Cyclooxygenase-2 downregulates inducible nitric oxide synthase
in rat intestinal epithelial cells
Osamu
Kobayashi1,
Hiroto
Miwa1,
Sumio
Watanabe2,
Masahiko
Tsujii3,
Raymond N.
Dubois4, and
Nobuhiro
Sato1
1 Department of Gastroenterology, Juntendo University School
of Medicine, Tokyo 113-8421; 2 Department of First Internal
Medicine, Akita University, Akita 010-8543; 3 Department of
First Internal Medicine, Osaka University, Osaka 565-0871, Japan; and
4 Department of Gastroenterology, Vanderbilt University
Medical Center, Nashville, Tennessee 37232
 |
ABSTRACT |
Cyclooxygenase-2 (COX-2) and
inducible nitric oxide synthase (iNOS) expression has been demonstrated
in inflamed intestinal mucosa. Although regulation of COX-2 and
iNOS expression has been studied extensively, the interplay between
these two enzymes remains unclear. Because they play crucial roles in
inflammation and/or carcinogenesis, we investigated whether COX-2
regulates iNOS expression and evaluated the effects of COX-2 inhibitor
and arachidonic acid (AA) on iNOS induction. The COX-2 gene coding
region was stably transfected into rat intestinal epithelial cells (RIE
sense cells). After interferon-
(IFN-
) and lipopolysaccharide
(LPS) administration, iNOS and COX-2 expression was evaluated by
Western blotting. PGE2 was measured by the enzyme
immunoassay (EIA) method. Expression of IFN response factor-1,
phosphorylated extracellular signal-related kinase-1 and -2, and
I
-B
was evaluated. Activator protein-1 and nuclear
factor-
B (NF-
B) were examined by gel mobility shift assay; a
supershift assay was performed to identify the NF-
B complex
components. JTE-522 or AA was added before IFN-
and LPS administration, and effects on iNOS and PGE2 induction were
evaluated by Western blotting or EIA. iNOS protein and mRNA expression
was inhibited in RIE sense cells. Although NF-
B activation was
suppressed and I
-B
protein was more stable, respectively, in RIE
sense cells, no difference was noted in other transcription factors. JTE-522 increased iNOS protein expression in RIE cells. We conclude that COX-2 suppressed iNOS expression in RIE cells through suppression of NF-
B by stabilizing I
-B
.
nuclear factor-
B; I
-B
; extracellular signal-related
kinase-1; extracellular signal-related kinase-2; activator protein-1; interferon response factor-1; cyclooxygenase-2 inhibitor
 |
INTRODUCTION |
CYCLOOXYGENASE
(COX), a key enzyme required for the synthesis of prostaglandins,
exists as two different isoforms, COX-1 and COX-2. Although COX-2 is
not detectable in most tissues, it is known to be regulated by
proinflammatory cytokines, mitogenic stimuli, and tumor promoters
(9, 11, 21, 24). On the other hand, inducible nitric oxide
synthase (iNOS), an enzyme that catalyzes the synthesis of NO from the
terminal guanidinonitrogen of L-arginine, is regulated by
bacterial endotoxins and cytokines [interleukin-
, tumor necrosis
factor-
, and interferon-
(IFN-
)] in a number of cell types
(7, 26, 29). In clinical studies (10, 11, 16, 37,
49), COX-2 is expressed not only in inflammatory tissue such as
intestinal mucosa of inflammatory bowel disease (IBD) patients but also
in neoplastic lesions of the colon. Similar to COX-2, iNOS is also
expressed in neoplastic lesions of the colon as well as in colonic
mucosa of IBD patients (1, 2, 17, 38). Moreover, there
have been several observations that suggest an interaction between
COX-2 and iNOS. Both enzymes were expressed in the same population of
colonic epithelial cells in ulcerative colitis patients, as well as in
Barrett's esophagus and esophageal adenocarcinomas (37,
50). In vitro, peroxynitrate (ONOO
), which is
derived from NO and O
, has been shown to activate
COX (35, 46). Triterpenoids, which are potent
anti-inflammatory agents, have been shown to suppress both iNOS and
COX-2 expression (40). However, the precise interactions between COX-2 and iNOS are not well defined. Accordingly, in this study, we investigated whether COX-2 affects iNOS expression in rat
intestinal epithelial (RIE) cells in which the COX-2 gene has been
stably transfected.
 |
MATERIALS AND METHODS |
Cell culture and treatment.
RIE cells were grown in DMEM with 10% heat-inactivated FCS, 100 U/ml
ampicillin, and 100 mg/ml streptomycin (Life Technologies, Rockville, MD).
Plasmid construction and stable transfection.
The coding region of the rat COX-2 was subcloned into the mammalian
expression vector PCB7. RIE cells were prepared as described previously
(41). The empty vector was also stably transfected to the
RIE cell as a control.
Extraction of protein and mRNA.
COX-2-transfected RIE cells (RIE sense), empty vector-transfected RIE
cells (mock), and COX-2-nontransfected cells (parental) were inoculated
onto 100-mm2 culture plates at a concentration of 5 × 106/plate. Those cells were grown in DMEM-10%
heat-inactivated FCS plus 100 U/ml ampicillin and 100 mg/ml
streptomycin at 37°C in 5% CO2 and 95% air. These cells
formed confluent monolayers by 72 h after inoculation and were
cultured for another 12 h without FCS. Recombinant rat IFN-
(10-1,000 U/ml; Genzyme, Cambridge, MA) and lipopolysaccharide
(LPS; 10 ng/ml-100 µg/ml; Disco Laboratory, Detroit, MI) were
added to the medium. At, 0, 10, 15, 20, 30, and 60 min and 3, 6, 12, and 24 h after the administration of IFN-
+ LPS, cells
were collected and whole cell lysates were prepared. mRNA was also
extracted using TRIzol reagent (Life Technologies) and an Oligotex mRNA
purification kit (Takara, Ostu, Japan), 3, 6, 12, and 24 h after
the administration of IFN-
and LPS.
RIE parental and sense cells were preincubated with JTE-522 (43,
44), a COX-2 selective inhibitor (Japan Tobacco)
(10
6, 10
5, and 10
4 M ) for 3 or 6 h before the administration of IFN-
(100 U/ml) and LPS (10 µg/ml). Whole cell lysates were extracted 12 h after the
administration of IFN-
and LPS.
PGE2 (Sigma Chemical, St. Louis, MO) (10
7,
10
6, 10
5, and 10
4 M) was also
added to the parental and sense cells and preincubated for 3 or 6 h before the administration of IFN-
(100 U/ml) and LPS (10 µg/ml).
Whole cell lysates were extracted 12 h after the administration of
IFN-
and LPS.
RIE parental and sense cells were preincubated with arachidonic acid
(AA; 0, 1, 5, and 20 mM) for 6 h. LPS (10 µg/ml) and IFN-
(100 U/ml) were added to the medium, and whole cell lysate was
extracted 12 h after the administration of IFN-
and LPS. Those
whole cell lysates and mRNA were stored at
80°C until further use.
Extraction of nuclear protein.
Nuclear proteins were extracted 0, 15, 30, 60, and 180 min after the
administration of IFN-
and LPS. Cells were suspended in buffer
A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, pH 8, 1 mM
dithiothreitol (DTT), and 0.5% Triton X-100] with protease inhibitor
Complete (Boehringer Mannheim, Mannheim, Germany) and were vortexed
every 5 min. After a 30-min incubation, they were centrifuged for 5 min
at 6,000 rpm and the supernatants were removed. The pellets were
resuspended in buffer B (20 mM HEPES, pH 7.9, 400 mM KCl,
1.5 mM MgCl2, 0.2 mM EDTA, and 1 mM DTT; Complete) and
vortexed every 10 min. After a 1-h incubation at 4°C, the samples
were centrifuged for 15 min at 15,000 rpm, and 15% volume of glycerol
was added to the supernatants. These nuclear protein extracts were
stored at
80°C until further use.
Western blotting.
The concentration of cytoplasmic or nuclear protein was determined by
the Bradford assay (Bio-Rad Laboratories, Hercules CA). Thirty
micrograms of cytoplasmic protein or 20 µg of nuclear protein were
diluted in SDS-PAGE loading buffer and boiled for 5 min and loaded to
7.5% or 12% SDS-PAGE gel. The proteins were transferred to the
Hybond-P positively charged nylon membrane (Amersham Life Science,
Arlington Heights, IL) using a Hofer semiphor (Pharmacia Biotech,
Piscataway, NJ) semidry blotting device. After nonspecific binding was
blocked with 3% Blotto (3% dry milk, 150 mM NaCl, 20 mM
Tris · HCl, pH 7.5, and 0.05% Triton X-100) overnight at room
temperature, the membranes were incubated with primary antibodies against COX-2, iNOS, IFN response factor-1 (IRF-1), phosphorylated extracellular signal-related kinase (ERK)-1 and -2, and I
-B
(Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies were added to
the Blotto solution at a dilution of 1:500 and incubated for 3 h
at room temperature. Excess primary antibody was removed by washing in
buffer three times for 10 min. Then the membranes were incubated with
the horseradish peroxidase-conjugated secondary IgG antibody at a
dilution of 1:2,000 in 3% Blotto solution for 50 min. Subsequently,
the membranes were washed three times at 20 min each with TBST (150 mM
NaCl, 20 mM Tris · HCl, pH 7.5, and 0.05% Triton X-100). The
membranes were then developed for 2 min with enhanced chemiluminescence
regents (Amersham Life Science).
Northern blotting.
Two micrograms of mRNA from both sense and parental RIE cells were
electrophoresed on 50% formaldehyde-agarose gels and transferred to
nylon membranes (Osmonics, Westboro, MA). Prehybridization and
hybridization were performed at 42°C using a 50% formamide solution.
Complementary DNA (cDNA) probes for rat iNOS and
-actin were
obtained by RT-PCR products derived from IFN-
and LPS-treated RIE
cells. Primers used for the PCR reaction of iNOS and
-actin were
5'-TAGAAACAACAGGAACCTACCA-3' and 5'-ACAGGGGTGATGCTCCCGGACA-3' and
5'-GCCCAGAGCAAGAGAGGCAT-3' and 5'-GGCCATCTCTTGCTCGAAGT-3', respectively. The PCR reaction conditions were as follows: 35 cycles;
denature (95°C, 1 min), annealing (58°C, 1 min), and extension (72°C, 1 min and 15 s). PCR products were subcloned into the
plasmid pCRTM2.1 using a TA cloning kit (Invitrogen, San
Diego, CA). DNA fragments for each cDNA probe were excised from the
plasmids using the restriction enzyme EcoR I and purified
using the QIAEX gel extraction kit (Qiagen, Chatworth, CA). Also, the
DNA sequence of the insert in the pCRTM2.1 was confirmed to
be that of rat iNOS and
-actin registered in GenBank by direct
sequence using autosequencer (ABI PRISM 310 genetic analyzer, PE
Biosystems, Foster, CA). The probes were radiolabeled with
D-[
-32P]CTP by the Prime-It II random
Primer labeling kit (Stratagene, La Jolla, CA).
Electrical mobility shift assay.
Consensus oligonucleotide for the NF-
B element
(5'-AGTTGAGGGGACTTTCCCAGG G-3', Santa Cruz Biotechnology) and activator
protein-1 (AP-1) enhancer (5'-CGCTTGATGACTCAGCCGGAA-3', Santa Cruz
Biotechnology) were end-labeled with T4 polynucleotide kinase in the
presence of [
-32P]ATP. Nuclear protein (10 µg) was
incubated with 32P-labeled probe for 15 min at 25°C. The
specificity of binding was determined by the addition of 100-fold
excess of same unlabeled oligonucleotide. After electrophoresis of the
reaction mixture, the intensities of the bands were compared. To
examine which components of NF-
B were affected, supershift assays
were performed using 2 µg of p65, RelB, C-Rel, p50, and p52
antibodies (Santa Cruz Biotechnology). Nuclear extracts of the IFN-
and LPS-stimulated parental cells were used for this experiment.
PGE2 assay.
Two hundred microliters of supernatant from parental and sense cells
were collected before and 6, 12, and 24 h after the administration of IFN-
(100 U/ml) and LPS (10 µg/ml). As an another experiment, both parental and sense cells were preincubated with JTE-522
(10
5 M) for 6 h before the administration of IFN-
and LPS. At 12 h after the administration of IFN-
and LPS, 200 µl of supernatant were collected. All supernatants were stored at
80°C, and PGE2 concentrations were measured using a
PGE2 enzyme immunoassay kit (Caymann Chemical, Ann Arbor,
MI). PGE2 per milligram of protein was determined by the
calculation of total PGE2 production and quantity of cell protein.
 |
RESULTS |
Induction of iNOS and COX-2 protein and mRNA in RIE parental and
sense cells by LPS and IFN-
.
Although IFN-
(10-1,000 U/ml) or LPS (10 ng/ml-100
µg/ml) does not induce iNOS expression individually (data not
shown), the combination of IFN-
(100 U/ml) and LPS (10 µg/ml)
treatment increased iNOS protein levels in RIE parental cells and
mock-transfected cells. Expression of iNOS protein (130 kDa), was
recognized at 3 h with maximal induction occurring at 6-12 h,
which decreased up to 24 h after the administration of IFN-
and
LPS. In contrast, only a faint band of iNOS was recognized 24 h
after treatment of RIE sense cells (Fig.
1A). COX-2 protein (72 kDa)
levels were barely affected by IFN-
and LPS treatment of RIE
parental cells, although COX-2 was highly expressed throughout the
duration of the experiment in RIE sense cells (Fig. 1B).

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Fig. 1.
Western blot analysis of whole cell lysate from rat intestinal
epithelial (RIE) mock-transfected, parental, and sense cells. After
stimulation by 10 µg/ml lipopolysaccharide (LPS) and 100 U/ml
interferon- (IFN- ), 30 µg of protein were loaded on each lane.
M, mock; P, parental; S, sense. A: blots were probed with
polyclonal antiserum to inducible nitric oxide synthase (iNOS).
B: blots were probed with polyclonal antiserum to
cyclooxygenase-2 (COX-2).
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|
Northern blot analysis showed that the 4.5-kb iNOS mRNA was expressed
in RIE parental cells 3 to 6 h after the administration of IFN-
and LPS. In COX-2 sense cells, only a faint band was recognized 3 h after treatment (Fig. 2A).
Northern blotting for COX-2 mRNA revealed two different-sized bands
(2.5 and 4.5 kb). The 2.5-kb band found in RIE sense cells represents
the product of the expression vector, and the 4.5-kb band found in both
RIE parental and sense cells represents the endogenous COX-2 mRNA induced by treatment with IFN-
and LPS, respectively. Endogenous COX-2 mRNA, induced by treatment with IFN-
and LPS, was much lower
than the 2.5-kb mRNA, a product of the expression vector. The
endogenous 4.5 kb COX-2 mRNA increased maximally from 3 to 6 h
after the administration of IFN-
and LPS (Fig. 2B).

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Fig. 2.
mRNA expression in RIE parental and sense cell after stimulation by
LPS (10 µg/ml) and IFN- (100 U/ml) was analyzed using Northern
blot analysis. mRNA (2 µg) was loaded on each lane. A:
blots were probed with radiolabeled iNOS or -actin cDNA.
B: blots were probed with radiolabeled COX-2 or -actin
cDNA.
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|
PGE2 production in RIE parental and sense cells by
IFN-
and LPS.
PGE2 production from parental and sense cells was measured
at 0, 6, 12, and 24 h after the administration of IFN-
(100 U/ml) and LPS (10 µg/ml). Though both parental and sense cells
produced PGE2, sense cells produced significant amount of
PGE2 compared with the parental cells. Sense cells produced
4.6-to 7-fold more PGE2 (572 ± 40 pg/mg protein;
mean ± SD) compared with parental cells (125 ± 14 pg/mg
protein; mean ± SD) 24 h after the administration of IFN-
and LPS (Fig. 3; P
0.001).
The significant difference of PGE2 production between
parental and sense cells reflects the expression level of COX-2 mRNA
and protein (Figs. 1B and 2B).

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Fig. 3.
PGE2 production from RIE parental and sense
cells after stimulation by 10 µg/ml LPS and 100 U/ml IFN- was
measured using PGE2 enzyme immunoassay kit.
PGE2 production from parental or sense cells (per mg
protein) was statistically analyzed by Student's t-test.
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IRF-1, phosphorylated ERK1 and ERK2 expression, and I
-B
degradation in RIE parental and sense cells by IFN-
and LPS.
Nuclear protein (20 µg) was prepared from RIE parental and sense
cells for the measurement of IRF-1 protein, a transcription factor that
regulates iNOS gene transcription and is induced by IFN-
.
IRF-1 protein was expressed from 30 to 180 min after the administration
of LPS and IFN-
(Fig. 4A).
Whole cell lysate (30 µg) was prepared from RIE parental and sense
cells to measure levels of phosphorylated ERK1 and ERK2 protein.
Phosphorylated ERK1 and ERK2 protein was expressed from 60 to 180 min
after the administration of IFN-
and LPS, then levels returned to
background (Fig. 4B). Whole cell lysate (30 µg) was also
prepared from RIE parental and sense cells for the measurement of
I
-B
cytoplasmic protein (38 kDa), which binds to NF-
B and
inhibits its transactivation. I
-B
protein was maximally expressed
in quiescent cells and attenuated from 15 to 20 min, but increased
again by 30 min in parental cells, whereas it was expressed
constitutively in sense cells (Fig. 4C).

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Fig. 4.
A: Western blot analysis of IFN response factor-1
(IRF-1) protein expression from IFN- + LPS-treated RIE parental
and sense cells. Nuclear extracts (20 µg) were loaded onto each lane.
Blots were probed with polyclonal antiserum to IRF-1, a
transcription factor upregulated by IFN- . B: Western
blot analysis of phosphorylated extracellular signal-related
kinase (ERK)-1 and -2 protein expression from IFN- + LPS-treated RIE parental and sense cells. Whole cell lysate (30 µg) was loaded onto each lane. Blots were probed with monoclonal
antiserum to phosphorylated ERK1 and ERK2, transcription factors
upregulated by the stimulation of IFN- and LPS. C:
Western blot analysis of I -B protein expression from IFN- + LPS-treated RIE parental and sense cells. Whole cell lysate (30 µg)
was loaded onto each lane. Blots were probed with polyclonal antiserum
to the I -B , an inhibitor of nuclear factor- B (NF- B).
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|
AP-1 and NF-
B activation in both RIE parental and sense cells.
The activation of nuclear AP-1 and NF-
B was examined by
electromobility shift assays. Although NF-
B was not transactivated without treatment, it was activated 30 min after stimulation by LPS and
IFN-
and then remained elevated for 180 min. Two bands, which
represent NF-
B, were observed (Fig.
5A), suggesting that two
different isoforms of this nuclear protein were present. To identify
each isoform, p65, RelB, C-Rel, p50, and p52 antibodies (Santa Cruz
Biotechnology) were used for additional supershift assays. The
intensity of these two bands decreased after the administration of p65
antibody, after which a supershift was observed. Although the
supershift was not clear, the intensity of the two bands decreased after administration of p50 antibody (Fig. 5B), indicating
that the two bands were likely to represent some combination of p65 and
p50 hetro- or homodimer. Both bands disappeared after
administration of cold competitor oligonucleotide (Fig. 5A).
AP-1 was also transactivated 30 min after treatment, and the intensity
of the bands increased further by 180 min after the
administration of IFN-
and LPS (Fig. 6).

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Fig. 5.
A: electrophoretic mobility shift assay of NF- B.
Nuclear extracts (10 µg) from IFN- + LPS-treated RIE parental
and sense cells were incubated for 15 min with 32P-labeled
oligo. Right lane, unlabeled NF- B consensus oligo
(100-fold) was added to sample to confirm the specificity of the
DNA-protein interaction. B: electromobility supershift assay
of NF- B probe by extracts from RIE parental cells treated with
IFN- + LPS for 60 min. Sample was not treated with antiserum
(lane 2). Samples were treated with 2 µg of antiserum to
p65 (lane 3), RelB (lane 4), C-Rel (lane
5), p50 (lane 6), and p52 (lane 7).
Lane 1, nonstimulated RIE parental cell.
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Fig. 6.
Electromobility shift assay for activator protein-1
(AP-1). Nuclear extracts (10 µg) from IFN- + LPS-treated RIE
parental and sense cells were incubated for 15 min with
32P-labeled oligo. Right lane, unlabeled AP-1
consensus oligo (100-fold) was added to sample as a competitor to
confirm the specificity of the DNA-protein interaction.
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Effects of a COX-2 inhibitor on expression of iNOS.
Because iNOS levels were decreased in RIE sense cells, we next
evaluated whether COX-2 inhibition also has an influence on iNOS
expression. JTE-522 (10
6, 10
5, and
10
4 M) was added to the medium of parental cells for
6 h, following which there was an increase in iNOS expression in a
dose-dependent manner. On the other hand, iNOS induction was not seen
at the concentration of 10
6 M in sense cells (Fig.
7A). The 3-h preincubation
with JTE-522 showed similar results to the 6-h preincubation (data not
shown). Next we examined whether upregulation of iNOS expression is
related to the concentration of PGE2. Although iNOS protein
was not induced in sense cells at the concentration of
10
6 M, JTE-522 obviously downregulated PGE2
production in sense cells at this concentration (Fig. 7B).
In other words, even if PGE2 production from sense cells
was suppressed, iNOS protein was not induced in JTE-522
(10
6 M)-treated sense cells, and a high concentration
(10
4 or 10
5 M) was needed for the induction
of iNOS protein.

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Fig. 7.
A: Western blot analysis of whole cell lysate
from RIE parental and sense cells. Both cells were preincubated with
JTE-522 (0, 10 6, 10 5, and 10 4
M) for 6 h. LPS (10 µg/ml ) and IFN- (100 U/ml) were added to
the medium, and whole cell lysate was extracted 12 h after
administration of IFN- + LPS. Protein (30 µg) was loaded on
each lane, and blots were probed with polyclonal antiserum to iNOS.
B: PGE2 production from RIE parental and sense
cells preincubated with various concentrations of JTE-522 (0, 10 6, 10 5, and 10 4 M) was
measured using PGE2 enzyme immunoassay kit. RIE parental
and sense cells were preincubated for 6 h with JTE-522.
IFN- + LPS was administered to the medium, and whole cell
lysate was extracted 12 h after the administration of IFN- + LPS. The effects of JTE-522 on the production of PGE2
were compared at various concentrations (0, 10 6,
10 5, and 10 4 M) and analyzed statistically
by Student's t-test.
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|
Effects of PGE2 or AA on expression of iNOS.
As COX-2 sense cells produce significant amounts of PGE2,
it was administrated to the parental cells (10
7,
10
6, 10
5, and 10
4 M
PGE2) to evaluate the effects of exogenous PGE2
on the induction of iNOS. PGE2 administered 3 and 6 h
before the administration of IFN-
and LPS had no effect on the
induction of iNOS protein (Fig. 8; 3 h data not shown). Because AA is the substrate of COX-2 and has some
effects on the enzymatic activity of COX-2 and eicosanoid production,
the effects of AA (1, 5, and 20 mM) on the induction of iNOS were also
evaluated. Though AA increased PGE2 induction (data not
shown), it did not decrease the induction of iNOS in parental cells nor
did it have any effects on sense cells (Fig. 9).

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Fig. 8.
Western blot analysis of whole cell lysate from RIE
parental cells treated with various concentrations of PGE2
(0, 10 7, 10 6, 10 5, and
10 4 M). Cells were preincubated with PGE2 for
6 h and then incubated with IFN- + LPS for an additional
12 h. Whole cell lysate was extracted, and 30 µg of protein were
loaded on each lane. Blots were probed with polyclonal antiserum to
iNOS.
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Fig. 9.
Western blot analysis of whole cell lysate from RIE
parental and sense cells. Both cells were preincubated with arachidonic
acid (0, 1, 5, and 20 mM) for 6 h. LPS (10 µg/ml) and IFN-
(100 U/ml) were added to the medium, and whole cell lysate was
extracted 12 h after the administration of IFN- + LPS.
Protein (30 µg) was loaded on each lane, and blots were probed with
polyclonal antiserum to iNOS.
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 |
DISCUSSION |
There are several studies that evaluate the relationship between
iNOS and COX-2. However, most of the previous work does not evaluate
how COX-2 influences iNOS expression. For instance, NO donor and NO
derivatives such as peroxynitrite (ONOO
) have been
reported to induce COX-2 (35, 46). Another report (45) demonstrated that low-dose nitric oxide induces COX-2
expression in macrophages in which NO was found to inhibit
apoptosis. Accordingly, we examined the effect of COX-2
expression on iNOS levels using COX-2- transfected RIE cell lines.
Stably transfected RIE cells overexpressed COX-2 as shown in Fig. 2 and
as previously reported (41). Moreover, PGE2,
the product of COX-2, was significantly induced from sense cells
compared with parental cells (Fig. 3). Overexpression of COX-2 using
the CMV promoter enabled us to evaluate the effect of COX-2 on iNOS
induction. Both of these enzymes are known to be regulated by NF-
B.
In fact, NF-
B has been shown to induce the expression of both COX-2
and iNOS (31, 32, 51, 53). Furthermore, we examined the
expression of iNOS and COX-2 protein using mock-transfected cells to
exclude the effect of stable transfection itself.
We used a combination of IFN-
and LPS treatment that induced iNOS
expression. Although each factor alone did not induce iNOS formation
(data not shown), the combination of both factors strongly induced
expression of iNOS, possibly by synergistic action. The combination of
IFN-
and LPS is known to be a potent inducer of iNOS (23,
47). Furthermore, IFN-
and LPS play an important role in the
pathogenesis of IBD, especially in Crohn's disease, suggesting that
they are also important in some inflammatory diseases (5, 12, 15,
36). In fact, we demonstrate that IFN-
and LPS potentially
induced iNOS formation in RIE parental cells and mock-transfected cells
(Fig. 1A). The different expression level of protein appears
to be due to the different expression of iNOS mRNA (Fig.
2A).
IFN-
and LPS administration resulted in high expression of COX-2
protein in the sense cells and weak expression of COX-2 protein in the
parental and mock cells (Fig. 1B). COX-2 protein expressed
in sense cells is due to the stable transfection of a COX-2 expression
vector and IFN-
- and LPS-induced endogenous protein. This
observation was also confirmed by the presence of two mRNA bands (2.5 and 4.5 kb; Fig. 2B). IFN-
and LPS treatment induced
COX-2 protein in parental RIE cells, where a single mRNA band (4.5 kb)
was found by Northern blot analysis (Fig. 2B). Therefore, the RIE sense cells expressed much more COX-2 than RIE parental cells.
As a result, COX2 overexpression downregulated iNOS induction (Figs.
1A and 2A), and COX-2 inhibition not only
increased iNOS protein expression in parental cells but also caused
induction of iNOS protein in sense cells (Fig. 7A). These
two results suggest that the underlying effect of COX-2 on iNOS
induction in RIE cells may be suppressive. On the other hand, many
studies demonstrate coexpression of COX-2 and iNOS, especially in
inflamed or neoplastic tissues (37, 40, 41, 49, 52).
Though RIE sense cells expressed nonphysiological amounts of COX-2 and
PGE2 compared with parental cells (Figs. 1B,
2B, and 3) in our experiments, the activity of COX-2 or
COX-2 products in inflamed mucosa in vivo may not be high enough to
repress the expression of iNOS compared with the RIE sense cells. Next
we examined the mechanism whereby COX-2 downregulates the expression of
iNOS. To examine this, we studied the effect of COX-2 on transcription
factors that regulate iNOS gene expression. There are several candidate transcription factors that are known to bind the 5' flanking region of
the iNOS gene. Because a combination of IFN-
and LPS was used for
the induction of iNOS in our study, we focused on IRF-1 (a transcription factor upregulated by IFN-
), NF-
B (a transcription factor upregulated by LPS), and AP-1 (a transcription factor that cooperates with NF-
B to upregulate iNOS transcription) (13, 19, 20, 25, 47, 53). Moreover, we examined phosphorylated p44
and p42 because ERK1 and ERK2 are known to be involved in the
regulation of iNOS by IFN-
and LPS (3, 13). Our results showed that the expression of IRF-1 protein, phosphorylation of ERK1
and ERK2, and activation of AP-1 was similar between sense and parental
cells (Figs. 4 and 6). However, NF-
B was less activated in sense
cells compared with parental cells, suggesting that COX-2 may repress
iNOS expression through inhibition of NF-
B. From those studies, we
did not determine whether COX-2 itself or the products of COX-2 inhibit
the transactivation of NF-
B. To elucidate the major factors
responsible for suppression of iNOS induction, we examined the effects
of exogenous PGE2, the product of COX-2, on the induction
of iNOS (Fig. 8). Furthermore, we examined whether reduction of
PGE2 increases iNOS induction. In sense cells, JTE-522 (10
5 and 10
4 M) blocked the effect of COX-2
on the expression of iNOS. On the other hand, 10
6 M of
JTE-522 did not lead to an increase in iNOS expression, although it
inhibited PGE2 induction from COX-2 sense cells (Fig. 7).
Because endogenous PGE2 did not have an effect on the
induction of iNOS (Fig. 8) and downregulation of PGE2 did
not immediately lead to induction of iNOS (Fig. 7A),
PGE2 might not be the main COX-2 product that regulates
iNOS induction. PGE2 receptors (EPs) have been reported to
localize to the perinuclear envelope and affect the induction of iNOS
(4), and other reports (8) have indicated
that exogenous PGE2 and PGI2 analogs suppress
iNOS protein expression by inhibiting NF-
B activation in
macrophages. Other recent reports (18, 34, 39)
demonstrate that peroxysome proliferator-activated receptor-
combined with its ligand, 15-deoxy
12,14
PGJ2, results in inhibition of the inflammatory response
and NF-
B activation. Therefore, the products of COX-2 or endogenous PGE may be responsible for iNOS suppression. One report
(27) showed that serum-activated NIH/3T3 cells expressed
COX-2 protein in the perinuclear region, suggesting the possibility
that COX-2 itself might interfere with the translocation of NF-
B.
Further study is needed to determine whether COX-2 itself or its
products inhibit the transactivation of NF-
B and iNOS induction,
although exogenous PGE2 did not have effects in our
experiments. Next we evaluated I
-B
protein levels to further
investigate the mechanisms responsible for the attenuation of NF-
B
transactivation. Our data demonstrated that expression of I
-B
protein was decreased in parental cells 15-20 min after IFN-
and LPS stimulation, whereas its expression was observed in sense cells
(Fig. 4). This finding indicates that COX-2 may stabilize I
-B
protein that inhibits transactivation of NF-
B. As already reported
(6, 42), the activation of NF-
B requires several steps:
1) phosphorylation of I
-B
, 2) degradation
of I
-B
via the ubiquitin proteosome pathway, 3)
translocation of NF-
B from the cytoplasm into the nucleus, and
4) activation of gene transcription. Because I
-B
protein was less attenuated in the sense cells in this study (Fig. 4),
COX-2 or its products might prevent the degradation of I
-B
protein.
Our results clearly show that the COX-2 protein itself or one of its
eicosanoid products decreases iNOS expression by suppressing transactivation of NF-
B. Together with previous findings that 1) iNOS expression levels correlate with the severity of
ulcerative colitis (17, 38), 2) NF-
B
upregulates many genes involved in inflammation (22, 25, 31, 32,
48, 51), 3) antisense oligo to the NF-
B p65
subunit abrogates experimental colitis (30), 4)
experimental colitis is exacerbated by a COX-2 inhibitor (33), and 5) exacerbation of IBD is caused by
the administration of nonsteroidal anti-inflammatory drugs
(14), COX-2 overexpression in RIE cells may play a
cytoprotective role in inflammatory diseases by reducing iNOS
expression or preventing the transactivation of NF-
B. Furthermore,
recent studies (28) support a cytoprotective role of
COX-2. In COX-2 knockout mice, intestinal inflammation induced by
dextran sodium sulfate was more severe compared with that in wild-type
mice (28). Therefore, the ability of COX-2 to reduce iNOS
induction may have clinical relevance in humans.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: N. Sato,
Department of Gastroenterology, Jutendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113-8421 (E-mail: osamu-k{at}med.juntendo.ac.jp).
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.
Received 11 August 2000; accepted in final form 4 May 2001.
 |
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