Tumour necrosis factor-alpha regulation of prostaglandin H synthase-2 transcription is not through nuclear factor-kappa B in amnion-derived AV-3 cells

Tumour necrosis factor (TNF)-alpha -stimulated prostaglandin (PG) E-2 biosy nthesis by amnion-derived AV3 cells is accompanied by increased prostagland in H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged . PGHS-2 promoter-reporter constructs (- 891/+9 and 5' deletions thereof) w ere prepared. The regions containing concensus nuclear factor kappaB (NF-ka ppaB) elements ( - 447/ - 438 and - 222/ - 213) did not enhance promoter ac tivity. Elements associated with both basal and TNF-alpha -stimulated expre ssion lie between bases - 52 and - 203. Site-directed mutagenesis of nuclea r factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of t he - 203/+9 construct by over 95 per cent. Electrophoretic mobility-shift a ssays using oligonucleotides derived from these sites demonstrated formatio n of specific DNA-protein complexes. Both NF-IL6 and CRE unlabelled oligonu cleotides inhibited complex formation with the NF-IL6 oligonucleotide probe . Unlabelled CRE oligonucleotide also effectively inhibited formation of th e complex with the CRE probe, but reduced effectiveness was observed when t he NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensu s NF-kappaB oligonucleotide failed to compete for either probe. TNF-alpha t reatment did not increase levels of these complexes. Thus NF-kappaB does no t enhance basal or TNF-alpha -responsive PGHS-2 transcription in amnion-der ived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regul ating PGHS-2 expression in these cells is indicated, but requires further c larification. (C) 2000 Harcourt Publishers Ltd.