Regulation of Nrf2 Transactivation Domain Activity

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. 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. 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PubMed Scopus Google Scholar), the of the regulation of Phase II or anti-oxidant enzymes has been the by which these signaling pathways Nrf2 transactivation activity are at three are the regulation of Nrf2 transactivation activity. The is that the protein be to the oxidative stress on its and these may a Nrf2 from the D. S. Jaiswal A.K. PubMed Scopus Google Scholar, Itoh K. N. Katoh Y. Yamamoto M. Talalay P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Mulcahy R.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is these in or of or be in the in the of Nrf2 from the can also be by kinase such as mitogen-activated protein or protein kinase in the of these T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Nrf2 and through M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the is that the of Nrf2 from in response to stress may dramatically its protein T. P.J. 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Igarashi K. N. M. Yamamoto M. E. 1997; 14: PubMed Scopus Google Scholar). The CREB-binding protein has been shown to a role as a nuclear coactivator a variety of transcription factors in various different pathways including CREB, and Recent studies indicated that can bind to the Nrf2 transactivation domain Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google or through of protein family M. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). have shown that can be by MAPK J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. S. A. P. A. D. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar, D. Y. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. J. I. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in the domain of by the MAPK to of its transcriptional activity D. A. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is highly that MAPK transcription through of factors or that bind to the Nrf2 transcription such as N. J. I. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), or protein P. S. C. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Previous studies from different have shown that MAPK signaling pathways can reporter or detoxifying genes 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, C. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. Pharmacol. PubMed Scopus Google Scholar). In this study, we the different regions of Nrf2 transactivation domain, their activities in the Gal4-Luc co-transfection assay and the effects of different MAPK on Nrf2 transactivation domain activity. The effects of MAPKs on Nrf2 transactivation also verified by the regulation of the expression of MAPKs in HepG2 cells. The regulation of expression is and is of the anti-oxidant genes in response to oxidative stress J. PubMed Scopus Google Scholar, E. M. P. E. C.J. Alam J. Biochem. J. PubMed Scopus Google Scholar). results demonstrated that ERK and JNK pathways activated Nrf2 transactivation domain while the p38 pathway its transactivation activity. The of MAPKs also to the up-regulation of Nrf2 transactivation activity through direct phosphorylation of the Nrf2 transactivation In this study, we that mutagenesis on Gal4-Nrf2-(113–251) effect on the basal transcription activity as well as the effect of the the transactivation activity of Nrf2 mediated by coactivator CBP. and HepG2 cells from and in with of amino and from and from of Gal4-Nrf2 and chimeras of the Nrf2 transactivation domain from the Nrf2 amino by using the of and and and and The and the sites of the by of which contains the The reporter also by from to the of and and in the and The of is The of is with and a by protein to the N-terminal of and from and of a from of and and activated as Y. Han J. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, Y. C. S. Han J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. M.A. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. M. M. PubMed Scopus Google Scholar, S. Y. E. P. S. 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Site-directed using the mutagenesis from The Gal4-Nrf2-(113–251) as the The the used the The is the is the is the The verified by at of and cells in at a of cells with expression using the by the In to of in and of in well to by using with and in activity to the In cells with and in reporter a activity with a The activity by activity or by protein in the cells in 2 The protein of by the and of protein using and to using a system The with in and at The with in in at three with the with at The protein using the system the the with and in at with and with at The in a to that are as fold activity to the in cells at are as of by the with of Gal4-Nrf2 the of the different of Nrf2 N-terminal amino acids a of Gal4-Nrf2 which are shown in transactivation activities in the Gal4-Luc reporter co-transfection assay system in HepG2 cells. a of the chimera Gal4-Nrf2-(1–370) the full-length transactivation domain showed very high which as with the In the transactivation activities of the chimeras Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) dramatically diminished as with that of in this that chimera Gal4-Nrf2-(113–251) transactivation activity that of of as shown in In order to the Gal4-Nrf2 chimera to the Nrf2 in HepG2 the effects of on the activity of and The results showed that of at of and can the transactivation activity of chimera while no significant on the demonstrated that chimera to Nrf2 in the HepG2 cells and its transactivation activity of the to with in the the the domain in its of no effects on its transactivation activity. also the much activity of as with which be to the effect of on with the electrophile at the transactivation activity of to while the no effect on the transactivation activity of not results suggested that the effects on the at of not Katoh Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google demonstrated that and the N-terminal acids of Nrf2 transactivation in the transcriptional of and and the high transactivation activities of and to be with Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google Scholar). The diminished activity of be by the of transactivation Katoh Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google indicated that the amino acids chimera and possesses a of transactivation the chimera Gal4-Nrf2-(1–126) in study its transactivation The this is not but may be to the with of the transactivation domain of Gal4-Nrf2 by MEKK1, TAK1-ΔN, and and Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar, C. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google have demonstrated that MAPK signaling pathways are in the regulation of genes in a manner. we investigated MAPKs also the activity of Nrf2 transactivation we investigated the effects of upstream MAPK kinase kinase in the Gal4-Luc reporter co-transfection assay shown in activated the activity of Gal4-Nrf2-(1–370) transactivation activity in a dose-dependent while the MEKK1, TAK1-ΔN, and ASK1 showed induction as with The effects of these upstream also verified by their regulation of protein expression using shown in of in HepG2 cells in induction of The induction of protein as with that in the reporter assay be to the that the reporter assay system is much more the basal of expression is very high in the HepG2 system of the to oxidative stress. the results demonstrated that these up-regulated Nrf2 transactivation shown in the a much transactivation activity the with potential MAPK phosphorylation sites on both we to MAPK shown in the transactivation activity of by the upstream to with the of MEKK1, which showed transactivation at the as with that of indicated that the at both of not be in the by of Gal4-Nrf2-(1–370) by ERK the effects of the three MAPK pathways on the regulation of Nrf2 transactivation the chimera investigated the effects of different ERK pathway on transactivation ERK pathways can be and with the role of upstream of activated or the Gal4-Nrf2-(1–370) transactivation activity in a dose-dependent of with MAPKs and showed synergistic of Gal4-Nrf2-(1–370) results with the induction of expression of Gal4-Nrf2-(1–370) by JNK JNK pathway of upstream MEKK1, and It has been shown that the p38 pathway can also with shown in of and activated the Gal4-Nrf2-(1–370) transactivation activity in a dose-dependent manner. Among the of these with showed a synergistic induction effect on Gal4-Nrf2-(1–370) transactivation activity The of these to Nrf2 transactivation activity also by the induction of expression of which showed induction of Gal4-Nrf2-(1–370) by p38 have that the p38 pathway the expression of phase II detoxifying enzymes S. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). we the Gal4-Nrf2-(1–370) transactivation activity also be regulated by the p38 Recent studies have shown that are p38 and Y. Han J. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, Y. C. S. Han J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. H. M. J. Han J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in results clearly demonstrated that of the p38 the transactivation activity of while the p38 showed the to the effects of p38 upstream activated the Gal4-Nrf2-(1–370) transactivation in showed a effect and the effects of and with p38 both showed robust synergistic effects with the p38 and effects with the p38 results suggested that the p38 pathways a negative role in the regulation of Nrf2 transactivation domain activity. The of Nrf2 transactivation activity by or with p38 also by the in protein expression and Site-directed the of Gal4-Nrf2-(113–251) and Its to shown the chimera a of transactivation activity as that of in response to the upstream has potential MAPK phosphorylation sites as with we chimera to the mutagenesis the 113–251 amino is the conserved MAPK phosphorylation the N. S. S. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), and with high potential phosphorylation also the and the mutagenesis study, and of and as well as their on the shown in these or in effect on the basal transactivation activities of these with of the induction of transactivation activity by not with the of induction the with no significant effects The of these to the induction by on demonstrated that the effect of on may not be through direct the transactivation of and by upstream MAPKs also suggested that the regulation of Nrf2 transactivation domain activity by MAPKs not be direct of and on of Nrf2 showed that the nuclear transcription coactivator can bind to the Nrf2 transactivation domain Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google Scholar, M. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar), and that the MAPK signaling such as ERK and can the transactivation activity of by phosphorylation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we Nrf2 transactivation activity by the activity of in the system as well as in the system as we C. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). shown in of the transactivation activity of Gal4-Nrf2-(1–370), not of with different of CBP, significant synergistic up-regulation of Gal4-Nrf2-(1–370) transactivation at of of and The synergistic effect of of and of by of of and not this the synergistic effect of and also in the system C. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). shown in of of and of showed synergistic induction of activities and these effects by of and not Taken together, these suggested that in the up-regulation of Nrf2 transactivation activity by Nrf2 is a of expression of Phase II detoxifying enzymes and anti-oxidant enzymes. the by which the induction of these genes Nrf2 Itoh K. N. Katoh Y. Ishii T. Igarashi K. J.D. Yamamoto M. 1999; PubMed Scopus Google that Nrf2 regulated by nuclear under The protein with the in the N-terminal domain of Nrf2 and in the M. Itoh K. T. H. K. Katoh Y. Y. Yamamoto M. 7: PubMed Scopus Google Scholar). studies by also that not regulates Nrf2 by its but can also its of M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells are to oxidative stress or xenobiotic the or can the of to a which may in the of Nrf2, or the of Nrf2. of these dramatically the of Nrf2 in the and kinase as PKC, PI3K, and by oxidative stress or can also Nrf2 transactivation activity. It has been that or can Nrf2 at the N-terminal at and its from its nuclear T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, T. Pickett C.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. A. S. T. J. PubMed Scopus Google studies have also the of the in Nrf2 nuclear K. H. Y. T. K. K. PubMed Scopus Google Scholar, Lee Mol. Pharmacol. PubMed Scopus Google Scholar). from also suggested the of MAPK pathways in transcription through Nrf2 T. P.J. Pickett C.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Mulcahy R.T. Toxicol. Sci. PubMed Scopus Google Scholar, Mulcahy R.T. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar, C. T.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). the regulation of Nrf2 transactivation activity by MAPKs is in that are many in MAPK pathway as well as the of potential pathway The study is the to the role of the different MAPK in Nrf2 transactivation activity. the Gal4-Nrf2 chimera and the Gal4-Luc reporter co-transfection assay we the activity of different Nrf2 transactivation domain The Gal4-Nrf2-(1–370) which contains the full-length Nrf2 transactivation domain, very high transactivation activity. In the Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) their transactivation the domain the domain, which been to transactivation activities Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google Scholar). The study also indicated that the transactivation activity of chimera Gal4-Nrf2-(1–370) be by the of in HepG2 cells. the Gal4-Nrf2-(1–370) to Nrf2 in of regulation by Gal4-Nrf2-(113–251) the the activity of Gal4-Nrf2-(113–251) as with Gal4-Nrf2-(1–370) in this study be by on The study also demonstrated that the three MAPK both ERK and JNK pathways up-regulated the activity of Nrf2 transactivation The negative role of p38 pathway on Nrf2 transactivation activity also demonstrated the by of and effects by their by the induction of the in the study is that both Gal4-Nrf2-(1–370) and Gal4-Nrf2-(113–251) showed very of induction on their transactivation activities by upstream Site-directed mutagenesis on Gal4-Nrf2-(113–251) showed or no effect on the basal transactivation activity as well as the induction by which suggested that Gal4-Nrf2-(113–251) transactivation activity not by direct and Mulcahy Mulcahy R.T. Toxicol. Sci. PubMed Scopus Google mutagenesis on potential consensus MAPK phosphorylation sites on the transactivation domain as well as the domain of full-length Nrf2. results also showed that these to the transactivation activity of Nrf2, which is with these that Nrf2 not be a direct of MAPK potential by which phosphorylation Nrf2 nuclear transactivation activity phosphorylation of of the or Nrf2 Among these potential is the nuclear transcriptional coactivator CBP, which is of the its to bind Nrf2 transactivation domain has been verified Y. Itoh K. E. M. A. Yamamoto M. 2001; PubMed Scopus Google Scholar). shown that by ERK or J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. S. A. P. A. D. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar, D. Y. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. J. I. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that phosphorylation of the domain of by the MAPK to of its transcriptional activity D. A. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these suggested the that the transactivation domain activity of Nrf2 by be by the CBP. study demonstrated that synergistic effect with in the transactivation activity of Nrf2 both in the system as well as in the which be by of and In we have demonstrated that transactivation domain plays a critical role the Nrf2 transactivation and that the ERK and JNK MAPK pathways Nrf2 transactivation domain activity while the p38 MAPK pathways negative Site-directed mutagenesis studies demonstrated that of the transactivation activity of Nrf2 transactivation domain region is not direct but at of the CBP, to the transactivation activity of Nrf2 by Taken together, that MAPK signaling Nrf2 through CBP. A. M. J. E. and H. the