The role of CCAAT/enhancer-binding protein β in the transcriptional regulation of COX-2 in human amnion

Human labour is associated with increased prostaglandin synthesis within the uterus by the action of the inducible type-2 cyclo-oxygenase enzyme (COX-2). A major source of prostaglandin is the fetal membranes, in particular the amnion, in which expression of COX-2 increases in late pregnancy and with labour. The COX-2 gene promoter contains several putative transcription factor binding sites including those for NF-κB, AP-1 and C/EBP and therefore has the features of a rapid response gene. We have previously shown that, in amnion, the NF-κB DNA-binding sites in the COX-2 promoter are essential for gene expression and that there is an increase in NF-κB activity in amnion with the onset of labour. In this study, we demonstrate that in primary human amnion cells, CCAAT/enhancer-binding protein β (C/ EBPβ) DNA-binding sites are crucial for the function of the COX-2 gene promoter. Three potential C/EBPβ DNA-binding sites were identified within the COX-2 promoter which were shown to bind to C/ EBPβ but not to C/EBPα, C/EBPδ, CREB (cAMP responsive element modulator) or CREM. Luciferase reporter constructs with site-directed mutagenesis of the three C/EBPβ sites in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. However, comparison of C/EBPβ protein levels and their DNA-binding activity from cells obtained before and after labour showed no significant differences. This suggests that although C/ EBPβ plays an essential constitutive role in the expression of COX-2, C/EBPβ may not be directly involved in its regulation in association with human labour.