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Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced COX-2 expression in human airway smooth muscle (HASM)
cells. These effects were not reversed by exogenous PGE2,
suggesting that they are prostanoid-independent. Indeed,
PGE2 also induced and enhanced IL-1-induced COX-2
expression. Peroxisome proliferator-activated receptor (PPAR) 
and
PPAR
(not PPAR) were expressed in HASM cells. PPAR activators
ciglitizone (Cig) and 15-Deoxy-
12,14-PGJ2
(15d-PGJ2), but not the PPAR activator WY-14643,
mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur,
NS-398, Cig, and 15d-PGJ2 alone, but not Indo and WY-14643,
elevated COX activity; however, neither enhanced IL-1-induced COX
activity. Pretreatment with dexamethasone suppressed COX-2 expression,
PGE2 release, and COX activity induced by NS-398, Cig,
IL-1, alone or in combination. Unlike IL-1, NS-398 and Cig did
not cause NF-
B (p65) nuclear translocation, nor did they further
enhance IL-1-induced NF-B translocation, but they stimulated
PPAR translocation. Indo, NS-398, Flur, and 15d-PGJ2,
but not WY-14643, induced transcriptional activity of a COX-2 reporter
construct containing the peroxisome proliferator response element
(PPRE) on their own and enhanced the effect of IL-1, but had no
effect on a COX-2 reporter construct lacking the PPRE. The results
suggest that COX-2 expression by NSAIDs is biologically functional,
prostanoid-independent, and involves PPAR activation, and provide
the first direct evidence that the PPRE in the promoter is required for
NSAID-induced COX-2 expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for
their anti-inflammatory and analgesic properties and inhibition of
COX activity, and the subsequent proinflammatory prostanoid generation
is an important mechanism of their action. However, NSAIDs have
other pharmacological effects, including inhibition of the
transcription factor NF-B that is critically involved in the
expression of several inflammatory genes (2, 3) and
activation of peroxisome proliferator-activated receptors (PPARs)
(4, 5, 6, 7).
PPARs are members of the nuclear receptor superfamily of
ligand-activated transcription factors and exist in three subtypes:
, (
), and (8). PPAR, predominantly
localized to the liver, orchestrates -oxidation of fatty acids.
PPAR, predominantly expressed in adipose tissue, regulates adipocyte
differentiation; the physiological function of PPAR is unclear
(8). PPARs are also expressed in other cells (5, 9), but their expression and functions in airways is unknown.
PPARs are activated by a heterogeneous group of structurally dissimilar
chemicals and the selectivity is activator concentration- and cell
type-dependent (8). The PGD2
metabolite
15-deoxy-12,14PGJ2
(15d-PGJ2) is a direct-binding ligand for PPAR
(10). Other PPAR activators include WY-14643 (selective
for PPAR), several NSAIDs, antidiabetic drugs (thiazolidinediones),
and fatty acids (e.g., AA and other eicosanoids) (8).
PPARs have complex regulatory effects on inflammatory responses.
PPAR-deficient mice show a prolonged inflammatory response
(11), whereas PPAR activation inhibits inducible NO
synthase, TNF-, IL-6, and IL-l expression (9, 12)
and prevents matrix metalloproteinase induction (13) in
stimulated monocytes/macrophages.
The effect of NSAIDs on COX-2 expression and the role of PPARs in this
process is controversial. Meade et al. (4) demonstrated
that COX inhibitors (NSAIDs), substrate (AA), and products
(PGD2, 15d-PGJ2, and
PGF2) induce COX-2 expression
transcriptionally in epithelial cells through PPARs, whereas Staels et
al. (5) found that PPAR activation by WY-14643
suppresses IL-1-induced COX-2 expression in human aortic smooth muscle
cells. It has also been shown that NSAIDs induce COX-2 expression but
inhibit mitogen-induced COX-2 expression in the colon cancer cell line
(HT-29) and macrophage cell line (RAW 264.7) (6), and that
COX-2 expression is regulated by a negative feedback loop mediated
through PPAR in the macrophage-like-differentiated U937 cells
(7). Therefore, COX-2 expression is likely to be regulated
in a cell-specific manner by different PPAR activators. However, no
studies have shown that the peroxisome proliferator response element
(PPRE) in the promoter region is required for COX-2 expression by
NSAIDs.
We and others have shown that human airway smooth muscle (HASM) cells
have important synthetic functions. In addition to the synthesis of
cytokines/chemokines (14, 15, 16) and growth factors
(17, 18), HASM cells express COX-2 and release large
quantities of prostanoids (mainly PGE2) upon
stimulation by proinflammatory cytokines and other mediators
(19, 20, 21). PGE2 produced from COX-2
inhibits cell proliferation (22), mediates IL-1- and
bradykinin-induced attenuation of cAMP generation in response to
2-adrenoceptor agonists (23, 24),
and acts as an autocrine regulator of IL-8 and vascular endothelial
growth factor release (15, 18), suggesting COX-2 induction
modulates airway smooth muscle function (25). In terms of
the effect of NSAIDs on COX-2 expression, Bonazzi et al.
(26) showed that COX inhibitor flurbiprofen (Flur) reduced
IL-1-induced COX-2 expression in HASM cells but did not perform
mechanistic studies.
To investigate the regulation of COX-2 expression and function by
NSAIDs in HASM cells and the role of PPAR activators in this process,
we studied the effect of NSAIDs on COX-2 expression in the presence or
absence of IL-1, a COX-2 inducer, and compared the effect of NSAIDs
with other PPAR activators. We show in this study that NSAIDs induce
COX-2 expression and enhance IL-1-induced COX-2 expression via a
prostanoid-independent mechanism involving PPAR, but not NF-B,
activation. We also show for the first time that deletion of the PPRE
in the COX-2 promoter abolishes the effect of NSAIDs. Paradoxically,
PGE2, the major COX product of these cells, also
induces COX-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DMEM, penicillin and streptomycin, L-glutamine,
amphotericin B, PGE2, SDS, PMSF, Triton X-100,
glycerol, acrylamide/bis-acrylamide, Tris, DTT, leupeptin, pepstatin,
2-ME, MTT, EGTA, polyclonal rabbit anti-mouse IgG coupled
with HRP, polyclonal goat anti-rabbit IgG with HRP,
anti-PGE2 serum, ciglitizone (Cig),
indomethacin (Indo), Flur, AA, dexamethasone (Dex), and other
unspecified chemicals were all purchased from Sigma-Aldrich (Poole,
U.K.). FCS was purchased from Sera-Lab (Loughborough,
U.K.). Recombinant human IL-1 was from R&D
Systems(Minneapolis, MN). NS-398, WY-14643, and
15d-PGJ2 were from Calbiochem-Novabiochem
(Nottingham, U.K.). Anti-human COX-2 andanti-COX-1 Abs were from
Cayman Chemical (Ann Arbor, MI). Abs against PPAR, (), ,
NF-B (p65), and IB were from Santa Cruz Biotechnology (Santa
Cruz, CA); [5, 6, 8, 11, 12, 14,
15(n)-3H]PGE2,
Rainbow-colored protein m.w. markers, ECL Western blotting detection
reagent, and Hyperfilm-ECL were from Amersham Pharmacia Biotech (Little
Chalfont, U.K.). Pure nitrocellulose membrane was from Gelman Sciences
(Northampton, U.K.). The Bio-Rad protein assay reagent was from Bio-Rad
(Hemel Hempstead, U.K.). FuGene 6 transfection reagent was from Roche
(East Sussex, U.K.). The dual-luciferase reporter assay system was from
Promega (Southampton, U.K.).
Cell culture
Primary cultures of HASM cells were prepared from explants of HASM as previously reported (19, 20). This protocol was approved by the Nottingham City Hospital Research Ethics Committee. Cells at passages 5 and 6 were used for all experiments. We have previously shown that the cells grown in this manner depict the immunohistochemical and light microscopic characteristics of typical HASM cells (19). HASM cells were also obtained from BioWhittaker (Wokingham, U.K.) and were used at passage 6.
Experiment protocols
The cells were cultured to confluence in DMEM supplemented with
10% FCS, the antibiotics penicillin (100 U/ml) and streptomycin (100
µg/ml), the antifungal amphotericin B (2.5 µg/ml), and
L-glutamine (4 mM) in humidified 5%
CO2/95% air at 37°C and growth-arrested in
serum-free medium for 24 h before experiments. Immediately before
each experiment, fresh serum-free medium was added. To test the effect
of NSAIDs and PPAR activators on their own, cells were treated with the
drugs for 4 h in most cases or for the times indicated in the
time-course experiments. To test the effect of NSAIDs and PPAR
activators on IL-1-induced COX-2 expression,
PGE2 release, and COX activity, they were added
30 min before the incubation with IL-1. The inhibitory effect of Dex
was assessed by preincubating the cells with Dex for 30 min before the
incubation with NSAIDs, PPAR activators, and IL-1, alone or in
combination, for a further 4 h. All the reagents were dissolved in
DMSO (final concentration 
0.4% v/v). In all the experiments, a group
of control cells were incubated with the drug vehicle for the same
period of time as the experimental cells.
PGE2 assay and COX activity
After experiments, the culture media in 24-well plates were collected and stored at -20°C until the determination of PGE2 content by RIA as described previously (19, 20). The sensitivity for PGE2 was 75 pg/ml. The anti-PGE2 antiserum had negligible cross-reactivity with other prostanoids (19) except 15d-PGJ2. COX activity was assayed functionally by washing the cells three times in PBS after the experiments and then incubating with AA (15 µM) for a further 15 min. These samples were subjected to RIA for PGE2 and the resulting PGE2 level was taken as an index of COX activity.
Preparation of whole-cell lysate
After treatment, cells in 24-well plates were washed twice with ice-cold PBS and incubated for 5 min with an extraction buffer (0.9% NaCl, 20 mM Tris-HCl, pH 7.6, 0.1% Triton X-100, 1 mM PMSF, 0.01% leupeptin) with gentle shaking. The samples were collected and centrifuged, and the protein concentration in the supernatant was determined using the Bio-Rad protein assay reagent.
Preparation of cytosolic and nuclear proteins
Nuclear and cytosolic extracts from the cells were prepared as described by Eickelberg et al. (27) with minor changes. After treatment, cells in 90-mm dishes were washed twice with ice-cold PBS and harvested in 1 ml of PBS with a cell scraper. The samples were centrifuged for 2 min at 1000 x g, and cell pellets were resuspended in 200 µl of low salt buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% IGEPAL CA-630, 10% glycerol, 1 mM PMSF, and 0.01% leupeptin). After 10 min of incubation on ice, the samples were centrifuged at 7000 x g for 30 min (4°C), and the supernatants were taken as cytosolic extracts. Nuclei were then resuspended in 100 µl of high salt buffer (as low salt buffer but with 420 mM NaCl and 20% glycerol), and nuclear proteins were extracted by shaking on ice for 30 min. Samples were then centrifuged at 7000 x g for 30 min (4°C), and the supernatants were taken as nuclear extracts.
Western blot analysis
Identification of the interested proteins were performed by
Western blotting analysis as described before (19, 20).
Briefly, protein samples (30 µg/track) were subject to
electrophoresis in 7.5% SDS-polyacrylamide gel. Separated proteins
were electroblotted to pure nitrocellulose membranes and the blot was
blocked for 2 h at room temperature with blocking buffer (wash
buffer with 8% fat-free dried milk powder). The blot was then
incubated with monoclonal anti-COX-2 Ab (1/2000 dilution with
blocking buffer), or polyclonal Abs against PPAR, (), ,
NF-B (p65), or IB (1/1000 dilution in blocking buffer) for
2 h, washed with wash buffer (PBS pH 7.4 with 0.3% Tween 20), and
incubated with HRP-conjugated secondary Abs (1/2000 dilution with
blocking buffer) for 1 h. The blot was washed again and then
incubated with ECL Western blotting detection reagent for 1 min and
finally exposed to Hyperfilm-ECL.
Plasmids
The 3.9-kb COX-2 firefly luciferase reporter construct (3.9-kb COX-2-Luc) in pGL3 vectors containing the putative PPRE sequence located between -3721 to -3707 (AGGCGACAGGTCA) upstream of the starting point of the COX-2 gene and the 3.5-kb COX-2 firefly luciferase reporter construct (3.5-kb COX-2-Luc) lacking the PPRE were kindly provided by T. McIntyre (University of Utah, Salt Lake City, UT) and have been previously described in detail (4, 28). The internal Renilla luciferase control vector pRL-SV40 was purchased from Promega.
Transfection of HASM cells and reporter assays
All transient transfections were performed using FuGene 6 (1 µg DNA:3 µl FuGene 6) according to the manufacturer’s recommended protocol. HASM cells were seeded at a density of 2 x 104 cells/well in 24-well plates and grown to 50–60% confluence. COX-2-Luc (3.9 kb) or 3.5-kb COX-2-Luc (0.4 µg) and pRL-SV40 (4 ng) were cotransfected into the cells. Transfection was conducted in serum-free and antibiotic-free medium for 5 h and the medium was then replaced with fresh serum-free medium and the cells were treated with test drugs for a further 4 h. The cells were then washed with PBS and lysed in lysis buffer (Promega). The activities of firefly and Renilla luciferase in the cellular extracts were measured using the dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega). Relative luciferase activity was obtained by normalizing the firefly luciferase activity against the internal control Renilla luciferase activity.
Cell viability
The toxicity of all the chemicals used in this study and their vehicle DMSO to HASM cells was determined by MTT assay in a separate series of experiments in 96-well plates, as described previously (19). Viability was set as 100% in control cells.
Statistical analysis
Results were expressed as the mean ± SEM of n determinations from HASM cells obtained from two donors. Student two-tailed t tests were used to determine the significant differences between the means. Values of p < 0.05 were accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among various NSAIDs we tested before, the nonselective COX
inhibitor Indo and the selective COX-2 inhibitor NS-398 block
IL-1-induced COX-2 activity in HASM cells (19). Thus,
we focused our study on these two NSAIDs. As previously demonstrated
(19), IL-1 treatment (1 ng/ml, 4 h) induced a
strong COX-2 protein expression in HASM cells. This was markedly
enhanced by pretreatment with Indo (1–100 µM), NS-398 (1–100 µM),
and another nonselective COX inhibitor Flur (10 µM) (Fig. 1A). Indo and NS-398 at 10
µM also time-dependently enhanced IL-1-induced COX-2 expression
(Fig. 1B), but COX-1 protein expression was not altered
(data not shown). Because the concentrations of 1 and 10 µM of Indo
and NS-398 are those required to block IL-1-induced
PGE2 synthesis in these cells (19),
it is possible that the enhanced COX-2 expression is a consequence of
PGE2 synthesis inhibition. If that is the case,
the addition of exogenous PGE2 would reverse the
enhancing effect of both Indo and NS-398, as PGE2
is the dominant prostanoid product by these cells (19).
However, contrary to this hypothesis, PGE2
pretreatment enhanced IL-1-induced COX-2 expression in a
concentration-dependent manner and did not reverse the effect of NSAIDs
(Fig. 1C), suggesting that the effect of NSAIDs on
IL-1-induced COX-2 expression is independent of their inhibition of
the enzyme activity. We then examined if these NSAIDs could cause COX-2
expression on their own and we found that treatment with Indo and
NS-398 (1 and 10 µM) for 4 h induced COX-2 expression compared
with the control (Fig. 2A),
and 10 µM of both Indo and NS-398 also caused COX-2 expression in a
time-dependent manner, which appeared at 1 h, peaked at 
2–8 h
after incubation, and declined thereafter (Fig. 2B). Flur
and exogenous PGE2 (10 µM, 4 h) also
induced COX-2 expression as shown in Fig. 2C.
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activators also enhance COX-2 expression
Because PPAR activators enhance COX-2 expression and a large
number of NSAIDs have recently been identified as PPAR activators
(4, 11), we studied the expression of PPARs in HASM cells.
We found that PPAR and PPAR, but not PPAR, were constitutively
expressed in HASM cells and that treatment with NS-398, the selective
PPAR activator WY-14643, or the selective PPAR activator Cig for
4 and 24 h did not alter the expression (Fig. 3A). Similar results were also
obtained with other NSAIDs, the selective PPAR activator
15d-PGJ2, and IL-1 (data not shown). We then
examined if PPAR activators could mimic the effect of NSAIDs on COX-2
expression and found that Cig and 15d-PGJ2 (1 and
10 µM, 4 h), but not WY-14643, caused COX-2 expression on their
own (Fig. 3B) and that pretreatment with Cig and
15d-PGJ2, but not WY-14643, resulted in a similar
enhancing effect on IL-1-induced COX-2 expression (Fig. 3C) as that of NSAIDs.
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We have previously demonstrated that COX-2 expression by IL-1
is associated with increase in PGE2 synthesis and
COX activity in HASM cells (19); thus, we tested if COX-2
expression induced by NSAIDs and PPAR activators was biologically
functional. After 4 h of incubation, HASM cells released low
levels of PGE2 in unstimulated conditions, which
was significantly inhibited by Indo (10 µM, p <
0.01), not affected by NS-398 and WY-14643, but enhanced by Cig (10
µM, p < 0.01) (Fig. 4A), suggesting that the basal
level PGE2 release is the result of COX-1
activity, whereas enhanced PGE2 release from
Cig-treated cells is the result of COX-2 expression. Although both Indo
and NS-398 caused COX-2 induction, PGE2 release
was not increased due to their inhibition of the induced enzyme. The
effect of another PPAR activator, 15d-PGJ2, on
PGE2 release could not be interpreted, as
15d-PGJ2 had cross-reaction with the
anti-PGE2 serum we used for RIA. Treatment
with IL-1 (1 ng/ml, 4 h) caused a marked increase in
PGE2 release, which was significantly reduced by
Indo (p < 0.001) and NS-398
(p < 0.01), increased by Cig
(p < 0.01), and not affected by WY-14643 (Fig. 4A), suggesting that the enhanced COX-2 expression by Cig is
biologically functional. Again, as would be expected, due to the
inhibition of the enzyme by the two NSAIDs, no increase on
PGE2 release from the cells pretreated with Indo
and NS-398 was observed despite the fact that both enhanced
IL-1-induced COX-2 expression (Fig. 1, A and
B).
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PGE2 release from AA was regarded as the index of
COX activity. As shown in Fig. 4B, Indo markedly reduced
(p < 0.001), whereas NS-398, Cig, and
15d-PGJ2 significantly increased
(p < 0.01, p < 0.01, and
p < 0.05, respectively) COX activity compared with
control cells, WY-14643 had no effect. IL-1 also increased COX
activity, which was significantly reduced by Indo
(p < 0.001) but not affected by NS-398,
WY-14643, Cig, and 15d-PGJ2 (Fig. 4B).
Flur also increased COX activity on its own (p
< 0.05) but had no effect on IL-1-induced COX activity (Fig. 4C). These results indicate that the COX-2 induced by
NS-398, Flur, and the PPAR activators alone is biologically
functional and that even though NSAIDs and PPAR activators enhance
IL-1-induced COX-2 expression, the enzyme activity in the context of
this study is not increased accordingly. Dex inhibits the induced COX-2 expression, PGE2 release, and COX activity
We have shown before that IL-1-induced COX-2 expression,
PGE2 release, and COX activity are inhibited by
Dex (19), an COX-2 transcription inhibitor. In this study,
we further investigated if the effects of NSAIDs and PPAR activators on
COX-2 could be inhibited by Dex. As shown in Fig. 5A, COX-2 expression (4 h) by
NS-398 (10 µM), Cig (10 µM), and IL-1 (1 ng/ml), individually or
in combination, was strongly suppressed by Dex (1 µM). Dex also
significantly inhibited the enhanced PGE2 release
caused by IL-1, Cig, and their combination (Fig. 5B,
p < 0.01, p < 0.05, p
< 0.001, respectively) and the enhanced COX activity caused by
IL-1, NS-398, Cig, and their combinations (Fig. 5C,
p < 0.01 for all). Similar results were also observed
with other Indo and Flur (data not shown).
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B translocation, but
cause PPAR translocation
As the transcriptional factor NF-B had been shown to be
critically involved in COX-2 expression (29), we went on
to examine whether NF-B activation was involved in the COX-2
expression by NS-398 and Cig. After treatment with either drug for 30
min, a slight reduction of IB in the cytosol was observed but
NF-B levels in both cytosol and nucleus remained unchanged (Fig. 6A). In contrast, IL-1
caused a clear reduction of both IB and NF-B in the cytosol
and a marked increase of NF-B in the nucleus. However, cotreatment
of either NS-398 or Cig with IL-1 did not alter the effect of
IL-1 (Fig. 6A). The results suggest that NS-398 and Cig
have no direct effect on NF-B activation in HASM cells and that
COX-2 expression by these two drugs is likely via a mechanism different
from that of IL-1. A possible mechanism would be the activation of
PPARs, which then interact with other transcriptional factors,
including NF-B, to regulate COX-2 expression. If so, NS-398, like
the PPAR activator Cig, should be able to translocate PPARs from
cytosol to the nucleus. We found indeed that treatment with NS-398 or
Cig, but not IL-1, for 30 min resulted in a significant loss of
PPAR in the cytosol and a marked increase in the nucleus (Fig. 6B), and that all three reagents caused a slight increase of
PPAR in the nucleus even though the loss of PPAR in the cytosol
was not significant (Fig. 6B). Because PPAR activator
WY-14643 did not exert any effect on COX-2, these data suggest that
PPAR activation may be involved in the regulation of COX-2
expression by NSAIDs in HASM cells.
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The 5'-regulatory region of human COX-2 gene contains a distal
PPRE (4, 28) that interacts with PPARs. To further assess
the role of PPAR activation in COX-2 expression by IL-1, NSAIDs, and
PPAR activators, two COX-2 firefly luciferase reporter constructs
were used to transfect the cells. The 3.9-kb COX-2 construct contains
the PPRE (-3721 to -3707) in addition to the proximal regulatory
elements required for induction by cytokines (30, 31) such
as NF-B (-223 to -214), NF-IL-6 (-132 to -124), and cAMP
response element (CRE) (-59 to -53), whereas the 3.5-kb COX-2
construct lacks the PPRE but retains the proximal regulatory elements
(4, 28). After cotransfection with 3.9-kb COX-2-Luc and
the internal control vector pRL-SV40, relative luciferase activity was
significantly induced by 10 µM of Indo (p <
0.01), NS-398 (p < 0.01), Flur
(p < 0.05), and 15d-PGJ2
(p < 0.001) alone, but not by WY-14643 (Fig. 7A); IL-1 (1 ng/ml) alone
also markedly increased COX-2 promoter activity and the effect was
further enhanced by Indo, NS-398, Flur (p <
0.01), and 15d-PGJ2 (p <
0.001), but not by WY-14643 (Fig. 7A). In contrast, the
3.5-kb COX-2-Luc lacking the PPRE was unresponsive to these NSAIDs and
15d-PGJ2, but was still fully induced by IL-1
(Fig. 7B). These results suggest that NSAIDs and PPAR
activators induce COX-2 expression via transcriptional regulation of
the COX-2 gene and that it is the presence of PPRE in the the distal
-3.5-kb to -3.9-kb region that confers responsiveness to these NSAIDs
and 15d-PGJ2.
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Cell viability after treatment with the chemicals used in this study was consistently >95% compared with cells treated with the vehicle.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced
COX-2 expression in a prostanoid-independent manner and that the effect
is mimicked by PPAR, but not PPAR, activators. Unlike IL-1,
NSAIDs and PPAR activators do not activate NF-B. The fact that
NSAIDs stimulated the activity of a COX-2 promoter construct which was
not seen when the PPRE was deleted provides the strongest evidence yet
that NSAIDs induce COX-2 via PPAR activation. This study is the first
to implicate PPARs in regulating any function of HASM cells.
NSAIDs can regulate COX-2 expression, but studies are conflicting. For
instance, meclofenamate, mefenamic acid, ibuprofen, NS-398, and
sulindac acid enhance COX-2 expression in mammary epithelial cells
(4); aspirin and sodium salicylate suppress
IL-1-induced COX-2 expression in endothelial cells
(32); flufenamic acid induces COX-2 expression but
inhibits mitogen-induced COX-2 expression in a colon cancer cell line
and macrophages (6). These data suggest that COX-2
expression is regulated differently by NSAIDs in different cell types.
In this study, we report that Indo, NS-398, and Flur all induce COX-2
expression and enhance IL-1-induced COX-2 expression independently
of prostanoid synthesis inhibition. Our results differ from those of
Bonazzi et al. (26) in HASM cells, who demonstrate that
Flur inhibits IL-1-induced COX-2 expression as a result of
PGE2 synthesis inhibition; however, they did not
study the effect of NSAIDs alone on COX-2 expression. The reasons for
the discrepancy are unclear. One possible explanation could be
different experimental and culture conditions. In their experiments,
the cells were treated at the same time with Flur and IL-1, whereas
in ours the cells were treated with NSAIDs for 30 min before IL-1
stimulation, allowing NSAIDs to exert their effects before IL-1.
This is supported by the fact that COX-2 expression by NSAIDs occurs
faster than that by IL-1 (Figs. 1B and 2B).
Alternatively, Flur may have a different effect on COX-2 expression in
different cells since the same authors also report that Flur, like
Indo, significantly increases COX-2 expression in corneal epithelial
cells (33), and different culture conditions may have an
effect on HASM cell PPAR expression and consequently their response to
NSAIDs.
The mechanisms of COX-2 regulation by NSAIDs vary between NSAIDs and cell type. Because the main effect of NSAIDs is believed to be COX inhibition, it is reasonable to speculate that COX-2 regulation by NSAIDs is via their inhibition on COX activity, whose end products exert a feedback on COX-2 expression. For instance, Indo up-regulates endotoxin-induced COX-2 expression by removing the negative feedback of prostanoids in J774 macrophages (34). Paradoxically, we have found in this study that PGE2, the main prostanoid produced by HASM cells, exerts a positive feedback effect on COX-2 expression, which is in agreement with the report of Bonazzi et al. (33) in rabbit corneal epithelial cells. Meade et al. (4) also demonstrate that NSAIDs up-regulate COX-2 expression independently of prostanoid inhibition in mammary epithelial cells. In these cases, the increased COX-2 expression by NSAIDs is clearly not a feedback mechanism because 1) it is not reversed by exogenous PGE2; 2) COX-1 expression is not altered (4); 3) NSAIDs do not up-regulate COX-2 expression equally, even though they all block prostanoid generation (4). Therefore, NSAID-induced COX-2 expression in this study is a direct effect mediated by a prostanoid-independent mechanism.
Recent work has shown that NSAIDs, fatty acids, and prostanoids,
compounds that are inhibitors, substrates, and products, respectively,
of COX activity, regulate gene expression via PPAR activation (6, 10, 35, 36). COX-2 expression is also induced by PPAR activators
(4, 33, 37), and it has been speculated that this occurs
via a PPRE in the COX-2 promoter region (4), although
studies deleting the PPRE and observing the effect of NSAIDs have not
been previously performed. Our study is the first to show that PPAR
and PPAR, but not PPAR, are expressed constitutively in HASM
cells, providing the basis for PPAR activation. Like other nuclear
receptors, PPARs are translocated from the cytosol to the nucleus upon
binding with activators such as 15d-PGJ2
(38). Indeed, we found that PPAR activators mimicked
the effect of NSAIDs on COX-2 expression and that PPAR, but not
NF-B, was translocated to the nucleus by PPAR activator Cig as
well as NS-398. Although NSAIDs are also PPAR activators, our
results suggest that they mainly activate PPAR in these cells, as
PPAR translocation results were less impressive, and PPAR
activation is not involved in COX-2 expression, as PPAR activator
WY-14643 did not have any effect. This is supported by the findings in
the current study that NSAIDs and PPAR activator
15d-PGJ2 induced COX-2 promoter activity on their
own and enhanced IL-1-induced COX-2 promoter activity only when PPRE
was included in the construct (3.9-kb COX-2-Luc). Collectively our data
demonstrate that NSAID-induced COX-2 expression is likely to be
mediated by PPAR, however, do not exclude possible interaction
between PPARs and NF-B. The fact that the more sustained and
enhanced increase of COX-2 expression was observed when NSAIDs were
used together with IL-1 (Fig. 1B) compared with NSAIDs
alone (Fig. 2B) suggests that this may result from the
transactivation of two transcription factors, PPAR by NSAIDs and
NF-B by IL-1 (29), or an interaction between PPAR
and NF-B (Fig. 8). Recent studies have
indeed shown that PPARs have cross-talk with transcription factors
NF-B (6, 39) and AP-1 (39, 40) and
CREB-binding protein/p300 (40).
|
-induced COX-2 expression in HASM cells. Because
PGE2 stimulates cAMP generation via either
EP2 or EP4 adenylyl
cyclase-coupled receptors (41) in HASM cells
(26) and there is a CRE in the COX-2 gene promoter
(42), it is likely that PGE2
up-regulates COX-2 expression by a cAMP-dependent positive feedback
loop (Fig. 8). This is supported by the findings that the
2-adrenoceptor agonists salbutamol and
salmeterol and the direct adenylyl cyclase activator forskolin also
induce COX-2 (our unpublished observation) and that
PGE2 up-regulates COX-2 gene expression
(43, 44) and potentiates mitogen-induced COX-2 expression
(9, 45, 46). Because PGE2
up-regulates COX-2 expression, NSAID inhibition of IL-1-induced
PGE2 production would, in theory, reduce COX-2
expression; however, because the effect is compensated by NSAID
activation of PPAR, COX-2 expression is, in fact, enhanced (Fig. 8).
Moreover, 15d-PGJ2, the natural ligand of
PPAR, is a metabolite of PGD2 and we have
shown that IL-1 stimulates PGD2 synthesis in a
COX-2-dependent manner in HASM cells (19), it is possible
that COX-2 expression is self-regulated in an autocrine manner by
PGE2 and 15d-PGJ2 via a
cAMP- and PPAR-dependent mechanism, respectively (Fig. 8).
We also demonstrated that the PPAR activator Cig significantly
increases PGE2 accumulation either alone or in
combination with IL-1, in line with the increase in COX-2
expression. No PGE2 increase could be detected
with NSAIDs because its synthesis was blocked. Because one possibility
that could account for the low PGE2 levels seen
in other studies (33, 37) is the lack of substrate AA and
because NSAIDs abolish PGE2 generation from
endogenous AA, the PGE2 levels could not reflect
the increase in COX-2 enzymatic activity under these conditions. To
measure COX activity more accurately, we examined COX activity by
measuring the PGE2 generation from exogenous AA
in the absence of NSAIDs. Under these conditions, COX activity was
significantly increased in 15d-PGJ2-, Cig-,
NS-398-, and Flur-treated cells compared with control cells. As far as
we are aware, this is the first evidence that COX-2 induced by NSAIDs
is biologically functional when the drugs are no longer present. The
reason that Indo did not increase COX activity even though they did
induce COX-2 expression can be explained by the fact that its
inhibition on COX-2 is time-dependent and irreversible (47, 48). However, despite the fact that both NS-398 and Flur are
also time-dependent, irreversible inhibitors of COX-2 (47, 49), our results clearly show that their effect can be washed
away, but the reason remains unknown. Increased COX activity was also
observed with IL-1; however, addition of NSAIDs and PPAR activators
did not further increase COX activity even though they did enhance
COX-2 expression. This is probably because IL-1 is a stronger
inducer of COX-2 than NSAIDs and PPAR activators and the COX activity
has reached its maximum. Alternatively, NSAIDs and PPAR activators
might indirectly inhibit cytokine-induced COX activity by modulating
other enzyme and signal transduction pathways that regulate prostanoid
metabolism (37).
In summary, we have demonstrated that NSAIDs induce COX-2 expression in
HASM cells through PPAR activation. The fact that NSAIDs are not
effective in treating airway inflammation in asthma suggests that the
inflammatory process is not mainly mediated by prostanoids and/or that
NSAIDs may intensify the inflammatory response by activating PPARs and
subsequently amplifying the response via COX-2 expression and
proinflammatory prostanoid production. Our observations may explain in
part the lack of efficacy of NSAIDs in the treatment of airway
inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linhua Pang, Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, University of Nottingham, Hucknall Road, Nottingham NG5 1PB, U.K. E-mail address: linhua.pang{at}nottingham.ac.uk
3 Abbreviations used in this paper: COX, cyclooxygenase; HASM, human airway smooth muscle; AA, arachidonic acid; NSAID, nonsteroidal anti-inflammatory drug; Indo, indomethacin; Flur, flurbiprofen; Cig, ciglitizone; Dex, dexamethasone; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; 15d-PGJ2, 15-deoxy-12,14-PGJ2; CRE, cAMP response element.
Received for publication August 20, 2002. Accepted for publication November 11, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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