NF-IL6 and CRE elements principally account for both basal and interleukin-1 beta-induced transcriptional activity of the proximal 528 bp 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 chloramphenic ol acetyl-transferase (CAT) reporter gene. These were transfected into amni on-derived AV3 cells. The region, -528 to -203, which includes NF-kappa B s ites, had little influence on CAT expression. The region, -203 and -52, how ever, was responsible for most of the basal promoter activity and also conf erred responsiveness to interleukin (IL)-1 beta (>3-times basal). Point mut ations of NF-IL6 and cAMP response element (CRE) in this region reduced bot h basal and IL-1 beta-stimulated production of CAT; dual mutation eliminate d IL-1 beta responsiveness. Factors in nuclear extracts from control or IL- 1 beta-stimulated AV3 cells specifically complexed the NF-IL6 and CRE seque nces. However, the NF-IL6 and CRE oligonucleotides cross-competed, suggesti ng a common factor. C/EBP beta was identified by supershift assay as intera cting with both sequences. To a lesser extent C/EBP alpha and delta also in teracted with the NF-IL6 site. However, CRE binding protein (CREB), was abs ent from the complex with the CRE. In conclusion, NF-IL6 and CRE elements p rincipally account in AV3 amnion cells for basal and IL-1 beta-inducible tr anscriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-kappa B elements play no substantial role. C/EBPs, particularly C/EBP b eta, are implicated in control of PGHS-2 transcription through the NF-IL6 a nd CRE sites.