Abstract 122: Activation of Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) Enhances Cyclooxygenase 2 Expression via Promoter Antioxidant Response Element in Preglomerular Vascular Smooth Muscle Cells (PGVSMCs)

Abstract

Nrf2 is a key cytoprotective transcription factor driving many antioxidant and anti-inflammatory genes expression via binding to antioxidant response elements (AREs) in promoters of targeting genes. However, it was reported that Nrf2 also increased expression of pro-inflammatory and pro-oxidative genes IL-6 and NOX4 through the same mechanism. We detected 2 putative ARE consensus sequences within 1500 nucleotides (nts) located in the genomic region upstream of the transcription start site in rat cyclooxygenase 2 (COX2) gene. Therefore, we hypothesized that activation of Nrf2-dependent AREs upregulates COX2 expression in rat PGVSMCs. Incubation of the cells for 24 hours with Nrf2 activator, tert-butylhydroquinone (tBHQ), did not significantly alter Nrf2 expression but dose-dependently increased COX2 gene and protein in a dose response manner (5-20μM). Twenty μM of tBHQ treatment enhanced expression of COX2 gene and protein by 10.7±2.2 and 9.2±3.4 fold (both p<0.01, N=3), COX2 activity (from COX2-based product of fluorescent resorufin) by 229.9±28.8% (p<0.005, N=6) but reactive oxygen species (ROS) generated in the cells (from oxidized dihydroethidium-based fluorescent intensity) were reduced by 35.1±10.2% (p<0.01, N=4). tBHQ also significantly promoted Nrf2 nuclear translocation by 86±7.4% (p<0.05, N=3) and potentiated Nrf2 activity by 387.5±19.2% (p<0.005, N=4) determined with cell fraction immunoblotting and Nrf2-ARE binding-based enzyme-linked immunosorbent assay. Nrf2 expression was dramatically knocked down with markedly decreased COX2 expression and activity in the cells transfected with Nrf2 specific siRNA and treated with tBHQ compared to those transfected with the scramble control siRNA and treated with tBHQ. Chromatin immunoprecipitation (CHIP)-based PCR analysis revealed an enhanced recruitment of Nrf2 to the endogenous COX2 promoter spanning the ARE (-417 to -426 nts) after 24 hour-tBHQ treatment but knockdown of Nrf2 gene with siRNA significantly diminished the tBHQ-mediated role, suggesting that there is a functional ARE located in COX2 promoter. In conclusion, these results show a novel role of Nrf2 in inducing COX2 expression through binding to promoter ARE in the absence of increased ROS in rat PGVSMCs.