Specific NF‐κB blockade selectively inhibits tumour necrosis factor‐α‐induced COX‐2 but not constitutive COX‐1 gene expression in HT‐29 cells

Abstract

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-κB super-repressor (Ad5IκB) to investigate the role of NF-κB in tumour necrosis factor-α (TNF-α)-mediated COX-2 gene expression in a colonic epithelial cell line. COX-1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ- or Ad5IκB-infected HT-29 cells with no apparent change following TNF-α exposure. COX-2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up-regulated after TNF-α stimulation in uninfected and Ad5LacZ-infected HT-29 cells. This induction was prevented in Ad5IκB cells. TNF-α increased prostaglandin E₂ production by 20-fold in Ad5LacZ-infected HT-29 cells compared with uninfected cells and was significantly inhibited in Ad5IκB-infected cells in agreement with the COX-2 mRNA findings. We conclude that NF-κB 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-κB super-repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors.