From the
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, United Kingdom, and the
Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom
Received for publication, February 4, 2003 , and in revised form, March 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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GSTP has recently been linked to the regulation of stress-induced cell signaling by inhibition of c-Jun N-terminal kinase (JNK) through direct protein-protein interaction of the GSTP1 monomer (7, 8). Inhibition of JNK by GSTP has been demonstrated in vitro using embryonic fibroblasts from mice with a GSTP-null (GstP1/P2(/)) genotype; moreover fibroblasts from null mice exhibited a higher basal JNK activity compared with those from wild type mice (7). The association between GSTP and JNK has also been demonstrated from recent studies that utilized apoptosis in cell lines as a measure of JNK activation. Ishisaki et al. (9) determined that the protection of dopaminergic neurons against dopamine-induced apoptosis was mediated by an increase in the expression of GSTP and the subsequent suppression of JNK activity. Conversely apoptosis was shown to increase in a human leukemia Jurkat cell line following etoposide treatment, which was attributed to the dimerization of GSTP1-1 resulting in the release of JNK from the GSTP·JNK complex and thus increased JNK activity (10). A second GST isoform, GST Mu 1-1, has also been discovered to regulate cell signaling by inhibition of apoptosis signal-regulating kinase 1 (11).
JNK is a mitogen-activated protein kinase that can activate a variety of signal cascades through its phosphorylation of transcription factors such as c-Jun and activating transcription factor 2 (12, 13). c-Jun, via homo- or heterodimerization (e.g. with Nrf2 (nuclear factor erythroid 2 p45-related factor 2) as demonstrated in transfected HepG2 cells (14, 15)), can control both the constitutive and inducible expression of phase II detoxication enzymes and antioxidant proteins through the activation of activator protein-1 (AP-1) binding sites or antioxidant-responsive elements (AREs) in the promoters of such genes (1419). In addition, the Nrf2-dependent induction of ARE-mediated gene expression by specific mitogen-activated protein kinases and apoptosis signal-regulating kinase 1 in HepG2 cells is augmented by the co-expression of JNK (2022). Examples of target genes include heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, UDP-glucuronosyltransferase (UGT), and the GSTs themselves (17, 2325).
We have previously utilized GSTP-null mice to investigate the functional (enzymatic) role of GSTP in chemical-induced toxicity, and it was established that they exhibited increased susceptibility to chemical carcinogen-induced skin tumorigenesis (26) but paradoxically showed increased resistance to acetaminophen hepatotoxicity (27). It is conceivable that the observed differential effect on chemical-induced toxicity might be related to the non-enzymatic role of GSTP as a regulator of JNK. Hence the aim of this study was to determine whether deletion of GstP1/P2 resulted in a phenotype with increased constitutive JNK signaling to establish whether GSTP regulates JNK activity in vivo. This was assessed by the measurement of basal JNK activity and AP-1 DNA binding activity both in the liver and lung of GstP1/P2(+/+) and GstP1/P2(/) mice. In addition, we have assessed the biological significance of such a phenomenon with respect to the expression of genes relevant to cell defense.
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EXPERIMENTAL PROCEDURES |
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AnimalsAll experiments were undertaken in accordance with criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the Animal Ethics Committee of the University of Liverpool. Transgenic mice in which both the P1 and P2 genes were deleted (GstP1/P2(/)) and wild type (GstP1/P2(+/+)) mouse lines, on a 129xMF1 background, were generated and maintained as reported previously (26).
Determination of JNK ActivityJNK activity was determined using a stress-activated protein kinase/JNK assay kit from New England Biolabs according to the manufacturer's instructions. Briefly, the kinase reaction was performed in the presence of excess ATP and was optimized for both time (30 min) and protein concentration (1 mg of liver extract/0.6 mg of lung extract). Reactions without tissue extract were used as negative controls. Samples were electrophoresed on 12% polyacrylamide gels, and c-Jun phosphorylation was selectively measured using a phospho-c-Jun antibody, which specifically measures JNK-catalyzed phosphorylation of c-Jun at Ser-63. Phospho-c-Jun band volumes were quantified by band densitometry (arbitrary units) using UVISoftTM software (UVITech UK) and compared with that of the c-Jun protein band, which was visualized by staining the nitrocellulose membrane with Ponceau Red.
Electrophoretic Mobility Shift AnalysisAP-1 DNA binding activity was determined by electrophoretic mobility shift analysis using a radiolabeled AP-1 consensus oligonucleotide. The double-stranded oligonucleotide containing the consensus AP-1 binding site (underlined) consisted of the following sequence: 5'-GCGTTGATGAGTCAGCCGGAA-3'. The double-stranded oligonucleotide containing a mutant AP-1 binding site (underlined) consisted of the following sequence: 5'-CGCTTGAGCTGTCAGCCGGAA-3'.
Mouse hepatic or lung nuclear protein extractions were carried out as described previously (28). Nuclear extracts (10 µg) from wild type or null mice (n = 4) were incubated with 35 fmol of [-32P]ATP-labeled oligonucleotides for 20 min at room temperature in a final reaction volume of 10 µl. Binding buffer contained 4% Ficoll, 20 mM HEPES (pH 7.5), 35 mM NaCl, 60 mM KCl, 0.01% Nonidet P-40, 2 mM dithiothreitol, and 0.5 µg of poly(dI-dC). For antibody supershift experiments, nuclear extracts (pooled samples from null mice) were incubated with 1 µlofthe corresponding antibody solution. DNA·protein complexes were separated by electrophoresis on 5% polyacrylamide, 0.5x Tris borate/EDTA gels, dried, and analyzed by autoradiography at 80 °C with Kodak Biomax film (Eastman Kodak Co.). AP-1 band volumes were quantified as described above.
Determination of Nrf2 Nuclear TranslocationHepatic Nrf2 nuclear translocation was determined by Western immunoblot analysis. Briefly, nuclear extracts (25 µg) were separated by electrophoresis on 7.5% SDS-polyacrylamide gels (Bio-Rad) and subsequently transferred to nitrocellulose membranes (Sigma). After incubation in blocking buffer (10% fat-free milk in Tris-buffered saline (pH 7.6) containing 0.15% Tween 20) for 1 h, the membrane was incubated with anti-Nrf2 antibody (1:2000 in Tris-buffered saline/Tween 20 containing 2% milk) for 1 h. Following multiple washes with Tris-buffered saline/Tween 20, the secondary antibody was added (peroxidase-conjugated goat anti-rabbit IgG, 1:2000 in Tris-buffered saline/Tween 20 containing 2% milk). Visualization of the protein-antibody conjugate was performed using chemiluminescence (Lightning Western blotting reagent, PerkinElmer Life Sciences).
Northern Blot AnalysisThe mRNA levels of genes relevant to cell defense were determined by Northern blot analysis. Total RNA was isolated from livers of GstP1/P2(+/+) or GstP1/P2(/) mice. Liver tissue (100 mg) was homogenized in TRI reagent (Sigma), and total cellular RNA was isolated according to the manufacturer's instructions. The concentration, purity, and integrity of the RNA was determined by spectrophotometry and agarose gel electrophoresis. RNA samples (30 µg) were subjected to electrophoresis in MOPS/EDTA buffer and transferred to nylon membranes (Amersham Biosciences). The membranes were prehybridized in ExpressHyb hybridization buffer (Clontech) and then hybridized in the same buffer for 4 h at 68 °C to a 32P-labeled cDNA probe directed against each gene. The probe was labeled using [-32P]dCTP by random primed synthesis. Following high stringency washes, the blots were subjected to autoradiography using BioMax film (Kodak) and quantified. The density of the ribosomal 18 S RNA band was used to normalize all other RNA values. cDNA probes were obtained for each gene by reverse transcription-PCR using mouse liver RNA as the template.
Preparation of MicrosomesLivers were removed from eight male GstP1/P2(+/+) or GstP1/P2(/) mice (3040 g) immediately after they were killed by cervical dislocation. Livers were pooled and homogenized in 2 volumes of ice-cold 67 mM potassium phosphate buffer (pH 7.5) containing 0.15 M potassium chloride. Microsomal fractions were prepared by differential centrifugation according to the method of Gill et al. (29). Microsomal protein concentrations were determined by the method of Lowry et al. (30).
High Performance Liquid ChromatographyHPLC was performed with an Ultracarb 5-µm C-8 column (25 x 0.46 cm, Phenomenex, Macclesfield, Cheshire, UK) connected to a Dionex ASI-100 automated sample injector (Dionex Ltd., Macclesfield, Cheshire, UK), a Dionex P580 pump, and a Dionex UVD170S UV detector. Data was processed by Chromeleon software (Dionex Ltd.). Metabolites were identified as chromatographic peaks of UV absorbance that were absent from control incubations (minus substrate or cofactor).
Determination of UGT ActivityUGT assays were carried out as described previously (31). Briefly, incubations contained 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 0.3 mg/ml Brij 58, GstP1/P2(+/+) or GstP1/P2(/) hepatic microsomes, and 3 mM UDP-glucuronic acid. UGT activity was determined using the probe substrates acetaminophen (APAP) (020 mM, 40-min incubation, 1.0 mg of protein), p-nitrophenol (010 mM, 10-min incubation, 0.2 mg of protein), and 17-estradiol (0500 µM, 15-min incubation, 0.1 mg of protein) and quantified by HPLC.
Aliquots (2050 µl) were eluted with acetonitrile (5 to 15 to 35%, 0 to 8 to 15 min for APAP; 30 to 50%, 0 to 10 min for p-nitrophenol; 30 to 30 to 70%, 0 to 5 to 15 min for 17-estradiol) in acetic acid (1%) at 1 ml/min. Peak area measurements of absorbance (280 or 254 nm for APAP) were used to quantify the respective glucuronides formed and were compared with authentic glucuronide standards. Incubations were performed on at least four separate occasions in duplicate. Kinetic parameters were calculated using GraFit software (Sigma).
Determination of Urinary and Hepatic APAP Glucuronide Levels following APAP AdministrationMale GstP1/P2(+/+) and GstP1/P2(/) mice (3040 g) (n ≥ 4) were anesthetized with sodium pentobarbitone (60 mg/kg, intraperitoneal), and penises were ligated with cotton to prevent loss of urine from the bladder. APAP (150 mg/kg) was administered by a single intraperitoneal injection in saline. Animals were killed by cervical dislocation at 0.5, 1, or 5 h after dosing, and urine was collected from the bladder by aspiration with a syringe. Livers were removed, homogenized in 67 mM potassium phosphate buffer (pH 7.5), and extracted with ice-cold acetonitrile (2 x 6 ml). Acetaminophen glucuronide (APAPG) levels in both urine and liver extracts were quantified by HPLC as described above. Glucuronide conjugation was confirmed by hydrolysis with H-2 -glucuronidase-arylsulfohydrolase at 37 °C for 3 h.
Effect of APAP Administration on Hepatic JNK Activity and HO-1 mRNA LevelsMale GstP1/P2(+/+) and GstP1/P2(/) mice (3040 g) (n = 34) were administered either saline (vehicle) or APAP (300 mg/kg) by a single intraperitoneal injection. Animals were killed by cervical dislocation 1 h (for JNK activity) or 5 h (for HO-1 mRNA levels) after dosing, and livers were removed and snap frozen in liquid nitrogen and stored at 80 °C. Determination of JNK activity or Northern blot analysis was carried out as described above.
Statistical AnalysisData are expressed as mean ± S.D. Student's unpaired t test and the Mann-Whitney test were used where parametric and nonparametric analyses were indicated, respectively. Statistical significance was taken at p values less than 0.05.
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RESULTS |
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Electrophoretic Mobility Shift Analysis of AP-1 DNA Binding Activity
To determine whether increased JNK signaling occurred as a consequence of the observed increase in JNK activity in the null mice, we measured AP-1 DNA binding by electrophoretic mobility shift analysis for liver and lung extracts. There was a significant 8-fold increase (p < 0.05) in hepatic AP-1 DNA binding activity (assessed by quantification of band volume) in GstP1/P2(/) compared with GstP1/P2(+/+) mice (Fig. 2). There was no statistically significant difference in AP-1 DNA binding activity between null and wild type mouse lung. The absence of a band following incubation with the mutant oligonucleotide or its supershift by the addition of antibodies against c-Fos, c-Jun, and phospho-c-Jun confirmed the band as an AP-1 complex.
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Nrf2 Nuclear Translocation
Western blot analysis revealed no difference in the basal nuclear translocation of Nrf2, assessed by band volume (arbitrary units), in the livers of GstP1/P2(+/+) (14,740 ± 3479, n = 12) or GstP1/P2(/) (15,543 ± 5520, n = 12) mice (data not shown).
Hepatic mRNA Levels of Potential Target Genes
To define the molecular consequences of enhanced JNK activity in the null animals, the mRNA levels of various genes, including certain phase II detoxication enzymes and antioxidant proteins, were determined by Northern blotting. There was a significant (p < 0.01) increase (70%) in the constitutive expression of HO-1 in liver from GstP1/P2(/) compared with GstP1/P2(+/+) mice (Fig. 3). Additionally the constitutive expression of Ugt1a6 mRNA was increased 42% (p < 0.05) in null animals compared with wild type (Fig. 3). There were no significant differences in the basal expression of manganese superoxide dismutase, microsomal epoxide hydrolase, or NAD-(P)H:quinone oxidoreductase 1 (Fig. 3), or Gsta1, cytochrome P450 1a1 (Cyp1a1), Cyp1a2, Ugt1a1, Ugt1a9, or Ugt2b5 between GstP1/P2(+/+) and GstP1/P2(/) mice (Fig. 4). The mRNA levels for Nrf2 were identical between phenotypes (data not shown).
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UGT Activities
To establish that hepatic UGT mRNA levels correlated with enzyme activity, the glucuronidation of selective probe substrates was determined.
AcetaminophenAPAP glucuronidation by GstP1/P2(+/+) or GstP1/P2(/) hepatic microsomes conformed to Michaelis-Menten kinetics (Fig. 5A); the mean apparent Km was 1.19 ± 0.1 and 1.06 ± 0.4 mM for GstP1/P2(+/+) and GstP1/P2(/) hepatic microsomes, respectively. There was a significant 2-fold increase (p < 0.05) for the Vmax of APAP glucuronidation by GstP1/P2(/) compared with GstP1/P2(+/+) hepatic microsomes (Table I). Under the HPLC conditions used, APAPG and APAP had retention times of 7.6 and 12.0 min, respectively.
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p-Nitrophenolp-Nitrophenol glucuronidation conformed to Michaelis-Menten kinetics; the mean apparent Km was 0.98 ± 0.42 and 1.29 ± 0.18 mM for GstP1/P2(+/+) and GstP1/P2(/) hepatic microsomes, respectively. Vmax was significantly increased 1.5-fold (p < 0.05) by GstP1/P2(/) hepatic microsomes (Table I). Under the HPLC conditions used 4-nitrophenyl glucuronide and p-nitrophenol had retention times of 3.6 and 10.9 min, respectively.
Estradiol17-Estradiol glucuronidation conformed to Michaelis-Menten kinetics. The mean apparent Km for C-3 glucuronidation was 85.68 ± 14.4 and 100.50 ± 24.4 µM and for C-17 glucuronidation (mean ± S.E.) was 19.16 ± 14.0 and 13.83 ± 8.6 µM for GstP1/P2(+/+) and GstP1/P2(/) hepatic microsomes, respectively. There was no difference in Vmax for 17
-estradiol glucuronidation at either C-3 or C-17 by GstP1/P2(+/+) or GstP1/P2(/) hepatic microsomes (Table I). 17
-estradiol-3-glucuronide, 17
-estradiol-17-glucuronide, and 17
-estradiol had retention times of 9.5, 10.8, and 16.8 min, respectively, by HPLC.
Urinary and Hepatic APAPG Levels following APAP Administration
There was a greater than 2-fold increase (p < 0.05) in APAPG levels (percentage of dose) in the urine of null mice, compared with wild type, following APAP administration (0.5 h) (Fig. 5B). After 5 h, APAPG levels were equal between phenotypes. APAPG levels (percentage of dose) in liver extracts were 1.53 ± 0.4% and 2.30 ± 1.0% after 0.5 h and 1.9 ± 0.9% and 3.2 ± 1.5% after 1 h for GstP1/P2(+/+) and GstP1/P2(/) mice, respectively.
Hepatic JNK Activity following APAP Administration
Treatment of GstP1/P2(+/+) mice with APAP (300 mg/kg, intraperitoneal, 1 h) resulted in a substantial increase (10-fold) in hepatic JNK activity compared with vehicle controls (Fig. 6A). Hepatic JNK activity between vehicle control and APAP-treated GstP1/P2(/) mice was identical, indicating an absence of induction. Additionally the higher JNK activity in APAP-treated wild type liver was similar in magnitude to the levels of activity observed basally in null liver (Fig. 6A).
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Hepatic HO-1 mRNA Levels following APAP Administration
Hepatic HO-1 mRNA increased to the same level in GstP1/P2(+/+) and GstP1/P2(/) mice following APAP administration (Fig. 6B). However, when compared with their respective vehicle controls, this represented a 10-fold (p < 0.05) and 2.6-fold (p < 0.05) induction of HO-1 in GstP1/P2(+/+) and GstP1/P2(/) mice, respectively. The difference in induction can be attributed to the significantly higher constitutive HO-1 mRNA levels in the livers of GstP1/P2(/) compared with GstP1/P2(+/+) mice (Figs. 3 and 6B).
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DISCUSSION |
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In normal male mouse liver GSTP content is high (500 µg/g) (32), and consequently, due to the fact that it is in excess of the Kd for the GSTP·JNK association (8), one might expect JNK to be totally inactive in vivo. However, the constitutive JNK activity in GstP1/P2(+/+) mice may result from the fact that under basal conditions cellular GSTP content consists of both monomers and homodimers of which only the former have been shown to inhibit JNK (7).
The increased JNK activity in the liver and lung of GSTP-null mice establishes the role of GSTP as a direct inhibitory regulator of JNK in vivo and is in agreement with previous in vitro studies using cultured GstP1/P2(/) mouse embryo fibroblasts (7). Consequently we have investigated a number of potential downstream molecular targets of JNK that are important for cell defense to establish whether, in the absence of GSTP, the increased constitutive JNK activity leads to enhanced JNK signaling and ultimately altered gene expression. One such downstream target is the AP-1 transcription factor complex, which is composed of either homo- or heterodimers of c-Jun, the major substrate for phosphorylation by JNK (12). Confirmation of increased JNK signaling in the null mice was established by the observed increase in AP-1 DNA binding activity and the subsequent increase in the mRNA of a murine AP-1 target gene, the antioxidant protein HO-1 (33, 34), in liver, the tissue that exhibited by far the greatest increase in constitutive JNK activity (3-fold). Previous studies (16, 35) have demonstrated that chemicals that initiate oxidative stress can induce HO-1 gene expression through activation of JNK.
In addition to HO-1, the enhanced hepatic JNK signaling in GstP1/P2(/) mice also resulted in the altered gene expression of the phase II detoxification enzyme UDP-glucuronosyltransferase 1A6. The promoter of murine Ugt1a6 is not fully characterized, and although an AP-1 binding site has not been identified thus far, sequence analysis of other UGT promoters has revealed potential binding sites for AP-1 (36). It is known that the constitutive and inducer-specific expression of murine Ugt1a6 is regulated by a xenobiotic response element and an ARE (37). To eliminate the possibility that the increased Ugt1a6 mRNA levels were a result of xenobiotic response element-mediated transcription (as a consequence of an increase in the levels of an endogenous ligand, normally detoxified by GSTP, for the aromatic hydrocarbon receptor) the expression of specific aromatic hydrocarbon genes was investigated. The absence of any differences in Cyp1a1, Cyp1a2, or Gsta1 mRNA levels between phenotypes indicates that the higher expression of Ugt1a6 mRNA was independent of xenobiotic response element-mediated transcription.
Mitogen-activated protein kinase pathways have been shown to induce ARE-mediated gene transcription (20) possibly through phosphorylation of Nrf2, which activates the ARE (38, 39). The promoters of HO-1 (40) and murine Ugt1a6 (17, 24) contain AREs that can be transcriptionally activated by Nrf2. Therefore, we investigated the expression of other Nrf2-driven target genes to determine whether the increased JNK signaling in GstP1/P2(/) mice resulted in a generalized enhancement of ARE-mediated gene transcription. However, there were no differences in the mRNA levels of enzymes whose constitutive expressions are regulated by ARE-mediated transcription, such as microsomal epoxide hydrolase (25), GSTA1 (25, 38), or UGT2B5 (homologous to rat UGT2B1) (40). The pattern of constitutive ARE gene expression, coupled with the identical hepatic Nrf2 nuclear protein levels between wild type and null mice, confirm the absence of any additional Nrf2 nuclear translocation in the null animals. These findings not only suggest that, in this context, JNK does not induce ARE-mediated gene transcription by phosphorylation of Nrf2 but that the basal level of cellular oxidative stress has not been increased (a condition that could give rise to the induction of Nrf2/ARE-responsive genes and of manganese superoxide dismutase (37)) in GstP1/P2(/) mice as a consequence of the deletion of a major antioxidant protein. It can, therefore, be concluded that the up-regulation of both HO-1 and Ugt1a6 in GstP1/P2(/) mice is unlikely to be the result of ARE activation due to enhanced Nrf2 nuclear translocation.
Upon its phosphorylation by JNK, c-Jun can heterodimerize with Nrf2 and thus initiate the transcription of specific genes, such as NAD(P)H:quinone oxidoreductase 1 and -glutamylcysteine synthetase, through the ARE (14, 15). Northern blot analysis suggested a non-statistically significant increase in NAD(P)H:quinone oxidoreductase 1 mRNA in the null livers, but it was found previously that there was no difference in protein levels of
-glutamylcysteine synthetase between the phenotypes (26). The impact of a particular complex on gene expression and function is variable (14, 15, 20, 21), and it is therefore possible that Nrf2·c-Jun heterodimers may be responsible for the modest up-regulation of Ugt1a6 observed in the null mice.
We have thus established that there is an increased constitutive expression of hepatic Ugt1a6 in GstP1/P2(/) mice in response to enhanced JNK cell signaling by a mechanism that may involve the transcription factor complexes AP-1 or Nrf2·c-Jun. Furthermore use of the selective UGT probe substrates p-nitrophenol (UGT1A6) and 17-estradiol (UGT1A1) (41) confirmed that the increase in mRNA correlated to an increased enzyme activity. This was also demonstrated by the 2-fold increase in the glucuronidation of another UGT1A6 substrate, acetaminophen, both in vitro and in vivo. In the previous study, the difference in the recovery of APAPG in the urine of wild type and null mice 3 h following the administration of the same non-hepatotoxic dose of APAP, although not statistically significant, supports the finding of an increased rate of APAP glucuronidation in the GstP1/P2-deficient mice (27). Recently it has been shown that p53-null mice exhibit increased constitutive UGT activity toward p-nitrophenol (UGT1A6) (42). Interestingly other studies utilizing mouse fibroblasts or human thyroid cells have determined that in the absence of p53 (p53/) there is higher basal JNK activity (43, 44).
GstP1/P2-null mice were established previously to be more susceptible to 7,12-dimethylbenzanthracene-induced skin tumorigenesis compared with their wild type counterparts (26). The absence of any changes in the expression of microsomal epoxide hydrolase and Cyp1a1 (enzymes that contribute to the bioactivation of 7,12-dimethylbenzanthracene to the ultimate carcinogenic metabolite (45, 46)), despite enhanced JNK signaling, is consistent with the previous conclusion that the increased susceptibility of GstP1/P2(/) mice to 7,12-dimethylbenzanthracene is due to the absence of functional GSTP leading to an inability to detoxify via GSH conjugation.
In contrast to 7,12-dimethylbenzanthracene, we have shown previously that the null mice exhibited increased resistance to acetaminophen hepatotoxicity, and, as induction of the AP-1 pathway by APAP is believed to be important in protecting against the cytotoxicity of this compound (47), we hypothesized that GSTP might potentiate hepatotoxicity through its inhibition of JNK (27). However, the induction of hepatic JNK activity and of HO-1 mRNA in GstP1/P2(+/+) mice following APAP administration suggests that the presence of GSTP does not affect the APAP-mediated induction of the AP-1 pathway. Furthermore the JNK activity achieved in the wild type mice after APAP is identical to the activity observed constitutively in the null mice due to the absence of GSTP. This suggests that the induction of JNK activity following APAP administration is a consequence of the absence of GSTP inhibition, which may be attributed to the dimerization of GSTP in order for it to function as a transferase enzyme for the conjugation of N-acetyl-p-benzoquinoneimine (the reactive metabolite of APAP) with GSH. Dimerization of GSTP results in the liberation of JNK from the GSTP·JNK complex (8) as has been demonstrated previously with etoposide (10). There was no induction of hepatic JNK activity in GstP1/P2(/) mice after APAP, which presumably reflects the fact that in the absence of GSTP the enzyme is constitutively fully active. Nevertheless HO-1 mRNA was induced after APAP treatment, compared with the basal level in null animals, which is likely to be a consequence of further AP-1 activity due to the increased expression of c-Fos and c-Jun (47) and/or the involvement of the signaling pathways of other transcription factors. It is clear from these studies of JNK activity that deletion of GSTP does not affect how the cell responds to the chemical stress induced by APAP; rather it primes the cell, through increased JNK signaling, to be better equipped to deal with such a chemical insult. Consequently the small changes in the constitutive expression of both hepatic HO-1 and UGT1A6 (genes that are known to protect against APAP hepatotoxicity (17, 24, 4852)) will contribute to the protection observed in GstP1/P2(/) mice against acetaminophen hepatotoxicity. However, these changes cannot fully explain the phenotypic differences in hepatic GSH levels seen previously (27).
The data presented in this study demonstrate not only the role of GSTP as a direct endogenous inhibitory regulator of JNK in vivo but also its role in regulating the constitutive expression of specific phase II detoxication enzymes and antioxidant proteins that are downstream molecular targets of the JNK signaling pathway (Fig. 7).
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, University of Liverpool, The Sherrington Bldgs., Ashton St., Liverpool L69 3GE, UK. Tel.: 44-151-794-5559; Fax: 44-151-794-5540; E-mail: bkpark{at}liverpool.ac.uk.
1 The abbreviations used are: GST, glutathione S-transferase; GSTP, GST Pi; JNK, c-Jun N-terminal kinase; Nrf2, nuclear factor erythroid 2 p45-related factor 2; AP-1, activator protein-1; ARE, antioxidant-responsive element; HO-1, heme oxygenase-1; UGT, UDP-glucuronosyltransferase; APAP, acetaminophen; APAPG, acetaminophen glucuronide; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropane sulfonic acid.
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REFERENCES |
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