Transcriptional regulation of cyclooxygenase-2 gene in pancreatic beta-cells

Prostaglandin E(2) (PGE(2)) has been shown to negatively affect pancreatic beta-cell function, and its inducible synthesis is mediated in part by cycloxygenase-2 (COX-2). Regulation of basal and inducible COX-2 gene expression in pancreatic beta-cells is not fully understood. In this report, we used pancreatic beta-cells (RINm5F) to explore the molecular mechanisms regulating COX-2 promoter activity. Through deletion analysis of a -907/+70-bp 5' upstream region of the mouse COX-2 gene, we identified an inhibition domain (-804/-371) and an activation domain (-371/+70). The highest promoter activity was seen when the promoter was reduced to -371 bp. Several cis-acting elements were selected for site-directed mutations in the activation domain. We identified three sites that were essential for basal COX-2 promoter activity: 1) CCAAT/enhancer-binding protein (C/EBP), 2) aryl hydrocarbon receptor (AhR), and 3) cAMP response element-binding protein (CREB). Single mutation of each individual site inhibited 70-80% of basal COX-2 promoter activity. Double mutation of the AhR and CREB-binding sites showed synergy in repressing COX-2 promoter activity as did mutation of all three sites. We demonstrated that the transcription factors from RINm5F nuclear extracts specifically bound to oligonucleotides containing C/EBP, AhR, or CREB consensus sites. Forskolin, an activator of adenyl cyclase, increased COX-2 promoter activity via the CREB site. COX-2 promoter activity was also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR activator, through the AhR site. Both forskolin and 2,3,7,8-tetrachlorodibenzo-p-dioxin increased COX-2 mRNA in a dose-dependent manner. We consider these three transcriptional regulators of COX-2 expression to be potential targets for the prevention of beta-cell damage mediated by PGE(2).