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Cyclooxygenase-2 downregulates inducible nitric oxide synthase in rat intestinal epithelial cells
O Kobayashi1, H Miwa, S Watanabe
1Department of Gastroenterology, Juntendo University School of Medicine, Tokyo 113-8421, Japan. osamu-k@med.juntendo.ac.jp
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-gamma (IFN-gamma) and lipopolysaccharide (LPS) administration, iNOS and COX-2 expression was evaluated by Western blotting. PGE(2) 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-kappaB (NF-kappaB) were examined by gel mobility shift assay; a supershift assay was performed to identify the NF-kappaB complex components. JTE-522 or AA was added before IFN-gamma and LPS administration, and effects on iNOS and PGE(2) induction were evaluated by Western blotting or EIA. iNOS protein and mRNA expression was inhibited in RIE sense cells. Although NF-kappaB 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-kappaB by stabilizing Ikappa-Balpha.
Insights
Cyclooxygenase-2 (COX-2) suppresses inducible nitric oxide synthase (iNOS) expression in intestinal cells by inhibiting nuclear factor-kappaB (NF-kappaB) activation. This interaction is crucial for understanding inflammation and carcinogenesis pathways.
Area of Science:
- Molecular Biology
- Gastroenterology
- Inflammation Research
Background:
- Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are present in inflamed intestinal mucosa.
- The precise interplay between COX-2 and iNOS in inflammation and carcinogenesis remains incompletely understood.
- Investigating the regulatory relationship between COX-2 and iNOS is critical for understanding inflammatory processes.
Purpose of the Study:
- To determine if COX-2 regulates iNOS expression.
- To evaluate the effects of a COX-2 inhibitor and arachidonic acid (AA) on iNOS induction.
- To elucidate the molecular mechanisms underlying the COX-2 and iNOS interaction.
Main Methods:
- Stable transfection of the COX-2 gene into rat intestinal epithelial cells (RIE sense cells).
- Stimulation with interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) to induce iNOS and COX-2 expression.
- Western blotting for protein analysis, enzyme immunoassay (EIA) for PGE(2) quantification, and gel mobility shift assays for transcription factor analysis (NF-κB, AP-1).
Main Results:
- iNOS protein and mRNA expression were significantly inhibited in RIE sense cells compared to controls.
- COX-2 expression suppressed NF-κB activation and increased IκBα protein stability.
- The COX-2 inhibitor JTE-522 increased iNOS expression in RIE cells, while arachidonic acid (AA) effects were also evaluated.
Conclusions:
- COX-2 plays a suppressive role in iNOS expression within intestinal epithelial cells.
- This suppression is mediated through the inhibition of NF-κB activation, likely by stabilizing IκBα.
- Findings provide novel insights into the molecular crosstalk between COX-2 and iNOS in the intestinal environment.