Abstract
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).
Highlights
Cyclooxygenase is a key enzyme that catalyzes the conversion of arachidonic acid to prostaglandins
Luciferase activity of reporter construct pCOX2(Ϫ40/ϩ70) was almost at the level of the pGL3 Basic vector. These results indicated that the region between Ϫ371 and Ϫ40 bp contains activation domains for basal COX-2 expression in -cells
The selective COX-2 inhibitor NS-398 has been demonstrated to partially restore glucose-stimulated insulin secretion in HIT cells and islets treated with IL-1 for 24 h [25] and to prevent low dose streptozotocin-induced diabetes in mice [29]
Summary
Experimental Reagents—Forskolin, 3-isobutyl-1-methylxanthine (IBMX), and RPMI 1640 medium with L-glutamine were obtained from Sigma. SuperFect transfection reagent and one-step RT-PCR kit were obtained from Qiagen. Transfections were performed using SuperFect reagent (Qiagen), following the manufacturer’s protocol. Each transfection was performed using 1.5 g of Firefly luciferase reporter construct DNA that contained various deletions and site-directed mutants of COX-2 promoter gene plus 0.1 g of an internal control Renilla luciferase reporter plasmid pRL-TK (Promega). 2.5 h after transfection, the medium was removed by aspiration and replaced with normal culture media containing 10% fetal bovine serum and antibiotics. Following an overnight recovery period, the transfected cells were cultured in medium with 0.2% BSA for 48 h. The cells were switched to 0.2% BSA medium for 48 h to maintain the same culturing condition as the transfected cells used for luciferase assays. Statistics—Student’s t test was used to evaluate statistical significance of differences between two groups. p Ͻ 0.05 was considered statistically significant
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