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Small Maf (MafG and MafK) Proteins Negatively Regulate Antioxidant Response Element-mediated Expression and Antioxidant Induction of the NAD(P)H:Quinone Oxidoreductase1 Gene

2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês

10.1074/jbc.m003531200

1083-351X

Saravanakumar Dhakshinamoorthy, Anil K. Jaiswal,

Vitamin C and Antioxidants Research

The antioxidant response element (ARE) is known to regulate expression and induction of NQO1, GST Ya, and other detoxifying enzyme genes in response to antioxidants and xenobiotics. The nuclear transcription factor Nrf2 and Nrf1 bind to the ARE and positively regulate expression and induction of the NQO1 and GST Ya genes. In this study, we demonstrate that overexpression of small Maf (MafG and MafK) proteins negatively regulate ARE-mediated expression and tert-butyl hydroquinone induction of the NQO1 and GST Ya genes in transfected Hep-G2 cells. In similar experiments, overexpression of small Maf proteins also repressed Nrf2-mediated up-regulation of ARE-mediated NQO1 and GST Ya genes expression in Hep-G2 cells co-transfected with Nrf2 and small Maf proteins. Band and supershift assays with the NQO1 gene ARE and nuclear proteins demonstrate that small MafG and MafK bind to the ARE as Maf-Maf homodimers and Maf-Nrf2 heterodimers. Therefore, Maf-Maf homodimers and possibly Maf-Nrf2 heterodimers play a role in negative regulation of ARE-mediated transcription and antioxidant induction of NQO1 and other detoxifying enzyme genes. In contrast to Maf-Nrf2, the Maf-Nrf1 heterodimers failed to bind with the NQO1 gene ARE and did not demonstrate the repressive effect in transfection assays. The antioxidant response element (ARE) is known to regulate expression and induction of NQO1, GST Ya, and other detoxifying enzyme genes in response to antioxidants and xenobiotics. The nuclear transcription factor Nrf2 and Nrf1 bind to the ARE and positively regulate expression and induction of the NQO1 and GST Ya genes. In this study, we demonstrate that overexpression of small Maf (MafG and MafK) proteins negatively regulate ARE-mediated expression and tert-butyl hydroquinone induction of the NQO1 and GST Ya genes in transfected Hep-G2 cells. In similar experiments, overexpression of small Maf proteins also repressed Nrf2-mediated up-regulation of ARE-mediated NQO1 and GST Ya genes expression in Hep-G2 cells co-transfected with Nrf2 and small Maf proteins. Band and supershift assays with the NQO1 gene ARE and nuclear proteins demonstrate that small MafG and MafK bind to the ARE as Maf-Maf homodimers and Maf-Nrf2 heterodimers. Therefore, Maf-Maf homodimers and possibly Maf-Nrf2 heterodimers play a role in negative regulation of ARE-mediated transcription and antioxidant induction of NQO1 and other detoxifying enzyme genes. In contrast to Maf-Nrf2, the Maf-Nrf1 heterodimers failed to bind with the NQO1 gene ARE and did not demonstrate the repressive effect in transfection assays. NAD(P)H:quinone oxidoreductase1 glutathioneS-transferase γ-glutamylcysteine synthetase antioxidant response element human NQO1 gene ARE tert-butyl hydroquinone reactive oxygen species polyacrylamide gel electrophoresis NAD(P)H:quinone oxidoreductase1 (NQO1)1 is a flavoprotein that catalyzes two-electron reductive metabolism and detoxification of quinones. This protects cells against quinone-induced oxidative stress and neoplasia (1Radjendirane V. Joseph P. Jaiswal A.K. Forman H.J. Cadenas E. Oxidative Stress and Signal Transduction. Chapman & Hall, New York1997: 441-475Crossref Google Scholar, 2Talalay P. Fahey J.W. Holtzclaw W.D. Prestera T. Zhang Y. Toxicol. Lett. 1995; 82–83: 173-179Crossref PubMed Scopus (438) Google Scholar). Higher levels of NQO1 gene expression were observed in liver, lung, colon, and breast tumors, when compared with normal tissues of the same origin (3Cresteil T. Jaiswal A.K. Biochem. Pharmacol. 1991; 42: 1021-1027Crossref PubMed Scopus (168) Google Scholar, 4Schlager J.J. Powis G. Int. J. Cancer. 1990; 45: 403-409Crossref PubMed Scopus (247) Google Scholar). NQO1 gene transcription is coordinately activated with other detoxifying enzyme genes in response to xenobiotics (e.g. β-naphthoflavone) and antioxidants (e.g. tert-butylhydroquinone (t-BHQ)) (5Rushmore T.H. Pickett C.B. J. Biol. Chem. 1993; 268: 11475-11478Abstract Full Text PDF PubMed Google Scholar, 6Dhakshinamoorthy S. Long II D.J. Jaiswal A.K. Curr. Top. Cell. Regul. 2000; 36: 201-216Crossref PubMed Scopus (169) Google Scholar). The other detoxifying enzyme genes that are coordinately induced with NQO1 include glutathioneS-transferases (GSTs), which conjugate hydrophobic electrophiles and ROS with glutathione (7Pickett C.B. Lu A.Y.H. Annu. Rev. Biochem. 1989; 58: 743-764Crossref PubMed Scopus (544) Google Scholar, 8Tsuchida S. Sato K. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 337-384Crossref PubMed Scopus (530) Google Scholar); UDP-glucuronosyl transferases, which catalyze the conjugation of glucuronic acid with xenobiotics and drugs for excretion (9Tephly T. Burchell B. Trends Pharmacol. 1990; 11: 276-279Abstract Full Text PDF PubMed Scopus (244) Google Scholar); epoxide hydrolase, which inactivates epoxides (10Oesch F. Gath I. Igarashi T. Glatt H.R. Thomas H. Arinc E. Schenkman J.B. Hodgson E. Molecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds. Plenum Publishing Corp., New York1991: 447-461Crossref Google Scholar); γ-glutamylcysteine synthetase (γ-GCS), which plays a role in glutathione metabolism (11Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar); ferritin-L gene, which plays an important role in iron storage (12Wasserman W. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5361-5366Crossref PubMed Scopus (630) Google Scholar); and heme oxygenase-1, which catalyzes the first and rate-limiting step in heme catabolism (13Choi A.M. Alam J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19Crossref PubMed Scopus (999) Google Scholar). The coordinated induction of these genes, including NQO1, protects cells against free radical damage, oxidative stress, and neoplasia. It is critical in achieving chemoprevention. Deletion mutagenesis studies of the human NQO1 gene promoter identified 24 base pairs of an antioxidant response element (ARE) between nucleotides −470 and −447. This region is required for basal expression and induction of NQO1 in response to β-naphthoflavone and t-BHQ (6Dhakshinamoorthy S. Long II D.J. Jaiswal A.K. Curr. Top. Cell. Regul. 2000; 36: 201-216Crossref PubMed Scopus (169) Google Scholar). ARE elements have also been found in the promoter region of the human NQO2 gene (14Jaiswal A.K. J. Biol. Chem. 1994; 269: 14502-14508Abstract Full Text PDF PubMed Google Scholar), the rat and mouse GST Ya subunit genes (15Rushmore T.H. King R.G. Paulson K.E. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3826-3830Crossref PubMed Scopus (385) Google Scholar, 16Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar, 17Rushmore T.H. Morton M.R Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar, 18Friling R.S. Bensimon A. Tichauer Y. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6258-6262Crossref PubMed Scopus (422) Google Scholar, 19Friling R.S. Bergelson S. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 668-672Crossref PubMed Scopus (213) Google Scholar), the rat GST P gene (20Okuda A. Imagawa M. Maeda Y. Sakai M. Muramatsu M. EMBO J. 1990; 9: 1131-1135Crossref PubMed Scopus (87) Google Scholar), the human γ-GCS gene (11Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), the ferritin-L gene (12Wasserman W. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5361-5366Crossref PubMed Scopus (630) Google Scholar), and the human heme oxygenase-1 gene (21Alam J. Stewart D. Touchard C. Boinapally S. Choi M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar). Analysis of the AREs from various genes revealed that they contain AP1/AP1-like elements arranged as inverse or direct repeats. This is followed by a GC box (22Jaiswal A.K. Biochem. Pharmacol. 1994; 48: 439-444Crossref PubMed Scopus (223) Google Scholar). Additional cis-elements and nucleotide sequences, flanking the core sequence, have been shown to contribute to the ARE-mediated expression and induction of detoxifying enzyme genes (12Wasserman W. Fahl W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5361-5366Crossref PubMed Scopus (630) Google Scholar, 23Xie T. Belinsky M. Xu Y. Jaiswal A.K. J. Biol. Chem. 1995; 270: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 24Prestera T. Holtzclaw W.D. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2965-2969Crossref PubMed Scopus (403) Google Scholar). The various AREs bind to a complex of nuclear proteins from cells of different origins (15Rushmore T.H. King R.G. Paulson K.E. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3826-3830Crossref PubMed Scopus (385) Google Scholar, 16Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar, 17Rushmore T.H. Morton M.R Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar, 18Friling R.S. Bensimon A. Tichauer Y. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6258-6262Crossref PubMed Scopus (422) Google Scholar, 19Friling R.S. Bergelson S. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 668-672Crossref PubMed Scopus (213) Google Scholar, 20Okuda A. Imagawa M. Maeda Y. Sakai M. Muramatsu M. EMBO J. 1990; 9: 1131-1135Crossref PubMed Scopus (87) Google Scholar, 21Alam J. Stewart D. Touchard C. Boinapally S. Choi M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, 22Jaiswal A.K. Biochem. Pharmacol. 1994; 48: 439-444Crossref PubMed Scopus (223) Google Scholar, 23Xie T. Belinsky M. Xu Y. Jaiswal A.K. J. Biol. Chem. 1995; 270: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 25Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar). Analysis of ARE-nuclear protein complexes have identified several nuclear transcription factors including c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1, Nrf2, YABP, ARE-BP1, MafK, Ah (aromatic hydrocarbon) receptor, and the estrogen receptor (15–32). Among these transcription factors, Nrf1 and Nrf2 have been shown to heterodimerize with c-Jun and up-regulate the ARE-mediated expression and induction of NQO1 in response to antioxidants and xenobiotics (26Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (914) Google Scholar, 31Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (481) Google Scholar). Recently, Nrf3, a third member of the Nrf family of transcription factors, was cloned and sequenced (33Kobayashi A. Ito E. Toki T. Kogame K. Takahashi S. Igarashi K. Hayashi N. Yamamoto M. J. Biol. Chem. 1999; 274: 6443-6452Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The transcription factor Nrf2 and Nrf1, which regulate the ARE-mediated expression and induction of NQO1, were also demonstrated to regulate the expression and induction of other detoxifying enzymes such as GST Ya, γ-GCS and heme oxygenase-1 (21Alam J. Stewart D. Touchard C. Boinapally S. Choi M.K. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, 26Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (914) Google Scholar, 31Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (481) Google Scholar, 34Jaiswal A.K. Hashimoto I. Tsuchida S. Shinkawa H. Yagihashi S. Suda T. Uitto J. Molecular Medicine:Novel Findings of Gene Diagnosis, Regulation of Gene Expression, and Gene Therapy. Elsevier Science, Amsterdam, the Netherlands1999: 103-112Google Scholar, 35Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 36Moninova H. Mulcahy R.T. Biochem. Biophys. Res. Commun. 1999; 261: 661-668Crossref PubMed Scopus (217) Google Scholar, 37Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Recently, Nrf2-MafK heterodimer was shown to bind to the ARE of a gene encoding a subunit of GST Ya (32Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh K. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3087) Google Scholar). It was suggested that Nrf2-MafK heterodimers up-regulate ARE-mediated expression and induction of the GST Ya gene. More recently, a repressive role for MafK and MafG in ARE-regulated expression of the γ-GCS gene was suggested (37Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Small Maf (MafG, MafK, and MafF) proteins are leucine zipper proteins that are known to repress, as well as activate, transcription of many eukaryotic genes including β-globin gene (38Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar, 39Kim M.J. Andrews N.C. Blood. 1997; 89: 3925-3935Crossref PubMed Google Scholar, 40Fujiwara K.T. Ashida K. Nishina H. Iba H. Miyajima N. Nishizawa M. Kawai S. Oncogene. 1993; 8: 2371-2380PubMed Google Scholar, 41Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). Their gene products are closely related to v-Maf, especially in the structure of the DNA-binding and leucine zipper domains. The products of these small Maf proteins, however, lack the amino-terminal domain of v-Maf (39Kim M.J. Andrews N.C. Blood. 1997; 89: 3925-3935Crossref PubMed Google Scholar, 40Fujiwara K.T. Ashida K. Nishina H. Iba H. Miyajima N. Nishizawa M. Kawai S. Oncogene. 1993; 8: 2371-2380PubMed Google Scholar, 41Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). This domain is required for the trans-acting activity of v-Maf (39Kim M.J. Andrews N.C. Blood. 1997; 89: 3925-3935Crossref PubMed Google Scholar, 40Fujiwara K.T. Ashida K. Nishina H. Iba H. Miyajima N. Nishizawa M. Kawai S. Oncogene. 1993; 8: 2371-2380PubMed Google Scholar, 41Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). Recently, small Maf proteins were shown to homodimerize and heterodimerize with Nrf1 and Nrf2 (42Marini M.G. Chan K. Casula L. Kan Y.W. Cao A. Moi P. J. Biol. Chem. 1997; 272: 16490-16497Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Maf-Maf homodimers repressed, yet Maf-Nrf heterodimers activated transcription of the MARE-regulated β-globin gene (42Marini M.G. Chan K. Casula L. Kan Y.W. Cao A. Moi P. J. Biol. Chem. 1997; 272: 16490-16497Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Based on the above studies, small Maf proteins are expected to play a role in hARE-mediated regulation of NQO1 gene expression. This role remains unknown. In addition, the relationship of MafG and MafK with Nrf2 and Nrf1, in the regulation of ARE-mediated expression and induction of NQO1 and other detoxifying enzyme genes, also remains unknown. In the present report, we demonstrate that overexpression of MafG and MafK, individually or in combination with Nrf2, repressed ARE-mediated expression and induction of NQO1 and GST Ya genes. NQO1 hARE gel shift assays, with in vitro translated MafG and MafK, individually and in combination with Nrf2, revealed that MafG and MafK proteins bind to the NQO1 hARE as homodimers and heterodimers with Nrf2. These results indicate that MafG and MafK homodimers and possibly MafG-Nrf2 and MafK-Nrf2 heterodimers negatively regulate NQO1 and other detoxifying enzyme gene expression and induction by antioxidants. In similar experiments, MafG and MafK failed to bind to the NQO1 gene ARE as heterodimers with Nrf1. The enzymes used in this study were purchased from Life Technologies, Inc. pcDNA3.1/V-5-His-TOPO and anti-V5 antibody were purchased from Invitrogen (Carlsbad, CA). α-Minimum essential medium was purchased from Life Technologies, Inc. Effectene transfection reagent was purchased from Qiagen (Valencia, CA). The Nrf2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). t-BHQ and all other chemicals were purchased from Sigma. The pGL2 promoter plasmid containing firefly luciferase gene, internal control plasmid pRL-TK that encodes Renillaluciferase, the dual luciferase assay kit, and the TNT T7/T3 coupled rabbit reticulocyte lysate system were obtained from Promega (Madison, WI). The Hybond ECL nitrocellulose membrane, ECL Western blot analysis kit, and Amplify NAMP1000 were purchased from Amersham Pharmacia Biotech. The NQO1 gene ARE, mutant ARE, and GST Ya ARE were subcloned in the pGL2 vector to generate reporter plasmids pGL2-hARE-Luc, pGL2-mhARE-Luc, and pGL2-GST Ya ARE-Luc. The nucleotide sequences of the various AREs are shown in Fig. 1. The sense and antisense oligonucleotides corresponding to the AREs were synthesized with eitherNheI/XhoI sites (NQO1 hARE and mutant hARE) orNheI/BamHI sites (GST Ya ARE). The oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase, and cloned at the respective sites in the pGL2 promoter vector. The GST Ya ARE was cloned at the NheI/BglII site. The various constructs were checked by DNA sequencing. The mouse Nrf2 and Nrf1 cDNAs were kindly provided by Dr. Jefferson Y. Chan (University of California, San Francisco, CA). The full-length Nrf2 and Nrf1 cDNAs were amplified by polymerase chain reaction and subcloned separately into the mammalian expression vector pcDNA 3.1 to make the expression plasmids pcDNA-Nrf2R, pcDNA-Nrf2C, pcDNA-Nrf1R, and pcDNA-Nrf1C. The chicken MafG and MafK cDNAs were provided by Dr. Makoto Nishizawa (The Scripps Research Institute, La Jolla, CA). The cDNAs encoding MafG and MafK were subcloned separately into the pcDNA 3.1 vector to generate the expression plasmids pcDNA-MafGR, pcDNA-MafGC, pcDNA-MafKR, and pcDNA-MafKC. The plasmids with an R suffix contain cDNAs in the reverse orientation, and those with the suffix C contain cDNAs in the correct orientation. The MafG and MafK cDNAs were also subcloned in-frame with the V5 epitope of the pcDNA 3.1 expression plasmid. These plasmids encode the V5-tagged MafG-V5 and MafK-V5 proteins. The V5 epitope contains 14 amino acids in the sequence Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr. Human hepatoblastoma (Hep-G2) cells were grown in six-well monolayer cultures containing α-minimum essential medium supplemented with fetal bovine serum (25Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar). The Effectene transfection reagent kit from Qiagen was used to perform the transfections by procedures as described in the manufacturer's protocol. Briefly, 0.5 μg of reporter constructs (hARE-Luc, mutant hARE-Luc, and GST Ya ARE-Luc) were mixed, individually and in combinations, with different concentrations of the pcDNA expression plasmids (Nrf2, Nrf1, MafG, and MafK) and transfected into Hep-G2 cells. The plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The various plasmids were mixed with DNA condensation buffer and Enhancer solution from the kit and incubated at room temperature for 5 min. This was followed by addition of Effectene reagent to the mixture. This mixture was incubated for 7 min at room temperature. The DNA-Enhancer-Effectene mixture was added dropwise onto the Hep-G2 cells and incubated at 37 °C with 5% CO2. 48 h after the transfection, the cells were washed with 1× phosphate-buffered saline and lysed in 1× passive lysis buffer from the kit. The dual-luciferase reporter assay system from Promega was used to assay the samples for luciferase activity as described in the manufacturer's protocol. First, the cell lysate was assayed for the firefly luciferase activity using 100 μl of the substrate LARII. Then 100 μl of the STOP & GLO reagent was added to quench the firefly luciferase activity and activate theRenilla luciferase activity, which was also measured. The assays were carried out in a Packard luminometer, and the relative luciferase activity was calculated as follows: 100,000/activity ofRenilla luciferase (in units) × activity of firefly luciferase (in units). Each set of transfections was repeated three times. For induction studies, the cells were treated with 200 μm t-BHQ, dissolved in Me2SO for 16 h and analyzed for luciferase activity by procedures as described above. The transfection experiments were performed using pcDNA-Maf and pcDNA-Maf-V5 constructs. Both sets of expression plasmids gave similar results. The presence or absence of V5 epitope-tagged to Maf proteins had no effect on their activity/function. Therefore, we have shown transfection data only on Maf proteins without V5 tag. The addition of V5 tag to Maf proteins enabled us to use antibodies against V5 peptide in Western and band and supershift assays. The nuclear extracts from Hep-G2 (Me2SO control and t-BHQ-treated (200 μm for 16 h)) cells were prepared by previously described procedures (25Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar). The Hep-G2 cells were co-transfected with pcDNA-Nrf2C and pcDNA-MafG-V5 in 1:1 ratio to overexpress Nrf2 and MafG-V5. These cells were treated with Me2SO and t-BHQ (200 μm for 16 h) and nuclear extract prepared by previously described procedures (25Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar). Thein vitro transcription/translation of the plasmids encoding Nrf1, Nrf2, MafG-V5, and MafK-V5 were performed using the TNT-coupled rabbit reticulocyte lysate system (Promega) by procedures as suggested in the manufacturer's protocol. Redivuel-[35S]methionine was substituted for methionine in the reactions. After the coupled transcription/translation, the proteins were checked for their correct size on a 10% PAGE and Western analysis. Briefly, 5 μl of the translated proteins were resolved on a 10% PAGE, treated with Amplify solution (NAMP 100; Amersham Pharmacia Biotech) to enhance the35S signal, dried, and exposed to x-ray film. In a similar experiment, the proteins were transferred onto a Hybond ECL nitrocellulose membrane and probed with Nrf2 and V5 antibodies. V5 antibodies were used to detect the V5 tagged Maf proteins. All of the in vitro translated proteins gave the expected sized products. The NQO1 gene ARE was end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. The labeled ARE was incubated with nuclear extracts or in vitro translated proteins. Band and supershift assays were then performed by previously described procedures (25Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar, 31Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (481) Google Scholar). 10 μg of the nuclear extract or equimolar concentrations of in vitro translated proteins were used in the gel shift and supershift experiments. The supershift assay with nuclear extracts used 20 μg of proteins. 1.5 μl of anti-V5 antibody and 3 μl of Nrf2 antibody or preimmune serum were used in the supershift assays. The transfection of Hep-G2 cells with expression plasmids pcDNA-MafG-V5, pcDNA-MafK-V5, and pcDNA-Nrf2(C) led to the overexpression of these respective proteins as determined by SDS-PAGE, Western analysis, and antibody probing (data not shown). Overexpression of the various proteins was in near linear range between 0.1 and 1.0 μg of plasmids used for transfecting Hep-G2 cells. In similar experiments, transfection of Hep-G2 cells with cDNA in reverse orientation did not result in overexpression of the respective proteins. Transfection of Hep-G2 cells with the hARE-Luc plasmid expressed luciferase activity (Fig. 2). Overexpression of MafG in Hep-G2 cells repressed hARE-mediated luciferase activity in transfected cells (Fig. 2 A, left panel). This repression was MafG concentration-dependent. The transfection of 1.0 μg of pcDNA-MafG(C) plasmid repressed 83% of hARE-mediated luciferase activity in transfected Hep-G2 cells. Interestingly, transfection of Hep-G2 cells with plasmid pcDNA-MafG(R) expressing antisense MafG RNA significantly increased the hARE-mediated luciferase activity (Fig. 2 A, left panel). The transfection of 0.5 μg of plasmid pcDNA-MafG(R) caused 3.4-fold increase in the hARE-mediated luciferase activity (Fig. 2 A, left panel). Contrary to MafG, overexpression of Nrf2 in Hep-G2 cells led to increased expression of the NQO1 gene ARE-mediated luciferase gene (Fig. 2 A). In a similar experiment, the Nrf2-mediated up-regulation of luciferase gene expression was repressed because of the overexpression of MafG (Fig. 2 A, right panel). This repression was also MafG concentration-dependent. Similar results were also observed with Hep-G2 cells overexpressing MafK alone or MafK with Nrf2 (Fig. 2 B). The overexpression of MafK repressed hARE-mediated luciferase gene expression and its activation by Nrf2. Replacement of the reporter plasmid hARE-Luc with mutant hARE-Luc resulted in the loss of basal expression and repression of the luciferase gene by MafG and MafK (Fig. 2 C). In related experiments, the overexpression of MafG and MafK also repressed GST Ya ARE-mediated luciferase gene expression and up-regulation by Nrf2 (Fig. 3). t-BHQ treatment of Hep-G2 cells, transfected with hARE-Luc and GST Ya ARE-Luc, increased luciferase gene by approximately 2-fold (Fig. 4). Interestingly, t-BHQ induction of hARE- and GST Ya ARE-mediated luciferase gene expression was also repressed in Hep-G2 cells that overexpressed MafG and MafK (Fig. 4). The repression of t-BHQ induced expression was more or less proportional to the repression of basal expression. In other words, overexpression of small Maf proteins inhibited the basal and t-BHQ induced luciferase activity equally.Figure 3Effect of overexpression of MafG and MafK on GST Ya gene ARE-mediated luciferase gene expression and its up-regulation by Nrf2. The Hep-G2 cells were co-transfected with reporter plasmid GST Ya ARE-Luc and expression plasmids pcDNA-MafG(C), pcDNA-Maf(R), pcDNA-MafK(C), pcDNA-MafK(R), and pcDNA-Nrf2(C) individually or in combinations as shown. The concentrations (μg) of the various plasmids used in the transfections are also shown. 0.01 μg of plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The cells were harvested 48 h after transfection and assayed for luciferase activity. The values represent the means ± S.E. of three independent transfection experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of overexpression of MafG and MafK on antioxidant induction of ARE-mediated luciferase gene expression.The Hep-G2 cells were co-transfected with reporter plasmid hARE-Luc or GST Ya ARE-Luc and expression plasmids pcDNA-MafG(C) and pcDNA-MafK(C) in concentrations as shown. 0.01 μg of plasmid pRL-TK encoding Renilla luciferase was used as the internal control in each transfection. The transfected cells were treated with Me2SO (control) or 200 μm of t-BHQ 32 h after transfection. The cells were harvested 16 h after the treatment and assayed for luciferase activity. The values represent the means ± S.E. of three independent transfection experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The pcDNA-Nrf2C, pcDNA-Nrf1, pcDNA-MafG-V5, and pcDNA-MafK-V5 plasmids were in vitro transcribed and translated with rabbit reticulocyte lysate system. SDS-PAGE analysis ofin vitro translated Nrf2, Nrf1, and V5-tagged MafG and MafK is shown in Fig. 5. The in vitro translated Nrf2 and Nrf1 proteins migrated between the 105- and 75-kDa standards. Nrf1 migrated slower than Nrf2. The in vitro translated MafG-V5 and MafK-V5 proteins moved faster than the 25-kDa molecular mass standard. MafK-V5 moved faster than MafG-V5 in SDS-PAGE. The in vitro translated proteins were confirmed by Western blotting and probing with specific antibodies (data not shown). The binding of in vitro translated MafG-V5, MafK-V5, Nrf2, and Nrf1 to the NQO1 gene ARE was determined by band and supershift assays (Fig. 6). MafG and MafK both bound to the hARE as Maf-Maf homodimers and Nrf2-Maf heterodimers (Fig. 6). The binding of homo- and heterodimers of Maf and Nrf2 were competed with cold hARE (data not shown). The presence of V-5-tagged MafG and MafK in Maf-Maf homodimers and Nrf2-Maf heterodimers were confirmed by supershift assays with anti-V5 antibodies (Fig. 6). The antibodies against Nrf2 also supershifted Nrf2-Maf heterodimers in experiments with Nrf2-MafG and Nrf2-MafK (Fig. 6) The results of band shift assays with hARE and nuclear extract from Hep-G2 cells treated with Me2SO (control) and t-BHQ are shown (Fig. 7 A). The results demonstrated two-shifted bands as observed with in vitrotranslated Nrf2 and Maf proteins (Fig. 6). The upper and lower shifted bands corresponded to the Nrf2-Maf heterodimers and Maf-Maf homodimers, respectively. It may be noteworthy that both the lower and upper bands are not exclusively due to Nrf2-Maf and Maf-Maf dimers. These bands are also expected to contain posi