NF-IL6 and CRE elements principally account for both basal and interleukin-1 beta-induced transcriptional activity of the proximal 528bp of the PGHS-2 promoter in amnion-derived AV3 cells: evidence for involvement of C/EBP beta.
Prostaglandin H synthase (PGHS)-2 promoter fragments (-528 to +9 bp and 5' unidirectional deletions thereof) were cloned upstream of the chloramphenicol acetyl-transferase (CAT) reporter gene. These were transfected into amnion-derived AV3 cells. The region, -528 to -203, which includes NF-kappa B sites, had little influence on CAT expression. The region, -203 and -52, however, was responsible for most of the basal promoter activity and also conferred responsiveness to interleukin (IL)-1 beta (>3-times basal). Point mutations of NF-IL6 and cAMP response element (CRE) in this region reduced both basal and IL-1 beta-stimulated production of CAT; dual mutation eliminated IL-1 beta responsiveness. Factors in nuclear extracts from control or IL-1 beta-stimulated AV3 cells specifically complexed the NF-IL6 and CRE sequences. However, the NF-IL6 and CRE oligonucleotides cross-competed, suggesting a common factor. C/EBP beta was identified by supershift assay as interacting with both sequences. To a lesser extent C/EBP alpha and delta also interacted with the NF-IL6 site. However, CRE binding protein (CREB), was absent from the complex with the CRE. In conclusion, NF-IL6 and CRE elements principally account in AV3 amnion cells for basal and IL-1 beta-inducible transcriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-kappa B elements play no substantial role. C/EBPs, particularly C/EBPbeta, are implicated in control of PGHS-2 transcription through the NF-IL6 and CRE sites.
Potter
S
S
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Faculty of Medical and Health Sciences, Auckland, New Zealand.
Mitchell
M D
MD
Hansen
W R
WR
Marvin
K W
KW
D000650:Amnion / Q000166:cytology; D015152:Blotting, Northern / Q000379:methods; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D051546:Cyclooxygenase 2; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics*; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D020218:Response Elements; D014158:Transcription, Genetic
D022782:CCAAT-Enhancer-Binding Protein-beta; D007375:Interleukin-1; D015850:Interleukin-6; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases