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J. Biol. Chem., Vol. 278, Issue 45, 44675-44682, November 7, 2003
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From the Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, July 15, 2003 , and in revised form, August 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-glutamylcysteinyl synthetase, and heme oxygenase-1, contribute significantly to cellular protection against redox cycling, oxidative stress, and neoplasia (reviewed in Refs. 1 and 2). Specific nuclear protein complexes bind to the AREs of various genes (reviewed in Refs. 1 and 2). Among these proteins, the role of Nrf2 (NF-E2-related factor-2) is the most clearly established (reviewed in Refs. 1 and 2). Nrf2 is a basic leucine zipper protein that does not homodimerize, but that requires heterodimerization with another leucine zipper protein to be active (reviewed in Refs. 1 and 2). c-Jun and small Maf proteins have been shown to heterodimerize with Nrf2; these complexes bind to the ARE and alter ARE-mediated gene expression (reviewed in Refs. 1 and 2).
A cytosolic inhibitor of Nrf2, Keap1/INrf2, was identified (3, 4). Under normal conditions, it is believed that INrf2 retains Nrf2 in the cytoplasm (3, 4). When cells are challenged by chemically induced oxidative stress, Nrf2 is released from INrf2. Nrf2 then translocates to the nucleus, resulting in the activation of ARE-mediated gene expression. Recently, reports have shown that the interaction of Nrf2 with INrf2 targets Nrf2 for proteasomal degradation (5–7). Nrf2 accumulates in the nucleus of cells from INrf2-deficient mice, in a manner similar to challenge with chemicals that induce oxidative stress (7).
In vitro studies have demonstrated that several kinases, including JNK1, MAPK, p38, and MEK, are involved in regulating ARE-mediated gene expression (8–11), yet other studies have implicated phosphatidylinositol 3-kinase and tyrosine kinases as the kinases responsible (12, 13). However, another report has disputed the involvement of p38 and MEK kinases and instead has indicated that protein kinase C (PKC) is the major kinase involved in antioxidant induction of ARE-mediated gene expression (14). In vitro studies from the same group showed preferential phosphorylation of a serine residue in the Neh2 domain of Nrf2 by PKC (15).
Our laboratory has long-term interest in Nrf2 regulation of ARE-mediated gene expression with special emphasis on NQO1 gene expression. Because of the conflicting reports on the kinases involved, we felt further studies were necessary. In addition, there is no information available on the kinase(s) involved in the induction of ARE-mediated human NQO1 gene expression in response to antioxidants. Furthermore, it is unknown if phosphorylation of Nrf2 is required only for its release from INrf2 or is required also for stabilization/accumulation of Nrf2 in the nucleus and transcriptional activation of ARE-mediated gene expression.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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chemically competent cells (Invitrogen). The forward primers for each mutation are as follows: Nrf2
PKC1, 5'-GTG TTT GAC TTT GCT CAG CGA CAG AAG GAC-3'; Nrf2PKC2, 5'-GTG CCC CTG GAG CTG TCA AAC AGA ACG GCC-3'; Nrf2PKC3, 5'-CCA ATG TGA AAA TGC AGC AAA AAA AGA AGT TCC-3'; Nrf2PKC4, 5'-GTG AAA AGA CAA ACA TGC AGC CCG CTT AGA GGC-3'; Nrf2PKC5, 5'-CCT TGT TCC CAA AGC TAA GAA GCC AGA TAC-3'; and Nrf2PKC6, 5'-CAA GAA GCC AGA TGC TAA GAA AAA CTA GG-3'. The reverse primers are the exact complements of the forward primers. The pcDNA-Neh2 and pcDNA-Neh2S plasmids expressing Neh2 and Neh2S domains, respectively, were created by PCR, followed by TA cloning into the pcDNA3.1 vector. pcDNA-Nrf2 was used as the template for Neh2, and pcDNA-Nrf2PKC1 was used as the template for the Neh2S domain. The same primer set was used to PCR both constructs: 5'-CCC TCA GCA TGA TGG ACT TGG-3' (forward) and 5'-GGC CGG CTG AAT TTG GGG AGG-3' (reverse). Kinase Inhibitor/Luciferase Reporter Assay—HepG2 cells were grown in monolayer cultures by procedures described previously (3). The cells were cotransfected with a reporter construct containing the human NQO1 gene ARE driving luciferase expression and a Renilla luciferase reporter as an internal control. Thirty-six h after transfection, the cells were pretreated with inhibitors to various kinases (PD98059 for MEK/ERK, SB23580 for p38, wortmannin for phosphatidylinositol 3-kinase, genistein for tyrosine kinase, and calphostin C and staurosporine for PKC) for 2 h. All the inhibitors were purchased from Calbiochem and were the highest purity available. The cells were treated with a combination of kinase inhibitor and 50 µM t-butylhydroquinone (t-BHQ) for 16 h. The cells were harvested, washed, homogenized, and analyzed for luciferase activity. The dual luciferase reporter assay from Promega (Madison, WI) and a Packard Topcount NXT luminometer (PerkinElmer Life Sciences) were used to measure luciferase activity according to the manufacturers' protocols.
NQO1 Activity—HepG2 cells were pretreated with calphostin C for 2 h, and then fresh medium was added with Me2SO, with 50 µM t-BHQ, or with 50 µM t-BHQ plus increasing concentrations of calphostin C. Two h after treatment, the cells were harvested; cytosolic extracts were prepared; and NQO1 activity was assayed by a previously described method (16). The cytosolic extracts from HepG2 cells treated with t-BHQ and with t-BHQ plus calphostin C were also resolved by SDS-PAGE and Western-blotted, and the NQO1 protein level was visualized using antibodies to NQO1 and actin.
Bacterial Expression and Purification of Nrf2—Wild-type Nrf2 and Nrf2PKC1 through Nrf2PKC6 mutants were subcloned into the pProExHTb vector from Invitrogen. These clones were transformed into subcloning efficiency DH5 competent cells (Invitrogen). The bacteria were grown at 37 °C and induced with 0.5 mM isopropyl-
-D-thiogalactopyranoside when they reached A590 = 0.5. The bacteria were grown for another 2 h at 37 °C and then harvested. His6-tagged Nrf2 was purified on a nickel column (QIAGEN Inc.) under denaturing conditions according to the manufacturer's protocol. Before Nrf2 was eluted, the column was washed with diminishing amounts of urea. Nrf2 was then eluted under native conditions with 500 mM imidazole.
In Vitro Kinase Assay—Equal amounts of purified Nrf2 or Nrf2PKC mutants were used as the substrate for purified PKC consisting primarily of the - and -isozymes (Promega). The purified proteins were incubated with the PKC enzyme and [-32P]ATP in PKC buffer (20 mM Hepes (pH 7.4), 1 mM dithiothreitol, 10 mM MgCl2, 1.7 mM CaCl2, and 0.1 mg/ml phosphatidylserine) for 1 h at 30 °C. The proteins were then resolved by SDS-PAGE and visualized by autoradiography.
Immunoprecipitation and Phospho-specific Antibodies—Hepa-1 cells were grown in 150-mm dishes. The cells were treated with Me2SO or 50 µM t-BHQ for 2 h, washed twice with phosphate-buffered saline, scraped into 10 ml of phosphate-buffered saline, and pelleted by centrifugation. Cytosolic and nuclear extracts were prepared as follows. The cell pellet was suspended in 10 volumes of isotonic buffer (0.15 M NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 7.4), 0.5% (v/v) Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 µM NaF, 1 mM Na3VO4, and 1 µM okadaic acid) and homogenized in a Dounce homogenizer 10 times with a loose pestle. The nuclei were pelleted at 3500 rpm at 4 °C. The cytosolic extract was removed, and the pelleted nuclei were lysed using radioimmune precipitation assay buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 50 µM NaF, 1 mM Na3VO4, and 1 µM okadaic acid). Both extracts were cleared by centrifugation at 12,000 rpm for 10 min. Nrf2 was immunoprecipitated from the nuclear extracts using anti-Nrf2 antibody (H-300, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Equal amounts of precipitate were run on an SDS-polyacrylamide gel, and Western blotting was performed with antibody specific to Nrf2 or phosphoserine (Sigma), phosphothreonine (Cell Signaling, Salem, MA), or phosphotyrosine (Cell Signaling). The Neh2 and Neh2S domains were pulled down using Ni2+-nitrilotriacetic acid-Sepharose beads (QIAGEN Inc.) according to the manufacturer's protocol.
Similar assays were also performed with Hepa-1 cells transfected with the pcDNA-Neh2-V5 and pcDNA-Neh2S-V5 plasmids in separate experiments. The cytosolic fractions from the transfected cells were prepared by the procedures described above. Cytosolic proteins (1 mg) were immunoprecipitated with anti-V5 antibodies. Equal amounts of immunoprecipitated proteins from the transfected cells were separated by 12% SDS-PAGE, Western-blotted, and probed with anti-V5 and anti-phosphoserine antibodies.
Nrf2 Localization and Immunofluorescence—Hepa-1 cells were grown on Lab-Tek II chamber slides. The cells were pretreated with 10 nM staurosporine for 1 h and then treated with 50 µM t-BHQ alone or with 50 µM t-BHQ plus 10 nM staurosporine for 2 h. The cells were fixed using formalin and permeabilized with cold acetone. Immunofluorescence was then performed using anti-Nrf2 antibody H-300 and fluorescein isothiocyanate-labeled secondary antibody (Rockland, Philadelphia, PA). The cells were stained with Hoechst stain to visualize the nuclei. A Nikon Eclipse TE2000-U fluorescent microscope fitted with a Photometrics CoolSnap CF camera and the appropriate filters was used to capture the fluorescent images.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3.5-fold induction over basal activity. This induction was inhibited by cotreatment with the PKC inhibitor staurosporine (Fig. 1). Similar results were also observed with a second PKC inhibitor, calphostin C (Fig. 1). Inhibitors of MEK/ERK (PD98059), p38 (SB23580), phosphatidylinositol 3-kinase (wortmannin), and tyrosine kinases (genistein) failed to block t-BHQ induction of the ARE (Fig. 2). The small amount of inhibition observed with phosphatidylinositol 3-kinase and p38 inhibitors was not significant (p > 0.1). Interestingly, the inhibitors of MEK/ERK and tyrosine kinases stimulated NQO1 ARE-mediated gene expression.
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Calphostin C was also able to inhibit the induction of NQO1 activity in vivo (Fig. 3). Untransfected HepG2 cells were treated with calphostin C or with calphostin C plus t-BHQ. The lysates were then assayed for NQO1 activity, and the amount of NQO1 was determined by Western blot analysis. Both NQO1 activity and the amount of NQO1 protein decreased in a dose-dependent manner in response to treatment with the PKC inhibitor.
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To determine whether PKC inhibitors affect the localization of Nrf2, immunofluorescence was used to follow the distribution of endogenous Nrf2 before and after treatment. Hepa-1 cells were grown on chamber slides; treated with Me2SO, with t-BHQ, with staurosporine, or with t-BHQ and staurosporine; fixed; and probed with anti-Nrf2 antibody (Fig. 4A). In control cells, Nrf2 was found localized in the cytoplasm and nucleus, although the fluorescence was weak. After treatment with t-BHQ, Nrf2 accumulated in the nucleus, resulting in a much stronger signal. The PKC inhibitor staurosporine was able to block the t-BHQ-induced nuclear accumulation of Nrf2. These results were also confirmed by Western blot analysis. Cytosolic and nuclear extracts were prepared from treated and untreated Hepa-1 cells. The extracts were resolved by SDS-PAGE and probed with anti-Nrf2 antibody (Fig. 4B). Nrf2 was hardly visible in any of the cytosolic extracts or in the nuclear extracts from control cells or from cells treated with staurosporine plus t-BHQ. However, there was an abundance of Nrf2 in the nucleus of t-BHQ-treated cells. These results coincide with the proposed model that Nrf2 is rapidly degraded in the cytosol as a result of the Nrf2/INrf2 interaction and is stabilized/accumulated in nuclei after chemically induced oxidative stress (5–7).
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Next, we studied the ability of PKC to phosphorylate Nrf2 in vitro. Histidine-tagged Nrf2 was overexpressed in a bacterial system and purified using nickel-coated Sepharose beads. This Nrf2 was used as the substrate in an in vitro kinase assay utilizing purified PKC as the enzyme (Fig. 5A). Nrf2 was phosphorylated by PKC in vitro. Staurosporine was able to inhibit the phosphorylation, whereas wortmannin, a phosphatidylinositol 3-kinase inhibitor, was not.
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To determine whether Nrf2 is phosphorylated in vivo, we utilized antibodies specific to phosphoserine, phosphothreonine, and phosphotyrosine. Hepa-1 cells were treated with Me2SO or t-BHQ. Nrf2 was immunoprecipitated from the cytosolic and nuclear extracts, and Western analysis was performed (Fig. 5B). This revealed that Nrf2 that accumulated in the nucleus in response to t-BHQ treatment was phosphorylated at a serine residue(s). Phosphorylation of Nrf2 in the untreated nuclear extracts could not be detected. It is possible that it is not phosphorylated or that the amount of protein was not sufficient to detect the phosphorylation. Likewise, we could not detect phosphorylation with anti-phosphothreonine or antiphosphotyrosine antibody. The question arises whether PKC inhibitors can block the phosphorylation. However, there was an insufficient amount of Nrf2 in the staurosporine-treated nuclear extracts to determine phosphorylation.
A Prosite search revealed seven putative PKC sites in Nrf2. Of these, four are serine residues.2 PKC site 1, Ser40, is located in the N-terminal Neh2 domain of Nrf2, the domain that interacts with INrf2 (2, 3). This PKC site is conserved across the species (human, rat, mouse, and chicken).2 Six Nrf2 mutants were made and are referred to as Nrf2PKC1 through Nrf2PKC6 (Fig. 6A). All seven PKC sites were mutated from serine/threonine to alanine. The two threonines located next to each other at positions 417 and 418 were mutated in the same construct, Nrf2PKC3 (Fig. 6A).
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Mutating the putative PKC sites individually did not have a significant effect on the activity of any of the mutants (Fig. 6B, data shown only for Nrf2PKC1). However, mutating the PKC site in the Neh2 domain resulted in INrf2 inhibiting Nrf2PKC1 more efficiently than wild-type Nrf2 (Fig. 6C). The inhibition of the other mutants by INrf2 was not affected (data not shown). In vitro kinase results revealed that Nrf2PKC1 was not phosphorylated by PKC (Fig. 6D).
To further study the effect of this mutation in vivo, constructs were created that would express the Neh2 domain or the Neh2 domain with the PKC1 serine mutation, Neh2S (Fig. 7A). Both domains were tagged with V5 and His6. HepG2 cells were transfected with increasing amounts of these constructs as well as the ARE-luciferase reporter. As expected, the wild-type Neh2 domain was able to compete with endogenous Nrf2 for INrf2. This resulted in a concentration-dependent increase in ARE-luciferase activity (Fig. 7B). At the highest dose, the Neh2 domain induced ARE activity to the same extent as t-BHQ in cells that were not transfected with the Neh2 domain. However, the Neh2S domain was much more efficient in inducing ARE activity. At the highest dose, it induced ARE activity almost twice as much as t-BHQ alone or the wild-type Neh2 domain.
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To demonstrate binding of the Neh2 and Neh2S domains to INrf2 and that the domains can cause the nuclear accumulation of Nrf2, Hepa-1 cells were transfected with an empty vector or with vector encoding the Neh2 or Neh2S domain. The cells were treated with Me2SO or t-BHQ, and cytosolic and nuclear extracts were made. Western analysis revealed that the Neh2 and Neh2S domains were able to cause the nuclear accumulation of Nrf2 (Fig. 7C, upper panel). Treatment with t-BHQ or expression of the Neh2 domain caused nearly an equal amount of Nrf2 accumulation in the nucleus. However, expression of the Neh2S domain caused a greater amount of Nrf2 to accumulate in the nucleus, consistent with the ARE activity data. The Neh2 and Neh2S domains were equally expressed (Fig. 7C, middle panel).
The Neh2 and Neh2S domains tagged with histidines were precipitated from the cytosolic extract using nickel-coated Sepharose beads. Western analysis using anti-INrf2 antibody revealed that both the Neh2 and Neh2S domains bound to INrf2 in uninduced cells (Fig. 7D). However, after t-BHQ treatment, the wild-type Neh2 domain no longer associated with INrf2. The interaction of the Neh2S domain with INrf2 was not affected by t-BHQ treatment.
Next, Nrf2 was immunoprecipitated from the nuclear extracts from the previous experiment (Fig. 7E). Western analysis demonstrated that only Nrf2 from cells induced with t-BHQ was phosphorylated at serine residues. Nrf2 that accumulated in the nucleus of cells transfected with either the Neh2 or Neh2S domain was not phosphorylated. It may be noteworthy that each lane contains an equal amount of immunoprecipitated Nrf2 in Fig. 7E. It is this reason that the increase in Nrf2 in the presence of t-BHQ in Fig. 7C is not visible in Fig. 7E. Further studies with Hepa-1 cells transfected with Neh2-V5 and Neh2S-V5 and anti-phosphoserine antibody demonstrated that Neh2-V5 and not Neh2S-V5 was phosphorylated (Fig. 7F).
Taken together, these results indicate that the phosphorylation of Nrf2 at Ser40 by PKC is required for Nrf2 to evade INrf2-mediated degradation. Phosphorylation is not required for Nrf2 to accumulate in the nucleus. Furthermore, the unphosphorylated form of Nrf2 is able to activate transcription of the ARE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Antioxidants and oxidative stress induce a battery of more than two dozen defensive genes, including NQO1, by an ARE-dependent mechanism (reviewed in Ref. 1). This coordinated induction of detoxifying genes is of critical importance for antioxidant action and chemoprevention (reviewed in Ref. 1). The transcription factor Nrf2 is known to bind to the NQO1 ARE as well as to the AREs of several other detoxifying enzyme genes and to activate their transcription in response to antioxidants and xenobiotics (reviewed in Ref. 1). Under normal conditions, Nrf2 is believed to be retained in the cytoplasm by a cytosolic inhibitor, INrf2 (3, 4). Recently, it has also been shown that INrf2 targets Nrf2 for proteasomal degradation (5–7). Treatment of cells with antioxidants disrupts the Nrf2/INrf2 interaction (3, 4). When Nrf2 is no longer associated with INrf2, it is stabilized and accumulates in the nucleus. It forms heterodimers with other leucine zipper proteins, leading to the coordinated activation of NQO1 and other detoxifying enzyme genes, including glutathione S-transferase Ya, -glutamylcysteinyl synthetase, and heme oxygenase-1 (22–25). c-Jun and small Maf proteins have been shown to be heterodimeric partners of Nrf2, leading to the activation of detoxifying genes (26, 27). However, the role of small Maf proteins is controversial. Other studies have shown that Nrf2-small Maf heterodimers repress ARE-mediated gene expression (23, 25). t-BHQ treatment does not affect the levels of Nrf2 mRNA (2). Therefore, we suspected that t-BHQ treatment leads to the modification of Nrf2 and that these modifications are critical for ARE-mediated induction of detoxifying enzyme genes.
It is interesting that inhibitors of MEK/ERK and tyrosine kinases activate ARE-mediated gene expression. It is possible that phosphorylation of Nrf2 by members of these kinase families facilitates the binding of Nrf2 to INrf2 and therefore degradation of Nrf2. Tyrosine kinases are involved in regulating cell growth, so it would not be surprising if this pathway is linked to defensive measures. Inhibition of cell growth could serve as a warning to the cell that the environmental conditions are unfavorable. The cell would then activate defensive genes, through the ARE, to limit cellular damage.
A recent report had demonstrated that Nrf2 is phosphorylated in vitro by PKC and that this affects the interaction of Nrf2 with INrf2 in vitro (14). However, the involvement of PKC had been contradicted by other reports (8–13). We confirm that PKC activity is critical for the induction of ARE-mediated gene expression by t-BHQ. Furthermore, we show for the first time in vivo evidence that phosphorylation of Ser40 is necessary for Nrf2 to dissociate from INrf2. Mutation of this residue in the Neh2 domain resulted in the Neh2S domain/INrf2 interaction being unresponsive to t-BHQ treatment.
Importantly, inducing Nrf2 nuclear accumulation by overexpressing the Neh2 domain resulted in an increase in ARE activity similar to that caused by t-BHQ induction. The mutant Neh2S domain induced ARE expression and Nrf2 accumulation much more efficiently than the wild-type Neh2 domain or t-BHQ. These data point to the fact that phosphorylation of Nrf2 is not required for it to activate transcription. We demonstrated this by showing that Nrf2 that accumulated in the nucleus in response to t-BHQ treatment was phosphorylated at a serine residue(s), but that Nrf2 that accumulated in the nucleus as a result of Neh2 overexpression was not. Clearly, it is the association of Nrf2 with INrf2 that is regulated by PKC, not Nrf2 stability or activity.
The role of PKC phosphorylation of Nrf2 in ARE-mediated gene expression is shown in Fig. 8. Two alternative hypotheses are depicted. Briefly, in Hypothesis I, Nrf2 is retained in the cytoplasm by INrf2. Antioxidants induce the expression of or activate PKC. PKC then phosphorylates Nrf2 that is bound to INrf2. This phosphorylation leads to the release of Nrf2 from INrf2 and the stabilization/nuclear accumulation of Nrf2. Phosphorylated Nrf2 binds to the ARE and activates ARE-mediated gene expression. This hypothesis is based on currently accepted results on Nrf2 interaction with INrf2, antioxidant-induced release of Nrf2 from INrf2, and nuclear localization of Nrf2 (3, 4). However, an alternative hypothesis that can not be ruled out is possible. In Hypothesis II, INrf2 is an inhibitor of unphosphorylated Nrf2. INrf2 binds to unphosphorylated Nrf2 and stimulates its degradation (5–7). Antioxidants/oxidants/other stresses induce/activate PKC, which phosphorylates free Nrf2. Phosphorylated Nrf2 escapes INrf2-mediated degradation and accumulates in the nucleus, where it activates ARE-mediated gene expression. Hypothesis II is based on several observations. First, INrf2 targets Nrf2 for proteasomal degradation (5–7). Second, PKC phosphorylates Nrf2 in the Neh2 domain, which binds to INrf2 (this study and Ref. 4). The binding of INrf2 to the Neh2 domain might interfere with the availability of Ser40 for phosphorylation by PKC. Third, Nrf2 does not require phosphorylation to accumulate in the nucleus and to activate ARE-mediated gene expression (this study). Future experiments should select one hypothesis and rule out the other.
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The data suggest that any mechanism that modifies Nrf2, allowing it to disrupt the Nrf2-INrf2 complex and/or to escape Nrf2 binding to INrf2, will result in the up-regulation of ARE-mediated gene expression. Recently, four sulfhydryl groups in INrf2 that are sensitive to the redox state of the cell were reported (29). Alteration of these sulfhydryl groups leads to the loss of the association of Nrf2 with INrf2, leading to the activation of ARE-mediated gene expression (28). It is possible that this mechanism is redundant to the phosphorylation of Nrf2 by PKC or that the two mechanisms work in concert. It is also possible that these two mechanisms are activated in a stimulus- and/or cell type-specific manner. Likewise, there could be other cell type-specific factors that disrupt the Nrf2-INrf2 complex or modify Nrf2, preventing its interaction with INrf2 in response to various stimuli.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-7691; Fax: 713-798-3145; E-mail: ajaiswal{at}bcm.tmc.edu.
1 The abbreviations used are: ARE, antioxidant response element; NQO1, NAD(P)H:quinone oxidoreductase-1; JNK1, c-Jun N-terminal kinase-1; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; t-BHQ, t-butylhydroquinone.
2 D. A. Bloom and A. K. Jaiswal, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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