was added to ANA-1 and RAW 264.7 cells for a 12-h incubation period (data not shown). In both cell lines, unstimulated controls and SNAP-treated cells did not exhibit iNOS mRNA or protein expression. Control and SNAP expression of OPN mRNA did not differ, and there was no detectable secreted OPN protein in either control or SNAP cells. These data indicate that exogenous NO alone cannot induce OPN mRNA or protein expression in either ANA-1 or RAW 264.7 cells.
OPN gene transcription in ANA-1 and RAW 264.7 cells
Nuclear run-on analysis was performed to determine whether the
NO-mediated increase in OPN mRNA levels was the result of increased
gene transcription in ANA-1 and RAW 264.7 cells (Fig. 4). The OPN transcriptional signal was
normalized with the -actin-positive control. Baseline OPN gene
transcription was found in control cells. In the presence of LPS, OPN
gene transcription was increased by over 7- and 6-fold in ANA-1 and RAW
264.7 cells, respectively (p < 0.01 vs
control). In LPS plus L-NAME-treated cells in
which NO production was ablated, OPN gene transcription was not
different from that of controls. OPN gene transcription was then
restored when SNAP was added with LPS plus L-NAME
(p = NS, LPS vs LPS plus
L-NAME plus SNAP). As expected, no signal was
noted in the negative control, PT-Adv. In ANA-1 macrophages, the
half-life of OPN mRNA was measured in the presence of actinomycin D (50
µg/ml)-associated inhibition of global gene transcription. There was
no difference in mRNA half-life detected between the control and LPS
treatment groups (data not shown). These data indicate that the
NO-mediated increase in OPN mRNA expression was the result of increased
OPN gene transcription.
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To determine whether increased OPN gene transcription is the
result of increased OPN promoter activity, transient transfection
analysis was performed in ANA-1 cells using a plasmid construct in
which the 856-bp OPN promoter was cloned upstream from a luciferase
reporter gene (Fig. 5). Luciferase
activity was normalized to that of -galactosidase activity
determined following cotransfection of a -galactosidase-CMV promoter
plasmid construct. In the presence of LPS, normalized luciferase
activity was 7-fold greater than that of unstimulated controls
(p < 0.01). OPN promoter activity in LPS plus
L-NAME-treated cells was not significantly
different from that of control or L-NAME-treated
cells. Restoration of NO levels in LPS plus
L-NAME plus SNAP-treated cells resulted in
luciferase activity that was 4-fold greater than that of controls
(p < 0.01). Interestingly, luciferase activity
in LPS-treated cells was significantly greater by 40% than that
noted in LPS plus L-NAME plus SNAP-treated cells
(p < 0.05). These results suggest that
LPS-induced NO production increases OPN promoter activity with
resultant increases in OPN mRNA and secreted protein levels.
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- and IL-1-mediated NO production induces OPN expression
To determine whether NO-mediated OPN expression was LPS specific,
ANA-1 murine macrophages were stimulated to produce NO by incubation
with IFN- (100 U/ml) or IL-1 (20 ng/ml). The concentrations of
IFN- and IL-1 were chosen to induce NO synthesis in
concentrations comparable to that seen with [LPS] = 100 ng/ml. Media
levels of nitrite were determined following a 12-h incubation.
Unstimulated control cells produced 18.1 ± 1.2 nmol nitrite/mg
protein. NO production was significantly increased in the presence of
LPS, IFN-, and IL-1: 74 ± 5.6 nmol/mg, 64.8 ± 4.7
nmol/mg, and 66.1 ± 3.8 nmol/mg, respectively
(p < 0.01 vs control for LPS, IFN-, and
IL-1). Addition of L-NAME with LPS, IFN-,
or IL-1 ablated NO production to a level that was not statistically
different from that of control cells. These data indicate that LPS,
IFN-, and IL-1 can each induce NO production in ANA-1
macrophages.
Northern blot analysis was then performed (Fig. 6). OPN mRNA was detected in control
cells. Stimulation with LPS, IFN-, or IL-1 resulted in a 9-, 10-,
and 9-fold increase in normalized OPN mRNA expression, respectively
(p < 0.01 vs control for LPS, IFN-, and
IL-1). Ablation of NO synthesis by addition of
L-NAME with LPS, IFN-. or IL-1 resulted in
a decrease in OPN mRNA to a level equivalent to that of unstimulated
controls. Reconstitution of NO levels by addition of SNAP (50 µM)
with L-NAME to LPS-, IFN--, or IL-1-treated
cells restored OPN mRNA expression to levels that were equivalent to
those noted in the presence of LPS, IFN-, or IL-1 alone.
Immunoblot analysis was then performed to determine levels of secreted
OPN protein (Fig. 7). The pattern of
protein expression paralleled that of OPN mRNA expression. No secreted
OPN protein was found in unstimulated controls. LPS, IFN-, or
IL-1 stimulation was associated with detectable OPN in the culture
media. Again, addition of L-NAME with LPS,
IFN-, or IL-1 resulted in ablation of the OPN protein signal.
Secreted OPN protein was then restored when SNAP was added with
L-NAME and LPS, IFN-, or IL-1. These data
indicate that endogenous NO synthesis, induced by LPS, IFN-, or
IL-1, is associated with OPN mRNA expression and protein secretion.
However, NO alone will not induce OPN; a second signal transduction
pathway that is common to LPS, IFN-, and IL-1 is required.
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The functional correlate of NO-mediated up-regulation of OPN
synthesis in endotoxin-stimulated ANA-1 macrophages was analyzed (Fig. 8). The hexapeptide, GRGDSP, blocks
binding of RGD-containing proteins, such as OPN, to cell surface
integrins (13). GRGDSP was used to determine the role of
secreted OPN in the setting of LPS-induced NO synthesis. In comparison
to unstimulated controls, GRGDSP (0–10 nM) was found to increase LPS
(100 ng/ml)-mediated nitrite production by over 2-fold in a
concentration-dependent fashion (ANOVA, p = 0.0001).
The converse experiment was also performed by adding exogenous OPN
protein (0–10 nM) with LPS to ANA-1 cells. In this setting, exogenous
OPN was found to maximally decrease nitrite levels by over 50% (ANOVA,
p = 0.0001). These data indicate that endogenously
synthesized OPN secreted into the extracellular milieu can act to
decrease LPS-mediated NO synthesis. The time course of the
GRGDSP-mediated increase in LPS-stimulated NO production was evaluated
using [GRGDSP] = 0, 0.1, 1.0, 5.0, and 10 nM (Fig. 9). There was a GRGDSP concentration- and
time-dependent increase in NO production. Increasing GRGDSP
concentration was associated with increased NO production. Maximal NO
production was seen in cells treated with 10.0 nM GRGDSP following
18–24 h of incubation. These data suggest that blockade of the OPN
integrin cell surface receptor with GRGDSP increases ANA-1 macrophage
NO production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or
IL-1, was also associated with increased expression of OPN mRNA and
protein. Delivery of NO alone in the form of SNAP did not induce OPN
expression. This suggests that OPN expression is dependent upon NO and
a second signal transduction pathway common to LPS, IFN-, and
IL-1. Transient transfection studies using an OPN promoter construct
indicate that OPN promoter activity is significantly increased in the
presence of LPS and NO. Finally, addition of a competitive inhibitor of
OPN receptor binding resulted in a significant concentration- and
time-dependent increase in LPS-induced NO synthesis. Conversely,
exogenous OPN added at the time of LPS treatment also decreased NO
production be over 50%. These results suggest that NO production acts
through the OPN promoter to up-regulate OPN gene transcription and
protein synthesis. NO may then feedback regulate its own production, in
part, through induction of OPN synthesis.
OPN is a highly hydrophilic and negatively charged sialoprotein of
298 aa that contains a Gly-Arg-Gly-Asp-Ser sequence. It is a
secreted protein with diverse regulatory functions, including cell
adhesion and migration, tumor growth and metastasis, atherosclerosis,
aortic valve calcification, and repair of myocardial injury. Its
expression is tissue specific and subject to regulation by many factors
(7, 15, 16, 17, 18). Constitutive expression of OPN is found in
bone, kidney, placenta, and nerve cells. Induced expression of OPN is
found in T cells, epidermal and bone cells, and macrophages in response
to PMA, 1,25-dihydroxyvitamin D, basic fibroblast growth factor,
TNF-, IL-1, IFN-, and endotoxin. Interestingly, OPN and iNOS are
induced in response to the many of the same agents, such as TNF-,
IL-1, IFN-, and LPS (1, 19).
Recently, the relationship between NO and OPN has been examined by a
number of investigators. Rollo et al. (10) demonstrated
that exogenous recombinant OPN protein was effective in blocking
RAW264.7 murine macrophage NO production and cytotoxicity toward the
NO-sensitive mastocytoma cells. Their work suggested that OPN in
extracellular fluid may protect certain tumor cells from
macrophage-mediated destruction by inhibiting the synthesis of NO.
However, these authors did not attempt to localize a potential cellular
source for OPN in this setting (10). Singh et al.
(11, 13) reported that a synthetic 20-aa OPN peptide
analogue decreased iNOS mRNA and protein levels in ventricular myocytes
and cardiac microvascular endothelial cells. Transfection of cardiac
microvascular endothelial cells with an antisense OPN cDNA increased
iNOS mRNA in response to IL-1 and IFN-, suggesting that
endogenous OPN inhibits NO production (11, 13). Lastly,
using an Ab directed against the OPN
V3 integrin receptor,
Attur and coworkers demonstrated that ligand binding results in a
trans-dominant inhibition of NO production in human
cartilage (20). Hwang and colleagues found that OPN
suppressed NO synthesis induced by IFN and LPS in primary mouse kidney
proximal tubule epithelial cells, suggesting a regulatory role for OPN
in the NO signaling pathway (21). These studies clearly
demonstrate that endogenous OPN can inhibit induction of iNOS and that
OPN is an important regulator of the NO signaling pathway and
NO-mediated cytoregulatory processes. However, the converse
relationship, the role of NO in the induction of OPN synthesis, has not
been well studied.
In this study, we demonstrate that NO feedback regulates its own
production by up-regulating OPN promoter activity in this murine
macrophage model of endotoxin-mediated expression of iNOS. Recently,
Takahashi and colleagues (22) also demonstrated that OPN
mRNA expression is increased by IFN-LPS-induced NO production in RAW
264.7 cells. However, the effect of NO upon OPN transcription was not
assessed (22). Although the regulation of iNOS has been
examined at many levels, little is known of its negative feedback
regulatory systems. In the mouse macrophage cell line RAW 264.7,
Albakri and Stuehr (23) have shown that endogenously
produced NO inhibits posttranslational assembly of dimeric iNOS by
down-regulating heme insertion and availability. In addition, NO can
directly inhibit catalysis by binding to the iNOS heme iron to form an
inactive iron-nitrosyl complex (23). Subsequently, Park
and colleagues have shown that NO inhibits DNA binding of NF-B, an
essential transcription factor, and down-regulates iNOS gene
transcription (24). In this regard, we have demonstrated
that NO S-nitrosylates a key active site cysteine residue in
the NF-B p50 DNA binding domain and inhibits subsequent DNA binding
and iNOS promoter activity in ANA-1 macrophages (25, 26).
Recently, Ratovitski and colleagues have used the yeast two-hybrid
screening technique to isolate two proteins that interact with iNOS to
prevent iNOS homodimer formation: NAP110 and kalirin (2, 3). Unfortunately, it is currently unknown whether NO itself
induces increased production of these regulatory proteins. The presence
of a system of NO-mediated regulation of iNOS suggests potential
targets for modulation of the NO-dependent components of the
inflammatory response.
A number of features of this OPN-iNOS regulatory system remain to be
clarified. The specific signaling pathway by which OPN binding to
V3 integrin receptors
ultimately modulates NO synthesis in endotoxin stimulated macrophages
is unclear. The human
V3 integrin receptor
was originally identified as a heterodimeric molecule with vitronectin
binding activity. Subsequent studies indicate that it has a broad
binding specificity and can mediate binding to fibronectin, fibrinogen,
and thrombospondin (18). RGD-containing peptides and
proteins modulate [Ca2+] transients in
osteoclasts (16). Osteopontin stimulates
gelsolin-associated src activity, leading to increased
gelsolin-associated phosphatidylinositol 3-kinase activity and
phosphatidylinositol-(3, 4, 5)-trisphosphate levels, which facilitate
actin filament formation, osteoclast motility, and bone resorption
(15, 16, 17). The equivalent pathway in macrophages has not
been extensively characterized. In a similar fashion, the mechanism by
which LPS and NO up-regulate OPN promoter activity is also unclear.
Analysis of the murine OPN promoter demonstrates the presence of
potential binding sites whose corresponding transcription factor
activities are modified by NO, such as AP-1 (15). NO may
induce binding of NO-sensitive transcription factors to the promoter or
an enhancer region. Alternatively, NO and LPS may induce changes in the
secondary and tertiary structure of the promoter. Studies addressing
transcription factor binding are ongoing in our laboratory.
This study demonstrates that OPN inhibits NO production in the setting
of endotoxin stimulation. OPN promoter activity and gene transcription
are significantly up-regulated in the presence of LPS-mediated NO
production. The existence of OPN as an example of an NO-dependent
negative feedback regulatory mechanism is unique. In addition, the
V3 integrin receptor
appears to transduce an inhibitory signal for the down-regulation of
LPS-induced NO synthesis. Inhibition of
V3 integrin
receptor-mediated functions may serve to be a potential target for
future therapeutic interventions for inflammatory conditions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul C. Kuo, Georgetown University Medical Center, 4 PHC, 3800 Reservoir Road NW, Washington, D.C. 20007.
3 Abbreviations used in this paper: iNOS, inducible NO synthase; SSH, suppression subtractive hybridization; L-NAME, NG-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetylpenicillamine; OPN, osteopontin.
Received for publication May 25, 2000. Accepted for publication October 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v and 3 integrin chains on murine lymphocytes. Proc. Natl. Acad. Sci. USA 93:14698.B binding to DNA. Biochem. J. 322:609.
B p50 binding kinetics by S-nitrosylation. Biochem. Biophys. Res. Commun. 238:703.[Medline]
B dependent gene transcription in ANA-1 murine macrophages. J. Immunol. 162:4101.This article has been cited by other articles:
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  F. W. Kiefer, M. Zeyda, J. Todoric, J. Huber, R. Geyeregger, T. Weichhart, O. Aszmann, B. Ludvik, G. R. Silberhumer, G. Prager, et al. Osteopontin Expression in Human and Murine Obesity: Extensive Local Up-Regulation in Adipose Tissue but Minimal Systemic Alterations Endocrinology, March 1, 2008; 149(3): 1350 - 1357. [Abstract] [Full Text] [PDF]
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 G. Chidlow, J. P. M. Wood, J. Manavis, N. N. Osborne, and R. J. Casson Expression of Osteopontin in the Rat Retina: Effects of Excitotoxic and Ischemic Injuries Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 762 - 771. [Abstract] [Full Text] [PDF]
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 K. X. Wang, Y. Shi, and D. T. Denhardt Osteopontin regulates hindlimb-unloading-induced lymphoid organ atrophy and weight loss by modulating corticosteroid production PNAS, September 11, 2007; 104(37): 14777 - 14782. [Abstract] [Full Text] [PDF]
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H. A. Arafat, A. K. Katakam, G. Chipitsyna, Q. Gong, A. R. Vancha, J. Gabbeta, and D. C. Dafoe Osteopontin Protects the Islets and {beta}-Cells from Interleukin-1 {beta}-Mediated Cytotoxicity through Negative Feedback Regulation of Nitric Oxide Endocrinology, February 1, 2007; 148(2): 575 - 584. [Abstract] [Full Text] [PDF]
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 C. Gao, H. Guo, Z. Mi, M. J. Grusby, and P. C. Kuo Osteopontin Induces Ubiquitin-Dependent Degradation of STAT1 in RAW264.7 Murine Macrophages J. Immunol., February 1, 2007; 178(3): 1870 - 1881. [Abstract] [Full Text] [PDF]
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S. T. Hikita, B. P. Vistica, H. R. Jones, J. R. Keswani, M. M. Watson, V. R. Ericson, G. S. Ayoub, I. Gery, and D. O. Clegg Osteopontin is proinflammatory in experimental autoimmune uveitis. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4435 - 4443. [Abstract] [Full Text] [PDF]
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 M. Schroeter, P. Zickler, D. T. Denhardt, H.-P. Hartung, and S. Jander Increased thalamic neurodegeneration following ischaemic cortical stroke in osteopontin-deficient mice Brain, June 1, 2006; 129(6): 1426 - 1437. [Abstract] [Full Text] [PDF]
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 J. Sodek, A. P. Batista Da Silva, and R. Zohar Osteopontin and Mucosal Protection Journal of Dental Research, May 1, 2006; 85(5): 404 - 415. [Abstract] [Full Text] [PDF]
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 M. Katzenellenbogen, O. Pappo, H. Barash, N. Klopstock, L. Mizrahi, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, L. A. Mitchell, et al. Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the mdr2-knockout mice. Cancer Res., April 15, 2006; 66(8): 4001 - 4010. [Abstract] [Full Text] [PDF]
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 A K Katakam, G Chipitsyna, Q Gong, A R Vancha, J Gabbeta, and H A Arafat Streptozotocin (STZ) mediates acute upregulation of serum and pancreatic osteopontin (OPN): a novel islet-protective effect of OPN through inhibition of STZ-induced nitric oxide production J. Endocrinol., November 1, 2005; 187(2): 237 - 247. [Abstract] [Full Text] [PDF]
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C. Gao, H. Guo, Z. Mi, P. Y. Wai, and P. C. Kuo Transcriptional Regulatory Functions of Heterogeneous Nuclear Ribonucleoprotein-U and -A/B in Endotoxin-Mediated Macrophage Expression of Osteopontin J. Immunol., July 1, 2005; 175(1): 523 - 530. [Abstract] [Full Text] [PDF]
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 C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
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 C. Gao, H. Guo, J. Wei, Z. Mi, P. Wai, and P. C. Kuo S-Nitrosylation of Heterogeneous Nuclear Ribonucleoprotein A/B Regulates Osteopontin Transcription in Endotoxin-stimulated Murine Macrophages J. Biol. Chem., March 19, 2004; 279(12): 11236 - 11243. [Abstract] [Full Text] [PDF]
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 K. Tanaka, J. Morimoto, S. Kon, C. Kimura, M. Inobe, H. Diao, G. Hirschfeld, J. M. Weiss, and T. Uede Effect of Osteopontin Alleles on {beta}-Glucan-Induced Granuloma Formation in the Mouse Liver Am. J. Pathol., February 1, 2004; 164(2): 567 - 575. [Abstract] [Full Text] [PDF]
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 M. R. Potter, S. R. Rittling, D. T. Denhardt, R. J. Roper, J. H. Weis, C. Teuscher, and J. J. Weis Role of Osteopontin in Murine Lyme Arthritis and Host Defense against Borrelia burgdorferi Infect. Immun., March 1, 2002; 70(3): 1372 - 1381. [Abstract] [Full Text] [PDF]
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M. Mavroidis and Y. Capetanaki Extensive Induction of Important Mediators of Fibrosis and Dystrophic Calcification in Desmin-Deficient Cardiomyopathy Am. J. Pathol., March 1, 2002; 160(3): 943 - 952. [Abstract] [Full Text] [PDF]
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 M. Mazzali, T. Kipari, V. Ophascharoensuk, J.A. Wesson, R. Johnson, and J. Hughes Osteopontin--a molecule for all seasons QJM, January 1, 2002; 95(1): 3 - 13. [Full Text] [PDF]
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C. V. Ramana, M. P. Gil, Y. Han, R. M. Ransohoff, R. D. Schreiber, and G. R. Stark Stat1-independent regulation of gene expression in response to IFN-gamma PNAS, June 5, 2001; 98(12): 6674 - 6679. [Abstract] [Full Text] [PDF]
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