Degradation of Transcription Factor Nrf2 via the Ubiquitin-Proteasome Pathway and Stabilization by Cadmium*

Daniel StewartDagger §, Erin KilleenDagger , Ryan NaquinDagger , Safdar AlamDagger , and Jawed AlamDagger §||

From the Dagger  Department of Molecular Genetics, Ochsner Clinic Foundation, New Orleans, Louisiana 70121, the § Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, and the  Department of Environmental Health Sciences, Tulane University School of Medicine, New Orleans, Louisiana 70112

Received for publication, September 9, 2002, and in revised form, November 12, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nrf2 mediates inducer-dependent activation of the heme oxygenase-1 (HO-1) gene (Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) J. Biol. Chem. 274, 26071-26078), but the mechanism by which HO-1 inducers regulate Nrf2 function is not known. Treatment of mouse hepatoma (Hepa) cells with 50 µM CdCl2 increased the amount of Nrf2 protein in a time-dependent manner; induction was observed within 30 min, prior to the accumulation of HO-1 mRNA. Cadmium did not significantly affect the steady-state level of Nrf2 mRNA or the initial rate of Nrf2 protein synthesis but increased the half-life of Nrf2 from ~13 to 100 min. Proteasome inhibitors, but not other protease inhibitors, enhanced the expression of Nrf2, and ubiquitinylated Nrf2 was detected after proteasome inhibition. Cycloheximide inhibited cadmium-stimulated Nrf2 expression and DNA binding activity and attenuated HO-1 mRNA accumulation. Conversely, proteasome inhibitors enhanced HO-1 mRNA and protein accumulation by a Nrf2-dependent mechanism. Together, these results indicate that Nrf2 is targeted for rapid degradation by the ubiquitin-proteasome pathway and that cadmium delays the rate of Nrf2 degradation leading to ho-1 gene activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species (ROS),1 which are detrimental to cellular structures and activities, are generated during the course of normal cellular metabolism and in response to noxious, exogenous stimuli such as heavy metals, UV irradiation, and bacterial toxins. Cells utilize both exogenous (e.g. vitamins and plant-derived phenolic antioxidants) and endogenous (e.g. catalase and superoxide dismutase) mechanisms to detoxify ROS. A continuing imbalance between ROS production and ROS detoxification, however, results in cellular oxidative stress. In an effort to maintain cellular homeostasis, cells respond to this imbalance in part by modulating the expression of a select set of genes that encode proteins with antioxidant and cytoprotective activities. Central to this response are the transcription factors that control the activation of these stress-responsive genes. Examples of such stress-responsive transcription factors include the well-characterized heat shock factors (1) and members of the AP-1 (2, 3) and NF-kappa B (3) families of proteins.

Recent studies from several laboratories have identified another potentially important stress-responsive transcription factor, Nrf2. Like the AP-1 constituents, Fos and Jun factors, Nrf2 is a basic-leucine zipper (bZIP) protein that functions as an obligate dimer (4, 5); the bZIP sequence of Nrf2 precludes homodimerization, and it dimerizes most prominently with "small" Maf proteins (5, 6) but also with other bZIP proteins, including Jun family members (7) and ATF4 (8). Such dimers bind to cis-elements with similar core sequences and are alternatively known as MAREs (Maf recognition elements) (9), AREs (antioxidant response elements) (10), and StREs (stress response elements) (11). Multiple Nrf2 target genes, almost all of which are inducible by various oxidants, electrophiles, or xenobiotics, have been identified: among others these include genes that encode phase II detoxification enzymes (12-17) such as NAD(P)H:quinone oxidoreductase, gamma -glutamylcysteine synthase, and glutathione S-transferase; heme oxygenase-1 (18); and thioredoxin (19). Individually and collectively, the select set of Nrf2-regulated proteins function to detoxify xenobiotics, reduce oxidized proteins, maintain cellular reducing equivalents, disrupt redox cycling reactions, and inhibit the generation of, or counteract the effects of, ROS. Given these activities of the target gene products, Nrf2 appears to be a key physiological regulator of the cellular adaptive response to oxidants and xenobiotics. Consistent with this idea, Nrf2-deficient mice are more prone to butylated hydroxytoluene-mediated pulmonary dysfunction (20), are susceptible to acetaminophen hepatotoxicity (21, 22), and exhibit a significantly higher burden of benzo[a]pyrene-induced gastric neoplasia and reduced chemoprotective efficacy (23). Moreover, macrophages derived from such mice exhibit reduced resistance to toxic electrophiles (24).

Although much is known about the regulation of Nrf2 target genes, the mechanisms by which xenobiotics and oxidants regulate Nrf2 activity are not well characterized and are under active investigation. Accumulating evidence (25-30) suggests that such activity is regulated at least in part at the level of sub-cellular compartmentalization. According to this model, under normal conditions, Nrf2 exists in an inactive, cytoplasm-localized state, in part or fully as a consequence of binding to the cytoskeleton-associated protein Keap1 (25, 26). Upon cellular stimulation by stress agents, the cytoplasmic retention mechanism is inactivated, and Nrf2 is transported to the nucleus by one or more as yet uncharacterized mechanisms but one that, under certain circumstances, may involve protein kinase C-mediated phosphorylation of Nrf2 (16). In the nucleus, Nrf2 heterodimers bind to response elements to regulate target gene transcription. Another control point may be at the level of DNA binding. Jaiswal and colleagues (7, 31) have shown that the association and DNA-binding activity of Nrf2·Jun dimers requires a cytosolic factor yet to be characterized. Whether such a factor is responsive to oxidative stress and how it regulates an ultimately nuclear activity are not known. Xenobiotics may also promote Nrf2 activity by stimulating nrf2 gene transcription. Kwak et al. (32) have recently described a positive feedback mechanism in which Nrf2 autoregulates its own expression in response to 3H-1,2-dithiole-3-thione via an ARE-like sequence within the nrf2 gene.

In this report, we describe a different mechanism of Nrf2 regulation. We show that Nrf2 is a highly labile protein, rapidly and specifically degraded by the ubiquitin-proteasome system, and that cadmium stimulates Nrf2 activity and subsequent activation of the ho-1 target gene at least in part by stabilization of the Nrf2 protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Oligonucleotides were synthesized by IDT, Inc., and radiochemicals were obtained from PerkinElmer Life Sciences. Antibodies against transcription factors (Nrf1, Nrf2, E2F1, JunD, and ATF2) were obtained from Santa Cruz Biotechnology. Inc., whereas anti-rat HO-1 was acquired from StressGen Biotech. Corp. Protease inhibitors were purchased from Calbiochem. Reagents for luciferase assays were acquired from Sigma Chemical Co. All other chemicals were of reagent grade.

RNA and Protein Blot Analyses-- Mouse hepatoma (Hepa) cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) containing 0.45% glucose, 10% fetal bovine serum (Mediatech), and 50 µg/ml gentamicin. Cells were routinely passaged every 3-4 days. Unless otherwise indicated, cells were typically seeded (4 × 106 cells/100-mm plate or 1 × 106 cells/60-mm plate) and cultured for 40-48 h in complete medium prior to treatment with agents in serum-free medium. Total RNA was isolated by the procedure of Chomczynski and Sacchi (33), and Northern or dot blot analyses were carried out as previously described (34, 35). Western blot analyses were carried out using either whole cell protein extracts or nuclear extracts (36). For detection of Nrf2, extracts were electrophoresed on a 4-12% gradient SDS-PAGE gel (Invitrogen), and proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight in Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk and then incubated with the primary antibody (1:1000 dilution) for 3 h. Treatment with the secondary antibody and antigen detection were carried out using the ECL system (Amersham Biosciences) according to the manufacturer's recommendation. Detection of HO-1, ATF2, JunD, and MafG was carried out as previously described (8, 36). Additional details are provided in the figure legends.

Pulse Labeling and Pulse-Chase Analyses-- Hepa cells were plated (5 × 105 cells/35-mm plate) and cultured until ~80% confluent. Cells were washed (2×) with pulse labeling media (methionine-free, Hepes-buffered (pH 7.5) Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum) and incubated at 37 °C for 15 min. The media was removed and replaced with pulse labeling media containing 0.1 mCi/ml [35S]methionine and the appropriate inducing agent. After labeling for appropriate time periods, the media was removed and the cells were washed (2×) with cold phosphate-buffered saline and lysed by the addition of radioimmune precipitation assay buffer (10 mM Tris-HCl (pH 7.4), 0.5% sodium deoxycholate, 1% Nonidet P-40, 400 mM NaCl, 0.1% SDS) containing 50 mM NaF, 2 mM EGTA, and 0.1 mM PMSF. Samples were collected by scraping, transferred to Eppendorf tubes, and sonicated (3× for 30 s each). Lysates were collected after centrifugation at 10,000 × g for 10 min at 4 °C. For pulse-chase experiments, labeling was carried out for 30 min and terminated by direct addition of cold methionine (20-fold molar excess) to the culture media. Cells were incubated for varying time periods prior to lysis. For immunoprecipitation, cell lysates were pre-cleared using 1 µg of pre-immune rabbit IgG and 20 µl of a 50% slurry of Sepharose-coupled Protein A. Pre-cleared lysates containing equivalent amounts of trichloroacetic acid-precipitable counts (~150 µg of protein) were incubated with 1 µg of anti-Nrf2 IgG at 4 °C with continuous rotation. After 18 h, a 20-µl slurry of Protein A-Sepharose was added, and incubation was continued for an additional 2 h. Immunocomplexes were pelleted by centrifugation, washed in RIPA buffer (4×), and resuspended in 40 µl of Laemmli sample buffer containing 1% beta -mercaptoethanol and 0.1 mM PMSF. The samples were heated at 100 °C for 4 min, and 15-µl aliquots were subjected to electrophoresis. Signals were detected and quantified using a phosphorimaging device (Packard Instruments).

Ubiquitinylation Assay-- Ubiquitinylation of Nrf2 and E2F1 was carried out by modification of the procedure of Treier et al. (37). Human embryo kidney (HEK) 293 cells (1 × 106 cells/60-mm plate) were transfected with DNA mixtures consisting of the following: empty vector or plasmids encoding Nrf2 or E2F1 (500 ng), empty vector or plasmid encoding His-tagged ubiquitin (500 ng), and a luciferase expression plasmid (25 ng) using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's recommendations. Twenty-four hours after transfection, the cells were treated with 10 µM MG-132 for 3 h and collected in phosphate-buffered saline. A portion (1/20) of the cells was lysed and assayed for luciferase activity (38). The remainder of the cells was lysed in lysis binding buffer (20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 8 M urea) containing 5 mM imidazole, and luciferase-equivalent portions were adsorbed onto Ni+2-charged agarose (25-µl packed volume). The resin was washed (3×) with lysis binding buffer containing 5 mM imidazole and then (3×) with lysis binding buffer containing 20 mM imidazole. Proteins were eluted within 30 µl of elution buffer (20 mM Tris-HCl (pH 6.7), 0.5 M NaCl, 8 M urea, and 200 mM imidazole) and subjected to electrophoresis and Western blot analysis as described above.

Electrophoretic Mobility Shift Assay-- Cells were treated with vehicle or 25 µM CdCl2 in the presence or absence of 5 µg/ml cycloheximide in serum-free medium for 3 h and then collected for preparation of whole cell extracts (36). A double-stranded oligonucleotide containing the sequence 5'-TTTTATGCTGTGTCATGGTT-3' (core StRE sequence is underlined) was used as probe in EMSA reactions using conditions previously reported (36). In antibody supershift assays, 2 µg of pre-immune IgG or anti-transcription factor IgG was added to the reaction mixture and incubated for 20 min at room temperature prior to electrophoresis.

Transfection and Luciferase Assays-- The construction of pE1-luc and pE1M789-luc has been described previously (35). Transient transfections of Hepa cells, preparation of cell extract, and measurement of reporter enzyme activities were carried out as previously reported (38, 39). Additional details are provided in the figure legends.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadmium Stimulates the Steady-state Level of Nrf2 Protein but Not of Nrf2 mRNA-- We have previously demonstrated the requirement for Nrf2 in cadmium-dependent ho-1 gene activation in several cell types, including Hepa cells (18, 35). Consistent with this role, here we demonstrate that cadmium also regulates Nrf2 expression. Treatment of Hepa cells with 50 µM CdCl2 increased the steady-state level of Nrf2 in a time-dependent manner to greater than 20-fold above basal values (Fig. 1). In our gel system, mouse Nrf2 migrates as an 88-kDa protein, significantly larger than the predicted size of 66 kDa. Accumulation of Nrf2 was detected within 30 min after cell treatment, well before the observable increase in HO-1 mRNA (typically between 1 and 2 h after exposure to CdCl2) (Ref. 34 and Fig. 2) or HO-1 protein (2 h) (Fig. 1). The induction of Nrf2 protein by cadmium is a specific response, because the expression of ATF2 and of other transcription factors such as JunD and CREB (8) is not affected by this agent.


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 1.   Cadmium stimulates expression of Nrf2 and HO-1 protein in Hepa cells. Cells were exposed to 50 µM CdCl2 for the indicated times. Western blot analyses were carried out as described under "Experimental Procedures" using whole cell extracts (30 µg of protein/lane) and antibodies directed against the indicated proteins. The Nrf2 protein band is marked by an arrow, and the migration of the molecular mass (kDa) markers is indicated.


View larger version (71K):
[in this window]
[in a new window]
 
Fig. 2.   Cadmium stimulates accumulation of HO-1 mRNA but not of Nrf2 RNA. Hepa cells were exposed to 50 µM CdCl2 for the indicated time and collected for RNA isolation. Total RNA (10 µg/lane) was electrophoresed, transferred to nylon membranes, and hybridized to the indicated cDNA probes.

In stark contrast to the effect on Nrf2 protein expression, exposure of Hepa cells to 50 M cadmium (for up to 6 h) did not significantly alter the steady-state level of Nrf2 mRNA (Fig. 2). As expected, cadmium treatment dramatically increased the level of HO-1 mRNA in a time-dependent manner. The level of ribosomal protein S3 mRNA, which was used as a control for RNA loading, was not significantly changed during the course of this treatment. Taken together, these results suggest that cadmium regulates Nrf2 expression primarily by one or more post-transcriptional mechanisms.

Effect of Cadmium on Nrf2 Synthesis and Stability-- Cadmium may increase the level of Nrf2 by regulating one or both of two general processes: the rate of Nrf2 synthesis or the rate of Nrf2 degradation. The rate of Nrf2 synthesis was monitored by pulse-labeling analysis, and typical results are shown in Fig. 3. [35S]Methionine-labeled Nrf2 steadily accumulated over a period of 15 min in unstimulated cells, and the rate of accumulation was not affected by cadmium (left panel). The apparent stimulation of Nrf2 synthesis by cadmium was first detected after 20 min of labeling and was more pronounced (~2.5-fold over control) after 30 and 60 min (right panel).


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3.   Cadmium does not affect the initial rate of Nrf2 synthesis. Pulse labeling analyses was carried out as described under "Experimental Procedures" in the absence (-) or presence (+) of 50 µM CdCl2 for the indicated times. The electrophoresis gel was dried and exposed to the phosphorimaging screen for 5 days. The migration of the molecular mass (kDa) markers is indicated.

To examine the role of protein stabilization in Nrf2 regulation by cadmium, we monitored the decay of basal and cadmium-induced Nrf2 protein in Hepa cells after inhibition of protein synthesis by cycloheximide. In the experiment depicted in Fig. 1, whole cell extracts were used to monitor both Nrf2 and the endoplasmic reticulum-localized HO-1 in the same sample. Subsequent studies using cytoplasmic and nuclear fractions demonstrated that basal and induced Nrf2 are localized exclusively in the nucleus, because the level of cytoplasmic Nrf2 is below the detection limit of the Western blot assay (data not shown). Consequently, in the following experiment, nuclear extracts were used to obtain a stronger Nrf2 signal in unstimulated cells. Nrf2 in the nuclei of untreated cells decayed rapidly after addition of cycloheximide and could not be detected after 40 min even after longer exposure of the membrane to the phosphorimaging screen (Fig. 4A). In unstimulated Hepa cells, the half-life of Nrf2 was calculated to be ~13 min. Cadmium-induced Nrf2, however, was more stable with an estimated t1/2 of nearly 100 min. The specificity of this response was demonstrated by the fact that cadmium did not appreciably affect the steady-state level or the rate of degradation of transcription factor JunD (Fig. 4B). Similar results were obtained by pulse-chase analysis (data not shown but see below). We interpret these results to indicate that cadmium stimulates Nrf2 expression primarily by attenuating the rate of Nrf2 degradation. Although we cannot completely exclude an effect on Nrf2 synthesis, it is likely that the apparent increase in the synthetic rate observed at the later time points (30 to 60 min) is a consequence of protein stabilization.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   Cadmium increases the stability of Nrf2 protein. Hepa cells were incubated in serum-free medium in the absence or presence of 50 µM CdCl2 for 2 h. The culture medium was then replaced with serum-free medium containing 100 µg/ml cycloheximide, and the cells were harvested at the indicated times (t) after addition of cycloheximide. Nuclear extracts (15 µg/lane) were subjected to Western blot analyses for Nrf2 (A) or JunD (B). The Nrf2 protein band is marked by an arrow, and the migration of the molecular mass markers is indicated. Whole cell extract (3.5 µg) from HEK293 cells transfected with a mouse Nrf2 expression plasmid was used as a positive control (+) for Nrf2. The Nrf2 signal was quantified by densitometry and represents the average of two experiments.

Proteasome Inhibitors Stimulate Nrf2 Expression-- Many labile regulatory proteins, including signal-activated transcription factors, are commonly degraded by the 26 S proteasome, a highly conserved, multiprotein proteolytic system (40). If Nrf2 is degraded by this pathway, then inhibition of the proteasome should result in higher levels of Nrf2. As shown in Fig. 5A, with treatment of Hepa cells with MG-132 (10 µM), lactacystin (20 µM), and PI 1 (20 µM), all selective inhibitors of the 26 S proteasome significantly increased the steady-state level of Nrf2. The most potent response was observed with MG-132; lactacystin and PI 1 were less effective, but unlike MG-132, further stimulated Nrf2 expression in the presence of 25 µM CdCl2. Inhibitors of serine proteases (PMSF), calpain (PD150606), serine/cysteine proteases (leupeptin), and lysozomal proteases (chloroquine) were ineffective (Fig. 5A and data not shown). Control experiments showed that none of the protease inhibitors stimulate expression of alpha -tubulin (Fig. 5A).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 5.   A, proteasome inhibitors stimulate Nrf2 expression. Hepa cells were treated with vehicle (Me2SO, DMSO), MG-132 (10 µM), lactacystin (20 µM), PI 1 (20 µM), PMSF (100 µM), or PD150606 (20 µM) in the presence or absence of 25 µM CdCl2 for the indicated times. Western blot analyses for Nrf2 and alpha -tubulin (Tub) were carried out as described under "Experimental Procedures" using whole cell extracts (30 µg of protein/lane). +, positive control for Nrf2. B, MG-132 increases Nrf2 stability. Hepa cells were pulse-labeled with [35S]methionine for 30 min in the absence (-) or presence (+) of 10 µM MG-132 and "chased" with cold methionine for the indicated time. Pulse-chase analysis was continued as described under "Experimental Procedures." The electrophoresis gel was dried and exposed to the phosphorimaging screen for 5 days. +, immunoprecipitate of [35S]methionine-labeled Nrf2 in cell extract from HEK293 cells transfected with a mouse Nrf2 expression plasmid.

The proteasome inhibitors did not affect the steady-state level of Nrf2 mRNA (data not shown). To directly demonstrate that induction of Nrf2 by the proteasome inhibitors results from inhibition of protein degradation, Nrf2 stability was assayed by pulse-chase experiments. Under normal conditions, [35S]methionine-labeled Nrf2 was rapidly degraded and barely detected 30 min after termination of labeling (Fig. 5B, "-"). In the presence of MG-132, labeled Nrf2 was maintained at constant levels up to the last time point tested. Taken together, these results indicate that Nrf2 is specifically degraded by the 26 S proteasome.

Ubiquitinylation of Nrf2-- Most, but not all, proteins selectively degraded by the 26 S proteasome are marked by prior, covalent ligation of ubiquitin molecules, a highly conserved ~8-kDa polypeptide, to the epsilon -amino group of lysine residues. The ubiquitin-conjugating system usually generates substrate proteins containing varying lengths of polyubiquitin chains resulting from linkage of successive ubiquitin molecules to the previous moiety (40). Identification of ubiquitinylated Nrf2 would suggest that this transcription factor is regulated via the ubiquitin-proteasome system. Because they are rapidly degraded by the proteasome, endogenous ubiquitinylated species of a specific protein are generally difficult to detect (37, 41). We, therefore, employed the strategy of Treier et al. (37), which involves cellular co- and overexpression of the target protein (e.g. Nrf2) and poly-His-tagged ubiquitin. His-tagged ubiquitin-conjugated proteins are subsequently purified and concentrated by affinity chromatography, and the ubiquitinylated target protein is detected immunologically. In a necessary modification, transfected cells were treated with MG-132 to inhibit proteasome activity prior to isolation of ubiquitinylated proteins. Transcription factor E2F1 was used as a positive control for this experiment, because it is known to be ubiquitinylated (42). As shown in Fig. 6, a ladder of mono- and polyubiquitinylated Nrf2 was readily detected in cells expressing Nrf2 and His-tagged ubiquitin (lane 4). No such species were detected when either of the corresponding expression plasmids was omitted (lanes 1-3). An analogous profile was observed with E2F1 (lanes 5-8). In theory, unconjugated proteins should not be detected in this assay as was the case for E2F1 (arrow, lane 8). Nonetheless, unconjugated Nrf2 (arrow, lanes 3 and 4) was observed, suggesting some affinity for the resin.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 6.   Ubiquitinylation of Nrf2 and E2F1. HEK293 cells were transfected with the indicated expression plasmids, and ubiquitinylation assays were carried out as described under "Experimental Procedures." The positions of the unconjugated Nrf2 and E2F1 are marked by arrows, and the mono-ubiquitinylated proteins are marked by asterisks. The migration of the molecular mass (kDa) markers is indicated.

Functional Consequence of Modulation of Nrf2 Levels-- That induction of Nrf2 is not simply an epigenetic phenomenon but represents an effect with functional consequences is suggested by data provided in Figs. 7 and 8. Consistent with the mode of regulation described above, the basal and cadmium-stimulated expression of Nrf2 was abrogated in the presence of the protein synthesis inhibitor cycloheximide (Fig. 7A). Under these conditions, accumulation of MafG, an Nrf2 dimerization partner, was not altered. Cycloheximide had a similar effect on Nrf2 at the level of DNA binding activity. Electrophoretic mobility shift assays using an oligonucleotide probe harboring an Nrf2 recognition site (i.e. StRE from the ho-1 gene), and whole cell extracts from untreated Hepa cells detected several StRE·protein complexes of similar intensity (Fig. 7B, lane 2). Treatment with cadmium substantially altered the electrophoretic profile resulting in one prominent complex (or several co-migrating complexes) (lane 4). Nrf2 appears to be a predominant factor in this complex, because the complex was quantitatively shifted with anti-Nrf2 antibodies (lane 7) but not with pre-immunized IgG (lane 6) or an antibody to the related StRE-binding protein, Nrf1 (lane 8). Consistent with induction of Nrf2 protein by cadmium and the abrogation of this response by cycloheximide, cycloheximide co-treatment abolished formation of the Nrf2·StRE complex (lane 5). Inhibition of cadmium-stimulated binding of Nrf2 to the StRE correlated with the attenuation of cadmium-induced HO-1 mRNA accumulation in response to cycloheximide (Fig. 7C).


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 7.   A, cycloheximide inhibits cadmium-induced Nrf2 expression. Hepa cells were exposed to vehicle (-) or 50 µM CdCl2 (+Cd) in the presence or absence of 5 µg/ml cycloheximide for 2 h. Western blot analyses were carried out on nuclear extracts (15 µg/lane) using anti-Nrf2 or anti-MafG antibodies. The migration of the molecular mass markers is indicated. B, cycloheximide inhibits cadmium-induced Nrf2·StRE complex formation. EMSA reactions were carried out as described under "Experimental Procedures" using extracts prepared from Hepa cells treated with vehicle or 50 µM CdCl2 (Cd) in the presence or absence of 5 µg/ml cycloheximide (CHX) for 3 h (lanes 2-5). Antibody-supershift EMSA reactions (lanes 6-8) were carried out with protein extract from +cadmium/-cycloheximide cells and with pre-immune IgG ("Ig") or antibodies directed against the indicated transcription factors. No extract was used in the reaction in lane 1. C, cycloheximide inhibits cadmium-induced HO-1 mRNA accumulation. Hepa cells were exposed to 25 µM CdCl2 in the absence or presence of 5 µg/ml cycloheximide for the indicated time and harvested for RNA isolation. Total RNA (5 µg/slot) was dot-blotted onto a nylon membrane and hybridized consecutively to the HO-1 and S3 probes. S3-normalized HO-1 mRNA levels (the average value from two experiments) are presented.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8.   Proteasome inhibitors stimulate HO-1 mRNA (A) and protein (B) accumulation. Hepa cells were treated for 3 (A) or 4 (B) h with vehicle (Me2SO, DMSO), MG-132 (10 µM), lactacystin (20 µM), PI 1 (20 µM), PD150606 (20 µM), PMSF (100 µM), leupeptin (10 µg/ml), or chloroquine (100 µM) in the absence (-Cd) or presence (+) of 50 µM CdCl2 as indicated. RNA and protein blot analyses were carried out as described under "Experimental Procedures" and the legends to Figs. 5 and 7. S3-normalized HO-1 mRNA levels (S/H; average value from four experiments) are presented (A). C, MG-132 stimulates Nrf2·StRE complex formation. EMSA reactions were carried out as described under "Experimental Procedures" using extracts prepared from Hepa cells treated with vehicle (-), 50 µM CdCl2 (Cd), or 10 µM MG-132 (MG) for 3 h (lanes 1-4). Antibody-supershift EMSA reactions were carried out pre-immune IgG or anti-Nrf2 (lanes 5 and 6). D, proteasome inhibitors stimulate ho-1 enhancer activity. Hepa cells were transfected with plasmids pE1-luc (E1) or pE1M789-luc (E1M789) and treated with vehicle (Me2SO, DMSO), 10 µM MG-132, 20 µM lactacystin, 20 µM PD150606, or 50 µM CdCl2 for 5 h. Luciferase activity normalized to that in vehicle-treated cells is presented. Each data point represents the average ± S.E. from three to four experiments.

In contrast to cycloheximide, proteasome inhibitors, which positively regulate Nrf2 expression (Fig. 5A), also induced HO-1 mRNA and protein accumulation (Fig. 8, A and B). The highest level of HO-1 mRNA induction (17-fold) was observed with MG-132, the most potent of the Nrf2 stimulatory proteasome inhibitors. PD150606, PMSF, leupeptin, and chloroquine, which do not affect Nrf2 expression, also did not induce HO-1 mRNA or protein levels (Fig. 7, A and B, and data not shown). The proteasome inhibitors did not further stimulate cadmium-dependent HO-1 mRNA accumulation, suggesting that these agents activate the ho-1 gene by similar mechanisms. Support for this supposition is provided by the observation that MG-132, like cadmium, promotes formation of the StRE·Nrf2 complex (Fig. 8C). Additionally, both MG-132 and lactacystin stimulated expression of a luciferase reporter gene under the control of the mouse ho-1 gene enhancer (E1) containing three StREs by ~3- and 2-fold, respectively (Fig. 8D). By comparison, cadmium enhanced luciferase activity by ~13-fold and the calpain inhibitor PD150606 was without effect. Mutation of the StREs into sequences incapable of binding Nrf2 (35) generates a variant E1 (E1M789) that was unresponsive to MG-132 or lactacystin and exhibited reduced sensitivity to cadmium. Taken together, the above results suggest that stabilization of Nrf2 by cadmium (or proteasome inhibitors) results in a functional transcription factor capable of binding to its target sequences and promoting transcription of target genes such as ho-1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously reported that cadmium does not regulate Nrf2 protein expression in Hepa cells (8). This conclusion was based on artifactual results obtained using extract preparation, sample electrophoresis, and Western blotting conditions that generated a weak Nrf2 signal masked by a more intense, co-migrating but nonspecific signal. The use of a lower percentage polyacrylamide gel and polyvinylidene difluoride membranes has allowed separation of the Nrf2 signal from the nonspecific signal and also significantly improved the signal-to-noise ratio. The new analysis reveals a robust induction of Nrf2 protein in response to cadmium and demonstrates that such induction is regulated primarily at the level of protein degradation/stabilization.

The present studies demonstrate that Nrf2 is a highly labile protein (t1/2 ~ 13 min) specifically degraded by the ubiquitin-proteasome system. This pathway is known to regulate the activity of various transcription factors, such as c-Jun, c-Fos, NF-kappa B, STAT1, and p53 (40), that are typically short-lived and often activated in response to extracellular stimuli. Furthermore, Molinari et al. (43) have shown that the rate of degradation of transcription activators by the proteasome correlates with the potency of one or more of the activation domains; Nrf2 contains a very powerful activation domain (8, 26, 36). The detailed mechanism by which transcription factors are marked and degraded by the ubiquitin-proteasome pathway is generally unique for each protein, but such regulation is often dependent on cis-acting sequences (i.e. specific sequence domains within the target protein) and may involve post-translational signals such as phosphorylation. One structural signal commonly found in rapidly degraded proteins is the PEST domain, which is defined as a hydrophilic stretch of 12 or greater amino acids, enriched in proline, glutamate, serine and threonine residues and flanked by positively charged residues. PEST sequences have been identified in many labile proteins, including ornithine decraboxylase, the NF-kappa B inhibitor Ikappa Balpha , and c-Fos (44, 45). Using the PESTfind algorithm (available at Vienna.at.embnet.org/htbin/embnet/PESTfind), we have identified several poor PEST candidates and one potential PEST sequence at position 350-380 (HSVESSIYGDPPPGFSDSEMEELDSAPGSVK) in mouse Nrf2. Serine and threonine residues within PEST domains are potential phosphorylation sites that may be necessary for degradation. For instance, specific phosphorylation of Ser-32 and Ser-36 within Ikappa Balpha is required for ubiquitinylation and subsequent degradation of Ikappa Balpha leading to activation of NF-kappa B (46, 47). Studies to determine the role of the putative Nrf2 PEST domain (or other cis-elements) and of (de)phosphorylation reactions in degradation of Nrf2 are in progress.

How does cadmium promote Nrf2 stability? In the case of hypoxia-mediated stabilization of hypoxia-inducible factor 1alpha , low oxygen tension reduces ubiquitinylation of hypoxia-inducible factor 1alpha (41). We have not, however, been able to detect a similar effect of cadmium on Nrf2 (data not shown) suggesting that cadmium may inhibit another step or steps within the degradation pathway. Whether such interference involves the PEST domain identified above and (de)phosphorylation reactions or other post-translational modifications remains to be determined.

Finally, it is worth pointing out that, although proteasome inhibitors are as or more effective than cadmium in stimulating Nrf2 levels, they are less potent inducers of HO-1 mRNA. One explanation for this discrepancy is that Nrf2 may not be the sole regulator of cadmium-dependent ho-1 gene activation and that other transcription factors, which may not be regulated by the proteasome, also contribute to this process. This possibility is supported by the observation that mutation of the StREs within E1 does not completely abolish cadmium-dependent transcription activity of the enhancer (Fig. 8D). Additional support for a non-StRE/Nrf2 mode of regulation is provided by Takeda et al. (48) who identified a cadmium response element that is distinct from, but in close proximity to, the StREs in the human ho-1 gene. The protein that binds to this site and presumably mediates the transcriptional response is distinct from Nrf2. An alternative, or additional, possibility for the above discrepancy is that the steady-state level of Nrf2 is not the sole determinant of Nrf2 activity. Such activity is likely to require post-translational modification, such as phosphorylation, which may occur optimally in response to cadmium or other oxidants but not during proteasome inhibition.

    ACKNOWLEDGEMENTS

We thank Drs. Stuart Orkin, Volker Blank, and Erik Flemington for generously providing the Nrf2 expression plasmid, the anti-MafG antibody, and plasmids encoding E2F1 and His-tagged ubiquitin, respectively.

    FOOTNOTES

* This work was supported by the Department of Energy cooperative agreement DE-FC26-00NT40843 and the Center for Bioenvironmental Research, Tulane and Xavier Universities (to B. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Molecular Genetics, Ochsner Clinic Foundation, 1516 Jefferson Highway, New Orleans, LA 70121. Tel.: 504-842-3314; Fax: 504-842-3381; E-mail: jalam@ochsner.org.

Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209195200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; HO-1, heme oxygenase-1; Nrf, NF-E2-related factor; AP-1, activator protein 1; NF-kappa B, nuclear factor kappa B; bZIP, basic region/leucine zipper; StRE, stress response element; ARE, antioxidant response element; EMSA, electrophoretic mobility shift assay; STAT1, signal transducers and activators of transcription 1; PI 1, proteasome inhibitor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Morano, K. A., and Thiele, D. J. (1999) Gene Expr. 7, 271-282[Medline] [Order article via Infotrieve]
2. Karin, M., Liu, Z., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240-246[CrossRef][Medline] [Order article via Infotrieve]
3. Karin, M., Takahashi, T., Kapahi, P., Delhase, M., Chen, Y., Makris, C., Rothwarf, D., Baud, V., Natoli, G., Guido, F., and Li, N. (2001) Biofactors 15, 87-89[Medline] [Order article via Infotrieve]
4. Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9926-9930[Abstract/Free Full Text]
5. Itoh, K., Igarashi, K., Hayashi, N., Nishizawa, M., and Yamamoto, M. (1995) Mol. Cell. Biol. 15, 4184-4193[Abstract]
6. Marini, M. G., Chan, K., Casula, L., Kan, Y. W., Cao, A., and Moi, P. (1997) J. Biol. Chem. 272, 16490-16497[Abstract/Free Full Text]
7. Venugopal, R., and Jaiswal, A. K. (1998) Oncogene 17, 3145-3156[CrossRef][Medline] [Order article via Infotrieve]
8. He, C. H., Gong, P., Hu, B., Stewart, D., Choi, M. E., Choi, A. M., and Alam, J. (2001) J. Biol. Chem. 276, 20858-20865[Abstract/Free Full Text]
9. Kataoka, K., Noda, M., and Nishizawa, M. (1994) Mol. Cell. Biol. 14, 700-712[Abstract]
10. Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639[Abstract/Free Full Text]
11. Choi, A. M., and Alam, J. (1996) Am. J. Respir. Cell Mol. Biol. 15, 9-19[Abstract]
12. Venugopal, R., and Jaiswal, A. K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14960-14965[Abstract/Free Full Text]
13. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. (1997) Biochem. Biophys. Res. Commun. 236, 313-322[CrossRef][Medline] [Order article via Infotrieve]
14. Moinova, H. R., and Mulcahy, R. T. (1999) Biochem. Biophys. Res. Commun. 261, 661-668[CrossRef][Medline] [Order article via Infotrieve]
15. Wild, A. C., Moinova, H. R., and Mulcahy, R. T. (1999) J. Biol. Chem. 274, 33627-33636[Abstract/Free Full Text]
16. Huang, H. C., Nguyen, T., and Pickett, C. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12475-12480[Abstract/Free Full Text]
17. Nguyen, T., Huang, H. C., and Pickett, C. B. (2000) J. Biol. Chem. 275, 15466-15473[Abstract/Free Full Text]
18. Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) J. Biol. Chem. 274, 26071-26078[Abstract/Free Full Text]
19. Kim, Y. C., Masutani, H., Yamaguchi, Y., Itoh, K., Yamamoto, M., and Yodoi, J. (2001) J. Biol. Chem. 276, 18399-18406[Abstract/Free Full Text]
20. Chan, K., and Kan, Y. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12731-12736[Abstract/Free Full Text]
21. Chan, K., Han, X. D., and Kan, Y. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4611-4616[Abstract/Free Full Text]
22. Enomoto, A., Itoh, K., Nagayoshi, E., Haruta, J., Kimura, T., O'Connor, T., Harada, T., and Yamamoto, M. (2001) Toxicol. Sci. 59, 169-177[Abstract/Free Full Text]
23. Ramos-Gomez, M., Kwak, M. K., Dolan, P. M., Itoh, K., Yamamoto, M., Talalay, P., and Kensler, T. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3410-3415[Abstract/Free Full Text]
24. Ishii, T., Itoh, K., Takahashi, S., Sato, H., Yanagawa, T., Katoh, Y., Bannai, S., and Yamamoto, M. (2000) J. Biol. Chem. 275, 16023-16029[Abstract/Free Full Text]
25. Kobayashi, M., Itoh, K., Suzuki, T., Osanai, H., Nishikawa, K., Katoh, Y., Takagi, Y., and Yamamoto, M. (2002) Genes Cells 7, 807-820[Abstract/Free Full Text]
26. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J. D., and Yamamoto, M. (1999) Genes Dev. 13, 76-86[Abstract/Free Full Text]
27. Zipper, L. M., and Mulcahy, R. T. (2002) J. Biol. Chem. 277, 36544-36552[Abstract/Free Full Text]
28. Sekhar, K. R., Spitz, D. R., Harris, S., Nguyen, T. T., Meredith, M. J., Holt, J. T., Guis, D., Marnett, L. J., Summar, M. L., and Freeman, M. L. (2002) Free Radic. Biol. Med. 32, 650-662[CrossRef][Medline] [Order article via Infotrieve]
29. Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., and Talalay, P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11908-11913[Abstract/Free Full Text]
30. Dhakshinamoorthy, S., and Jaiswal, A. K. (2001) Oncogene 20, 3906-3917[CrossRef][Medline] [Order article via Infotrieve]
31. Jeyapaul, J., and Jaiswal, A. K. (2000) Biochem. Pharmacol. 59, 1433-1439[CrossRef][Medline] [Order article via Infotrieve]
32. Kwak, M. K., Itoh, K., Yamamoto, M., and Kensler, T. W. (2002) Mol. Cell. Biol. 22, 2883-2892[Abstract/Free Full Text]
33. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
34. Alam, J., Shibahara, S., and Smith, A. (1989) J. Biol. Chem. 264, 6371-6375[Abstract/Free Full Text]
35. Alam, J., Wicks, C., Stewart, D., Gong, P., Touchard, C., Otterbein, S., Choi, A. M., Burow, M. E., and Tou, J. (2000) J. Biol. Chem. 275, 27694-27702[Abstract/Free Full Text]
36. Gong, P., Hu, B., Stewart, D., Ellerbe, M., Figueroa, Y. G., Blank, V., Beckman, B. S., and Alam, J. (2001) J. Biol. Chem. 276, 27018-27025[Abstract/Free Full Text]
37. Treier, M., Staszewski, L. M., and Bohmann, D. (1994) Cell 78, 787-798[Medline] [Order article via Infotrieve]
38. Alam, J. (2000) in Functional Analysis of the Heme Oxygenase-1 Gene Promoter: Vol. 1. Current Protocols in Toxicology (Maines, M. D. , Costa, L. G. , Reed, D. J. , Sassa, S. , and Sipes, I. G., eds) , pp. 9.7.1-9.7.21, John Wiley & Sons, Inc., New York
39. Gong, P., Stewart, D., Hu, B., Li, N., Cook, J., Nel, A., and Alam, J. (2002) Antioxid. Redox Signal. 4, 249-257[CrossRef][Medline] [Order article via Infotrieve]
40. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve]
41. Sutter, C. H., Laughner, E., and Semenza, G. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4748-4753[Abstract/Free Full Text]
42. Campanero, M. R., and Flemington, E. K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2221-2226[Abstract/Free Full Text]
43. Molinari, E., Gilman, M., and Natesan, S. (1999) EMBO J. 18, 6439-6447[Abstract/Free Full Text]
44. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem. Sci. 21, 267-271[CrossRef][Medline] [Order article via Infotrieve]
45. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368[Medline] [Order article via Infotrieve]
46. Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciechanover, A., and Ben-Neriah, Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10599-10603[Abstract]
47. Chen, Z., Hagler, J., Palombella, V. J., Melandri, F., Scherer, D., Ballard, D., and Maniatis, T. (1995) Genes Dev. 9, 1586-1597[Abstract]
48. Takeda, K., Ishizawa, S., Sato, M., Yoshida, T., and Shibahara, S. (1994) J. Biol. Chem. 269, 22858-22867[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.