NF-IL6 and CRE elements principally account for both basal and interleukin-1β-induced transcriptional activity of the proximal 528 bp of the PGHS-2 promoter in amnion-derived AV3 cells: evidence for involvement of C/EBPβ

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-κB 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)-1β (>3-times basal). Point mutations of NF-IL6 and cAMP response element (CRE) in this region reduced both basal and IL-1β-stimulated production of CAT; dual mutation eliminated IL-1β responsiveness. Factors in nuclear extracts from control or IL-1β-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/EBPβ was identified by supershift assay as interacting with both sequences. To a lesser extent C/EBPα and δ 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-1β-inducible transcriptional activity of the proximal 528 bp of the PGHS-2 promoter, while NF-κB elements play no substantial role. C/EBPs, particularly C/EBPβ, are implicated in control of PGHS-2 transcription through the NF-IL6 and CRE sites.