Tumour Necrosis Factor-α Regulation of Prostaglandin H Synthase-2 Transcription is not through Nuclear Factor-κB in Amnion-derived AV-3 Cells

Abstract

Tumour necrosis factor (TNF)-α-stimulated prostaglandin (PG) E2biosynthesis by amnion-derived AV3 cells is accompanied by increased prostaglandin H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged. PGHS-2 promoter–reporter constructs (−891/+9 and 5′ deletions thereof) were prepared. The regions containing concensus nuclear factor κB (NF-κB) elements (−447/−438 and −222/−213) did not enhance promoter activity. Elements associated with both basal and TNF-α-stimulated expression lie between bases −52 and −203. Site-directed mutagenesis of nuclear factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of the −203/+9 construct by over 95 per cent. Electrophoretic mobility-shift assays using oligonucleotides derived from these sites demonstrated formation of specific DNA–protein complexes. Both NF-IL6 and CRE unlabelled oligonucleotides inhibited complex formation with the NF-IL6 oligonucleotide probe. Unlabelled CRE oligonucleotide also effectively inhibited formation of the complex with the CRE probe, but reduced effectiveness was observed when the NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensus NF-κB oligonucleotide failed to compete for either probe. TNF-α treatment did not increase levels of these complexes. Thus NF-κB does not enhance basal or TNF-α-responsive PGHS-2 transcription in amnion-derived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regulating PGHS-2 expression in these cells is indicated, but requires further clarification.