Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells.
OBJECTIVE
Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation 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 colonic epithelial cells.


METHODS
In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent 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-performance liquid chromatography.


RESULTS
The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase 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-kinase; 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 activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, England. s.a.weaver@bath.ac.uk
S A
SA
Weaver
M P
MP
Russo
K L
KL
Wright
G
G
Kolios
C
C
Jobin
D A
DA
Robertson
S G
SG
Ward
D000730:Androstadienes / Q000494:pharmacology; D018938:Caco-2 Cells; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000276:immunology; D019073:HT29 Cells; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D018793:Interleukin-13 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*; D007527:Isoenzymes / Q000235:genetics*; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D018793:Interleukin-13; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D015847:Interleukin-4; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D019869:Phosphatidylinositol 3-Kinases; D015232:Dinoprostone; D000077191:Wortmannin