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Am J Physiol Gastrointest Liver Physiol 281: G688-G696, 2001;
0193-1857/01 $5.00
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Vol. 281, Issue 3, G688-G696, September 2001

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

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


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