The Human Peroxiredoxin 6 (Prdx6) Promoter Activity is Stimulated by H 2 O 2

Abstract

Peroxiredoxin 6 (Prdx6), the only member of the Prdx family that contains a single conserved cysteine residue, is an important lung anti-oxidant enzyme. To study the human Prdx6 promoter, we used A549, a human lung epithelial cell line which expresses both Prdx6 and Nrf2, an oxidative stress-responsive transcription factor. Treatment of A549 cells with 200μM H2O2 for 12 h increased Prdx6 mRNA levels 2.5 fold compared with control. 1524 bp of the human Prdx6 promoter region obtained by PCR was cloned into the pSEAP2-Basic vector (BD, Bioscience) for analysis using secreted alkaline phosphatase as a reporter gene. Promoter clone S82, obtained from BAC clone 638L8, contains a single A to T transversion at position −273 compared to the Genbank sequence. Potential antioxidant response elements (AREs) exist at positions −401 to −393 and −387 to −349. AREs have been shown to bind Nrf2. The pSEAP2-Basic plasmid containing the promoter was transfected into A549 cells. A plasmid expressing β-galactosidase from the SV40 promoter was used as a transfection control. We measured SEAP activity after transfection for 24 h and treated the cells with 400μM H2O2 for another 8 hours. Prdx6 promoter activity increased by about 2.5 fold in the presence of H2O2. These data indicate that oxidative stress can induce an increased transcriptional response from the Prdx6 promoter. [PO1-HL-19737]