title: Tumour necrosis factor-alpha regulation of prostaglandin H synthase-2 transcription is not through nuclear factor-kappaB in amnion-derived AV-3 cells.
abstract: Tumour necrosis factor (TNF)-alpha-stimulated prostaglandin (PG) E(2)biosynthesis 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 kappaB (NF-kappaB) elements (-447/-438 and -222/-213) did not enhance promoter activity. Elements associated with both basal and TNF-alpha-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-kappaB oligonucleotide failed to compete for either probe. TNF-alpha treatment did not increase levels of these complexes. Thus NF-kappaB does not enhance basal or TNF-alpha-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.
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Faculty of Medical and Health Sciences, 85 Park Road, Grafton, Auckland, New Zealand.
authors.forename: W R
authors.initials: WR
authors.lastname: Hansen
authors.forename: K W
authors.initials: KW
authors.lastname: Marvin
authors.forename: S
authors.initials: S
authors.lastname: Potter
authors.forename: M D
authors.initials: MD
authors.lastname: Mitchell
mesh_terms: D000650:Amnion / Q000201:enzymology*; D002460:Cell Line; D000242:Cyclic AMP; D015232:Dinoprostone / Q000096:biosynthesis; D004586:Electrophoresis; D005260:Female; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000502:physiology*; D009841:Oligonucleotides / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020218:Response Elements; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
chemical_list: D015850:Interleukin-6; D016328:NF-kappa B; D009841:Oligonucleotides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone