Regulation of Nrf2 Transactivation Domain Activity
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m401368200
ISSN1083-351X
AutoresGuoxiang Shen, Vidya Hebbar, Sujit Nair, Changjiang Xu, Wenge Li, Wen Lin, Young-Sam Keum, Jiahuai Han, Michael A. Gallo, A.-N. Tony Kong,
Tópico(s)Glutathione Transferases and Polymorphisms
ResumoTranscription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes as well as anti-oxidative enzymes. In this study, we investigated the transactivation potential of different Nrf2 transactivation domain regions by using the Gal4-Nrf2 chimeras and Gal4-Luc reporter co-transfection assay system in HepG2 cells. The results indicated that chimera Gal4-Nrf2-(1–370), which contains the full transactivation domain showed very potent transactivation activity. The high transactivation activity of Gal4-Nrf2-(113–251) and the diminished transactivation activities of chimera Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) suggested that the Nrf2 N-terminal 113–251 amino acids region is critical in maintaining its transactivation activity. Overexpression of upstream MAPKs such as Raf, MEKK1, TAK1-ΔN, and ASK1 up-regulated the transactivation activities of Gal4-Nrf2-(1–370) and Gal4-Nrf2-(113–251) in a dose-dependent manner. Further investigation on the effects of the three MAPK pathways on Nrf2 transactivation domain activity demonstrated that both ERK and JNK signaling pathways stimulated the Gal4-Nrf2-(1–370) transactivation activity while the p38 pathway played a negative role. Site-directed mutagenesis studies on potential MAPK phosphorylation sites of Gal4-Nrf2-(113–251) showed no significant effect on its basal transactivation activity or the fold of induction by Raf. Interestingly, the nuclear transcription coactivator CREB-binding protein (CBP), which can bind to Nrf2 transactivation domain and can be activated by ERK cascade, showed synergistic stimulation with Raf on the transactivation activities of both the chimera Gal4-Nrf2-(1–370) and the full-length Nrf2. Taken together, this study clearly demonstrated that different segments of Nrf2 transactivation domain have different transactivation potential and different MAPKs have differential effects on Nrf2 transcriptional activity. It also suggested that the up-regulation of Nrf2 transactivation domain activity by upstream MAPKs such as Raf may not be mediated by direct phosphorylation of the Nrf2 transactivation domain, but rather by regulation of the transcriptional activity of coactivator CBP. Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes as well as anti-oxidative enzymes. In this study, we investigated the transactivation potential of different Nrf2 transactivation domain regions by using the Gal4-Nrf2 chimeras and Gal4-Luc reporter co-transfection assay system in HepG2 cells. The results indicated that chimera Gal4-Nrf2-(1–370), which contains the full transactivation domain showed very potent transactivation activity. The high transactivation activity of Gal4-Nrf2-(113–251) and the diminished transactivation activities of chimera Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) suggested that the Nrf2 N-terminal 113–251 amino acids region is critical in maintaining its transactivation activity. Overexpression of upstream MAPKs such as Raf, MEKK1, TAK1-ΔN, and ASK1 up-regulated the transactivation activities of Gal4-Nrf2-(1–370) and Gal4-Nrf2-(113–251) in a dose-dependent manner. Further investigation on the effects of the three MAPK pathways on Nrf2 transactivation domain activity demonstrated that both ERK and JNK signaling pathways stimulated the Gal4-Nrf2-(1–370) transactivation activity while the p38 pathway played a negative role. Site-directed mutagenesis studies on potential MAPK phosphorylation sites of Gal4-Nrf2-(113–251) showed no significant effect on its basal transactivation activity or the fold of induction by Raf. Interestingly, the nuclear transcription coactivator CREB-binding protein (CBP), which can bind to Nrf2 transactivation domain and can be activated by ERK cascade, showed synergistic stimulation with Raf on the transactivation activities of both the chimera Gal4-Nrf2-(1–370) and the full-length Nrf2. Taken together, this study clearly demonstrated that different segments of Nrf2 transactivation domain have different transactivation potential and different MAPKs have differential effects on Nrf2 transcriptional activity. It also suggested that the up-regulation of Nrf2 transactivation domain activity by upstream MAPKs such as Raf may not be mediated by direct phosphorylation of the Nrf2 transactivation domain, but rather by regulation of the transcriptional activity of coactivator CBP. In order to survive a variety of environmental or intracellular stress, mammalian cells have developed robust cellular defensive systems to protect themselves from oxidative or electrophilic stress. Among these defensive enzymes are the detoxifying systems, including phase II drug metabolizing enzymes, such as glutathione S-transferase, NADP(H):quinone oxidoreductase, UDP-glucuronosyltransferase (1Zhang Y. Talalay P. Cho C.G. Posner G.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2399-2403Crossref PubMed Scopus (1507) Google Scholar, 2Morse M.A. Stoner G.D. Carcinogenesis. 1993; 14: 1737-1746Crossref PubMed Scopus (407) Google Scholar, 3Chan K. Han X.D. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4611-4616Crossref PubMed Scopus (643) Google Scholar), and anti-oxidant enzymes, such as heme oxygenase-1 (HO-1) 1The abbreviations used are: HO-1, heme oxygenase-1; MAPK, mitogen-activated protein kinase; Nrf2, NF-E2-related factor 2; ARE, anti-oxidant responsive element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CREB, cAMP responsive-binding protein; CBP, CREB-binding protein; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SFN, sulforaphane; EpRE, electrophile response element.1The abbreviations used are: HO-1, heme oxygenase-1; MAPK, mitogen-activated protein kinase; Nrf2, NF-E2-related factor 2; ARE, anti-oxidant responsive element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CREB, cAMP responsive-binding protein; CBP, CREB-binding protein; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SFN, sulforaphane; EpRE, electrophile response element. and γ-glutamylcysteine synthetase (γGCS) (4Primiano T. Sutter T.R. Kensler T.W. Adv. Pharmacol. 1997; 38: 293-328Crossref PubMed Scopus (99) Google Scholar, 5Prestera T. Talalay P. Alam J. Ahn Y.I. Lee P.J. Choi A.M. Mol. Med. 1995; 1: 827-837Crossref PubMed Google Scholar, 6Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar, 7Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Previous studies have shown that these enzymes are coordinately regulated through a consensus cis-element called anti-oxidant responsive element (ARE) or electrophile response element (EpRE) at their 5′-flanking promoters (8Rushmore T.H. Pickett C.B. J. Biol. Chem. 1990; 265: 14648-14653Abstract Full Text PDF PubMed Google Scholar, 9Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: 15097-15104Abstract Full Text PDF PubMed Google Scholar, 10Favreau L.V. Pickett C.B. J. Biol. Chem. 1991; 266: 4556-4561Abstract Full Text PDF PubMed Google Scholar). Recent extensive studies have demonstrated that transcription factor NF-E2-related factor 2 (Nrf2) plays a critical role in the constitutive and inducible expression of genes encoding these defensive enzymes in response to oxidative and xenobiotic stress (11Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3146) Google Scholar, 12Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (522) Google Scholar, 13Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 14Kwak M.K. Itoh K. Yamamoto M. Sutter T.R. Kensler T.W. Mol. Med. 2001; 7: 135-145Crossref PubMed Google Scholar, 15McMahon M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar). Nrf2 belongs to the CNC (Cap-N-Collar) family of transcription factors and possesses a highly conserved basic region-leucine zipper (bZip) structure (16Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1205) Google Scholar). Its important role in the regulation of the expression of many mammalian detoxifying and anti-oxidant enzymes under oxidative or electrophilic stress has been verified in various Nrf2-deficient mice experiments, in which the expression of these enzymes are dramatically abolished and the Nrf2 knockout mice are much more susceptible to carcinogen-induced toxicity and carcinogenesis (12Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (522) Google Scholar, 17Chan J.Y. Kwong M. Biochim. Biophys. Acta. 2000; 1517: 19-26Crossref PubMed Scopus (261) Google Scholar, 18Enomoto A. Itoh K. Nagayoshi E. Haruta J. Kimura T. O'Connor T. Harada T. Yamamoto M. Toxicol. Sci. 2001; 59: 169-177Crossref PubMed Scopus (629) Google Scholar, 19Hayes J.D. Chanas S.A. Henderson C.J. McMahon M. Sun C. Moffat G.J. Wolf C.R. Yamamoto M. Biochem. Soc. Trans. 2000; 28: 33-41Crossref PubMed Scopus (285) Google Scholar). As a sensor for regulating the ARE-mediated gene expression in response to oxidative stress, Nrf2 is sequestered in the cytoplasm of the cells by a cytoskeleton-related protein called Keap1 that interacts with the N-terminal Neh2 domain of Nrf2 (20Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2738) Google Scholar). But Nrf2 may also exist in the nucleus under homeostatic resting conditions for basal transcription of Nrf2-mediated genes. When cells are exposed to oxidative or electrophilic stress, Nrf2 appears to be liberated from the Keap1-Nrf2 complex and translocates into the nucleus (21Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (434) Google Scholar, 22Nakaso K. Yano H. Fukuhara Y. Takeshima T. Wada-Isoe K. Nakashima K. FEBS Lett. 2003; 546: 181-184Crossref PubMed Scopus (276) Google Scholar), thereby activating Nrf2-dependent gene transcription. Although the understanding of the regulation of Nrf2-dependent Phase II or anti-oxidant enzymes has been greatly enhanced, the exact mechanisms by which these oxidative/electrophile stress-triggered signaling pathways regulate Nrf2 transactivation activity are still unclear. Currently, at least three mechanisms are proposed for the regulation of Nrf2 transactivation activity. The first mechanism proposed is that the cysteinerich Keap1 protein might be able to directly sense the oxidative stress via thiol modification on its cysteine residues, and these modifications may cause a conformational change thereby releasing Nrf2 from the complex (23Bloom D. Dhakshinamoorthy S. Jaiswal A.K. Oncogene. 2002; 21: 2191-2200Crossref PubMed Scopus (92) Google Scholar, 24Dinkova-Kostova A.T. Holtzclaw W.D. Cole R.N. Itoh K. Wakabayashi N. Katoh Y. Yamamoto M. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11908-11913Crossref PubMed Scopus (1583) Google Scholar, 25Zipper L.M. Mulcahy R.T. J. Biol. Chem. 2002; 277: 36544-36552Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). However, it is debatable whether these in vitro conditions or concentrations of oxidants or electrophiles would be achievable in the in vivo situation. Furthermore, the release of Nrf2 from the complex can also be triggered by some kinase signals such as mitogen-activated protein kinases (MAPKs) or protein kinase C (PKC) in the absence of these modifying agents (21Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (434) Google Scholar). Under homeostasis conditions, Nrf2 undergoes Keap1-dependent and -independent degradation through proteasome (26McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar), so the second mechanism proposed is that the release of Nrf2 from Keap1 in response to oxidative/electrophilic stress may dramatically increase its protein stability (27Nguyen T. Sherratt P.J. Huang H.C. Yang C.S. Pickett C.B. J. Biol. Chem. 2003; 278: 4536-4541Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar), resulting in the accumulation of Nrf2 in the cells and enhancement of Nrf2-dependent transcription activity. A third alternative hypothesis proposed is that the transactivation activity of Nrf2 can also be regulated by specific kinase signals activated by the oxidative stress. These kinases, such as MAPKs (27Nguyen T. Sherratt P.J. Huang H.C. Yang C.S. Pickett C.B. J. Biol. Chem. 2003; 278: 4536-4541Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 28Zipper L.M. Mulcahy R.T. Toxicol. Sci. 2003; 73: 124-134Crossref PubMed Scopus (211) Google Scholar), PI3K (22Nakaso K. Yano H. Fukuhara Y. Takeshima T. Wada-Isoe K. Nakashima K. FEBS Lett. 2003; 546: 181-184Crossref PubMed Scopus (276) Google Scholar), or PKC (29Huang H.C. Nguyen T. Pickett C.B. J. Biol. Chem. 2002; 277: 42769-42774Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar) could directly (phosphorylation on Nrf2) or indirectly regulate Nrf2 transactivation activity by either affecting its nuclear translocation and/or its ability of binding to and activating the transcription of its target genes (21Huang H.C. Nguyen T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12475-12480Crossref PubMed Scopus (434) Google Scholar, 30Zipper L.M. Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; 278: 484-492Crossref PubMed Scopus (215) Google Scholar). Once in the nucleus, Nrf2 not only can bind to the specific ARE sequence on the target genes, but also can bind to other trans-acting factors that can coordinately regulate gene transcription with Nrf2. Among these trans-acting factors, the small Maf protein MafG and MafK have been reported to dimerize specifically with Nrf1 and Nrf2 (31Toki T. Itoh J. Kitazawa J. Arai K. Hatakeyama K. Akasaka J. Igarashi K. Nomura N. Yokoyama M. Yamamoto M. Ito E. Oncogene. 1997; 14: 1901-1910Crossref PubMed Scopus (94) Google Scholar). The CREB-binding protein (CBP) has been shown to play a key role as a nuclear coactivator for a wide variety of transcription factors involved in various different pathways including CREB, AP-1, p53, and NFκB. Recent studies indicated that CBP can bind directly to the Nrf2 transactivation domain (32Katoh Y. Itoh K. Yoshida E. Miyagishi M. Fukamizu A. Yamamoto M. Genes Cells. 2001; 6: 857-868Crossref PubMed Scopus (367) Google Scholar) or through another member of p160 protein family ARE-binding protein-1 (33Zhu M. Fahl W.E. Biochem. Biophys. Res. Commun. 2001; 289: 212-219Crossref PubMed Scopus (114) Google Scholar). Furthermore, previous reports have shown that CBP can be phosphorylated by MAPK (34Liu Y.Z. Chrivia J.C. Latchman D.S. J. Biol. Chem. 1998; 273: 32400-32407Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 35Ait-Si-Ali S. Carlisi D. Ramirez S. Upegui-Gonzalez L.C. Duquet A. Robin P. Rudkin B. Harel-Bellan A. Trouche D. Biochem. Biophys. Res. Commun. 1999; 262: 157-162Crossref PubMed Scopus (120) Google Scholar, 36Liu Y.Z. Thomas N.S. Latchman D.S. Neuroreport. 1999; 10: 1239-1243Crossref PubMed Scopus (48) Google Scholar, 37See R.H. Calvo D. Shi Y. Kawa H. Luke M.P. Yuan Z. J. Biol. Chem. 2001; 276: 16310-16317Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 38Gusterson R. Brar B. Faulkes D. Giordano A. Chrivia J. Latchman D. J. Biol. Chem. 2002; 277: 2517-2524Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 39Sang N. Stiehl D.P. Bohensky J. Leshchinsky I. Srinivas V. Caro J. J. Biol. Chem. 2003; 278: 14013-14019Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) and, in particular, the C-terminal activation domain of CBP was phosphorylated by the MAPK cascades leading to enhancement of its transcriptional activity (38Gusterson R. Brar B. Faulkes D. Giordano A. Chrivia J. Latchman D. J. Biol. Chem. 2002; 277: 2517-2524Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It is highly possible that MAPK signals could regulate Nrf2-mediated gene transcription through posttranslational modification of CBP and/or other unidentified transacting factors or co-activators that bind to the Nrf2 transcription machinery, such as p300 (39Sang N. Stiehl D.P. Bohensky J. Leshchinsky I. Srinivas V. Caro J. J. Biol. Chem. 2003; 278: 14013-14019Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), or peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP) (40Misra P. Owuor E.D. Li W. Yu S. Qi C. Meyer K. Zhu Y.J. Rao M.S. Kong A.N. Reddy J.K. J. Biol. Chem. 2002; 277: 48745-48754Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Previous studies from different laboratories have shown that MAPK signaling pathways can regulate ARE-containing reporter or detoxifying genes via Nrf2-dependent mechanisms (6Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar, 41Yu R. Chen C. Mo Y.Y. Hebbar V. Owuor E.D. Tan T.H. Kong A.N. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 42Owuor E.D. Kong A.N. Biochem. Pharmacol. 2002; 64: 765-770Crossref PubMed Scopus (404) Google Scholar). In this study, we first dissected the different regions of Nrf2 transactivation domain, tested their activities in the Gal4-Luc co-transfection assay system, and then examined the effects of different MAPK signals on Nrf2 transactivation domain activity. The effects of MAPKs on Nrf2 transactivation acitivity were also verified by the regulation of the endogenous HO-1 expression level after overexpression of corresponding MAPKs in HepG2 cells. The regulation of ARE-containing HO-1 gene expression is Nrf2-dependent, and HO-1 is one of the most readily induced anti-oxidant genes in response to oxidative stress signals (43Maines M.D. FASEB. J. 1988; 2: 2557-2568Crossref PubMed Scopus (1567) Google Scholar, 44Balogun E. Hoque M. Gong P. Killeen E. Green C.J. Foresti R. Alam J. Motterlini R. Biochem. J. 2003; 371: 887-895Crossref PubMed Scopus (889) Google Scholar). Our results demonstrated unequivocally that ERK and JNK pathways positively activated Nrf2 transactivation domain activity, while the p38 pathway inhibited its transactivation activity. The involvement of MAPKs also led us to investigate whether the up-regulation of Nrf2 transactivation activity was through direct phosphorylation of the Nrf2 transactivation domain. In this study, we observed that site-directed mutagenesis on Gal4-Nrf2-(113–251) had little effect on the basal transcription activity as well as the stimulatory effect of RafBXB. On the other hand, Raf-BXB substantially enhanced the transactivation activity of Nrf2 mediated by Nrf2-interacting coactivator CBP. Cell Culture, Antibody, and Chemicals—Human hepatoma HepG2 cells were obtained from ATCC (Manassas, VA) and maintained in F-12 medium supplemented with 10% fetal bovine serum, 1.17 g/liter of sodium bicarbonate, 0.1 units/ml insulin, 0.5× minimal essential medium amino acid, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were split every 3 days. Sulforaphane was purchased from LKT Laboratories (St. Paul, MN). Goat polyclonal HO-1 and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Generation of Gal4-Nrf2 Chimeras and Expression Constructs—Four chimeras of the Nrf2 transactivation domain were amplified from the Nrf2 template (589 amino acids) by polymerase chain reaction using the following sets of primers, N1: 5′-GGTACCTGGATTTGATTGACATACTT-3′ (sense) and 5′-TCTAGAGTGACTGAAACGTAGCCGAAGAAACCTC-3′ (antisense); N2: 5′-GGTACCCGTTTGTAGATGACAATGAGGTT-3′ (sense) and 5′-TCTAGACTGTCAACTGGTTGGGGTCTTCTGTGGAGAG-3′ (antisense); N3: 5′-GGTACCTTCTTAATGCTTTTGAGGAT-3′ (sense) and 5′-TCTAGATTCCAGGGGCACTATCTAGCTCTTC-3′ (antisense); N4: 5′-GGTACCTGGATTTGATTGACATACTT-3′ (sense) and 5′-TCTAGATTCCAGGGGCACTATCTAGCTCTTC-3′ (antisense). The amplified fragments were inserted into pCR2.1 (Invitrogen) vector and then subcloned into the KpnI/XbaI sites of the pSG424 vector (kindly provided by Dr. Anning Lin, University of Chicago), which contains the Gal-4 DNA binding domain. The Gal4-luciferase reporter plasmid was also kindly provided by Dr. Anning Lin. Full-length human Keap1 cDNA (GenBank™, BC002417) was purchased from IMAGE Clone Consortium (IMAGE number: 3163902). PCR primers were designed to include the entire open reading frame of Keap1 and XhoI and HindIII overhangs were added in the sense and antisense primer, respectively. The sequence of sense primer is: 5′-T TTT CTC GAG ATG CAG CCA GAT CCC AGG CCT-3′ (XhoI site is underlined). The sequence of antisense primer is: 5′-TTTT AAG CTT ACA GGT ACA GTT CTG CTG GTC-3′ (HindIII site is underlined). An 1872-bp PCR product was amplified, purified, digested with XhoI/HindIII, and inserted into a Ds-Red-express-N1 vector (Clontech) by ProteinTech Group (Chicago, IL). A coral Discosoma sp. red fluorescent protein (Ds-Red) tag was added to the N-terminal of Keap1. Active ERK5 (pCMV-HA-ERK5) and active MEK5 (pCMV-HA-MEK5) were kindly obtained from Dr. Jack Dixon and Dr. Kun-Liang Guan (University of Michigan, Ann Arbor, MI). Constitutively active MEK1 (DNEE-MEK1-pCDNA3) was a gift from Dr. Rony Seger (The Weizmann Institute of Science). Wild-type and dominant-negative p38α, p38β, p38γ, p38δ, and constitutively activated pCDNA3-MKK6b(E) were described as previously (45Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (352) Google Scholar, 46Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 47Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (275) Google Scholar, 48Mertens S. Craxton M. Goedert M. FEBS Lett. 1996; 383: 273-276Crossref PubMed Scopus (134) Google Scholar, 49Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 50Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). CBP was kindly provided by R. H. Goodman (Vollum Institute, Oregon Health Sciences University). All other plasmids used in this study have been described previously (41Yu R. Chen C. Mo Y.Y. Hebbar V. Owuor E.D. Tan T.H. Kong A.N. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 51Yu R. Mandlekar S. Lei W. Fahl W.E. Tan T.H. Kong A.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Site-directed Mutagenesis—Amino acid replacements were performed using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The construct Gal4-Nrf2-(113–251) served as the template. The following oligonucleotides containing the appropriate codon exchange (underlined) were used for the procedure. The forward primer for S148A is 5′-GCTACTAATCAGGCTCAGGCACCTGAAACTTCTGTTG-3′; the forward primer for S199A is 5′-GAGACTACCATGGTTCCAGCTCCAGAAGCCAAACTGAC-3′; the forward primer for S243A is 5′-CAGCAGCATCCTCGCCACAGAAGACCC-3′. All manipulations were performed following the manufacturer's instructions. The mutations were verified by DNA sequencing at CMG DNA sequencing facility (University of Florida). Transient Transfection and Reporter Gene Activity Assays—HepG2 cells were plated in 6-well plates at a density of 4.0 × 105 cells/well. Twenty-four hours after plating, cells were transfected with expression plasmids using LipofectAMINE 2000 (Invitrogen) following the protocol provided by the manufacturer. In brief, cell culture medium was changed to OPTI-MEM medium before each transfection, 0.5 μg of pRSV-β-galactosidase plasmid was cotransfected for transfection efficiency normalization in each transfection, and total amount of DNA transfected in each well was adjusted to 4 μg by using empty vector pCDNA3.1. Cells were incubated with transfection mixtures for 5 h and then cultured in fresh F-12 medium for an additional 36 h before harvesting. Luciferase activity was determined according to the manufacturer's protocol (Promega, Madison, WI). In brief, cells were washed once with ice-cold phosphate-buffered saline and harvested in 1× reporter lysis buffer. After centrifugation, a 10-μl supernatant was assayed for luciferase activity with a Sirius luminometer (Berthold Detection Systems). The luciferase activity was normalized by β-galactosidase activity or by protein concentration in the CBP-related experiments. Western Blotting—HepG2 cells were harvested 36 h after transient transfection in whole cell lysis buffer (10 mm Tris-HCl, pH 7.9, 250 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 0.5% Triton X-100, 10% glycerol, 1× proteinase inhibitor mixture, 1 mm phenylmethylsulfonyl fluoride, 100 μm Na3VO4, 5 μm ZnCl2, 2 mm indole acetic acid). The protein concentration of cell lysates was determined by the Bradford method, and 30 μg of protein was resolved using 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane using a semi-dry transfer system (Fisher). The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBST, containing 20 mm Tris-HCl, pH 7.6, 8 mg/ml NaCl, and 0.2% Tween-20) at room temperature for 1 h. The membrane was then probed with polyclonal goat anti-HO-1 antibody (1:200 dilution) in 3% nonfat milk (dissolved in TBST) at 4 °C overnight. After washing three times with TBST, the membrane was blotted with peroxidase-conjugated bovine anti-goat antibody (1:5000 dilution) at room temperature for 1 h. The protein was detected using the ECL system (Amersham Biosciences). After developing the film, the membrane was washed with TBST twice and stripped in buffer (62.5 mm Tris-HCl, pH 6.5, 10% SDS, 100 mm β-mercaptoethanol) for 30 min at 50 °C, washed twice again with TBST, and then re-probed with anti-actin antibody (1:1000) overnight at 4 °C. The actin-specific band was detected in a manner similar to that described for HO-1. Statistics—Values are given as fold activity relative to the luciferase in unstimulated cells (set at 1). Values are expressed as mean ± S.D. of experiments. Statistical analysis was performed by the two-tailed Student's t test for unpaired data, with p value <0.05 considered statistically significant. Transactivation Potential of Different Gal4-Nrf2 Chimeras—To delineate the biological functions of the different domains of Nrf2 N-terminal 1–370 amino acids region, a series of Gal4-Nrf2 fusion constructs, which are schematically shown in Fig. 1A were generated. Their transactivation activities were examined in the Gal4-Luc reporter co-transfection assay system in HepG2 cells. At a transfection dose of 5 ng, the chimera Gal4-Nrf2-(1–370) (N4) containing the full-length transactivation domain showed very high Gal4-luciferase activity, which was about 100-fold as compared with the vector control (Fig. 1B). In contrast, the transactivation activities of the truncated chimeras Gal4-Nrf2-(1–126) (N1) and Gal4-Nrf2-(230–370) (N3) were dramatically diminished as compared with that of N4. An interesting finding in this experiment was that another truncated chimera Gal4-Nrf2-(113–251) (N2) had transactivation activity even higher than that of N4 (1000-fold of vector control) as shown in Fig. 1B. In order to verify whether the Gal4-Nrf2 chimera acts similarly to the Nrf2 in HepG2 cells, the effects of Keap1 on the activity of N2 and N4 were tested. The results (Fig. 1C) showed that coexpression of Keap1 at doses of 25 and 50 ng can significantly suppress the transactivation activity of chimera N4, while no significant suppression was observed on the N2 chimera. These observations demonstrated that chimera N4 behaved similarly to Nrf2 in the transfected HepG2 cells and its transactivation activity was suppressed because of the binding to Keap1 with subsequent retention in the cytoplasm. On the other hand, because N2 lacked the Keap1 binding domain ETGE in its N terminus, coexpression of Keap1 had no effects on its transactivation activity. This also explained the much higher activity of N2, as compared with N4, which could be due to the suppressing effect of endogenous Keap1 on N4. As expected, treatments with the electrophile sulforaphane (SFN) at 12.5 μm increased the transactivation activity of N4 to about 2-fold (Fig. 1C), while the same SFN treatments had no effect on the transactivation activity of N2 (data not shown). These results suggested that SFN treatments reversed the Keap1 suppression effects on N4; however, the reversal was never complete even at higher concentrations of SFN (data not shown). Katoh et al. (32Katoh Y. Itoh K. Yoshida E. Miyagishi M. Fukamizu A. Yamamoto M. Genes Cells. 2001; 6: 857-868Crossref PubMed Scopus (367) Google Scholar) previously demonstrated that Neh4 and Neh5 domains within the N-terminal (amino acids 1–317) of Nrf2 possessed independent
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