Department of Pharmacology, K4/554 Clinical Sciences Center, 600 Highland Avenue, Madison, Wisconsin 53792
Received November 25, 2002; accepted December 22, 2002
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ABSTRACT |
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Key Words: EpRE; phosphorylation; Erk, p38.
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INTRODUCTION |
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Glutamate cysteine ligase modulatory (GCLM), also known as GCS1
-glutamylcysteine synthetase, light subunit, is transcriptionally regulated through an Electrophile Response Element (EpRE) in response to various oxidative stresses (Cai et al., 1997
; Moinova and Mulcahy, 1998
; Wild et al., 1999
). EpREs or EpRE-like sequences are also present in the promoters of numerous phase-II genes such as NQO1 (NADPH-Quinone Oxio Reductase 1) and glutathione S-transferases (Favreau and Pickett, 1991
; Rushmore et al., 1990
, 1991
; Rushmore and Pickett, 1990
; Wasserman and Fahl, 1997
) as well as other antioxidant genes including both GCL genes and heme-oxygenase 1 (Alam et al., 1999
; Kim et al., 2001
). The presence of EpREs in the promoters of multiple detoxification genes results in a coordinated induction of these protective enzymes in response to pro-oxidants or electrophiles such as pyrrolidine dithiocarbamate (PDTC) (Prestera et al., 1993
). Treatment of HepG2 cells with PDTC results in time- and dose-dependent increases in GCLM expression in a mechanism mediated through the EpRE (Mulcahy et al., unpublished data; Wild and Mulcahy, 1999
). Therefore, investigations into the mechanism of PDTC induction of GCLM will yield information that can be applied toward a better understanding of other EpRE-regulated genes and enzymes.
The core consensus sequence of the EpRE was originally established by Rushmore et al.(1991) as an 11-bp sequence: 5'-G/ATGAG/CnnnGCG/A-3'. This core sequence was later expanded by Wasserman and Fahl to include additional flanking sequences, resulting in the 20-bp element: 5'-TMAnnRTGAYnnnGCRwwww-3' (Wasserman and Fahl, 1997
). The EpRE sequence bears a high degree of similarity to the Maf response element (MARE-5'-TGCTGAGTCAGCA-3'), suggesting that similar transcription factors may bind both EpREs and MAREs (Igarashi et al., 1994
). Subsequent studies have confirmed that heterodimers consisting of Cap-N-Collar family members (Nrf1, Nrf2, NFE2-p45) and other b-zip transcription factors (Fos, Jun, Mafs) can bind at both MAREs and EpREs (Alam et al., 1999
; Dhakshinamoorthy and Jaiswal, 2000
; Igarashi et al., 1994
; Jeyapaul and Jaiswal, 2000
; Kim et al., 2001
; Moinova and Mulcahy, 1998
; Myhrstad et al., 2001
; Venugopal and Jaiswal, 1996
, 1998
; Wild et al., 1999
).
Results of recent experiments using Nrf2 knockout mice, Nrf2 overexpression systems, and Nrf2 dominant negative mutants have demonstrated that Nrf2 is involved in both basal and induced expression of multiple EpRE-containing genes encoding detoxification enzymes, as well as the genes for human GCLC (glutamate cysteine ligase catalytic) and GCLM (Chan and Kan, 1999; Chan and Kwong, 2000
; Chan et al., 2001
; Enomoto et al., 2001
; Hayes et al., 2000
; Ishii et al., 2000
; Itoh et al., 1997
; Kwak et al., 2001
; McMahon et al., 2001
; Ramos-Gomez et al., 2001
). Nrf2 is normally sequestered in the cytoplasm of cells bound to Keap1, a cytoskeletal binding protein related to the Drosophila actin-binding Kelch protein, which under basal conditions anchors Nrf2 in the cytoplasm (Itoh et al., 1999
). Itoh et al. demonstrated that after an oxidative/electrophilic stress, Nrf2 translocates from the cytoplasm to the nucleus and transactivates EpRE-containing genes. The mechanism by which the Nrf2/Keap1 complex senses the oxidative/electrophilic stress and triggers Nrf2 release and subsequent nuclear translocation is still unclear. It has been proposed that Keap1, which is rich in reactive cysteines, may directly sense the oxidative insult via thiol modification and undergo a conformational change that releases Nrf2 (Itoh et al., 1999
).
An alternate hypothesis is that Nrf2 release from Keap1 is mediated by specific kinases that are activated after an oxidative stress. Both protein kinase C (PKC) and PI3K have been demonstrated to regulate the nuclear translocation of Nrf2 (Huang et al., 2000, 2002
; Lee et al., 2001
). Mitogen-activated protein kinases (MAPK) have also been demonstrated to be involved in Nrf2 binding to an EpRE sequence (Zipper and Mulcahy, 2000
). A cross-species comparison of Nrf2 amino acid sequence identified several conserved consensus MAPK phosphorylation sites in Nrf2. We hypothesized that these conserved phosphorylation sites might function in Nrf2 activation in one of several ways: (a) phosphorylation of Nrf2 by MAPK at conserved sites could be required for release of Nrf2 from Keap1, (b) MAPK phosphorylation could be involved in Nrf2 nuclear translocation, or (c) direct phosphorylation of Nrf2 by MAPK could be required for Nrf2/DNA binding and subsequent transactivation. We generated Nrf2 mutants lacking the conserved MAPK phosphorylation sites and examined the effect of these Nrf2 mutants on Nrf2/Keap1 release and Nrf2 binding/transactivation of the GCLM reporter transgene. The results demonstrate that the PKC and PI3K pathways do not mediate GCLM upregulation in response to PDTC in HepG2 cells. Furthermore, while nuclear translocation of Nrf2 is decreased following MAPK inhibition, suggesting MAPK-dependent phosphorylation events may be required for Nrf2 nuclear translocation, it is unlikely that Nrf2 itself is a direct target for MAPK phosphorylation as mutation of the conserved MAPK consensus phosphorylation sites in Nrf2 failed to alter Nrf2 transactivation of GCLM gene expression or Nrf2 interaction with the cytoskeletal-binding protein, Keap1.
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MATERIALS AND METHODS |
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Cell culture and transient transfection.
HepG2 cells were maintained in Dulbeccos modified Eagle medium, supplemented with 10% (v/v) fetal bovine serum and 50 µg/ml gentamycin. HepG2 cells were obtained from ATCC and are derived from a human hepatocellular carcinoma. Cells of liver origin are highly metabolic and express high levels of many detoxification enzymes including GCLM (Gipp et al., 1995), thereby facilitating the study of mechanisms of transcriptional regulation. Cells were transfected using a standard calcium phosphate and glycerol shock procedure as described previously (Moinova and Mulcahy, 1998
; Wild and Mulcahy, 1999
). Thirty-six h after transfection, the medium was replaced with fresh medium containing PDTC and/or specific inhibitors, as indicated in figure legends. In experiments with kinase inhibitors, cells were pretreated with the inhibitor for 1 h prior to a 6-h exposure to 100 µM PDTC (dissolved in dH2O at 1000 x stock) or an 18-h exposure to tert-butyl hydroquinone (tBHQ) (dissolved in DMSO at 1000 x stock). After treatment of cells with the indicated agents and/or inhibitors, cells were lysed in 1X lysis buffer (Promega), and extracts were assayed for luciferase activity, as described by the manufacturer. Luciferase activity was monitored using a Monolight 2010 luminometer. Transfection experiments were normalized to ß-gal expression by cotransfection of pRSV-ß-gal (A. Jaiswal, Baylor College of Medicine). Experiments were performed in triplicate and results were expressed as luciferase/ß gal. The mean of at least 23 independent experiments (± standard deviation, or standard error, respectively) are presented. Students t-test was performed and p values < 0.05 were considered significant.
Immunofluorescence, GFP imaging, and Western blotting.
For immunocytochemistry experiments, HepG2 cells were grown on sterile glass cover slips in 6-well plates and pretreated with 25µM PD98059 and/or SB202190, or with vehicle (DMSO) for 1 h prior to a 6-h exposure to PDTC. The coverslips were washed with PBS before fixation in 1.4% formaldehyde for 3060 min at 4°C. Cells were washed with 0.2% Triton X-100 in PBS three times for 5 min at room temperature, in order to permeabilize the cells. The coverslips were then incubated with 2% goat serum/PBS for 13 h at 4°C, and incubated overnight at 4°C with Nrf2 antibody diluted 1:200 in 2% goat serum/PBS. The coverslips were washed with PBS prior to incubation with a 1:200 dilution of anti-rabbit-FITC antibody (ZyMax). Coverslips were washed, mounted on glass slides using fluorescent mounting medium (Dako), and visualized by fluorescence microscopy. For experiments using the GFP-Nrf2 construct, HepG2 cells were transiently transfected as described above. Thirty-sixforty-eight h after transfection, cells were stained with 5 µg/ml Hoescht #33342 for 10 min at 37°C, washed with PBS, and visualized with a fluorescence microscope to determine subcellular localization of GFP-Nrf2.
Western blots were performed using nuclear extracts prepared from HepG2 cells pretreated with SB202190 and/or PD98059 before exposure to PDTC, as described previously (Zipper and Mulcahy, 2000). 25 µg of nuclear protein was separated on 7% acrylamide gels and transferred to nitrocellulose membranes using a Trans-blot Semi-Dry Transfer Cell (Biorad). Membranes were blocked in 5% nonfat dry milk/TBS/Tween 0.1% for 13h at room temperature before incubation with 1:200 dilution of Nrf2 antibody (Santa Cruz) overnight at 4°C. Membranes were washed in TBS/Tween 0.1% and incubated with anti rabbit-HRP (Pierce) before development with SuperSignal chemiluminescent substrate (Pierce). Nonspecific bands recognized by the Nrf2 antibody served as loading controls for each of the nuclear extracts. The Nrf2 standard was produced by incubating purified GST-Nrf2 protein with thrombin protease (Calbiochem) at 22° C for 20 h. An aliquot of the cleaved GST-Nrf2 products was run in a lane alongside the nuclear extracts to identify the Nrf2-specific band.
Site-directed mutagenesis.
Site-directed mutagenesis was performed with commercially available kits (Stratagene and Promega) using the following primers as the sense strand (specific bases mutated are indicated in lowercase):
Primers were synthesized at the University of Wisconsin Biotechnology Center. All mutations were confirmed by sequence analysis.
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RESULTS |
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Lee et al.(2001) have demonstrated that in IMR-32 neuroblastoma cells the PI3 kinase pathway is the major signaling cascade involved in EpRE gene induction. In IMR-32 cells, PDTC and tBHQ induction of GCLM are reduced ~50% by LY294002, suggesting PI3K mediates partial induction in this cell line (Fig. 2A
p, < 0.05). In contrast, in HepG2 cells, the PI3K pathway inhibitors were ineffective at blocking PDTC (Figs. 2B
and 2C
) or tBHQ (Fig. 2C
) induction of GCLM. PDTC treatment of HepG2 cells induced GCLM reporter gene activity ~twofold. Preincubation with the PI3K inhibitor wortmanin, prior to PDTC exposure, had no effect on the magnitude of PDTC induction (Fig. 2B
). Similarly, pretreatment of HepG2 cells with either 25 or 50 µM LY294002 had no effect on PDTC or tBHQ induction of the GCLM luciferase transgene. PDTC increased expression of the luciferase constructs ~twofold, and tBHQ induced ~fivefold, even in the presence of the inhibitor (Fig. 2C
).
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A comparison of the primary amino acid sequence for chicken, rat, mouse, and human Nrf2 identified three potential phosphorylation sites (P-X-S/T-P or minimally S/T-P) conserved in all four species and three additional conserved sites in the mouse, rat, and human proteins (Fig. 3). A series of Nrf2 mutant proteins were developed by mutating the serine in each conserved phosphorylation site to an alanine. Functional properties of the mutant Nrf2 proteins were then compared with those of wild-type Nrf2 to determine if disruption of one or more MAPK phosphorylation sites modulated Nrf2 function in vivo.
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In HepG2 cells overexpressing Nrf2, PDTC exposure failed to further increase GCLM reporter transgene expression. In contrast, GCLM reporter expression was elevated ~2.6-fold following PDTC exposure in cells expressing both Keap1 and wild-type Nrf2, signifying that PDTC treatment had resulted in release of Nrf2 from Keap1. The level of GCLM expression obtained from PDTC-mediated Nrf2 release from Keap1 was not as great as that observed in cells overexpressing Nrf2 alone. These results are consistent with previous reports which also demonstrate that in the presence of overexpressed Keap1, exposure to oxidants/electrophiles only partially restores Nrf2 transactivation potential (Itoh et al., 1999; Sekhar et al., 2002
). Reporter gene expression following PDTC exposure was equivalent in cells expressing wild-type Nrf2, the S561A, or M6 Nrf2 mutants, indicating that these sites have no direct influence on the dynamics of the Nrf2 binding to Keap1 or on Nrf2 release following PDTC exposure.
Since mutation of potential MAPK phosphorylation sites present in Nrf2 failed to alter Nrf2-mediated transactivation of reporter genes or the dynamics of Keap1/Nrf2 interactions, we examined the effect of the MAPK inhibitors on Nrf2 nuclear translocation. Increased nuclear levels of Nrf2 were detected by Western blotting of nuclear extracts prepared from PDTC-treated cells (Figs. 6A and 6B
). When PDTC-treated HepG2 cells were preincubated with 25 µM of the Erk pathway inhibitor, PD98059 (a dose previously shown to reduce induction by PDTC, Zipper and Mulcahy, 2000
), levels of nuclear Nrf2 were reduced dramatically (Fig. 6A
). Preincubation with the p38 inhibitor, SB202190, only slightly decreased Nrf2 levels evident in the nucleus following exposure to PDTC (Fig. 6B
). Simultaneous incubation with both inhibitors prior to PDTC exposure reduced the level of nuclear Nrf2 to that of untreated controls (Fig. 6B
).
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DISCUSSION |
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In 1999, Itoh et al., reported that Nrf2 was regulated by nuclear exclusion (Itoh et al., 1999). In unstimulated cells, Nrf2 is localized in the cytoplasm bound to the cytoskeletal binding protein Keap1, which itself contains double-glycine-repeat domains (also called Kelch repeats) capable of binding the actin cytoskeleton (Adams et al., 2000
; Lecuyer et al., 2000
, Zipper and Mulcahy, 2002
). In response to an oxidative challenge, Nrf2 is released from Keap1 repression and accumulates in the nucleus. Itoh et al.(1999)
have hypothesized that electrophiles or reactive oxygen species (ROS) may react with one or more of the multiple cysteines on Keap1, resulting in a conformational change that liberates Nrf2. More recent experiments have resulted in the identification of several cysteines that are modified by oxidative insult and are implicated in Nrf2 release from Keap1 (Dinko-Kostova et al, 2002
).
Alternatively, Nrf2 or Keap1 may serve as a substrate for one or more of the many kinase pathways that are activated following exposure to oxidants/electrophiles. Many different candidate pathways have been suggested to be involved in EpRE/ARE gene induction by pro-oxidants such as tBHQ and PDTC. The results presented here demonstrate that in HepG2 cells, PDTC induction of a GCLM reporter transgene can be inhibited by the general PKC inhibitor, staurosporine. However, more specific PKC inhibitors failed to effect PDTC induction. Since staurosporine also inhibits other protein kinases, its effect on GCLM gene expression cannot be attributed solely to PKC. Both RO-32-0432 and RO-31-8425, however, are specific inhibitors of multiple PKC isoforms. The failure of the more specific PKC inhibitors to alter GCLM gene expression suggests PKC is not mediating PDTC induction of GCLM. These results conflict with the findings of Huang et al.(2000) who observed that the specific PKC inhibitor RO-32-0432 blocked NQO1 gene induction by ~60% in HepG2 cells. One potential explanation for this difference could be related to differences in the reporter transgene employed in the respective studies. We utilized the GCLM luciferase reporter, which contains 1.9 kb of promoter sequence, whereas Huang et al. used a 31-bp EpRE enhancer derived from the NQO1 promoter. Furthermore, Huang et al.(2000)
exposed cells to tBHQ, whereas we induced expression with PDTC. It is conceivable that these two classes of inducing agents activate different kinase cascades as a consequence of generation of distinct biochemical signals. In support of this explanation, we have observed that PDTC induction of the GCLM reporter can be prevented by preincubating cells with the antioxidant N-acetyl cysteine (NAC) prior to PDTC exposure. However, NAC pretreatment is ineffective at inhibiting tBHQ induction of the same luciferase reporter construct (Mulcahy et al., unpublished data). The activation of different kinase cascades by different inducers (PDTC and tBHQ) could explain why PKC inhibitors are effective against tBHQ induction but not PDTC induction of GCLM.
We also tested the ability of PI3K inhibitors to effect GCLM gene induction HepG2 cells and IMR-32 cells. PI3K inhibitors were unable to prevent induction of the GCLM luciferase transgene by either tBHQ or PDTC in HepG2 hepatoblastoma cells. However, in IMR-32 neuroblastoma cells, the PI3K inhibitor LY294002 decreased GCLM induction by ~50%. These observations are in agreement with Lee et al.(2001), who observed that the PI3K pathway mediates NQO1 induction through the EpRE sequence in IMR-32 cells. We previously demonstrated that inhibition of Erk and/or p38 kinases reduced or eliminated PDTC induction of GCLM transcription and also decreased Nrf2 binding to the GCLM EpRE (Zipper and Mulcahy, 2000
). Collectively, these data suggest that the activation of multiple kinase cascades can culminate in activating EpRE-containing genes, although the specific cascade responsible can vary in a cell line-specific manner. In the case of PDTC induction of GCLM expression in HepG2 cells, activation of the Erk and p38, but not PKC or PI3K pathways mediates signal transduction, culminating in upregulation of the GCLM gene.
The decreased Nrf2-EpRE binding detected after MAPK inhibition suggested the possibility that Nrf2 might be a direct downstream target of MAPKs, as has been demonstrated for other transcription factors such as c-jun. In in vitro studies, purified Nrf2 protein was phosphorylated by immunoprecipitated Erk 1 and 2 kinases (data not shown). Although Nrf2 has not been previously identified as a MAPK substrate, our in vitro phosphorylation studies, coupled with the presence of multiple conserved MAPK phosphorylation sites within Nrf2, lend support to the hypothesis that Nrf2 could be a substrate of activated MAPKs. MAPK-dependent phosphorylation of Nrf2 could contribute to EpRE-mediated GCLM gene activation by at least 3 distinct mechanisms following oxidative/electrophilic stress: (1) phosphorylation of one or more MAPK sites on Nrf2 might mediate Nrf2 release from Keap1; (2) Nrf2 phosphorylation may be required for nuclear translocation; or (3) phosphorylation could be a prerequisite for DNA-binding and/or transactivation. We reasoned that if Nrf2 phosphorylation was required to affect the release of Nrf2 from Keap1, individual Nrf2 proteins harboring mutations in one or more of the potential MAPK phosphorylation sites might differ in their ability to mediate activation of EpRE reporter transgenes. However, no differences among the various Nrf2 mutants and wild-type Nrf2 were detected with respect to Keap1 binding or reporter gene induction following PDTC exposure, effectively discounting a role for these conserved MAPK sites in Nrf2 binding and release from Keap1.
Once released from Keap1, in response to an as yet unidentified signal, Nrf2 has been shown to translocate from the cytoplasm to the nucleus. It has not yet been determined whether post-translational modification of Nrf2 is required for this step of the signal transduction pathway. Nrf2 was detected in the cytoplasm and nucleus of untreated HepG2 cells by immunofluorescence. The presence of endogenous Nrf2 in the nucleus differs from previous reports, which failed to detect nuclear Nrf2 in untreated cells (Huang et al., 2000), but is consistent with Western blots (Fig. 6
) and our previous super-shift analyses, which detected Nrf2 in nuclear extracts prepared from control cells (Zipper and Mulcahy, 2000
). Following PDTC treatment, there was a significant decrease in Nrf2 fluorescence evident in the cytoplasm, but a corresponding increase in nuclear fluorescence was difficult to appreciate by microscopy. However, Western blots did reveal a significant increase in nuclear Nrf2 protein following PDTC exposure. Both the decrease in cytoplasmic Nrf2 fluorescence (observed with immunofluorescence) and the nuclear Nrf2 accumulation (measured with Western blots) were inhibited by PD98059, and, to a lesser extent, by inhibition of p38 with SB202190. Simultaneous treatment with both inhibitors abrogated Nrf2 translocation, consistent with our previous report that combination of the two inhibitors effectively blocked GLC gene induction and binding of Nrf2 to the GCLM EpRE (Zipper and Mulcahy, 2000
).
Collectively these data suggest that the Erk and p38 pathways mediate signal transduction, culminating in upregulation of the GCLM subunit gene (and presumably other EpRE-regulated genes) following PDTC exposure, at least in part by regulating the nuclear translocation of Nrf2. While Erk or p38 inhibition prevents accumulation of Nrf2 in the nucleus following PDTC exposure, this effect is not likely to be related to Nrf2 phosphorylation per se, since other experiments described in this report indicate that the mutated Nrf2 proteins were as effective at transactivating reporter transgenes as was wild-type Nrf2.
The exact mechanism(s) by which Erk regulates Nrf2 nuclear localization and transcriptional activation has yet to be elucidated. We recently discovered that Keap1 dimerization is required to sequester Nrf2, and that disruption of the Keap1 dimer results in Nrf2 release, nuclear translocation and subsequent activation of Nrf2 target genes (Zipper and Mulcahy, 2002). Disruption of the Keap1 complex was not dependent on Erk activation, suggesting MAPKs act downstream of Nrf2 release from Keap1. Collectively, our data suggest that Keap1 and Nrf2 are not direct targets of MAPK phosphorylation, nor is MAPK phosphorylation the trigger for Nrf2 release from Keap1. However, MAPK activation is involved in Nrf2 nuclear translocation.
We have developed a model (Fig. 8) that illustrates some potential mechanisms by which phosphorylation could regulate Nrf2 nuclear entry. Once Nrf2 is released from Keap1 (a mechanism that is independent of Erk activation, Zipper and Mulcahy, 2002
), Erk-mediated phosphorylation of a Nrf2 chaperone or some other type of accessory protein might be required for Nrf2 nuclear translocation (Fig. 8
, pathway B). Precedence for such a mechanism of MAPK-regulated nuclear translocation exists in the case of nuclear factor of activated T cells (NFAT). Calcium signals induce NFAT nuclear translocation in a process regulated by calcineurnin, which is cotransported with NFAT to form a transcriptionally active complex. Recent work has demonstrated that c-Jun N-terminal kinase (Jnk) regulates NFAT nuclear translocation through phosphorylation of calcineurnin, the NFAT chaperone protein (Chow et al., 2000
; Gomez del Arco et al., 2000
).
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NOTES |
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