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-1 beta 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 mu M). In addition, iNOS protein induction was confirmed by Western blot. 4 Actinomycin D (5 mu g ml(-1)) inhibited cytokine-induced iNOS activity, protein expression and mRNA level. Pyrrolidine dithiocarbamate (PDTC: 10-200 mu M) and 3,4 dichloroisocoumarin (0.1-100 mu M) reduced cytokine-induced iNOS activity and protein expression at both day 10 and 15 after confluence. PDTC also decreased iNOS mRNA levels, suggesting NF-kappa B 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 mu M, 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 mu M) 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.