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DJ-1, a cancer- and Parkinson’s disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2 Casey M. Clements*, Richard S. McNally*, Brian J. Conti*, Tak W. Mak†‡, and Jenny P.-Y. Ting*‡ *Department of Microbiology–Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295; and †The Campbell Family Institute for Breast Cancer Research, University Health Network, 620 University Avenue, Suite 706, Toronto, ON, Canada M5G 2C1 Contributed by Tak W. Mak, August 22, 2006 DJ-1͞PARK7, a cancer- and Parkinson’s disease (PD)-associated Nrf2 is a member of the cap ’n’ collar family of basic leucine protein, protects cells from toxic stresses. However, the functional zipper transcription factors that regulate the expression of many basis of this protection has remained elusive. We found that loss antioxidant pathway genes (reviewed in ref. 10). Nrf2 is main- of DJ-1 leads to deficits in NQO1 [NAD(P)H quinone oxidoreductase tained at basal levels in cells by binding to its inhibitor protein, 1], a detoxification enzyme. This deficit is attributed to a loss of Keap1 (11, 12). Keap1 is a BTB (Broad complex, Tramtrack, Nrf2 (nuclear factor erythroid 2-related factor), a master regulator Bric-a-Brac) domain-containing protein that targets Nrf2 for of antioxidant transcriptional responses. DJ-1 stabilizes Nrf2 by ubiquitination by Cul3͞Roc-1, leading to its constitutive degra- preventing association with its inhibitor protein, Keap1, and Nrf2’s dation (13–16). Upon exposure to oxidative stress, xenobiotics, subsequent ubiquitination. Without intact DJ-1, Nrf2 protein is or electrophilic compounds, Nrf2 protein is stabilized and unstable, and transcriptional responses are thereby decreased translocates to the nucleus (17). There, it forms heterodimers both basally and after induction. This effect of DJ-1 on Nrf2 is with other transcription regulators, such as small Maf proteins, present in both transformed lines and primary cells across human and induces the expression of antioxidant genes (18, 19). Nrf2 and mouse species. DJ-1’s effect on Nrf2 and subsequent effects on drives the expression of detoxification enzymes, such as NQO1 antioxidant responses may explain how DJ-1 affects the etiology of [NAD(P)H quinone oxidoreductase 1] and Hmox-1, and en- both cancer and PD, which are seemingly disparate disorders. zymes that generate antioxidant molecules, such as glutathione Furthermore, this DJ-1͞Nrf2 functional axis presents a therapeutic (20, 21). Nrf2 function and the expression of its regulated genes, target in cancer treatment and justifies DJ-1 as a tumor biomarker. including NQO1, have been implicated in the risk and͞or prevention of both cancer and PD (22–27). oxidative stress ͉ PARK7 ͉ NQO1 ͉ Keap1 ͉ neurodegeneration In this study, we find that DJ-1 is required for the expression of several genes, including the prototypic Nrf2-regulated anti- oxidant enzyme NQO1. We report here that DJ-1 is indispens- xidative stress has been implicated as a major contributing able for Nrf2 stabilization by affecting Nrf2 association with Ofactor in a wide variety of ailments. Cancer, cardiovascular Keap1, an inhibitor protein that promotes the ubiquitination and disease, neurodegenerative disorders, and aging all are associ- degradation of Nrf2. These findings implicate DJ-1’s effects on ated with increased oxidative stress in tissues. Such stress results Nrf2 in the development of Parkinson’s disease and cancer and from the accumulation of oxidative species due to their meta- present potential therapeutic targets. bolic generation and environmental exposures. These oxidative species are detoxified by a gambit of antioxidant enzymes and Results molecules. The balance between oxidative species generation siRNA-Mediated Knockdown of DJ-1 and Affymetrix GeneChip Anal- and removal determines the oxidative stress on a given tissue. ysis. To explore DJ-1’s function, we examined its effect on global Not surprisingly, therefore, cellular responses to oxidative stress gene expression. DJ-1 expression was reduced by siRNA in H157 are major determinants of disease susceptibility, particularly in non-small-cell lung carcinoma cells (Fig. 1). The characterization tissues that are sensitive to oxidative stress, such as in the central of the antibody used to verify DJ-1 expression is shown in Fig. nervous system. Genetic defects in oxidative responses lead to 6, which is published as supporting information on the PNAS neurodegenerative diseases. Examples include mutations in web site. The first DJ-1 siRNA (referred to as siDJ-1#1) caused SOD1 (superoxide dismutase 1) that lead to ALS (1) and loss of a modest decrease in DJ-1, whereas siDJ-1#2 caused a profound DJ-1, which leads to early onset Parkinson’s disease (PD) with decrease. RNA samples from cells with siDJ-1#1, two control high penetrance (2). scrambled oligomers (siCTL), and one mock-transfected sample DJ-1 was initially described as a putative oncogene that is able were subjected to GeneChip profiling (Affymetrix, Santa Clara, to transform cells weakly on its own and more strongly in CA). To ensure that changes warranted further study, we combination with Ras (3). DJ-1 is expressed at high levels in primary lung and prostate cancer biopsies (4, 5), and its expres- sion correlates negatively with clinical outcomes in nonsmall cell Author contributions: C.M.C., T.W.M., and J.P.-Y.T. designed research; C.M.C., R.S.M., and lung carcinoma patients (6). The DJ-1 protein affects cell B.J.C. performed research; T.W.M. contributed new reagents͞analytic tools; C.M.C., R.S.M., and B.J.C. analyzed data; and C.M.C. and J.P.-Y.T. wrote the paper. survival, in part, by modulating cellular signaling cascades such CELL BIOLOGY as PTEN͞phosphatidylinositol 3-kinase͞Akt (6) and altering The authors declare no conflict of interest. p53 activity (7). Additionally, we and others have previously Freely available online through the PNAS open access option. shown that DJ-1 expression in cancer cell lines conveys protec- Abbreviations: ARE, antioxidant response element; tBHQ, tert-butylhydroquinone; MEF, mouse embryonic fibroblast; PD, Parkinson’s disease. tion against stresses, including chemotherapy, oxidative stress, Data deposition: The microarray data reported in this paper have been deposited in the endoplasmic reticulum stress, and proteosome inhibition (4, 8, Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov͞geo (accession no. 9). The mechanism by which DJ-1 imparts this protection GSE5519). remains unknown. We report here that DJ-1 is required for the ‡To whom correspondence may be addressed. E-mail: [email protected] or activity of Nrf2 (nuclear factor erythroid 2-related factor), a jenny[email protected]. master regulator of response to oxidative stress. © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607260103 PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15091–15096 Downloaded by guest on October 1, 2021 moter (33), which is a prototypic target of the antioxidant tran- A C scription factor Nrf2 (20). With this fact in mind, we used the siCTL siDJ-1 siDJ-1#1 NTC #2 1000 tfsearch algorithm (as in ref. 34) to search for putative AREs within DJ-1 1,000 bp upstream of the transcriptional start site of the genes identified in Fig. 2. Seven of 17 genes that were changed by Ͼ3-fold G3PDH by siDJ-1 contained an ARE-like sequence (TMAnnRTGAY- nnnGCRwwww) in their promoters (Fig. 2, rightmost column). We then reanalyzed our microarray data with respect to Nrf2 and found B that several Nrf2-regulated genes were altered in the absence of Relative DJ-1 siCTL siDJ-1 #1siDJ-1 #2 Expression mRNA DJ-1 (Fig. 7, which is published as supporting information on the 0 DJ-1 siCTL siDJ1 siDJ1 siMOCK PNAS web site). All array data have been deposited in the Gene #1 #2 Expression Omnibus online repository. Actin DJ-1 Is Required for Nrf2-Mediated Transcription. To verify the Fig. 1. siRNA-mediated knockdown of DJ-1 and GeneChip analysis. (A) microarray data, we used NQO1 as a prototypic target gene of End-point RT-PCR of H157 cells transfected with control siRNA (siCTL) or two DJ-1. Real-time PCR analysis shows that siDJ-1#2 reduced DJ-1 different siRNA targeting DJ-1 (siDJ-1#1 and siDJ-1#2). The DJ-1 RT-PCR gel is and NQO1 by Ͼ80%. However, Nrf2 mRNA expression was not presented as a negative image so bands can be more easily visualized. NTC is changed (Fig. 3A), indicating that NQO1 expression differences a nontemplate control. (B) Western blot analysis of siRNA-transfected H157 are not due to a reduction of Nrf2 mRNA. To determine whether cells demonstrating DJ-1 knockdown at the protein level. (C) Quantitative DJ-1 affects NQO1 gene transcription by means of Nrf2 func- real-time PCR of DJ-1 mRNA after siRNA transfection. Relative mRNA quanti- tation is normalized to 18S rRNA expression. siDJ-1#2 reduced DJ-1 expression tion, we used a reporter construct, pGL2-ARE, which contains to a greater degree than siDJ-1#1, whereas transfection with either a scram- the firefly luciferase gene under the control of an ARE from the bled nonspecific oligomer siRNA or transfection reagent alone (siMOCK) did human NQO1 promoter (Fig. 3B). This construct was tested in not affect DJ-1 expression. the absence or presence of DJ-1. The liver cell line Huh7 was used because Nrf2 activity can be induced in these cells by the nontoxic food preservative tert-butylhydroquinone (tBHQ) (35). stringently filtered expression to exclude differences Ͻ3-fold and Cells were treated with either 50 M tBHQ or DMSO vehicle any genes having spots with a raw signal intensity of Ͻ500 units control, and luciferase activity was measured (Fig.