Original Contribution
Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element

https://doi.org/10.1016/j.freeradbiomed.2008.09.027Get rights and content

Abstract

Peroxiredoxin 6 (Prdx6) is a unique antioxidant enzyme that can reduce phospholipid and other hydroperoxides. A549 cells, a human lung-derived cell line, express both Prdx6 and Nrf2, a transcription factor that binds to antioxidant-response elements (AREs) and promotes expression of antioxidant genes. Treatment of A549 cells with 500 μM H2O2 increased Prdx6 mRNA levels 2.5-fold, whereas treatment with 400 μM H2O2 or 200 μM tert-butylhydroquinone (t-BHQ) triggered a corresponding 2.5-fold increase in reporter gene activity in A549 cells transfected with the pSEAP2:Basic vector (BD Bioscience), containing 1524 nucleotides of the human Prdx6 promoter region. Deletion of a consensus ARE sequence present between positions 357 and 349 before the start of transcription led to a striking decrease in both basal and H2O2- or t-BHQ-induced activation in A549 cells and H2O2-induced activation in primary rat alveolar type II cells. Cotransfection with Nrf2 stimulated the Prdx6 promoter in an ARE-dependent manner, whereas it was negatively regulated by Nrf3. siRNA targeting Nrf2 down-regulated reporter gene expression, whereas siRNA targeting the Nrf2 repressor, Keap1, up-regulated it. Binding of Nrf2 to the ARE sequence in chromatin was confirmed by PCR after chromatin immunoprecipitation. These data demonstrate that the ARE within the Prdx6 promoter is a key regulator of basal transcription of the Prdx6 gene and of its inducibility under conditions of oxidative stress.

Section snippets

Chemicals and reagents

Minimal essential medium (MEM) was purchased from Life Technologies (Grand Island, NY, USA), OptiMEM was from Invitrogen (Carlsbad, CA, USA), and H2O2 and tert-butylhydroquinone (t-BHQ) were from (Sigma). pSEAP2 alkaline phosphatase reporter vectors and an alkaline phosphatase activity assay kit were from BD Bioscience (San Jose, CA, USA); competent cells, restriction enzymes, and the Beta-Glo assay system were from Promega (Madison, WI, USA). All chemicals used were at least analytical grade.

Cell culture

Expression of Prdx6 and Nrf2 in lung epithelial A549 cells

A549 cells were examined to determine whether Prdx6 and the transcription factor Nrf2 are expressed in this cell line. RNA was detected using RT PCR and clearly showed expression of both Prdx6 and Nrf2 (Fig. 1A). Prdx6 protein expression was demonstrated by Western blot, using recombinant Prdx6 as a control (Fig. 1B).

Prdx6 mRNA levels and promoter activity of Prdx6 are increased by H2O2

A549 cells were exposed to H2O2 at concentrations ranging from 50 to 1000 μM for 12 h. There was a concentration-dependent increase in Prdx6 mRNA expression with H2O2 treatment as

Discussion

In contrast to the other peroxiredoxins, Prdx6 contains a single conserved cysteine and utilizes GSH as the redox cofactor to reduce lipid hydroperoxides [9]. Unlike the GSH peroxidases, Prdx6 is not a selenoprotein but uses cysteine as its catalytic center. In view of its antioxidant properties, it is not surprising that oxidative stress induced Prdx6 expression [22]. However, the mechanism of such up-regulation has not been addressed previously.

In this study, we examined the human Prdx6

Acknowledgments

We are grateful to Dr. Anil K. Jaiswal (University of Maryland School of Medicine) for kindly providing the cDNAs encoding Nrf2 and Nrf3, Dr. Shyam Biswal (Johns Hopkins University) for the gifts of siRNA, and Drs. Tatyana Milovanova and Jonni Moore for assistance with cell viability studies. This work was supported by HL P01-75587. I.C. was supported as an NRSA Postdoctoral Fellow by HL T32-07748. This work has been presented in part at the Experimental Biology Meetings of 2005, 2006, and 2007.

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