Abstract

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.