Regulation of induction of nitric oxide synthase and the inhibitory actions of dexamethasone in the human intestinal epithelial cell line, Caco-2: influence of cell differentiation.

1. The inducible isoform of nitric oxide synthase (iNOS) may be involved in the pathogenesis of inflammatory bowel disease. Using the human intestinal epithelial cell line, Caco-2, iNOS expression, regulation and sensitivity to the glucocorticoid, dexamethasone after cytokine exposure and its relationship to the degree of differentiation has been studied. 2. NOS activity, assessed by NO2- and NO3- release, was time-dependently increased after exposure to interferon gamma alone or in combination with interleukin-1beta and tumour necrosis factor alpha. 3. Cytokine-induced iNOS activity was increased with days in culture over 20 days and number of passages, suggesting iNOS up-regulation during enterocyte-like differentiation. This activity was inhibited by the selective iNOS inhibitor 1400 W (0.1 - 100 microM). In addition, iNOS protein induction was confirmed by Western blot. 4. Actinomycin D (5 microg ml(-1) inhibited cytokine-induced iNOS activity, protein expression and mRNA level. Pyrrolidine dithiocarbamate (PDTC: 10 - 200 microM) and 3,4 dichloroisocoumarin (0.1 - 100 microM) reduced cytokine-induced iNOS activity and protein expression at both day 10 and 15 after confluence. PDTC also decreased iNOS mRNA levels, suggesting NF-kappaB involvement in its transcription at these times. 5. The tyrphostins A25 and B42 reduced cytokine-induced iNOS activity at both day 10 and 15 after confluence, indicating the JAK-2 kinase is also involved at these times. The tyrphostins also reduced the iNOS protein expression. 6. Dexamethasone (0.1 - 10 microM, for 24 h) reduced cytokine-induced iNOS activity at day 15 and 20 after cell confluence, but not at day 5 or 10. 7. Dexamethasone (5 microM) decreased cytokine-induced iNOS protein expression at day 10 as well as at day 15 after confluence. 8. These findings indicate that iNOS induction and its inhibition by dexamethasone in this human intestinal epithelial cell line is dependent on the degree of differentiation.