Cytokine-induced Prostaglandin E2 Synthesis and
Cyclooxygenase-2 Activity Are Regulated Both by a Nitric
Oxide-dependent and -independent Mechanism in Rat
Osteoblasts in Vitro*
Francis J.
Hughes,
Lee D. K.
Buttery
§,
Mika V. J.
Hukkanen
,
Ann
O'Donnell,
Jacques
Maclouf¶, and
Julia M.
Polak
From the Department of Periodontology, Faculty of Clinical
Dentistry, St. Bartholomews and the Royal London School of Medicine and
Dentistry, London E1, United Kingdom,
Department of
Histochemistry, Imperial College School of Medicine, Hammersmith
Campus, London W12 0NN, United Kingdom, and ¶ U384 INSERM,
Hopital Laribsiere, 75475 Paris, Cedex 10, France
 |
ABSTRACT |
Osteoblasts respond to stimulation with
interleukin-1 (IL-1), tumor necrosis factor-
(TNF-
), and
interferon-
(IFN-
) by production of nitric oxide and
prostaglandins (PGs). In this study the relationship between nitric
oxide and PG synthesis was investigated after cytokine stimulation of
cultured rat osteoblasts. IL-1, TNF-
, IFN-
, and exogenous sodium
nitroprusside, a nitric oxide donor, all stimulated
PGE2 production in a dose-dependent
manner. PGE2 production was blocked by
L-nitro-arginine methyl ester, an inhibitor of nitric oxide
production, after IFN-
stimulation and was partially blocked after
TNF-
stimulation. However, IL-1-induced PGE2 was
unaffected. Similarly, expression of the cyclooxygenase-2 protein was
stimulated by cytokines, and IFN-
-induced expression was again
blocked by L-nitro-arginine methyl ester. In contrast, all
cytokines induced the cyclooxygenase-2 mRNA expression
independently of nitric oxide production, although exogenous sodium
nitroprusside was able to induce the cyclooxygenase-2 mRNA in the
absence of cytokines. The results show that nitric oxide can induce PG
synthesis and cyclooxygenase-2 expression and may regulate
cyclooxygenase-2 expression at both transcriptional and
post-transcriptional levels. In addition, the data show the existence
of both nitric oxide-dependent and -independent pathways of
PG synthesis after cytokine stimulation of osteoblasts. The results
suggest that nitric oxide may be an important mediator of PG production
in inflammatory bone diseases.
 |
INTRODUCTION |
The factors that may regulate bone metabolism during inflammation
are of considerable importance in understanding the pathogenesis of a
number of common inflammatory diseases, including rheumatoid arthritis
and osteoarthritis and the periodontal diseases, and may suggest novel
therapeutic approaches to control of bone destruction seen in these
conditions. The effects of cytokines such as interleukin-1 (IL-1),1 tumor necrosis
factor-alpha (TNF-
), and interferon-
(IFN-
) on bone cells have
been studied extensively in the past and have complex effects on bone
metabolism (for review, see Ref. 1). These cytokines may act directly
on osteoblasts to regulate their activity and also regulate
osteoclastic bone resorption indirectly via their interaction with
osteoblasts. IL-1 is a potent inducer of bone resorption (2, 3) and has
a number of different effects on osteoblast metabolism in
vitro, including inhibition of bone formation and stimulation or
inhibition of proliferation, alkaline phosphatase activity, and
collagen synthesis depending on the cell type and the culture
conditions used (4-10). TNF-
also stimulates bone resorption and
can inhibit markers of osteoblast activity such as alkaline phosphatase
and collagen synthesis (10, 11). In contrast, IFN-
specifically
inhibits IL-1-induced bone resorption and can directly inhibit cell
proliferation and alkaline phosphatase activity in osteoblast cultures
(12, 13).
There is strong evidence that some of the effects of these cytokines on
bone cells may be mediated by the induction of prostaglandin synthesis.
In particular, inhibition of prostaglandin synthesis can partially
reverse the effects of IL-1 on both osteoclastic and osteoblastic
activity (4, 8, 14-16). Prostaglandins are potent stimulators of
osteoclastic bone resorption and have wide-ranging effects on
osteoblast metabolism directly by their interaction with cell surface
receptors on the osteoblasts (17). Prostaglandins are produced by the
action of cyclooxygenase enzymes on arachidonic acid after its release
by the enzyme phospholipase A2. Two isoforms of
cyclooxygenase have been described, namely cyclooxygenase-1 (prostaglandin H synthase-1) and cyclooxygenase-2 (COX-2, prostaglandin H synthase-2). COX-1 is constitutively expressed by many cells, whereas
COX-2 expression may be induced by a range of stimuli. For example,
IL-1 is a potent inducer of COX-2 expression in a number of cell types,
including osteoblasts (18, 19).
Recent studies have demonstrated the production of nitric oxide (NO) by
osteoblasts after stimulation with IL-1, TNF-
, and IFN-
by the
induction of expression of the inducible NO synthase enzyme (iNOS)
(20-25). Furthermore, these studies have suggested that the production
of NO may also mediate some of the effects of these cytokines on bone
metabolism. The evidence suggests that NO may both stimulate and
inhibit bone resorption in a biphasic manner and inhibit osteoblastic
activity (21, 22, 24-27). In addition, osteoblastic cells may also
produce NO and prostaglandin (PG) after mechanical deformation (28).
During acute and chronic inflammatory processes in vivo,
both iNOS and COX-2 may be co-expressed in an apparently co-ordinated
way (29). Taken together these findings raise the question of the
possible relationship between NO and prostaglandin synthesis in the
regulation of bone metabolism.
In the present study we have investigated the relationship between NO
and PG synthesis in osteoblasts after cytokine stimulation. Specifically, the aim of the study was to investigate the hypothesis that NO mediates prostaglandin synthesis and induction of COX-2 expression in osteoblast cultures isolated from fetal rat calvariae after stimulation with the cytokines IL-1, TNF-
, and IFN-
.
 |
MATERIALS AND METHODS |
Cell Cultures--
Primary osteoblast-enriched cultures were
isolated by sequential enzymatic digestion of neonatal rat calvariae as
described previously (30, 31). Briefly, the calvariae were dissected from neonatal Wistar rats and digested with collagenase for periods of
10, 10, 20, 20, and 20 min sequentially. The cells isolated from the
final three digests were plated into 75-cm2 flasks for
24 h, released by trypsin treatment and pooled, replated at a cell
density of 6 × 103 cells/cm2, and
cultured until confluent for experiments. Cells were cultured in
modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and fungizone (Life Technologies). For experiments cells were stimulated with recombinant human IL-1
(specific activity, 2.8 × 108 units/mg), recombinant murine IFN-
(specific
activity, 1 × 107 units/mg), and recombinant human
TNF-
(specific activity, 1 × 107 units/mg; all
obtained from Genzyme Ltd., West Malling, Kent, UK). To block NO
production, cultures were treated with the arginine analogue
L-nitro arginine methyl ester (L-NAME, 1 mM) or in some experiments with
N-nitro-L-arginine 1 mM). In some
experiments cultures were treated with the NO donor sodium
nitroprusside (SNP), with exogenous PGE2, or with the
cyclooxygenase inhibitor indomethacin (all obtained from Sigma).
Prostaglandin and Nitrite Assays--
Confluent cultures in
96-well plates were treated for 24 h with cytokines in the
presence or absence of the inhibitor of NO production
L-NAME at 1 mM concentration. In some
experiments cells were stimulated with exogenous PGE2
(Sigma) or the NO donor SNP. Culture medium was then collected and
either used immediately or stored for periods of up to 1 week at
20 °C for assay of production of PGE2 by enzyme
immunoassay using a commercially available assay kit (Amersham
Pharmacia Biotech).
NO production was assayed by measurement of nitrite present in the
culture medium using the Griess reaction consisting of 0.2%
sulfanilamide and 2% N-(1-naphthyl)ethylenediamine in 1% H2PO4 (32). Nitrite measurement was determined
by reading absorbance at 590 nm using a 96-well plate reader.
All experiments were carried out in triplicate in a minimum of two
independent experiments.
Immunocytochemical Staining for COX-2 and iNOS
Enzymes--
Confluent cultures grown in 75-cm2 flasks
were stimulated for 24 h with cytokines either singly or in
combination and in the presence or absence of L-NAME (1 mM). Culture media were then decanted, and the cells were
immediately fixed with a 1% solution of paraformadehyde in PBS for 20 min. After several washes in PBS, cell cultures were immunostained by
the avidin-biotin-peroxidase complex method. Endogenous peroxidase was
blocked by immersing slides in 0.03% hydrogen peroxide in methanol for
30 min, followed by washing in PBS (three washes, 10 min each). After
blocking nonspecific binding by incubating in 3% normal goat serum for 20 min, sections were blotted and incubated overnight at 4 °C with
rabbit antibodies to murine macrophage iNOS (21) and COX-2 (33), both
diluted 1:1000 in PBS containing 0.05% bovine serum albumin and 0.01%
sodium azide. Sections were washed in PBS and then successively
incubated with biotinylated goat antiserum to rabbit IgG (Vector
Laboratories, Burlingame, CA) diluted 1:100 in PBS/bovine serum albumin
and freshly prepared avidin-biotin-peroxidase complex (Vectastain,
Vector Laboratories), for 30 and 60 min, respectively. Peroxidase
activity was revealed using the diaminobenzidine-hydrogen peroxide method.
Northern Blots--
Osteoblast total RNA was isolated by the
Ultraspec (Biotecx Laboratories) isolation method. Total RNA (20 µg)
was separated on a 1% agarose-formaldehyde gel, transferred to a nylon
membrane (Hybond-N, Amersham Pharmacia Biotech), and immobilized by
baking at 80 °C for 4 h. Membranes were prehybridized in
ExpressHyb solution (CLONTECH) for 30 min. The
membranes were then hybridized for 1 h at 37 °C with continuous
shaking with 50 ng/ml 32P-labeled (T4 polynucleotide kinase
labeling kit, Promega) murine COX-2 oligonuclotide probe (R & D
systems) in fresh ExpressHyb solution. After stringency washes in
3 × SSC, 0.1% SDS and 0.1% SSC, 0.1% SDS, the membranes were
exposed to autoradiographic film (Reflection NEF-485, DuPont NEN) for
48 h at
70 °C. Loading of RNA was normalized between lanes by
subsequent probing of blots with
-actin.
To determine whether NO affected RNA stability, cultures were treated
with actinomycin-D to block new RNA synthesis, and RNA was quantified
by Northern analysis after 0, 1.5, and 3 h.
Blots were quantified (Scion Image, Scion Corp.) to determine mean
absorbancies of mRNA bands (corrected for background).
Western Blots--
Cells were grown to confluence in
75-cm2 flasks and stimulated for 24 h with cytokines
either alone or in combination and in the presence or absence of
L-NAME (1 mM). Cells were also incubated with
SNP (1 mM). Total cellular proteins were extracted by
freeze-thawing the cells in 0.05 M Tris buffer, pH 7.2, containing 5 mM dithiothreitol, 1 µg/ml leupeptin, 10 µg/ml chymostatin, 1 µg/ml pepstatin, 40 µg/ml bestatin, and 50 µg/ml N-
-p-tosyl-L-lysine
chloromethyl ketone (all purchased from Sigma). After centrifugation at
15,000 × g for 20 min, the supernatant was collected
and stored at
80 °C. After separation of the homogenates (20 µg
of total protein) by electrophoresis on 7.5% SDS-polyacrylamide gels,
proteins were electroblotted on to nitrocellulose membranes. Specific
proteins were detected using polyclonal rabbit antibodies to iNOS and
COX-2, followed by anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Protein bands were revealed by
enhanced chemiluminesence (Amersham Pharmacia Biotech) and autoradiography.
Blots were quantified (Scion Image) to determine mean absorbancies of
protein bands (corrected for background).
Data Analysis--
Data from assays were used to calculate
mean ± S.D. and tested for significant differences by one-way
analysis of variance and Tukey's multiple comparisons test.
 |
RESULTS |
Role of NO in Cytokine-stimulated PGE2
Production
To investigate whether NO is involved in cytokine-stimulated
PGE2 production, cells were stimulated with cytokines in
the presence or absence of the inhibitor of NO production
L-NAME, and PGE2 was determined by enzyme
immunoassay. 24-h stimulation of cells with IL-1
, TNF-
, or
IFN-
resulted in a marked, dose-dependent induction of
PGE2 release into the culture medium. When normalized for
equimolar concentrations of cytokines, IL-1
showed the greatest and
IFN-
the least activity. The addition of L-NAME had no
significant effect on IL-1-induced PGE2 production.
However, L-NAME consistently blocked IFN-
-induced
PGE2 production. The stimulatory effect of TNF-
on
PGE2 production was partially blocked by
L-NAME, although overall these data were not statistically
significant (Fig. 1).

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Fig. 1.
Effects of IL-1 , TNF- , and IFN- with
and without 1 mM L-NAME on production of
PGE2. Values are mean ± S.D.; n = 3. *, different from control values; #, significant effect of
L-NAME; p < 0.05.
|
|
Combinations of cytokines were also tested in further experiments (Fig.
2). Addition of IFN-
to both TNF-
and to low (10 units/ml) concentrations of IL-1 showed synergistic
increases in PGE2 activity, which were largely blocked by
L-NAME. However, there was no evidence of additional
effects of either TNF-
or IFN-
when added to higher
concentrations of IL-1
(
100 units/ml), and L-NAME
alone did not affect PGE2 production. When the NO donor SNP
was added to the cultures, again PGE2 production was
induced in a dose-dependent manner (Fig.
3).

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Fig. 2.
Effects of combinations of IL-1
(units/ml), TNF- (ng/ml), and IFN- (units/ml) with and without 1 mM L-NAME on production of PGE2.
Values are mean ± S.D.; n = 3. *,
different from control values; #, significant effect of
L-NAME; p < 0.05.
|
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Fig. 3.
Effects of SNP on PGE2
production. Values are mean ± S.D.; n = 3. *, different from control values; p < 0.05.
|
|
Cytokine Stimulation of NO Production
To confirm that cytokines stimulated NO production the
accumulation of nitrite, one of the stable end products of NO
metabolism, was measured in culture supernatants. TNF-
and IFN-
produced small but significant increases in nitrite production, and
combinations of all cytokines showed strong synergistic increases in
nitrite production, which were inhibited by addition of
L-NAME (Fig. 4). Addition of
indomethacin did not inhibit cytokine-induced nitrite, and addition of
exogenous PGE2 did not affect nitrite production (Fig.
5).

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Fig. 4.
Representative data of effects of IL-1
(100 units/ml), TNF- (1 ng/ml), and IFN- (100 units/ml) with and
without 1 mM L-NAME on nitrite
accumulation. Values are mean ± S.D.; n = 3. *, different from control values; p < 0.05.
|
|

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Fig. 5.
Effects of PGE2 and cytokines
with or without indomethacin (Indo) on nitrite
accumulation. Values are mean ± S.D.; n = 3.
|
|
Expression of NOS and COX-2 after Cytokine Stimulation
Intracellular Expression of Enzymes--
COX-2 and iNOS enzyme
expression in osteoblasts after stimulation was determined by
immunohistochemistry on cultured cells. Cells stimulated with IL-1,
TNF-
, or IFN-
showed strong immunoreactivity for both iNOS and
COX-2 in ~10% of all cells. A combination of all three cytokines
together increased the number of cells stained to ~60%. Staining for
iNOS was not affected if the cytokine-stimulated cells were
additionally incubated with L-NAME. Immunoreactivity for
COX-2, however, was inhibited by the presence of L-NAME,
such that staining seen in cells incubated with IFN-
and
L-NAME was comparatively much weaker than that seen with
the cytokine alone. L-NAME had no effect on the
immunoreactivity of cells stimulated with IL-1 (Fig.
6).

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Fig. 6.
Immunocytochemical detection of iNOS and
COX-2 expression. a, iNOS expression stimulated by IFN- .
b, iNOS expression stimulated by IL-1. c, COX-2
expression stimulated by IL-1. d, COX-2 expression
stimulated by IL-1 and L-NAME. e, COX-2
expression stimulated by IFN- . f, COX-2 expression after
stimulation by IFN- in the presence of L-NAME
|
|
Total Expression of Enzymes--
Total COX-2 and iNOS enzyme
expression was quantified by Western blots. The rabbit antibodies to
iNOS and COX-2 reacted with protein bands corresponding to ~130 and
70 kDa, respectively, in homogenates of osteoblasts stimulated with
IL-1
, TNF-
, or IFN-
, either alone or in combination (Fig.
7). These bands correspond to the known
molecular masses of rodent iNOS and COX-2. The antibodies did not
recognize any protein bands in control, unstimulated cells. The
presence of L-NAME had no effect on cytokine-stimulated
expression of iNOS, nor did it affect IL-1
-stimulated expression of
COX-2. However, the signal produced for COX-2 protein in cells
stimulated with IFN-
in the presence of L-NAME was
consistently much weaker than that seen with the cytokine alone. The
expression of COX-2 by osteoblasts was also stimulated in a
dose-dependent manner by the NO donor SNP. The reduced
expression of COX-2 seen in cells stimulated by IFN-
in the presence
of L-NAME was partially abolished if the cells were
additionally incubated with SNP for the last 12 h of incubation.
Analysis of the absorbancies of the COX-2 protein bands revealed a
2-3-fold reduction in the amount of protein expressed by cells
stimulated by IFN-
in presence of L-NAME compared with
the cytokine alone. Furthermore, densitometric analysis of COX-2
protein in cells stimulated with IFN-
in the presence of L-NAME and increasing concentrations of SNP revealed a
gradual recovery of COX-2 protein expression (Fig. 7).

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Fig. 7.
Western blots after stimulation of cells for
24 h. a, iNOS expression after stimulation with IL-1,
TNF- , and IFN- . b, COX-2 expression after stimulation
with IFN- or IL-1 with or without L-NAME. c,
COX-2 expression after stimulation with IFN- with or without
L-NAME, IFN- with L-NAME, and SNP for last
12 h and continuous SNP only. d, quantification of
COX-2 expression after stimulation with IFN- or IL-1 with or without
L-NAME. Values are mean ± S.D.; n = 3 separate blotting experiments. *, difference from IFN- values;
p < 0.01. e, quantification of COX-2
expression after stimulation with IFN- with or without
L-NAME, IFN- with L-NAME and SNP for the
last 12 h, and continuous SNP only. Values are mean ± S.D.;
n = 3 separate blotting experiments. *, difference
from IFN- values; p < 0.01.
|
|
Expression of COX-2 mRNA--
The effects of NO on expression
of COX-2 mRNA were determined by Northern blots. Osteoblast
cultures exposed to IL-1
, TNF-
, or IFN-
for 0, 6, and 15 h showed induction of a single band of ~4.4 kb corresponding to the
expected size of COX-2 mRNA (Fig. 8).
The cytokine-stimulated induction of COX-2 mRNA was unaffected by
the presence of L-NAME for all cytokines tested. However,
exogenous SNP stimulated the expression of COX-2 in a
concentration-dependent manner. In cultures treated with
actinomycin D there was a small decline in RNA levels by 3 h after
both IL-1 and IFN-
treatment. However, addition of L-NNA
did not affect the decline in RNA levels (Fig.
9).

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Fig. 8.
Northern blots for COX-2 and -actin
mRNA. a, stimulated for 15 h with IFN- or IL-1
with or without L-NAME and with SNP. b,
quantification of COX-2 and -actin mRNA (ratio). Values are
mean ± S.D.; n = 3 separate blotting experiments.
*, difference from 1 mM SNP; p < 0.01.
|
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Fig. 9.
Northern blots for COX-2 after treatment with
actinomycin D subsequent to IFN (a) or IL-1 (b)
with or without N-nitro-L-arginine stimulation.
c, quantification of COX-2 and -actin mRNA (ratio).
Solid bar, 0 h; striped bar, 1.5 h; gray
bar, 3 h after additional actinomycin D. Values are mean ± S.D.; n = 3 separate blotting experiments.
|
|
 |
DISCUSSION |
The mechanisms of action of cytokines in modulating tissue
responses during inflammation have been extensively studied and may be
useful targets for therapeutic control of tissue damage occurring
during chronic inflammatory diseases. Cytokines such as IL-1, TNF-
,
and IFN-
stimulate both PG and NO synthesis in osteoblasts by
regulation of the inducible isoforms of the cyclooxygenase (COX-2) and
nitric oxide synthase (iNOS) enzymes, respectively. Inhibition of PG
synthesis by indomethacin may abrogate the effects of, for example,
IL-1 in regulating osteoblast metabolism and osteoclastic bone
resorption (8, 14, 15), and inhibition of NO production by
L-arginine analogues has been shown to abrogate both
stimulatory and inhibitory effects of IL-1, TNF-
, and IFN-
on
both osteoblast and osteoclast activities (21, 22, 24-27). These data
suggest the importance of both PG and NO production as mediators of the
actions of these cytokines in regulating bone metabolism during
inflammatory disease processes.
Previous studies have shown that NO may increase PG synthesis by
activation of the COX-2 protein (34-38). NO is known to react with
iron-containing enzymes, resulting in their activation, and in this
respect the heme-containing COX-2 enzyme is a potential target for the
direct action of NO. Conversely, production of PG has been reported to
increase or decrease NO production in a biphasic manner in murine
macrophages and mesangial cells (39, 40).
Effects of NO on PGE2 Synthesis--
The data from the
present study suggest that NO may regulate PG synthesis at a number of
different levels of control and furthermore suggest the existence of
both NO-dependent and -independent pathways of PG synthesis
in osteoblasts after cytokine stimulation. This conclusion is reached
by the finding that 24-h stimulation of cells with IL-1
, TNF-
, or
IFN-
resulted in a dose-dependent increase in
PGE2 production, the major PG type synthesized by osteoblasts. The suggestion of the existence of both
NO-dependent and -independent pathways is supported by our
findings that IL-1
-induced PGE2 synthesis was
independent of NO synthesis, as judged by the lack of effect of added
L-NAME, whereas L-NAME almost totally inhibited
IFN-
-stimulated PGE2 synthesis. The effects of
L-NAME on TNF-
-stimulated PGE2 synthesis
were less conclusive, consistently producing a partial inhibitory
effect, but overall this was found not to be statistically significant.
Combinations of TNF-
and IFN-
showed a synergistic effect on
PGE2 production, which was largely reversed by
L-NAME. In addition, exogenous NO provided by treatment
with the NO donor SNP also stimulated PGE2 production. IL-1
was the most potent inducer of PGE2 production of
the cytokines tested. In support of its important role in stimulating
PGE2 synthesis, L-NAME did not inhibit
synthesis of PG in combinations of cytokines that included IL-1
, and
in these experiments addition of both TNF-
and IFN-
did not
significantly increase PG production over IL-1
stimulation alone.
Regulation of COX-2 Protein Expression by NO after Cytokine
Stimulation--
Investigation of COX-2 protein expression by
immunohistochemistry and Western blot analysis gave results consistent
with the data for PGE2 production. First, all cytokines and
exogenous SNP induced COX-2 protein expression after 24-h stimulation.
Addition of L-NAME did not affect IL-1
-induced COX-2
expression but markedly inhibited IFN-
-induced protein expression,
and the effect of L-NAME was reversible by addition of SNP
for the final 12 h of stimulation. In other experiments we found
that COX-1 expression was not regulated by cytokines or NO donors (data
not shown).
Regulation of COX-2 mRNA Expression by NO after Cytokine
Stimulation--
In contrast to the findings of both PGE2
and COX-2 protein expression, the data suggest that NO was not required
for cytokine-induced COX-2 mRNA expression. Measurement of steady
state levels of mRNA for COX-2 by Northern blot demonstrated the
induction of COX-2 mRNA by each of the cytokines tested, but this
was not affected by addition of L-NAME. Despite this, NO
was also able to induce COX-2 mRNA expression, as shown by the
observation that exogenous SNP also induced COX-2 mRNA. Taken
together these data suggest that although NO can induce COX-2
expression, cytokine-induced COX-2 mRNA was independent of NO production.
Mechanisms of NO Regulation of COX-2 Enzyme Activity--
Although
the effects of NO on the activation of COX-2 enzyme were not directly
examined in these studies, it is possible that at least part of the
effect of NO on PGE2 production by IFN-
(and TNF-
)
could be attributable to enzyme activation. However, COX-2 activity was
not further activated by NO in osteoblast cultures already exposed to
IL-1
. In addition to effects of NO on COX-2 enzyme activation, which
have been previously described (35, 36), the present data demonstrate
the regulation of PG synthesis by NO at both the transcriptional and
post-transcriptional levels. First, exogenous SNP increased COX-2
mRNA levels in a dose-dependent manner. Second,
although NO was not required for cytokine-induced COX-2 mRNA
expression, L-NAME inhibited IFN-
-induced COX-2 protein expression. These data suggest that NO acts at a post-transcriptional or translational level to increase COX-2 protein expression and PG
synthesis in addition to any effects it may have on COX-2 enzyme activity. However, there was no evidence that NO was acting by altering
RNA stability.
In contrast to the data presented here, an earlier study did not detect
any effects of the NO donor
S-nitroso-N-acetyl-penicillamine on
PGE2 production by mouse calvaria organ cultures (41). This variation from our results is not clear but might reflect the different
culture systems and conditions used for stimulation in the two studies.
However, recent reports have suggested that PGE2 production
by osteoblastic cells after mechanical deformation may be
NO-dependent and mediated by production of low levels of NO
by activation of the constitutively expressed endothelial NOS isoenzyme
(28, 42). These findings are consistent with the data here, first in
showing PGE2 production by NO, and second, more
specifically, by suggesting that relatively low levels of NO are able
to regulate PG synthesis, as seen in the current studies with the use
of combinations of single cytokines, particularly IFN-
.
In summary, the results of the study presented here demonstrate that NO
can induce PG synthesis and can regulate COX-2 expression both at the
levels of gene transcription and by post-transcriptional mechanisms in
cultured osteoblastic cells. The data further demonstrate that although
IL-1
-induced PGE2 production was independent of NO
production, IFN-
induced COX-2 protein expression, and
PGE2 production was dependent on its ability to induce NO
production. Although IL-1 shows a greater potency in inducing
PGE2 production when compared with IFN-
, these results
suggest that NO may play a significant role in PG-mediated bone
resorption in inflammatory bone disease processes. In addition, the
ability of NO to produce PGE2 may be an important mechanism
in regulating bone metabolism in other circumstances, such as in
mediating responses to mechanical loading of bone.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K Morgan for advice with design
of the oligonucleotide probe for COX-2 and Prof. Sir John Vane and
Prof. Salvador Moncada for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by Medical Research Council Project
Grant G9409774MA and the Dunhill Medical Trust. Cell culture facilities were partly provided by a generous donation from the Maurice Wohl Charitable Foundation.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.
§
To whom correspondence should be addressed: Dept. of
Histochemistry, Imperial College School of Medicine, Hammersmith
Campus, Du Cane Rd., London W12 0NN, UK. Tel.: 44-181-383-2151; Fax:
44-181-743-5362; E-mail: l.buttery{at}rpms.ac.uk.
The abbreviations used are:
IL, interleukin; COX-2, cyclooxygenase-2; NO, nitric oxide; iNOS, inducible nitric oxide
synthase; L-NAME, L-nitro arginine methyl
ester; SNP, sodium nitroprusside; TNF, tumor necrosis factor; PG, prostaglandin; PBS, phosphate-buffered saline.
 |
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