A genomic screen for activators of the antioxidant response element Yanxia Liu†, Jonathan T. Kern‡, John R. Walker§, Jeffrey A. Johnson‡, Peter G. Schultz§¶ʈ, and Hendrik Luesch†ʈ †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 (sent for review December 13, 2006) The antioxidant response element (ARE) is a cis-acting regulatory activation, translocates into the nucleus and transcriptionally acti- enhancer element found in the 5؅ flanking region of many phase II vates ARE-dependent genes after recruiting Maf proteins (2). The detoxification enzymes. Up-regulation of ARE-dependent target upstream regulatory mechanisms by which ARE-activating signals genes is known to have neuroprotective effects; yet, the mechanism are linked to Nrf2 remain to be fully elucidated. It has been of activation is largely unknown. By screening an arrayed collection demonstrated that reactive sulfhydryl groups of Keap1 are sensors of Ϸ15,000 full-length expression cDNAs in the human neuroblas- for induction of phase II genes (13), leading to the proposal that the toma cell line IMR-32 with an ARE-luciferase reporter, we have Nrf2/Keap1 interaction represents a cytoplasmic sensor for oxida- identified several cDNAs not previously associated with ARE activa- tive stress. However, 1-phosphatidylinositol 3-kinase (PI3K), tion. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and MAPKs, and protein kinase C (PKC) have also been implicated in dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse- ARE activation (14–17), suggesting that multiple signaling mech- derived cortical neurons. Overexpression of SQSTM1 and DPP3 in anisms may be involved. A deeper understanding of the molecular IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear trans- mechanisms governing the response to oxidative stress is necessary location and led to increased levels of NAD(P)H:quinone oxidoreduc- to exploit this signaling pathway therapeutically. Consequently, we tase 1, a protein which is transcriptionally regulated by the ARE. When have carried out a genome-wide, high-throughput screen to identify transfected into IMR-32 neuroblastoma cells that were depleted of previously unrecognized ARE activators. Genome-scale screens of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 this type with spatially arrayed cDNA and siRNA libraries have were unable to activate the ARE or induce NAD(P)H:quinone oxi- proven powerful for the functional analysis of various pathways doreductase 1 expression, indicating that the ARE activation upon involved in inflammation, cancer, insulin signaling, and other ectopic expression of these cDNAs is mediated by NRF2. Studies with biological processes (18). pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Results Overexpression of these cDNAs conferred partial resistance to hydro- High-Throughput Screen of Arrayed cDNA Library for ARE Activators. gen peroxide or rotenone-induced toxicity, consistent with the in- A cDNA library consisting of Ϸ15,000 full-length human expres- duction of antioxidant and phase II detoxification enzymes, which can sion cDNAs arrayed in 384-well plates was screened for activators protect from oxidative stress. This work and other such studies may of the ARE (19). The cDNA collection, covering approximately provide mechanisms for activating the ARE in the absence of general half of the human genome, was obtained from OriGene Technol- oxidative stress and a yet-unexploited therapeutic approach to de- ogies (Rockville, MD); each cDNA was cloned downstream of a generative diseases and aging. CMV promoter. The library was transfected into IMR-32 human neuroblastoma cells along with a human NQO1-ARE reporter genome-wide screen ͉ oxidative stress ͉ neuroprotection construct consisting of the luciferase gene under the control of the ARE-containing promoter, as described in ref. 20. The IMR-32 cell xidative stress is implicated in the pathogenesis of many line has been used as a cellular model of oxidative stress (3, 14, 20). Oage-related diseases, including neurodegenerative disorders, After 48 h, luminescence was detected in each well as a measure of such as Alzheimer’s and Parkinson’s disease, and aging itself (1). In ARE activity (Fig. 1A). The screen was carried out in duplicate to humans, the antioxidant response element (ARE) regulates the evaluate the reproducibility of the results, and a constitutively active expression of a number of cytoprotective antioxidant enzymes and PI3K construct (21), which is known to activate the ARE in IMR-32 scavengers, which contribute to the endogenous defense against cells (14), was used as a positive control (five-fold average activation oxidative stress. The ARE is a cis-acting regulatory enhancer over background). cDNAs that showed high activation and high NEUROSCIENCE element (core sequence: 5Ј-GTGACnnnGC-3Ј) found in the 5Ј reproducibility were selected for further analysis (Fig. 1B, lower flanking region of many phase II detoxification enzymes and is right corner, green). activated by reactive oxygen species, as well as other electrophilic agents (2). Genes regulated by the ARE include heme oxygenase-1, GSTs, and NAD(P)H:quinone oxidoreductase 1 (NQO1). It has Author contributions: J.T.K., J.A.J., P.G.S., and H.L. designed research; Y.L., J.T.K., J.R.W., been shown that activation of the ARE protects neuroblastoma and H.L. performed research; and P.G.S. and H.L. wrote the paper. cells, astrocytes, and neurons from oxidative damage (3–5). The authors declare no conflict of interest. The molecular mechanism of ARE activation is largely unknown. Abbreviations: ARE, antioxidant response element; NQO1, NAD(P)H:quinone oxidoreductase However, the central transcription factor involved in the induction 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, of phase II enzymes has been shown to be NF-E2-related factor 2 tert-butylhydroquinone. (Nrf2). Nrf2 knockout mice show reduced expression of Data deposition: The microarray data reported in this paper has been deposited in the Gene glutathione biosynthetic genes (6) and GSTs (7), diminished de- Expression Omnibus database (accession no. GSE6451). toxification capabilities (8), decreased responsiveness to chemo- ʈTo whom correspondence may be addressed. E-mail: [email protected]fl.edu or schultz@ protective agents (9), and enhanced susceptibility to oxidative scripps.edu. stress-induced cell death (10–12). Conversely, Nrf2 overexpression This article contains supporting information online at www.pnas.org/cgi/content/full/ in vitro and in vivo protects from oxidative stress (10, 11). Nrf2 is 0700898104/DC1. bound to the cytoplasmic repressor protein Keap1 and, upon © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700898104 PNAS ͉ March 20, 2007 ͉ vol. 104 ͉ no. 12 ͉ 5205–5210 Downloaded by guest on October 1, 2021 terized prosurvival gene products. SQSTM1 is induced by oxidative stress and mediates diverse signaling pathways associated with cell stress, survival, and inflammation (22–24). It colocalizes with protein aggregates found in Parkinson’s, Pick’s, and Alzheimer’s disease (25–27), and was recently reported to prevent huntingtin- induced cell death (28). Bcl-xL regulates the outer mitochondrial membrane channel opening and consequently controls the produc- tion of reactive oxygen species and cytochrome c release by mitochondria (29). Overexpression of Bcl-xL has been shown to inhibit 6-hydroxydopamine-induced death in a Parkinson’s disease- like model in a neuroblastoma cell line (30). The longer isoform of MCL1 is a viability-promoting member of the BCL2 family that reduces cell damage-induced release of mitochondrial cytochrome c. It exerts its antiapoptotic properties by inhibiting mitochondrial Ca2ϩ signals (31). Two other cDNAs encode transcription- associated factors (DBP and TORC1). DBP is a basic leucine zipper transcription factor (32); TORC1 (MECT1) is a cAMP-responsive element binding protein-dependent transcriptional coactivator. The fusion product of TORC1 with MAML2 has transforming, cAMP-responsive element binding protein-dependent activity (33) and is required for the growth of mucoepidermoid salivary gland tumors (34). DPP3 is a zinc metallo-exopeptidase with broad substrate specificity implicated in various disease processes, such as Fig. 1. Genome-wide cDNA overexpression screen for ARE activators. (A) General high-throughput screening procedure. Approximately 15,000 expres- cancer and inflammation (35–37). One cDNA, SFRS10, encodes a sion cDNAs, normalized and arrayed in 384-well plates, were transfected into splicing factor involved in exon 10 splicing of tau (38, 39), a IMR-32 human neuroblastoma cells along with an ARE–luciferase reporter microtubule-stabilizing protein that is hyperphosphorylated and construct. After 48-h incubation, luciferase activity was assessed by measuring forms