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 of 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 sought 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-1beta and IFN-gamma and analyzed for iNOS upregulation and NO production in the presence of various aminosalicylates. 5-ASA, but not 4-ASA, dose-dependently inhibited NO production by both cell lines [IC50 (mM) DLD-1 =4.5, Caco-2BBe =2. 5]. 5-ASA also inhibited the expression 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-induced nuclear translocation of NF-kappaB or expression of IRF-1, transactivating 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 activation and is independent of IRF-1 and NF-kappaB. Since NO is an important final effector of mucosal injury in inflammatory bowel disease, these findings may have implications for the clinical efficacy of 5-ASA.
Kennedy
M
M
Divisions of Critical Care and Pulmonary Medicine and Allergy, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Wilson
L
L
Szabo
C
C
Salzman
A L
AL
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D018938:Caco-2 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D019804:Mesalamine / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D014158:Transcription, Genetic / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007375:Interleukin-1; D009569:Nitric Oxide; D019804:Mesalamine; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II