Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells.
Cyclo-oxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid pathway. While COX-1 is mostly constitutively expressed, the COX-2 isoform is inducible by proinflammatory cytokines. We used an adenoviral vector containing an NF-kappaB super-repressor (Ad5IkappaB) to investigate the role of NF-kappaB in tumour necrosis factor-alpha (TNF-alpha)-mediated COX-2 gene expression in a colonic epithelial cell line. COX-1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ- or Ad5IkappaB-infected HT-29 cells with no apparent change following TNF-alpha exposure. COX-2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up-regulated after TNF-alpha stimulation in uninfected and Ad5LacZ-infected HT-29 cells. This induction was prevented in Ad5IkappaB cells. TNF-alpha increased prostaglandin E2 production by 20-fold in Ad5LacZ-infected HT-29 cells compared with uninfected cells and was significantly inhibited in Ad5IkappaB-infected cells in agreement with the COX-2 mRNA findings. We conclude that NF-kappaB activation is critical in mediating COX-2, but not COX-1 gene expression in HT-29 cells. Selective inhibition of COX-2 expression with the NF-kappaB super-repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors.
Jobin
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Departments of Medicine, Microbiology, Immunology and the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27599, USA.
Morteau
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Han
D S
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Balfour Sartor
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R
D000256:Adenoviridae; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008565:Membrane Proteins; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D013268:Stimulation, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone