5-aminosalicylic acid inhibits iNOS transcription in human intestinal epithelial cells

The mechanism by which 5-aminosalicylic acid (5-ASA) reduces mucosal injury in colitis is undefined. In murine cells, 5-ASA modulates the expression o f the inducible nitric oxide (NO) synthase, a potential mediator of colitic injury, but its effect on the human isoform is unknown. Given the lack of conserved regulation of iNOS expression in rodent and human systems, we sou ght to test the effect of 5-ASA on the expression of human iNOS in cultured enterocytes. Transformed human intestinal epithelial cells, DLD-1 and Caco -2BBe, were stimulated by IL-1 beta and IFN-gamma and analyzed for iNOS upr egulation and NO production in the presence of various aminosalicylates. 5- ASA, but not 4-ASA, dose-dependently inhibited NO production by both cell l ines [IC50 (mM) DLD-1 =4.5, Caco-2BBe =2.5]. 5-ASA also inhibited the expre ssion of iNOS protein and mRNA and blocked cytokine-induced transcriptional upregulation of the iNOS gene. 5-ASA (1-5 mM) had no effect on cytokine-in duced nuclear translocation of NF-kappa B or expression of IRF-1, transacti vating factors which regulates the human iNOS enhancer. We conclude that 5- ASA inhibits iNOS expression and NO production at therapeutically relevant concentrations. The inhibition occurs at the level of transcriptional activ ation and is independent of IRE-1 and NF-kappa B. Since NO is an important final effector of mucosal injury in inflammatory bowel disease, these findi ngs may have implications for the clinical efficacy of 5-ASA.