Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14960–14965, December 1996 Pharmacology Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene RADJENDIRANE VENUGOPAL AND ANIL K. JAISWAL* Department of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 Communicated by Y. W. Kan, University of California, San Francisco, CA, August 5, 1996 (received for review February 21, 1996) ABSTRACT Twenty-four base pairs of the human anti- (c-Jun, Jun-D, and Jun-B) form homodimers among them- oxidant response element (hARE) are required for high basal selves and heterodimers with c-Fos and Fra (Fra1 and Fra2) transcription of the NAD(P)H:quinone oxidoreductase1 proteins and bind to the TRE (a single AP1 binding site) and (NQO1) gene and its induction in response to xenobiotics and regulate the expression of target genes (6). Nrf1 and Nrf2 are antioxidants. hARE is a unique cis-element that contains one recently identified NF-E2-related factors that bind to the perfect and one imperfect AP1 element arranged as inverse sequence GCTGAGTCATGATGAGTCA (7, 8). The Nrf1 repeats separated by 3 bp, followed by a ‘‘GC’’ box. We report and Nrf2 contain 742 and 589 amino acids, respectively. Both here that Jun, Fos, Fra, and Nrf nuclear transcription factors these proteins display marked homology to the NF-E2, which bind to the hARE. Overexpression of cDNA derived combi- regulates expression of globin genes in developing erythroid nations of the nuclear proteins Jun and Fos or Jun and Fra1 cells through interaction with upstream AP1-like recognition repressed hARE-mediated chloramphenicol acetyltransferase sites (9). Like Jun and Fos, Nrf1 and Nrf2 are bZIP proteins (CAT) gene expression in transfected human hepatoblastoma (7, 8). hARE is a unique element. This is because, the hARE (two (Hep-G2) cells. Further experiments suggested that this re- AP1- or AP1-like elements and a GC box) and not the TRE pression was due to overexpression of c-Fos and Fra1, but not (a single AP1 element) is responsive to xenobiotics and due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) antioxidants (3). The Jun and Fos transcription factors are proteins in all the possible combinations were more or less known to bind to the human NQO gene hARE but not with ineffective in repression or upregulation of hARE-mediated 1 rat NQO1 gene ARE even though both elements contained gene expression. Interestingly, overexpression of Nrf1 and highly conserved sequences (4, 5). Therefore, the actual Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells proteins that bind to both hARE and rat ARE and mediate the significantly increased CAT gene expression from reporter expression and induction of the NQO1 gene in human and rat plasmid hARE-thymidine kinase-CAT in transfected cells that tissues were predicted to be other than Jun and Fos proteins were inducible by b-naphthoflavone and tert-butyl hydroqui- (4, 5). We reasoned that these other proteins have to be none. These results indicated that hARE-mediated expression AP1-related because both hARE and rat ARE contain AP1 of the NQO1 gene and its induction by xenobiotics and and AP1-like elements. This prompted us to search the antioxidants are mediated by Nrf1 and Nrf2. The hARE- literature on AP1-related proteins. Interestingly, we found that mediated basal expression, however, is repressed by overex- tissue specific expression of Nrf1 and Nrf2 transcription pression of c-Fos and Fra1. factors was identical to the tissue specific expression of NQO1 gene regulated by hARE (7, 8, 10). In addition, we found that the binding site for Nrf1 and Nrf2 was very similar to hARE NAD(P)H:quinone oxidoreductase1 (NQO1) catalyzes reduc- tive detoxification of quinones and its derivatives and protect (4, 7, 8). These results encouraged us to determine the role of the cells against redox cycling and oxidative stress (1). Twenty- Nrf1 and Nrf2 proteins in the regulation of hARE-mediated expression of NQO and other detoxifying enzyme genes. four base pairs of the human antioxidant response element 1 In the present report, we demonstrate that overexpression of (hARE) were characterized previously in the promoter of the combinations of nuclear proteins Jun and Fos or Jun and Fra1 human NQO1 gene that are essential for high basal transcrip- down regulated hARE-mediated gene expression. We also tion and induction in response to xenobiotics and antioxidants show that downregulation of hARE-mediated chlorampheni- including b-naphthoflavone (b-NF), tert-butyl-hydroquinone col acetyltransferase (CAT) gene expression in transfected (t-BHQ), and hydrogen peroxide (2–4). Similar cis-elements cells is due to overexpression of c-Fos and Fra1, but not due have also been characterized in the promoters of the genes to Jun proteins. We further demonstrate that the nuclear encoding rat NQO1, rat and mouse glutathione S-transferase protein Nrf1 binds to the hARE. The overexpression of Nrf1 (GST) Ya subunit, and rat GST P (4, 5). The current hypoth- and Nrf2 proteins individually and in combination upregulated esis is that coordinated induction of several defensive gene both the expression of hARE-mediated CAT gene expression products may provide the required protection for cells against and induction by b-NF and t-BHQ. environmental insults and oxidative stress (4). The NQO gene hARE contains one perfect and one 1 MATERIALS AND METHODS imperfect AP1 binding site arranged as inverse repeats sepa- rated 3 bp apart followed by ‘‘GC’’ box (3, 4). The AP1 binding Plasmid Construction. The construction of hARE-, mutant site [also known as 12-O-tetradecanoylphorbol-13-acetate hARE- and TRE–thymidine kinase (tk)–CAT plasmids have (TPA) response element or TRE] was characterized previ- ously in the promoter regions of the collagenase and metal- Abbreviations: NQO1, NAD(P)H:quinone oxidoreductase1;ARE, lothionein genes, and is required to activate their expression in antioxidant response element; hARE, human ARE; TRE, TPA response to TPA (6). The products of protooncogenes Jun (12-O-tetradecanoylphorbol-13-acetate) response element; b-NF, b-naphthoflavone; t-BHQ, tert-butyl hydroquinone; GST, glutathione S-transferase; RSV, Rous sarcoma virus; CAT, chloramphenicol The publication costs of this article were defrayed in part by page charge acetyltransferase, TK, thymidine kinase; LNCX, eukaryotic expres- payment. This article must therefore be hereby marked ‘‘advertisement’’ in sion vector; b-gal, b-galactosidase. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 14960 Downloaded by guest on October 4, 2021 Pharmacology: Radjendirane and Jaiswal Proc. Natl. Acad. Sci. USA 93 (1996) 14961 been described (3). In addition, the plasmid pNQO1CAT0.837 are also described (2). The plasmid eukaryotic expression vector (LNCX), LNCX–c-Jun, LNCX–Jun-D, LNCX–Jun-B, LNCX–c-Fos and LNCX–Fra1 were obtained from Kevin Ryder (Fox Chase Cancer Center, Philadelphia) (11). These plasmids were all derived by inserting the respective murine genes into the polylinker region of LNCX, so that each gene is expressed via the cytomegalovirus promoter. The human Nrf1 and mouse Nrf2 cDNA were gift from Jefferson Y. Chan (University of California, San Francisco). The 2.6-kb EcoRI insert of Nrf1 cDNA was subcloned at HpaI site of LNCX to generate LNCX–Nrf1R and LNCX–Nrf1C. The Nrf1 cDNA was also subcloned at the EcoRI site of pMT2 to generate pMT2–Nrf1R and pMT2–Nrf1C. The Nrf1 cDNA subcloned in the pMT2 vector is transcribed by major adenovirus late promoter upon transfection in COS1 cells (12). The 1.5-kb SalI–NheI fragment of mouse Nrf2 cDNA was subcloned in expression vector LNCX to generate LNCX–Nrf2R and LNCX–Nrf2C. In the above described expression plasmids, R denotes the reverse orientation of the cDNA insert, and C denotes the correct orientation of the cDNA insert. Cell Culture. Mouse hepatoma (Hepa-1), mouse embryonal carcinoma (F9), human hepatoblastoma (Hep-G2), human cervical carcinoma (HeLa), and monkey kidney (COS1) cells were grown in monolayer cultures as described (2, 3). Supershift Assays. The nuclear extracts from various cells were prepared according to the procedure of Kadonaga and Tjian (13). The supershift assays with hARE, nuclear extracts FIG. 1. Band and supershift assays. (A) Nucleotide sequences of from various cell lines and Jun, Fos, and Nrf1 antibodies were human NQO1 gene hARE, mutant hARE and human collagenase performed by procedures as described (2, 3). The supershifted gene TRE are shown. The mutant hARE contained mutations in the Jun, Fos, and Nrf1 bands were observed in all the cell lines 39 AP1 element. This mutation is known to significantly reduce the hARE-mediated CAT gene expression and b-NF induction (3). (B) tested. However, their intensity varied depending on the The nuclear extract from Hepa-1 cells was incubated with preimmune degree of cross-species crossreactivity and abundance. serum and antibodies against murine c-Jun, Jun-D, c-Fos, and human Cotransfection of Reporter and Expression Plasmids. Ten Nrf1 and supershift assays performed with the 32P-end labeled hARE. micrograms of the reporter plasmids TRE–tk–CAT, hARE– Only shifted (SB) and supershifted (SSB) bands are shown. tk–CAT, mutant hARE–tk–CAT, and pNQO1CAT0.837 were cotransfected alone or with various expression plasmids (Jun, shift assays with hARE and Hepa-1 nuclear extracts indicated Fos, Fra, and Nrf) individually and in several combinations to presence of Jun, Fos, and Nrf1 in the complex (Fig. 1B). study the role of these transcription factors in the regulation of To determine the role of Jun, Fos, Fra1, Nrf1, and Nrf2 hARE-mediated NQO1 gene expression. Five micrograms of nuclear factors in the hARE-mediated regulation of NQO1 the Rous sarcoma virus (RSV)–b-galactosidase (b-gal) plas- gene expression, we cotransfected hARE–tk–CAT plasmid mid was included in each transfection to normalize the trans- with plasmids expressing these proteins individually and in fection efficiency.