Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells

Background & Aims: Cyclooxygenase (COX)-2 is up-regulated in most colonic c ancers and in inflammatory bowel disease in which tumor necrosis factor (TN F)-alpha is believed to play a central role. There has been recent speculat ion on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF -alpha and its role in the regulation of genes controlled by NF-kappaB, We investigated the regulatory role of PI 3-kinase on COX-2 expression in colo nic epithelial cells. Methods: In HT-29 and Caco-2 colonic epithelial cells , COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1 al pha as observed by Northern and Western analyses. COX-2 activity was assess ed by measuring prostaglandin E-2 (PGE(2)) production by enzyme-linked immu nosorbent assay, NF-kappaB binding activity was assessed by electrophoretic mobility shift assay, PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performan ce liquid chromatography. Results: The PI 3-kinase inhibitor wortmannin up- regulated induced COX-2 expression in a concentration-dependent manner in b oth HT-29 and Caco-2 cells, An alternative PI 3-kinase inhibitor, LY294002, caused upregulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells a t concentrations of less than or equal to1 mu mol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, downregulated HT-29 COX-2 mRNA, protein, and PGE(2) production. NF-kappaB binding activity was unaltered by PI 3-ki nase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly, Conclusions: COX-2 is negatively regulated by PI 3-kina se; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding a ctivity. TNF-alpha can activate PI 3-kinase directly in addition to inducin g COX-2.