A genomic screen for activators of the antioxidant response element

^†Department of Medicinal Chemistry, University of Florida, 1600 Southwest Archer Road, Gainesville, FL 32610;
^‡Department of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705;
^§Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; and
^¶Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Peter G. Schultz, January 31, 2007 (received for review December 13, 2006)

Abstract

The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5′ flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of ≈15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.

Footnotes

^‖To whom correspondence may be addressed. E-mail: luesch{at}cop.ufl.edu or schultz{at}scripps.edu
Author contributions: J.T.K., J.A.J., P.G.S., and H.L. designed research; Y.L., J.T.K., J.R.W., and H.L. performed research; and P.G.S. and H.L. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The microarray data reported in this paper has been deposited in the Gene Expression Omnibus database (accession no. GSE6451).
This article contains supporting information online at www.pnas.org/cgi/content/full/0700898104/DC1.
Abbreviations:

ARE,

antioxidant response element;

NQO1,

NAD(P)H:quinone oxidoreductase 1;

Nrf2,

NF-E2-related factor 2;

NRF2,

human NF-E2-related factor;

PI3K,

1-phosphatidylinositol 3-kinase;

qPCR,

quantitative PCR;

RT,

reverse transcription;

SQSTM1,

sequestosome 1;

tBHQ,

tert-butylhydroquinone.