An antioxidant response element in the human peroxiredoxin 6 (Prdx6) promoter is necessary for induction by H 2 O 2

Abstract

Peroxiredoxin 6 (Prdx6) is the only member of the Prdx family that uses a single conserved cysteine in its peroxidase activity and can reduce lipid hydroperoxides. H2O2 induces Prdx6 mRNA in several experimental systems. 1524 bp of the human Prdx6 promoter region were cloned into the pSEAP2-Basic vector (BD, Bioscience) and transfected into A549 cells. The cells were treated with 400μM H2O2 for 24 h after transfection and secreted alkaline phosphatase (SEAP) activity was measured 8 h later. A plasmid expressing β-galactosidase from the SV40 promoter served as a transfection control. Basal transcriptional activity of the full-length construct increased by ~ 2.5-fold in the presence of H2O2. Potential antioxidant response elements (AREs), shown elsewhere to bind the transcription factor Nrf2, exist at positions −401 to −393 and −357 to −349. Co-transfection with Nrf2 stimulated the Prdx6 promoter while Nrf3 negatively regulated the promoter activity. The region at −357 to −349 bp perfectly matched the ARE consensus sequence. Deletion of this element led to a striking decrease in both basal and H2O2-induced activity in A549 cells as well as in rat primary alveolar type II cells and abolished Nrf2-activation upon co-transfection. These data demonstrate that the ARE within the Prdx6 promoter is a key regulator of basal transcription of the Prdx6 gene, as well as its inducibility under conditions of oxidative stress. [PO1-HL-19737].