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Originally published In Press as doi:10.1074/jbc.M501014200 on April 29, 2005

J. Biol. Chem., Vol. 280, Issue 26, 25267-25276, July 1, 2005
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Selective Induction of the Tumor Marker Glutathione S-Transferase P1 by Proteasome Inhibitors*

Hiroko Usami{ddagger}, Yuri Kusano{ddagger}, Takeshi Kumagai{ddagger}§, Shigehiro Osada¶, Ken Itoh||, Akira Kobayashi||, Masayuki Yamamoto||, and Koji Uchida{ddagger}**{ddagger}{ddagger}

From the {ddagger}Graduate School of Bioagricultural Sciences and the **Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan, the Graduate School of Pharmacological Sciences, Osaka University, Osaka 565-0871, Japan, and the ||Center for Tsukuba Advanced Research Alliance and the Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan

Received for publication, January 27, 2005 , and in revised form, April 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs). Transcription factor Nrf2, which is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein-1) under unstimulated conditions, regulates the induction of phase 2 enzymes. In this study, to explore the role of the proteasome in the detoxification response, we tested the effect of proteasome inhibitors such as MG132, clasto-lactacystin {beta}-lactone, and lactacystin on the induction of GST isozymes and found that these inhibitors selectively induced the class Pi GST isozyme (GST P1). Down-regulation of the proteasome by antisense oligonucleotides or RNA interference indeed resulted in significant up-regulation of GST P1, suggesting that a decline in the proteasome activity could be directly or indirectly linked to the induction of GST P1. From the functional analysis of various deletion constructs of the upstream regulatory region of the GST P1 promoter, GST P1 enhancer I was identified as the response element for proteasome inhibition. Overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 not only by the proteasome inhibitor, but also by phase 2-inducing isothiocyanate, suggesting that there may be a process of GST P1 induction distinct from other phase 2 gene induction mechanisms. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Xenobiotic metabolizing enzymes play a major role in regulating the toxic, oxidative damaging, mutagenic, and neoplastic effects of chemical carcinogens. Mounting evidence has indicated that the induction of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs)1 results in protection against toxicity and chemical carcinogenesis, especially during the initiation phase. The GSTs are a family of enzymes that catalyze the nucleophilic addition of the thiol of GSH to a variety of electrophiles (for a review, see Ref. 1). In addition, the GSTs bind with varying affinities to a variety of aromatic hydrophobic compounds. It is now generally accepted that the GSTs are encoded by at least five different gene families. Four of the gene families (classes Alpha, Mu, Pi, and Theta) encode the cytosolic GSTs, whereas the fifth encodes a microsomal form of the enzyme. It has been shown that the induction of GST is associated with the reduced incidence and multiplicity of tumors (2, 3). The induction of phase 2 enzymes such as GSTs is reported to be evoked by an extraordinary variety of chemical agents, including Michael reaction acceptors, diphenols, quinones, isothiocyanates, peroxides, vicinal dimercaptans, and others (4-6). With few exceptions, these agents are electrophiles, and accordingly, many of these inducers are substrates for the phase 2 detoxification enzymes.

The transcriptional activation of the phase 2 enzymes has been traced to a cis-acting transcriptional enhancer called an antioxidant response element (ARE) (7) or, alternatively, the electrophile response element (8). Nrf2, a member of the NF-E2 family of nuclear basic leucine zipper transcription factors, binds to the ARE and accelerates the transcription of cognate genes (9-11). Under basal conditions, Keap1 (Kelch-like ECH-associated protein-1), a recently identified protein associated with the actin cytoskeleton, binds very tightly to Nrf2, anchors this transcription factor in the cytoplasm, and targets it for ubiquitination and proteasome degradation, thereby repressing the ability of Nrf2 to induce phase 2 detoxification enzyme genes (12-17). Inducers disrupt the Keap1-Nrf2 complex by modifying two (Cys273 and Cys288) of the 25 cysteine residues of Keap1 (18), allowing Nrf2 to translocate to the nucleus, where, in heterodimeric combinations with other basic leucine zipper proteins, it binds to the ARE of the phase 2 genes and accelerates their transcription.

In this study, we investigated the molecular mechanism involved in the induction of phase 2 detoxification enzymes and found that the induction of the class Pi GST isozyme (GST P1) is, at least in part, attributable to the disruption of the proteasome-dependent regulatory mechanism of protein turnover. The proteasome is a large multisubunit protease complex that selectively degrades intracellular proteins (19-21). Most of the proteins removed by these proteases are tagged for destruction by ubiquitination. The proteasome plays a role in controlling cellular processes such as metabolism and the cell cycle through the signal-mediated proteolysis of key enzymes and regulatory proteins. It also operates in the stress response by removing abnormal proteins and in the immune response by generating antigenic peptides. Our findings therefore suggest that down-regulation of the proteasome may represent an important indirect action of anti-carcinogenic chemicals that can contribute to their protective effects against chronic diseases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—MG132 and lactacystin were obtained from Peptide Institute, Inc. (Osaka, Japan). clasto-Lactacystin {beta}-lactone was obtained from Wako Pure Chemical Industries, Ltd. (Osaka). Succinyl-LLVY-methylcoumarin amide (MCA) for the chymotrypsin activity and t-butoxycarbonyl-LSTR-MCA for the trypsin activity of the proteasome were obtained from Peptide Institute, Inc. Benzyloxycarbonyl-LLE-{beta}-nitroanilide, a substrate of the peptidylglutamyl-peptide hydrolase activity of the proteasome, was obtained from Sigma. Anti-GST A1, anti-GST A4, anti-GST A5, and anti-GST P1 polyclonal antibodies were kindly provided by Dr. Kimihiko Satoh (Hirosaki University). The anti-ubiquitin polyclonal antibody was obtained from Biomega Co. (Foster City, CA). The antisense oligonucleotide for the proteasome C2 subunit was obtained from Sigma: sense, 5'-CACCATGTTTCGAAA-3'; and antisense, 5'-TTTCGAAACATGGTG-3'. 6-Methylsulfinylhexyl isothiocyanate (6-HITC) was a kind gift of Dr. Yasujiro Morimitsu (Ochanomizu University). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka).

Cell Culture—RL34 cells were obtained from the Japanese Cancer Research Resources Bank. The cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 588 µg/ml L-glutamine, and 0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2.

Proteasome Activity—The peptidase activity of the proteasome was measured using three peptidase activities (chymotrypsin-like, trypsinlike, and peptidylglutamyl-peptide hydrolase) with fluorogenic peptides as substrates. Cells transfected with antisense oligodeoxynucleotides or small interfering RNA (siRNA) were washed twice with phosphate-buffered saline and homogenized in 0.8% digitonin (pH 7.8) containing 2 mM EDTA for 10 min at 37 °C. Afterward, the lysates were centrifuged at 5000 rpm for 5 min, and the supernatants were collected as whole cell extracts. The activities of the proteasome were measured as follows. Whole cell extracts were incubated with 200 µM fluorogenic peptide substrates for 30 min at 37 °C in 100 µl of 100 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol. After incubation, the reaction mixture was added to 2 ml of 100 mM Tris-HCl (pH 9.0) and 100 µl of 10% SDS, followed by measurement of the fluorescence of the cleavage products using a Hitachi Model F-2000 fluorescence spectrophotometer. The fluorescent cleavage products of succinyl-LLVY-MCA and t-butoxycarbonyl-LSTR-MCA were detected at an emission wavelength of 460 nm and an excitation wavelength of 380 nm, and the fluorescent group of benzyloxycarbonyl-LLE-{beta}-nitroanilide was detected at an emission wavelength of 410 nm and an excitation wavelength of 335 nm.

The proteasome activity was also evaluated using RL34 cells stably transfected with the Proteasome Sensor VectorTM (Clontech, Palo Alto, CA). The ZsProSensorTM protein accumulated through the inhibition of the proteasome (resulting in a strong green emission signal) was measured using a Coulter® Epics XLTM flow cytometer (Beckman Coulter, Inc.).

Western Blot Analysis—The homogenates prepared from the cells were treated with SDS sample buffer and immediately boiled for 5 min. The protein concentrations were determined using the BCA protein assay kit (Pierce). Twenty µg of the proteins were separated by SDS-PAGE in the presence of 2-mercaptoethanol and electrotransferred onto a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). To detect the immunoreactive proteins, we used horseradish peroxidase-conjugated anti-rabbit IgG and ECL blotting reagents (Amersham Biosciences).

Reverse Transcription (RT)-PCR—Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol and spectrophotometrically quantified. Total RNAs (5 µg) were reverse-transcribed into cDNA and used for RT-PCR analysis (Qiagen Inc., Hilden, Germany). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard. The PCR products were separated on a 1% agarose gel, and the positive signals were quantified by densitometry analysis after staining with ethidium bromide.

Luciferase Assay—We used a promoterless luciferase plasmid vector (pGVB; Nippon Gene Co., Ltd., Toyama, Japan) with the minimal promoter sequence of the rat GST P1 gene (-50 to +37 bp) containing a GC box and a TATA box (22). A 3.0-kb fragment between -2.5 kb and +59 bp of the gene for GST P1 was inserted into the SacI-HindIII site of the plasmid vector and designated -2.5GST. A series of 5'-deletion mutants were constructed from -2.5GST using the appropriate restriction enzymes. The following sequences were also utilized for the reporter transfection assay: GPE1, 5'-GTCAGTCACTATGATTCAGCAAC-3'; and two mutated forms of GPE1 (with the mutated positions underlined), 5'-GTCAGTCACTACGATTCAGCAAC-3' (mGPE1) and 5'-GTCAGTCGCTATGATTCAGCAAC-3' (mmGPE1). These were prepared as described previously (23). For the reporter transfection assay, RL34 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and seeded at 4 x 104 cells/well in 6-well dishes 2-18 h before transfection. The cells were transfected with plasmids using Lipofectamine 2000TM (Invitrogen) according to the manufacturer's instructions. Twenty-four h after transfection, the cells were treated with a proteasome inhibitor (0.5 µM). After 24 h, a luciferase assay was performed using the Dual-Luciferase assay system (Nippon Gene Co., Ltd.) following the supplier's protocol, followed by measurement using a mini Lumar LB9560 (Berthold Technologies GmbH, Bad Wildbad, Germany). Transfection efficiencies were routinely normalized by the activity of cotransfected Renilla luciferase (5 µg). Normally, three independent experiments were performed (each carried out in duplicate), and the means ± S.E. are presented.

Transient Transfection of Nrf2—Mouse full-length Nrf2 cDNA was subcloned into the pcDNA3 vector (Invitrogen). An expression vector for mutant Nrf2 lacking both the Neh4 and Neh5 domains (required for the transactivation activity of Nrf2) was created by site-directed mutagenesis. Transient transfections were performed using Lipofectamine 2000TM according to the manufacturer's protocols. Briefly, 1 µg of plasmid DNA and 2 µl of Lipofectamine 2000TM were each diluted with 250 µl of Opti-MEM I (Invitrogen). After 5 min at room temperature, they were combined and incubated for 20 min. Reaction mixtures were overlaid on the cell culture for 6 h. The medium was then changed to fresh medium containing 5% fetal bovine serum, maintained for another 24 h, and subjected to the experiment.

Stable Transfection with the Wild-type and Dominant-negative Forms of Keap1 in RL34 Cells—Mouse full-length Keap1 cDNA was subcloned into the pcDNA3 vector. Mutant keap1 genes containing alanine codons substituted for specific cysteine codons were constructed. In the experiments reported here, we focused our attention on two cysteine residues within the N-terminal region of Keap1 (Cys23 and Cys38), seven cysteine residues within the intervening region (Cys226, Cys241, Cys249, Cys257, Cys273, Cys288, and Cys297), one cysteine residue within the double glycine repeat (Cys319), and three cysteine residues within the C-terminal region (Cys613, Cys622, and Cys624). The four cysteine residues within the intervening region of Keap1 (Cys257, Cys273, Cys288, and Cys297) were selected on the basis of a recent report that these cysteine residues preferentially react with thiol-specific chemicals in vitro (24). Transient transfections were performed using Lipofectamine 2000TM according to the manufacturer's protocols. In these experiments, 1 x 106 RL34 cells were incubated with DNA transfection reagent mixture (2 µg of DNA/4 µl of Lipofectamine 2000TM) in 0.5 ml of serum-free Opti-MEM I at 37 °C. After 6 h of incubation, 1 ml of the complete medium was added, and the cells were cultured for 18 h. Thereafter, stable transfectants were isolated by culture in selection medium containing 700 µg/ml G418 for ~3 weeks. A single clone of the stably transfected cells was isolated and expanded. Several G418-resistant stable clones were maintained in regular growth medium containing 700 µg/ml G418.

RNA Interference (RNAi) Using siRNA—The following siRNAs were generated by Invitrogen: rat proteasome {beta}5 subunit nucleotides 480-504, 5'-GCACCAUGAUCUGUGGCUGGGAUAA-3'; rat proteasome Rpn2, 5'-AAUUUGGCCCAGUUAGUGGCUCUGG-3'; and rat Nrf2 nucleotides 465-489, 5'-AAUAUCCAGGGCAAGCGACUCAUGG-3'. As a control, we used StealthTM RNAi negative control duplexes (Invitrogen). RL34 cells were seeded at 80% density the day before transfection. Cells were transfected with Lipofectamine 2000TM; 2.5 µl of siRNA stock (20 µM) and 5 µl of Lipofectamine 2000TM were each diluted with 250 µl of Opti-MEM I. After 5 min at room temperature, they were combined and incubated for 20 min. The reaction mixtures were overlaid on the cell culture for 6 h. The medium was then changed to fresh medium containing 5% fetal bovine serum.



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  		FIG. 1.
Proteasome inhibitors selectively induce GST P1 expression in RL34 cells. A, MG132 (0.1 µM); B, clasto-lactacystin {beta}-lactone (LC {beta}; 1 µM); C, lactacystin (1 µM). The cells were treated with the inhibitors for different time intervals as indicated, and whole cell lysates (20 µg) were subjected to immunoblot analysis for the detection of each GST isozyme. In A-C, the left panels are representative immunoblots of three separate experiments, and the right panels represent averaged data quantified by densitometry of immunoblots expressed as -fold increase in GST isozymes; the level of each GST isozyme in cells at time 0 is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments. , GST P1; {circ}, GST A1; {blacktriangleup}, GST A4; {triangleup}, GST A5.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Selective Induction of GST P1 by Proteasome Inhibitors—Our results showed that phase 2 enzyme inducers, including a sulforaphane analog (6-HITC), gave rise to significant downregulation of proteasome activities and the accumulation of ubiquitinated proteins (Supplemental Fig. S1). This and the previous findings that the phase 2-inducible electrophilic aldehydes and ketones are inhibitors of the proteasome (25-28) led us to the assumption that proteasome down-regulation might be associated with the induction of phase 2 detoxification enzymes. Hence, to explore the role of the proteasome in the phase 2 response, we examined whether well established proteasome inhibitors such as MG132, clasto-lactacystin {beta}-lactone, and lactacystin could induce GST isozymes. As shown in Fig. 1, these inhibitors hardly induced the class Alpha GST isozymes such as GST A1, GST A4, and GST A5, whereas they significantly induced class Pi GST P1 (Fig. 1, A-C). In addition, RT-PCR analysis also demonstrated the selective up-regulation of GST P1 gene expression in cells treated with clasto-lactacystin {beta}-lactone (Fig. 2). These data suggest that proteasome inhibition may result in the selective induction of GST P1 gene expression.

Effect of Proteasome Down-regulation on GST P1 Induction—To establish whether proteasome inhibition is functionally associated with the induction of GST P1, we examined the effect of proteasome down-regulation on GST P1 expression using antisense oligonucleotides and RNAi. As shown in Fig. 3A, the proteasome C2 subunit antisense oligonucleotide treatment resulted in a modest decline in the chymotrypsin, trypsin, and peptidylglutamyl-peptide hydrolase activities of the proteasome. In addition, the antisense oligonucleotide-induced down-regulation of the proteasome was also confirmed by a fluorescent reporter assay for proteasome activity using the Proteasome Sensor VectorTM (Fig. 3B). We evaluated the functional impact of proteasome dysfunction on the accumulation of ubiquitinated proteins in response to antisense oligonucleotide treatment and observed that the steady-state level of ubiquitinated proteins was higher in the antisense oligonucleotide-treated cells than in the control cells (Fig. 3C). A significant increase in the levels of GST P1 was observed, accompanied by a significant decline in the proteasome activities and accumulation of ubiquitinated proteins (Fig. 3D). Thus, the level of proteasome inhibition observed in the cells seemed to be inversely correlated with the accumulation of ubiquitinated proteins and the induction of GST P1.

To further examine the effect of the reduced expression of a proteasome subunit in RL34 cells, we utilized RNAi to block the expression of individual subunits of the 26 S proteasome in RL34 cells. We selected two representative subunits of the 26 S proteasome as targets for RNAi: one 20 S proteasome subunit ({beta}5) and one 19 S proteasome subunit (Rpn2). As shown in Fig. 4(A and B), the siRNA of the proteasome {beta}5 subunit resulted in corresponding changes in the proteasome activities. The reduced expression of the proteasome subunit promoted increased expression of GST P1 (Fig. 4C). Similar results were also obtained with the siRNA of the proteasome subunit Rpn2 (Fig. 5). These results establish that a decline in the proteasome activity could be directly or indirectly linked to an increase in the levels of GST P1 mRNA.



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  		FIG. 2.
Effect of a proteasome inhibitor on the induction of phase 2 detoxification enzymes in RL34 cells. Left panel, induction of phase 2 detoxification enzymes by clasto-lactacystin {beta}-lactone. The cells were treated with 1 µM clasto-lactacystin {beta}-lactone for different time intervals as indicated. Semiquantitative RT-PCR was performed using total RNA isolated from RL34 cells treated with the proteasome inhibitor. NQO1, NAD(P)H:(quinone-acceptor) oxidoreductase-1. Right panel, quantitation of the GST mRNA levels after normalization for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The results are shown as the means ± S.D. for two independent experiments. , GST P1; {circ}, GST A1; {blacktriangleup}, GST A4; {triangleup}, GST A5.

 
Involvement of GST P1 Enhancer I (GPEI)—To determine whether proteasome dysfunction stimulates the promoter activity of the GST P1 gene, we examined the effect of clasto-lactacystin {beta}-lactone on the transient expression of the firefly luciferase reporter gene harboring the 5'-flanking region (-2.5 kb to +59 bp) of the GST P1 gene in RL34 cells (Fig. 6A). As shown in Fig. 6B, -2.5GST, containing the 5'-flanking region, showed low but detectable luciferase activity, and the proteasome inhibitor markedly stimulated it by ~6-fold, indicating that the region contains an element responsible for clasto-lactacystin {beta}-lactone. To identify the element responsible for the clasto-lactacystin {beta}-lactone-stimulated promoter activities, we utilized a series of deletion mutants and observed that deletion to -2.15 kb (-2.15GST) led to the complete loss of the basal and clasto-lactacystin {beta}-lactone responsiveness (Fig. 6B). These results indicate that the proteasome inhibitor response element of the GST P1 gene is located between -2.5 and -2.15 kb upstream from the transcription start site of the GST P1 gene. Deletion to -0.395 kb (-395GST) and -0.091 kb (-91GST) barely changed the luciferase activity of clasto-lactacystin {beta}-lactone.

In the restricted region between -2.5 and -2.15 kb, there is a strong enhancer element (GPEI) whose core sequence consists of a palindromic dyad of a 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-like sequence motif (29). Previous studies have indicated that the 5'-end nucleotides of the two inverted TRE-like sequences (5'-TCAGTCACTATGATTCA-3') in GPEI are important for their strong enhancer activity (29, 30). To examine whether this region (GPEI) has a responsive function for GST P1 expression by proteasome inhibitors, two point mutants, mGPE1 (5'-GTCAGTCACTACGATTCAGCAAC-3') and mmGPE1 (5'-GTCAGTCGCTATGATTCAGCAAC-3'), were prepared. As shown in Fig. 6C, clasto-lactacystin {beta}-lactone stimulated the luciferase activity by ~3-fold in cells transfected with GPEI, whereas mutation of either of the TRE-like elements completely abolished the basal and clasto-lactacystin {beta}-lactone-induced stimulation of the promoter activity of GPEI. These data establish that GPEI represents the response element for proteasome inhibition and that both TRE-like elements are essential for stimulation of promoter activity.

Effect of Nrf2 Overexpression on GST P1 Induction by a Proteasome Inhibitor—It has been suggested that the Nrf2-Keap1 pathway regulates a battery of detoxifying and antioxidant genes (12-17). We indeed observed that treatment with the proteasome inhibitors clasto-lactacystin {beta}-lactone and MG132 resulted in the accumulation of Nrf2 in RL34 cells (Fig. 7, A and B). To examine the role of Nrf2 in GST P1 induction stimulated by the proteasome inhibitors, we analyzed the effect of transient overexpression of Nrf2 on GST P1 induction by the proteasome inhibitors. As shown in Fig. 7C, overexpression of wild-type Nrf2 exerted significant up-regulation of the basal level of GST P1, whereas the GST P1-inducing ability of clasto-lactacystin {beta}-lactone was abrogated. We also observed that the GST P1 inducibility of the proteasome inhibitor was barely affected by the expression of dominant-negative Nrf2. In addition, to examine the effect of reduced expression of Nrf2 in RL34 cells, we utilized RNAi of Nrf2 and observed that the reduced expression of Nrf2 promoted increased expression of GST P1 (Fig. 7D).

Effect of Keap1 Overexpression on GST P1 Induction by a Proteasome Inhibitor—To further examine the role of the Nrf2-Keap1 pathway in GST P1 induction, we established Keap1-overexpressing derivatives of RL34 cells by stable transfection of wild-type and mutant Keap1, in which all of the cysteines except for those in the double glycine repeat were mutated to alanine. As this Keap1 mutant cannot mediate the proteasome degradation of Nrf2 (data not shown), its overexpression is expected to induce constitutive activation of Nrf2. In fact, overexpression of dominant-negative Keap1 significantly up-regulated the basal level of GST P1 (Fig. 8). Overexpression of wild-type Keap1 had no effect on both basal and inhibitor-induced GST P1 expression. Moreover, the GST P1-inducing ability of the proteasome inhibitors was completely abrogated by the overexpression of dominant-negative Keap1. These data suggest that the induction process of GST P1 by proteasome inhibitors may be, at least in part, different from that of other phase 2 enzymes.

Effect of Nrf2 and Keap1 Overexpression on the Induction of GST Isozymes by the Sulforaphane Analog 6-HITC—Finally, we examined the effect of Nrf2 and Keap1 overexpression on the induction of GST isozymes by phase 2-inducing electrophiles. In this study, we used 6-HITC, an analog of sulforaphane (4-methylsulfinylbutyl isothiocyanate) isolated from broccoli, as a representative electrophile (31). To examine the role of Nrf2 and Keap1 in GST P1 induction stimulated by 6-HITC, we first analyzed the effect of the transient overexpression of Nrf2 on GST P1 induction by this isothiocyanate. As shown in Fig. 9A, the transient overexpression of wild-type Nrf2 significantly increased the 6-HITC-stimulated induction of class Alpha GST isozymes (GST A1, GST A4, and GST A5), whereas dominant-negative Nrf2 abrogated both the basal and 6-HITC-induced expression of class Alpha GST isozymes. However, consistent with the results in Fig. 7, the basal and 6-HITC-induced expression of GST P1 was not influenced by the overexpression of wild-type or dominant-negative Nrf2. We then examined the effect of Keap1 overexpression on the induction of GST isozymes by 6-HITC. As shown in Fig. 9B, treatment of empty vector-transfected cells with 6-HITC led to a significant elevation of the levels of the class Alpha GST isozymes and the class Pi GST isozyme (GST P1) compared with the levels observed in 6-HITC-untreated cells. Overexpression of wild-type Keap1 markedly suppressed both the basal and 6-HITC-stimulated induction of the class Alpha GST isozymes; however, overexpression of wild-type Keap1 had little influence on the effect of 6-HITC on the induction of GST P1. Dominant-negative Keap1 overexpression resulted in a dramatic increase in the basal levels of the GST isozymes and in the disappearance of the effect of 6-HITC. Thus, the failure of Nrf2 and Keap1 to affect the induction of GST P1 was also confirmed in cells treated with the phase 2-inducing electrophile.



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  		FIG. 3.
Effect of the proteasome C2 subunit antisense oligonucleotide on the induction of GST P1. RL34 cells were transfected the sense (S) and antisense (AS) oligonucleotides for 6 h. A, effect of the proteasome C2 subunit antisense oligonucleotide on proteasome activity. The peptidase activity of the proteasome was measured using three peptidase activities (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase) with fluorogenic peptides as substrates and is expressed as a percent of the activity of control cells without transfection. White bars, 0.25 µM sense oligonucleotide; hatched bars, 0.125 µM antisense oligonucleotide; black bars, 0.25 µM antisense oligonucleotide. s, succinyl; boc, t-butoxycarbonyl; z, benzyloxycarbonyl; {beta}NA, {beta}-nitroanilide. B, effect of the proteasome C2 subunit antisense oligonucleotide on proteasome activity evaluated by a fluorescent reporter assay. The proteasome activity was evaluated using RL34 cells stably transfected with the Proteasome Sensor VectorTM. Left panel, inhibition of proteasome by an authentic proteasome inhibitor, clasto-lactacystin {beta}-lactone (LC {beta}; 1 µM); right panel, inhibition of the proteasome by the proteasome C2 subunit antisense oligonucleotide. C, immunoblot analysis of ubiquitinated proteins in cells subjected to the proteasome C2 subunit antisense oligonucleotide. D, immunoblot analysis of GST P1 in cells subjected to the proteasome C2 subunit antisense oligonucleotide. Upper panel, representative immunoblot analysis; lower panel, averaged data quantified by densitometry of immunoblots expressed as -fold increase in GST P1. The level of GST P1 in the sense oligonucleotide-transfected cells is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments. Whole cell lysates were subjected to immunoblot analysis for the detection of ubiquitinated proteins (C) and GST P1 (D).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There has been considerable interest in the properties of the class Pi GST isozyme (GST P1), particularly in the relationship to carcinogenesis and human cancer. GST P1 is almost undetectable in normal rat hepatocytes and is markedly overexpressed in hepatic foci arising spontaneously or in animals treated with carcinogens (32). A number of studies have indeed shown that GST P1 is the predominant GST isozyme found in human cancer tissue and that it sometimes serves as a putative tumor marker of both rodent and human disease (33-37). On the other hand, GST P1 is also known to be the enzyme with the highest activity in the detoxification of carcinogens, such as the ultimate carcinogenic metabolite of benzo[a]pyrene. Berhane et al. (38) have reported that GST P1 is more effective in the detoxification of electrophilic {alpha},{beta}-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism than other GSTs. Indeed, deletion of the GST P1 gene from mice has been reported to result in increased skin tumorigenesis (39). These findings suggest that GST P1 is an important determinant in cancer susceptibility and that its elevated synthesis is required to prevent toxic compounds from accumulating in the cells.

In this study, we have shown that the induction of GST P1 may be attributable to the disruption of the proteasome-dependent regulatory mechanism of protein turnover. This finding is consistent with our observations that phase 2 enzyme inducers, including a sulforaphane analog (6-HITC), give rise to significant down-regulation of proteasome activities and the accumulation of ubiquitinated proteins (Supplemental Fig. S1). In addition, it has also been reported that GST P1-inducible electrophilic aldehydes and ketones are potential inhibitors of the proteasome (25-28). In contrast to these observations, however, the treatment of cells with a proteasome inhibitor has been shown to prevent rapid degradation of Nrf2, leading to the enhanced expression of the downstream gene encoding {gamma}-glutamylcysteine ligase (40). Kwak et al. (41) have also demonstrated that an anti-carcinogenic chemical induces the expression of a broad range of proteasome subunits encompassing both the catalytic core (20 S proteasome) and the ATP-dependent regulatory core (19 S proteasome). We observed that the Nrf2 protein was significantly up-regulated by proteasome inhibitors (Fig. 7). Meanwhile, despite the significant up-regulation of Nrf2 by proteasome inhibitors, there were no differences in the mRNA levels of phase 2 enzymes, including GST A1, GST A4, and GST A5, whose constitutive expression is regulated by ARE-mediated transcription (Fig. 1). Moreover, overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 by proteasome inhibitors (Fig. 7). The failure of Nrf2 and Keap1 overexpression to affect the induction of GST P1 was also observed in RL34 cells treated with a sulforaphane analog, although the overexpression significantly modulated the induction of other GST isozymes by the isothiocyanate (Fig. 8). It can therefore be speculated that the mechanism of the up-regulation of GST P1 may not be identical to that of other phase 2 enzymes and antioxidant proteins whose induction is ascribed primarily to the activation of the Nrf2-Keap1 detoxification pathway. This hypothesis is supported by the previous observations that (i) there are no significant differences in N,N-diethylnitrosamine carcinogenicity between homozygous and heterozygous female Nrf2 knockout mice compared with wild-type females and (ii) distinct GST P1-positive single cell populations are induced in the livers of homozygous and heterozygous female Nrf2 knockout mice compared with wild-type mice (42).



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  		FIG. 4.
RNAi of the 20 S proteasome {beta}5 subunit induces GST P1. A, changes in proteasome activity in RL34 cells subjected to siRNA of the proteasome {beta}5 subunit. The peptidase activity of the proteasome was measured using three peptidase activities (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase) with fluorogenic peptides as substrates. s, succinyl; boc, t-butoxycarbonyl; z, benzyloxycarbonyl; {beta}NA, {beta}-nitroanilide. B, effect of the specific siRNA of the proteasome {beta}5 subunit on proteasome activity evaluated by a fluorescent reporter assay. The proteasome activity was evaluated using RL34 cells stably transfected with the Proteasome Sensor VectorTM. C, effects of specific siRNAs on the mRNA levels of the proteasome {beta}5 subunit and GST P1 in RL34 cells. Semiquantitative RT-PCR was performed using total RNA isolated from RL34 cells treated for 6 h with the indicated siRNAs as described under "Experimental Procedures" (upper panel). The results are representative of three separate experiments. The relative proteasome {beta}5 subunit and GST P1 mRNA levels were determined by quantitative analysis (lower panel). Averaged data quantified by densitometry of RT-PCR are expressed as -fold increase in the proteasome {beta}5 subunit and GST P1; the level of the proteasome {beta}5 subunit and GST P1 in cells at 48 h (Control) is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
The GST P1 gene structure has been intensively studied (1, 22, 29, 30, 43). It has been shown that the 5'-upstream region of the GST P1 gene contains a TRE and a related enhancer element termed GPEI with characteristics similar to those of ARE. GPEI contains a dyad of TRE-like elements (22, 29) identified as cis-elements required for the basal and inducible expression of GST P1 (29, 30, 44). Transgenic rat experiments have suggested that the high expression of GST P1 during hepatocarcinogenesis occurs due to a trans-mechanism mediated by common factors (45). It is known that the GST P1 gene has a negative regulatory element, a silencer that is located 400 bp upstream from the cap site (22). We have shown here that proteasome inhibitors stimulated the promoter activity of the 5'-flanking region of the GST P1 gene and then induced GST P1 mRNA and protein in RL34 cells (Fig. 6B). It also appeared that this stimulation required the specific region containing GPEI (Fig. 6C). These data therefore suggest that the molecular mechanism of GST P1 expression by proteasome inhibitors may be closely related to the fundamental process of early hepatocarcinogenesis in rats.



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  		FIG. 5.
RNAi of the 26 S proteasome subunit Rpn2 induces GST P1. A, changes in proteasome activity in RL34 cells subjected to siRNA of Rpn2. The peptidase activity of the proteasome was measured using three peptidase activities (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase) with fluorogenic peptides as substrates. s, succinyl; boc, t-butoxycarbonyl; z, benzyloxycarbonyl; {beta}NA, {beta}-nitroanilide. B, effect of the specific siRNA of Rpn2 on proteasome activity evaluated by a fluorescent reporter assay. The proteasome activity was evaluated using RL34 cells stably transfected with the Proteasome Sensor VectorTM. C, effects of specific siRNAs on the mRNA levels of Rpn2 and GST P1 in RL34 cells. Semiquantitative RT-PCR was performed using total RNA isolated from RL34 cells treated for 6 h with the indicated siRNAs as described under "Experimental Procedures" (upper panel). The results are representative of three separate experiments. The relative Rpn2 subunit and GST P1 mRNA levels were determined by quantitative analysis (lower panel). Averaged data quantified by densitometry of immunoblots are expressed as -fold increase in Rpn2 and GST P1; the level of Rpn2 and GST P1 in cells at 24 h (Control) is defined as 1.0. The results are shown as the mean ± S.D. for three separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 



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  		FIG. 6.
Stimulation of the promoter activity of GST P1 by a proteasome inhibitor. A, structures of the GST P1 promoter. Luc, luciferase. B, activation of GST P1 reporter genes by a proteasome inhibitor. The luciferase reporter gene harboring the 5'-flanking region (-2.5 kb to +59 bp) of the GST P1 gene was transfected into RL34 cells and treated with 0.5 µM clasto-lactacystin {beta}-lactone (LC{beta}). C, determination of the DNA element required for the induction GST P1 expression by the proteasome inhibitor. The luciferase construct (5 µg) was transfected into RL34 cells and treated with (black bars) or without (hatched bars) 1 µM clasto-lactacystin {beta}-lactone. The sequence of GPE1 is 5'-GTCAGTCACTATGATTCAGCAAC-3'; the sequences of the two mutated forms of GPE1, mGPE1 and mmGPE1, are 5'-GTCAGTCACTACGATTCAGCAAC-3' and 5'-GTCAGTCGCTATGATTCAGCAAC-3' (with the mutated positions underlined), respectively.

 
It has been shown that the enhancer (GPEI) of GST P1 expression is regulated by multiple factors, including activator protein-1, which is known to be a heterodimer composed of the products of the jun and fos oncogenes (46). Our previous study has indeed demonstrated that c-Jun is involved in the formation of nuclear protein-GPEI complexes induced by an electrophile (26). Consistent with these findings, we have shown that clasto-lactacystin {beta}-lactone dramatically stimulated the phosphorylation of c-Jun in a time-dependent manner (Supplemental Fig. S2). On the other hand, a number of transcription factors such as c-Jun are phosphorylated in vitro by distinct members of the MAPK families triggered by a large variety of extracellular stresses. MAPKs are important cell signaling components that convert various extracellular signals into intracellular responses through serial phosphorylation cascades. Now three distinct but parallel MAPK cascades have been identified in mammalian cells. It has been established that the activation of MAPKs culminates in the phosphorylation of downstream cytosolic and nuclear substrates, ultimately leading to changes in gene expression. We have observed that clasto-lactacystin {beta}-lactone potently induced the phosphorylation of p46 and p54 JNK and ERK (ERK1 and ERK2) and moderately induced the phosphorylation of p38 (Supplemental Fig. S2). Moreover, to establish the possible connection between MAPK activation and GST P1 induction by proteasome inhibitors, we examined the effect of pharmacological inhibitors of MAPK on the induction of GST P1 and observed that SB203580 and PD98059, specific inhibitors of p38 and MEK, respectively, were ineffective, whereas SP600125, a selective JNK inhibitor, significantly attenuated GST P1 expression induced by clasto-lactacystin {beta}-lactone (Supplemental Fig. S3). It is therefore likely that JNK is responsible for GST P1 gene expression induced by proteasome inhibitors.



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  		FIG. 7.
Effect of Nrf2 overexpression on GST P1 induction by a proteasome inhibitor. A, induction of Nrf2 by clasto-lactacystin {beta}-lactone (1 µM). B, induction of Nrf2 by MG132 (0.1 µM). In A and B, an equal amount of protein (20 µg) from whole cell extracts was analyzed by Western blotting with anti-Nrf2 antibody. C, effect of transient overexpression of the wild-type (WT) and dominant-negative (DN) forms of Nrf2 on the induction of GST P1 by clasto-lactacystin {beta}-lactone (LC{beta}). RL34 cells were transiently transfected with either the wild-type or dominant-negative Nrf2 construct as described under "Experimental Procedures" (upper panel). Empty vectors were used in control transfection experiments. Twenty-four h after transfection, RL34 cells were treated with or without 1 µM clasto-lactacystin {beta}-lactone for 24 h, and then whole cell lysates (20 µg) were subjected to immunoblot analysis for GST P1. The results are representative of three separate experiments. The relative GST P1 protein levels were determined by quantitative analysis (lower panel). Averaged data quantified by densitometry of immunoblots are expressed as -fold increase in GST P1; the levels of GST P1 in empty vector (pcDNA3)-treated cells (without clasto-lactacystin {beta}-lactone) is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments. D, effect of the specific siRNA of Nrf2 on the mRNA levels of GST P1 in RL34 cells. Semiquantitative RT-PCR was performed using total RNA isolated from RL34 cells treated for 6 h with the siRNA as described under "Experimental Procedures" (upper panel). The relative Nrf2 and GST P1 mRNA levels were determined by quantitative analysis (lower panel). Averaged data quantified by densitometry of RT-PCR are expressed as -fold increase in Nrf2 and GST P1; the level of Nrf2 in control cells (without clasto-lactacystin {beta}-lactone) is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 



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  		FIG. 8.
Effect of Keap1 overexpression on GST P1 induction by a proteasome inhibitor. A, effect of overexpression of the wild-type (WT) and dominant-negative (DN) forms of Keap1 on the induction of GST P1 by clasto-lactacystin {beta}-lactone (LC{beta}). Cells were stably transfected with the wild-type and dominant-negative forms of Keap1 as described under "Experimental Procedures" (upper panel). The results are representative of three separate experiments. The relative GST P1 mRNA levels were determined by quantitative analysis (lower panel). B, effect of overexpression of the wild-type and dominant-negative forms of Keap1 on the induction of GST P1 by MG132 (upper panel). The results are representative of three separate experiments. The relative GST P1 mRNA levels were determined by quantitative analysis (lower panel). In A and B, averaged data quantified by densitometry of immunoblots are expressed as -fold increase in GST P1; the level of GST P1 in empty vector (pcDNA3)-treated cells (without proteasome inhibitor) is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments.

 



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  		FIG. 9.
Effect of Nrf2 and Keap1 overexpression on the induction of GST isozymes by the sulforaphane analog 6-HITC. A, chemical structure of 6-HITC. B, effect of transient overexpression of the wild-type (WT) and dominant-negative (DN) forms of Nrf2 on the induction of GST isozymes by 6-HITC. RL34 cells were transiently transfected with either the wild-type or dominant-negative Nrf2 construct as described under "Experimental Procedures" (upper panels). Empty vectors were used in control transfection experiments. Twenty-four h after transfection, RL34 cells were treated with or without 10 µM 6-HITC for 24 h, and whole cell lysates (20 µg) were subjected to immunoblot analysis for GST isozymes. The results are representative of three separate experiments. The relative GST levels were determined by quantitative analysis (lower panels). C, effect of overexpression of the wild-type and dominant-negative forms of Keap1 on the induction of GST isozymes by 6-HITC. Cells were stably transfected with the wild-type and dominant-negative forms of Keap1 as described under "Experimental Procedures" (upper panels). The results are representative of three separate experiments. The relative GST levels were determined by quantitative analysis (lower panels). In B and C, averaged data quantified by densitometry of immunoblots are expressed as -fold increase in each GST isozyme; the level of GST isozymes in empty vector (pcDNA3)-treated cells (without 6-HITC) is defined as 1.0. The results are shown as the means ± S.D. for three separate experiments.

 
The association between GST P1 and JNK has also been demonstrated from recent results that GST P1 is linked to the regulation of stress-induced cell signaling by the inhibition of JNK through direct protein-protein interaction of GST P1 (47, 48). The inhibition of JNK by GST P1 has been demonstrated in vitro using embryonic fibroblasts from mice with a GST P1-null genotype; moreover, fibroblasts from null mice exhibit higher basal JNK activity compared with those from wild-type mice (47). The recent study by Elsby et al. (49) not only demonstrated the role of GST P1 as a direct inhibitor of JNK in vivo, but also suggested its role in regulating the constitutive expression of specific downstream molecular targets of the JNK signaling pathway.

In summary, we have found that proteasome inhibitors are specific inducers of GST P1 in rat liver epithelial RL34 cells. Down-regulation of the proteasome by antisense oligonucleotides or RNAi resulted in significant up-regulation of GST P1. From the functional analysis of various deletion mutant genes, GPEI was identified as the response element for proteasome inhibition. Strikingly, overexpression of the wild-type and dominant-negative forms of Keap1 and Nrf2 had little effect on the induction of GST P1, suggesting the involvement of an alternative signaling mechanism, such as the JNK/c-Jun pathway, in GST P1 gene expression induced by proteasome inhibitors. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.


   FOOTNOTES
 
* This work was supported in part by a research grant from the Ministry of Education, Culture, Sports, Science, and Technology and by the Center of Excellence Program in the 21st Century (Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1-S3. Back

§ Recipient of a research fellowship from the Japan Society for the Promotion of Science. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Lab. of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Tel.: 81-52-789-4127; Fax: 81-52-789-5741; E-mail: uchidak{at}agr.nagoya-u.ac.jp.

1 The abbreviations used are: GSTs, glutathione S-transferases; ARE, antioxidant response element; MCA, methylcoumarin amide; 6-HITC, 6-methylsulfinylhexyl isothiocyanate; siRNA, small interfering RNA; RT, reverse transcription; RNAi, RNA interference; GPEI, glutathione S-transferase P1 enhancer I; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Toshihiko Osawa (Nagoya University) for fruitful discussions. We also thank Drs. Kimihiko Satoh and Yasujiro Morimitsu for providing antibodies against GSTs and 6-HITC, respectively.



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 RESULTS
 DISCUSSION
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D. L. Terriff, C. L. Chik, D. M. Price, and A. K. Ho
Proteasomal Proteolysis in the Adrenergic Induction of Arylalkylamine-N-Acetyltransferase in Rat Pinealocytes
Endocrinology, November 1, 2005; 146(11): 4795 - 4803.
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