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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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-gamma (IFN-gamma ) 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 Ikappa -Balpha was evaluated. Activator protein-1 and nuclear factor-kappa B (NF-kappa B) were examined by gel mobility shift assay; a supershift assay was performed to identify the NF-kappa B complex components. JTE-522 or AA was added before IFN-gamma 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-kappa B activation was suppressed and Ikappa -Balpha 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-kappa B by stabilizing Ikappa -Balpha .

nuclear factor-kappa B; Ikappa -Balpha ; extracellular signal-related kinase-1; extracellular signal-related kinase-2; activator protein-1; interferon response factor-1; cyclooxygenase-2 inhibitor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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-beta , tumor necrosis factor-alpha , and interferon-gamma (IFN-gamma )] 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<UP><SUB>2</SUB><SUP>−</SUP></UP>, 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.


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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-gamma (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-gamma  + 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-gamma 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-gamma (100 U/ml) and LPS (10 µg/ml). Whole cell lysates were extracted 12 h after the administration of IFN-gamma 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-gamma (100 U/ml) and LPS (10 µg/ml). Whole cell lysates were extracted 12 h after the administration of IFN-gamma 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-gamma (100 U/ml) were added to the medium, and whole cell lysate was extracted 12 h after the administration of IFN-gamma 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-gamma 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 Ikappa -Balpha (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 beta -actin were obtained by RT-PCR products derived from IFN-gamma and LPS-treated RIE cells. Primers used for the PCR reaction of iNOS and beta -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 beta -actin registered in GenBank by direct sequence using autosequencer (ABI PRISM 310 genetic analyzer, PE Biosystems, Foster, CA). The probes were radiolabeled with D-[alpha -32P]CTP by the Prime-It II random Primer labeling kit (Stratagene, La Jolla, CA).

Electrical mobility shift assay. Consensus oligonucleotide for the NF-kappa 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 [gamma -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-kappa 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-gamma 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-gamma (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-gamma and LPS. At 12 h after the administration of IFN-gamma 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of iNOS and COX-2 protein and mRNA in RIE parental and sense cells by LPS and IFN-gamma . Although IFN-gamma (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-gamma (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-gamma 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-gamma 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-gamma (IFN-gamma ), 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).

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-gamma 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-gamma and LPS, respectively. Endogenous COX-2 mRNA, induced by treatment with IFN-gamma 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-gamma 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-gamma (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 beta -actin cDNA. B: blots were probed with radiolabeled COX-2 or beta -actin cDNA.

PGE2 production in RIE parental and sense cells by IFN-gamma and LPS. PGE2 production from parental and sense cells was measured at 0, 6, 12, and 24 h after the administration of IFN-gamma (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-gamma 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-gamma 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.

IRF-1, phosphorylated ERK1 and ERK2 expression, and Ikappa -Balpha degradation in RIE parental and sense cells by IFN-gamma 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-gamma . IRF-1 protein was expressed from 30 to 180 min after the administration of LPS and IFN-gamma (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-gamma 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 Ikappa -Balpha cytoplasmic protein (38 kDa), which binds to NF-kappa B and inhibits its transactivation. Ikappa -Balpha 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-gamma  + 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-gamma . B: Western blot analysis of phosphorylated extracellular signal-related kinase (ERK)-1 and -2 protein expression from IFN-gamma  + 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-gamma and LPS. C: Western blot analysis of Ikappa -Balpha protein expression from IFN-gamma  + 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 Ikappa -Balpha , an inhibitor of nuclear factor-kappa B (NF-kappa B).

AP-1 and NF-kappa B activation in both RIE parental and sense cells. The activation of nuclear AP-1 and NF-kappa B was examined by electromobility shift assays. Although NF-kappa B was not transactivated without treatment, it was activated 30 min after stimulation by LPS and IFN-gamma and then remained elevated for 180 min. Two bands, which represent NF-kappa 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-gamma and LPS (Fig. 6).


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Fig. 5.   A: electrophoretic mobility shift assay of NF-kappa B. Nuclear extracts (10 µg) from IFN-gamma  + LPS-treated RIE parental and sense cells were incubated for 15 min with 32P-labeled oligo. Right lane, unlabeled NF-kappa B consensus oligo (100-fold) was added to sample to confirm the specificity of the DNA-protein interaction. B: electromobility supershift assay of NF-kappa B probe by extracts from RIE parental cells treated with IFN-gamma  + 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-gamma  + 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.

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-gamma (100 U/ml) were added to the medium, and whole cell lysate was extracted 12 h after administration of IFN-gamma  + 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-gamma  + LPS was administered to the medium, and whole cell lysate was extracted 12 h after the administration of IFN-gamma  + 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.

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-gamma 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-gamma  + 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-gamma (100 U/ml) were added to the medium, and whole cell lysate was extracted 12 h after the administration of IFN-gamma  + LPS. Protein (30 µg) was loaded on each lane, and blots were probed with polyclonal antiserum to iNOS.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B. In fact, NF-kappa 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-gamma 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-gamma and LPS is known to be a potent inducer of iNOS (23, 47). Furthermore, IFN-gamma 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-gamma 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-gamma 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-gamma - 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-gamma 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-gamma and LPS was used for the induction of iNOS in our study, we focused on IRF-1 (a transcription factor upregulated by IFN-gamma ), NF-kappa B (a transcription factor upregulated by LPS), and AP-1 (a transcription factor that cooperates with NF-kappa 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-gamma 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-kappa B was less activated in sense cells compared with parental cells, suggesting that COX-2 may repress iNOS expression through inhibition of NF-kappa B. From those studies, we did not determine whether COX-2 itself or the products of COX-2 inhibit the transactivation of NF-kappa 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-kappa B activation in macrophages. Other recent reports (18, 34, 39) demonstrate that peroxysome proliferator-activated receptor-gamma combined with its ligand, 15-deoxy Delta 12,14 PGJ2, results in inhibition of the inflammatory response and NF-kappa 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-kappa B. Further study is needed to determine whether COX-2 itself or its products inhibit the transactivation of NF-kappa B and iNOS induction, although exogenous PGE2 did not have effects in our experiments. Next we evaluated Ikappa -Balpha protein levels to further investigate the mechanisms responsible for the attenuation of NF-kappa B transactivation. Our data demonstrated that expression of Ikappa -Balpha protein was decreased in parental cells 15-20 min after IFN-gamma and LPS stimulation, whereas its expression was observed in sense cells (Fig. 4). This finding indicates that COX-2 may stabilize Ikappa -Balpha protein that inhibits transactivation of NF-kappa B. As already reported (6, 42), the activation of NF-kappa B requires several steps: 1) phosphorylation of Ikappa -Balpha , 2) degradation of Ikappa -Balpha via the ubiquitin proteosome pathway, 3) translocation of NF-kappa B from the cytoplasm into the nucleus, and 4) activation of gene transcription. Because Ikappa -Balpha protein was less attenuated in the sense cells in this study (Fig. 4), COX-2 or its products might prevent the degradation of Ikappa -Balpha 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-kappa B. Together with previous findings that 1) iNOS expression levels correlate with the severity of ulcerative colitis (17, 38), 2) NF-kappa B upregulates many genes involved in inflammation (22, 25, 31, 32, 48, 51), 3) antisense oligo to the NF-kappa 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-kappa 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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