ToxSci Advance Access originally published online on November 22, 2006
Toxicological Sciences 2007 96(2):206-213; doi:10.1093/toxsci/kfl175
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PI3K, RSK, and mTOR Signal Networks for the GST Gene Regulation
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, South Korea
1 To whom correspondence should be addressed. Fax: +822 (872)-1795. E-mail: sgk{at}snu.ac.kr.
Received September 21, 2006; accepted November 20, 2006
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ABSTRACT |
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 TOP ABSTRACT GST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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The induction of glutathione S-transferases (GST) represents not only cell detoxification and survival but also cancer prevention. In response to various extracellular stimuli, expression of the gene has been shown to be regulated coordinately by activating the transcription factors in a transcriptional or posttranscriptional manner. Cytoprotective agents induce GST and concomitantly activate the PI3K-Akt/ERK-RSK1-mTOR pathways that activate the transcription factors favoring cell viability. The mechanistic basis and cell signaling for the induction of GST induction by prooxidants and toxicants may be different from that by cytoprotective agents. This paper summarizes the molecular mechanisms of the transcriptional induction of the GST gene orchestrated by a series of transcription factors that recruit coactivators or corepressors.
Key Words: CCAAT-enhancer binding protein; glutathione S-transferases; NF-E2-related factor2; hepatocyte nuclear factors; mammalian target of rapamycin; peroxisome proliferator-activated receptors; phosphatidylinositol 3-kinase.
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GST EXPRESSION AS A RHEOSTAT OF CELL VIABILITY AND DEATH |
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TOPABSTRACTGST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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Phase II detoxifying enzymes catalyze the conjugating reactions between xenobiotics and endogenous compounds. Among the phase II enzymes, the glutathione S-transferases (GSTs) are responsible for the cellular metabolism as well as for the detoxification of several electrophilic intermediates such as carcinogenic compounds. The genes in alpha-, mu-, pi-class of GSTs are induced by chemopreventive agents, which contribute to anticarcinogenic activity through multiple mechanisms (Kensler, 1997
; Yang et al., 2001). Also, alpha-class GSTs play an important role as antioxidant enzymes modulating stress-induced signaling pathways (Yang et al., 2001). All classes of GSTs catalyze the conjugation of compounds with glutathione (GSH) mainly in the liver and in the extrahepatic organs to a much lesser extent. The GSH conjugates can either be excreted in bile intact or be converted to mercapturic acids in the kidney and be excreted in the urine (Vos et al., 1991). The conjugation of substances with GSH is different from their conjugation with amino acids. This is because the substrates of GSH conjugation include an enormous range of electrophilic xenobiotics or xenobiotics that can be biotransformed to electrophiles (Salinas and Wong, 1999). Therefore, the induction of GST represents not only cell detoxification and survival but also cancer prevention, which is supported by the observation that a loss of GST protection increases the susceptibility of hepatocytes to chemical-induced genotoxicity during chemical-induced carcinogenesis (Smith et al., 1977).
GST is induced by a group of chemoprotective agents such as oltipraz, sulforaphane (Prestera and Talalay, 1995) and 3H-1,2-dithiole-3-thione (Kwak et al., 2001). In addition, various growth factors (e.g., insulin, insulin-like growth factor) and peroxisome proliferator–activated receptors (PPAR) 
agonists increase the level of GST expression (Kang et al., 2002; Park et al., 2004b). Moreover, the exposure of animals or cells to various toxicants or prooxidants (e.g., tert-butylhydroquinone [t-BHQ], butylhydroxyanisole, thiazoles and sulfur amino acid deprivation) induces GST and other phase II enzymes (Kim and Cho, 1996). Indeed, the induction of GST by a series of methylthiazoles accompanies various tissue injuries (Kim and Cho, 1996). The observations showing the induction of GSTs by toxicants and chemoprotective agents (Thimmulappa et al., 2002) highlight the need to determine the molecular basis for the induction of GST by xenobiotics. This article covers a part of a larger study aimed at investigating the mechanisms for the GST gene induction by chemoprotectants and toxicants. The identification of the transcription factors and the characterization of the cell signaling pathways would explain the differences between chemoprotective agents and electrophilic toxicants for the induction of GSTs.
GSTA2 was chosen as a representative GST gene because the promoter region consists of diverse DNA binding elements. In particular, the gene contains the binding sites for NF-E2–related factor 2 (Nrf2), CCAAT-enhancer binding protein (C/EBPß), hepatocyte nuclear factors (HNFs), PPARs, arylhydrocarbon receptor, putative phenobarbital-responsive element, and glucocorticoid receptor. In response to various extracellular stimuli, expression of the gene has been shown to be regulated coordinately by activating the transcription factors in a transcriptional or posttranscriptional manner. This paper summarizes the molecular mechanisms of the transcriptional induction of the GSTA2 gene orchestrated by a series of transcription factors that recruit coactivators or corepressors.
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TRANSCRIPTION FACTORS FOR GST EXPRESSION |
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TOPABSTRACTGST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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NF-E2–Related Factor 2
The antioxidant response element (ARE) was identified to be a cis-acting element that is responsible for GST expression (Rushmore et al., 1991
). Because of the high similarity of the ARE binding site to that of the activating protein-1 (AP-1, GTGACNNNGC vs. TGACTCA), it was initially suggested that AP-1, which is composed of Jun and Fos, was the transcription complex responsible for the expression of the phase II enzymes (Friling et al., 1992). Nrf1/2 is a basic leucine zipper transcription factor that heterodimerizes with small Maf proteins during oxidative stress, and binds to the ARE site (Itoh et al., 1995). The signals activated by the electrophilic chemicals or oxidative stress stimulate the transduction of the Nrf2 activity interacting with ARE. Previous studies have shown that a GSH deficiency and the exposure of cells to reactive oxygen species (ROS) (e.g., peroxynitrite) or prooxidants (e.g., phenolic antioxidants) enhance the induction of GST by activating Nrf2 binding to the ARE of the GSTA2 gene (Kang et al., 2000, 2001, 2002). Because basal Nrf2 activity is essential for ARE-dependent gene transcription, many of the genes encoding the antioxidant enzymes are not induced by electrophiles in Nrf2-deficient cells or animals (Ramos-Gomez et al., 2001).
The activation of Nrf2 involves the phosphorylations by cellular kinases. Phosphatidylinositol 3-kinase (PI3K) has been identified as a kinase that is essential for the nuclear translocation of Nrf2 and Nrf2 DNA binding (Kang et al., 2000, 2002). Pickett and colleagues reported that the protein kinase C (PKC) pathway contributes to the activation of ARE by the quinoid prooxidants (Huang et al., 2002), and later identified the PKC-directed phosphorylation of Nrf2 as a key step before its nuclear accumulation (Bloom and Jaiswal, 2003). Furthermore, Yoshida's group reported that Nrf2 phosphorylation is mediated by TPA-insensitive atypical PKC
(Numazawa et al., 2003). The mitogen-activated protein kinases (MAPKs), namely, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase, are other important cellular signal networks that have been examined for Nrf2 activation. It was reported that PD98059, a MAPK kinase (MEK) inhibitor, blocked the sulforaphane-induced activation of ERK and the ARE-mediated reporter activity in HepG2 cells (Yu et al., 1999). Yu et al. reported that the inhibition of ERK reduced the activity of quinone reductase. In contrast, these observations show that PD98059 failed to inhibit the ARE binding activity. Later, PD98059 was identified as an inducer of GSTA2, which activates another activating transcription factor, C/EBPß (Kang et al., 2003b). The lack of MAPK involvement in the activation of Nrf2 is consistent with the report by Pickett's group (Huang et al., 2002). In their study, neither the MEK inhibitor nor the p38 kinase inhibitor altered Nrf2 phosphorylation in HepG2 cells. Therefore, it is likely that neither ERK nor p38 kinase regulates the activation of Nrf2 by prooxidants.
Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1) is the regulatory factor that binds to Nrf2. Early studies have shown that the oxidation of the sulfhydryl moiety in Keap1 allows the release of Nrf2 from its complex, leading to the accumulation of Nrf2 in the nucleus and Nrf2-ARE–mediated gene transactivation (Wakabayashi et al., 2004; Zipper and Mulcahy, 2002). Recently, Velichkova and Hasson (2005) suggested that Keap1 and Nrf2, which are complexed in the cytoplasm for Nrf2 degradation under various resting states, translocate to the nucleus for the transcription of the ARE-containing target genes when oxidative stress inactivates the nuclear export signal of Keap1.
PI3K-Akt initiates the cell survival signal and catalyzes the downstream phosphorylation of glycogen synthase kinase-3ß (GSK3ß) to inhibit its activity. GSK3ß is a serine-threonine kinase that is involved not only in the glycogen metabolism but also in the apoptosis mediated by oxidative stress (Koh et al., 2005). Salazar et al. (2006) provided evidence showing that GSK3ß phosphorylates Nrf2 both in vitro and in vivo. Therefore, it is likely that the induction of the phase II genes is linked to PI3K-Akt-GSK3ß. Another study also showed that the phosphorylation of Nrf2 by GSK3ß leads to the exclusion of Nrf2 from the nucleus, attenuating the expression of the phase II enzymes thereby reducing its protective effects against oxidants. This is consistent with previous observations that Nrf2 activity is stimulated by treating the cells with insulin, which activates PI3K and Akt (Kang et al., 2002). This suggests that PI3K-Akt and its downstream cell signals contribute to the induction of GST, which in turn enhances the cell viability.
CCAAT-Enhancer Binding Protein-ß
C/EBPs are the essential master transcription factors in the liver and other major organs, whose binding to the DNA elements promotes the transactivation of the target genes (Kang et al., 2003a). In particular, C/EBPß plays important roles in regulating the expression of various hepatocyte-specific genes, particularly those associated with cell survival or proliferation (Buck and Chojkier, 2003). In response to environmental signals, C/EBPß is regulated by the mechanism of posttranslational modifications including phosphorylation. Several cellular kinases including p90-ribosomal S6-kinase-1 (RSK1), PKC, PKA, GSK3ß, Ras-MAPK, and cyclin-dependent kinase II (CDKII) phosphorylate C/EBPß (Lee and Kim, 2006). Moreover, the signaling pathways activated downstream from the cell surface receptors increase C/EBPß phosphorylation in its activation domain, which contributes to its target gene transcription. RSK1, PKA, and PKC phosphorylate the rat form of C/EBPß at Ser105 in the N-terminal domain, eliciting transcriptional gene activation. ERK downstream from Ras phosphorylates Thr235 in the human form of C/EBPß (analogous to Thr189 in the rat form). In addition, PKC and CDKII induce the Ser240 and Ser227 phosphorylations of rat C/EBPß in the C-terminal region, respectively, and promote its DNA binding activity. GSK3ß, a downstream kinase of Akt, also phosphorylates Thr188 in the human form of C/EBPß, which is analogous to Thr189 in the rat form, for the progress of adipogenesis (Park et al., 2004a). GSK3ß also phosphorylates Ser185 of the rat C/EBPß.
RSK1-mediated C/EBPß activation has been implicated in cell survival responding to liver damage (Buck et al., 1999). The activation of RSK1 downstream from the TGF
receptor induces the phosphorylation of C/EBPß at specific residues (e.g., Thr217 in the mouse form). The phosphorylation of the Thr217 residue in mouse C/EBPß (Thr266 in the human form) appears to be essential for inducing the target gene. Because the rat form of C/EBPß has evolved with a double mutation and lacks the phosphoacceptor, it has a compensatory Ser105, whose phosphorylation is also catalyzed by RSK1. Therefore, Ser105 in rat C/EBPß and the functionally analogous residues, Thr217 and Thr266 in mouse and human forms, respectively, are the critical phosphoacceptors that are responsible for gene transactivation. Our research results suggest that C/EBPß activation by an oltipraz treatment involves Ser105 phosphorylation in the rat form, or Thr217/266 phosphorylations in the mouse and human forms. However, oltipraz treatment does not enhance Thr189 phosphorylation in C/EBPß, which is catalyzed by Ras-MAPK or CDK (Lee and Kim, 2006). Oltipraz treatment increases the level of C/EBPß phosphorylated at Ser105, which can bind to cAMP response element-binding protein (CREB)-binding protein (CBP), inducing the acetylation of histone for gene transactivation. The C/EBPß gene contains its own binding site(s) in its promoter (GenBank178567). The key role of the Ser105 phosphorylation of C/EBPß for the induction of the GSTA2 gene is also supported by the finding that oltipraz treatment specifically increases the expression of C/EBPß but not C/EBP and C/EBP. Therefore, it is highly likely that the induction of C/EBPß by oltipraz treatment after the initial activation of preexisting C/EBPß helps activate gene induction. Specific mutagenesis analysis of C/EBPß strengthens the conclusion that Ser105 phosphorylation plays an important role in C/EBPß-mediated gene expression. Therefore, the specific phosphorylation of C/EBPß by certain chemopreventive agents contributes to its cytoprotective effects.
The PI3K pathway affects cell growth, survival, and motility. Previously, it was shown that PI3K regulates the nuclear localization of C/EBPß, which is an essential step for its activation (Kang et al., 2003a). The activation C/EBPß induced by an oltipraz treatment also depends on PI3K. In general, the full activation of RSK1 requires phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is a constitutively active kinase downstream of PI3K (Casamayor et al., 1999). After activation by PDK1, RSK1 translocates to the nucleus (Roux et al., 2003). Hence, the inhibition of PI3K hinders the ability of oltipraz treatment to induce the nuclear translocation of RSK1. Either the chemical inhibition of PI3K or the stable transfection of the p85 subunit almost completely blocked Ser105 phosphorylation in C/EBPß, which is consistent with the PI3K dependence of RSK1 activation. This concurs with a previous observation (Kang et al., 2003a) and also with a report (Roux et al., 2003) showing that the full activation of RSK1 requires PDK1 downstream of PI3K. Overall, the RSK1-mediated Ser105 phosphorylation of C/EBPß by oltipraz treatment requires the basal PI3K activity.
In primary cultured hepatocytes, RSK1 is capable of regulating C/EBPß Ser105 phosphorylation (Buck et al., 1999). Oltipraz treatment also enhances the phosphorylation of mouse or human C/EBPß at Thr217 or Thr266, which is catalyzed by RSK1 (Lee and Kim, 2006). The role of human RSK1 in the induction of the C/EBPß-mediated gene is also supported by the siRNA knockdown of RSK1. The activation of C/EBPß by the specific phosphorylation would result in a conformational change, which would promote its DNA binding for gene induction. The finding that oltipraz treatment activates C/EBPß through the PI3K-RSK1 pathway highlights the role of the signaling pathway in the cytoprotective functions.
Previously, it was reported that the ERK1/2, p38 kinase, and JNK pathways were not responsible for the C/EBPß-mediated GST induction by oltipraz (Kang et al., 2003a). In an additional study, the extent of the increase in C/EBPß phosphorylation or its gene transactivation by oltipraz in MAPK kinase 1 (MKK1)–deficient cells was similar to that in the control cells, indicating that oltipraz treatment could stimulate C/EBPß phosphorylation independently of MKK1-ERK activation. In general, the activation of ERK1/2 is essential for RSK1 activation by growth factors such as epidermal growth factor (EGF) (Roux et al., 2003). The observations that the C/EBPß phosphorylation by oltipraz treatment was only weakly affected by the chemical inhibition of MKK1-ERK1/2 and that RSK1 regulated C/EBPß phosphorylation indicates that the chemical activation of RSK1 may not require the constitutive activity of ERK1/2. This is also supported by previous observations that oltipraz treatment does not activate MAPKs for C/EBPß activation.
RSK1 activation by growth factors requires ERK docking near the C-terminus region (Roux et al., 2003). The C-terminal kinase domain of RSK1 catalyzes the Ser380 phosphorylation of RSK1 (i.e., autophosphorylation). The activation of ERK initiates a series of activating processes of RSK1, which includes the autophosphorylation of RSK1. It was found that RSK1 expression in the lysates was unaffected by oltipraz treatment. In additional experiments, it was observed that Ser380 phosphorylation in RSK1 was increased by oltipraz treatment, which persisted for up to 24 h. The increase in RSK1 activity by oltipraz was similar to that of its own Ser380 phosphorylation. The observation that MKK1-ERK1/2 inhibition did not alter the oltipraz-inducible Ser105 phosphorylation of C/EBPß shows that the mechanistic basis of RSK1 activation by oltipraz may be different from that by growth factors.
Hepatic Nuclear Factor-1
Hepatic nuclear factor-1 (HNF1) is a dimeric transcriptional regulator containing a homeodomain, and one of the most important transactivators of liver-specific gene transcription. HNF1, but not HNF1ß, is expressed in hepatocytes (Schrem et al., 2002) and the extrahepatic tissues such as the kidney, intestine, and pancreatic islets (Parrizas et al., 2001). The homodimer of HNF1 is found in the liver, whereas heterodimers are detected in other organs. A number of genes are positively regulated by an interaction between HNF1 and the respective cis-acting HNF1 binding element in the promoters of target genes, which include albumin, aldolase B, fibrinogen, -lipoprotein AII and B, glucose-6-phosphatase, ferrochelatase, and cytochrome P450IIE1 (CYP2E1) (Borlak and Thum, 2001; Schrem et al., 2002). In contrast, the dominant negative mutant of HNF1 induces mitochondrial hyperpolarization, the release of cytochrome c, and the activation of caspases-3 and -9 (Wobser et al., 2002).
The suppression of HNF1 activates the apoptotic cell death machinery through alterations in gene expression and mitochondrial dysfunction (Wobser et al., 2002). Mice lacking HNF1 die around the time of weaning after suffering from progressive wasting syndrome with significant liver enlargement. This supports the concept that HNF1 is a transcription factor that is essential for cell survival. In addition, the inhibition of HNF1 increases the sensitivity to other toxic stimuli (e.g., high glucose-induced apoptosis) except for ceramide.
The regulatory region localized to nucleotides –867 to –857 bp in the promoter region of the GSTA2 gene has been identified as an HNF recognition element (HRE) (Rushmore et al., 1990). The expression of GST and many other genes (e.g., albumin) requires HNF1 for their maximal transcription activity (Schrem et al., 2002). The HNF binding site was found to contribute to the maximum basal expression of the gene. Further studies support the functional role of HRE inhibition in the ceramide-induced repression of GSTA2, which was verified by luciferase reporter and promoter deletion analyses (Park et al., 2004b). The crucial role of activating HNF1 binding to the HRE was also demonstrated by reporter assays using an HRE-deleted mutant. Multiple coactivators of HNF1 and other HNFs have been previously identified, which include CBP, p300, and p300/CBP-associated factor (p/CAF) (Schrem et al., 2002). The physical interaction of HNF1 with CBP, p/CAF, steroid receptor coactivator-1, and steroid receptor coactivator-3 increases the HNF1-dependent transcription of a genome-integrated promoter (Ma et al., 1999). Sphingolipid is involved in the downregulation of certain genes such as cytochrome P450s. For example, the cellular accumulation of ceramide leads to CYP2C11 repression (Chen et al., 1995). Stimulation of HNF1 and other transcription factors degradation by ceramide may be responsible for the transcriptional repression of the cytochrome P450s (Chen et al., 1995). In general, the cellular responses to ceramide culminate in increased apoptosis and decreased cell proliferation and differentiation (Kolesnick and Fuks, 2003). Hence, ceramide, as a signaling molecule for apoptosis, antagonizes HNF1-mediated GSTA2 expression (Park et al., 2004c). On the other hand, oltipraz, as a GSTA2 inducer, enhances the nuclear accumulation and DNA binding of HNF1 (Park et al., 2004d), suggesting that an increase in cell viability by oltipraz treatment is associated with the activation of HNF1 and HNF1-mediated induction of GST.
Ceramide decreases the transcriptional activity of C/EBPß as a result of a decrease in its phosphorylation (Sprott et al., 2002). Because ceramide stimulates the degradation of C/EBPß and HNF1 (Park et al., 2004d), the apoptosis of cells in response to ceramide might be mediated by the repression of C/EBPß, HNF1, or other important transcription factors that are essential for the cell viability.
Either t-BHQ or ß-naphthoflavone enhances the phosphorylation Nrf2, which increases its stability and transactivation activity (Nguyen et al., 2003). The finding that ceramide inhibits the activity of Nrf2 (Park et al., 2004c) indicates that ceramide represses GSTA2 expression at least in part through the inhibition of the basal Nrf2 activity. Therefore, ceramide mediates the degradation of the activating transcription factors that are essential for gene induction. The ability of ceramide to decrease the essential transcription factors mirrors the observed GST repression. Overall, the repression of GST by toxic stimuli probably leads to a decrease in cell viability, involving changes in the behavior of the activating transcription factors.
Peroxisome Proliferator–Activated Receptors
Peroxisome proliferators–activated receptors (PPARs) are well-characterized transcription factors that are members of the nuclear hormone receptor superfamily (Dubuquoy et al., 2002). There are three PPAR subtypes, PPAR, PPARß, and PPAR, which have distinct tissue distributions. PPAR is mainly found in the liver, heart, and kidney, whereas PPARß is expressed ubiquitously. PPAR is mainly expressed in the adipose tissue and in the liver to a lesser extent. Although the basal expression of PPAR in the liver is relatively low, hepatic PPAR is significantly upregulated by PPAR activators and under certain physiological conditions (e.g., obesity) for the expression of several PPAR-responsive genes. PPAR is also an important target for the development of new drugs that are aimed at preventing or treating cancer. A deficiency in PPAR can be a significant risk factor for carcinogenesis (Sporn et al., 2001). Ligands for PPAR have been shown to inhibit carcinogenesis in various experimental models, while inducing the differentiation of tumorigenic cells. The activated PPAR forms a heterodimer with the retinoid X receptor (RXR) and the heterodimer complex formed then binds to the specific PPAR response elements (PPRE) in the target gene promoters (Kliewer et al., 1992). The induction of the PPAR target genes might explain the regulation of cell survival, growth, and differentiation.
Studies have been conducted to determine the role of PPAR and RXR activation in GSTA2 gene expression (Park et al., 2004b). In the study, 15-deoxy-(12,14)-prostaglandin J2 (PGJ2) and thiazolidinedione PPAR ligands were used as the PPAR agonists. The combined treatment of the PPAR agonist and 9-cis retinoic acid (RA) synergistically enhanced the activities of Nrf2 and C/EBPß, which are key transcription factors for GSTA2 induction. This study identified the PPAR-binding site cluster in the GSTA2 gene as being a functionally active PPRE-responsive enhancer module.
It has been shown that either PGJ2 or RA induces GSTA2 along with Nrf2 and C/EBPß activation. Compared with PGJ2 or RA alone, the combination treatment synergistically promotes the induction of GSTA2 by increasing the Nrf2 and C/EBPß activities. Thiazolidinediones including troglitazone, rosiglitazone, and pioglitazone are synthetic PPAR agonists. When used in combination with RA, all of the agents potentiated the induction of GSTA2 (Park et al., 2004b). PGJ2 + RA increased the binding of the PPAR and RXR heterodimer to the putative PPREs in the GSTA2 promoter. Specific mutations in these multiple PPRE sites resulted in the complete loss of its responsiveness to the activators (Park et al., 2004b). This suggests that these binding sites function as a PPAR response enhancer module (PPREM). The transactivation of PPREM by the PPAR and RXR heterodimer was verified by the potentiated induction of GSTA2 in the cells treated with PGJ2 + RA after transfection with the plasmids encoding PPAR1 and RXR (Park et al., 2004b). Therefore, the PPAR and RXR heterodimer promotes the induction of GSTA2 by activating PPREM in the gene, as well as by promoting Nrf2 and C/EBPß activation.
Cooperative Interactions of Activating Transcription Factors
Both the ARE and C/EBP-binding sites play important roles in the transactivation of the GSTA2 gene, as evidenced by the results of the PPRE-deleted promoter-luciferase assays (Park et al., 2004b). The complete blockage of the PPAR and RXR-mediated induction of the GSTA2 gene due to a deletion mutation of either the ARE or C/EBP-binding sites confirms our previous observation that the simultaneous binding of Nrf2 and C/EBPß to their response elements are essential for full gene transactivation. A blockage of the ligand-dependent transcriptional response as a result of a mutation of the respective PPRE binding site suggests that multiple putative PPREs function as a module in which the nuclear binding sites in close proximity to each other are essential for the full responsiveness of the ligands. In addition, the essential role of the activation of HNF1 binding to its binding site was demonstrated by the luciferase reporter experiment. Collectively, the results show that the formation of the transcriptional protein complexes on the binding sites is essential for full responsiveness to chemical inducers.
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PI3K-RSK-mTOR SIGNALING AND TRANSCRIPTION FACTOR ACTIVATION |
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TOPABSTRACTGST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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Extracellular stimuli, such as growth factors, cytokines, chemicals, or matrix proteins transduce signals through the tyrosine kinase cascades. For example, TGF
binds to the EGF receptor to induce autophosphorylation and a conformational change, which is recognized by the adaptor molecules. Ras is a small GTPase that is activated with increased guanine nucleotide exchange activity by guanine nucleotide exchange factor, and propagates the signals to Raf, MEK1, and ERK. The phosphorylated tyrosine kinase receptor also induces the recruitment of PI3K through its PH domain. In response to PIP3, which is a second messenger generated by PI3K or PDK1, various cellular kinases including Akt, p70 ribosomal S6 kinase (S6K), and RSK1 are phosphorylated. In particular, RSK1 is phosphorylated by PDK1 in the N-terminal kinase domain as well as by ERK in the C-terminus region (Casamayor et al., 1999; Roux et al., 2003).
The members of the RSK family play a key role in mitogen-activated cell growth, differentiation, or cell survival. Among the RSK isoforms, RSK1 is a major form expressed in the liver, muscle, and fat (Moller et al., 1994). RSK1 contains two distinct functionally active kinase domains, and the N-terminal kinase of the activated RSK1 phosphorylates the cellular protein substrates including C/EBPß, CREB, c-Fos, and I
B (Lee and Kim, 2006). RSK1 activation by the growth factor requires ERK docking near the C-terminus region. Activation of the C-terminal kinase domain leads to autophosphorylation in the linker region. Another PDK1-mediated phosphorylation in the activation loop of the N-terminal kinase domain allows the full activation of RSK1 that is capable of phosphorylating the target proteins.
The mammalian target of rapamycin (mTOR) mediates cell growth and proliferation in response to various mitogens, nutrients, and cellular energy levels (Roux et al., 2004). Stimulation by growth stimuli (i.e., insulin and EGF) leads to the activation of the PI3K-Akt and Ras-Raf-MEK1/2-ERK1/2-RSK1 pathway. The ATP-regulated LKB1/AMP-activated protein kinase pathway controls mTOR in addition to directly sensing the ATP levels in the cells by mTOR itself (Chiang and Abraham, 2005). The tuberous sclerosis complex1/2 (TSC1/2) complex is a negative regulator of mTOR, which inhibits Rheb activity, and is controlled by signaling through the growth, nutrient and energy level. The phosphorylation of TSC2 by Akt or RSK1 inactivates the TSC1/2 complex, whereas its phosphorylation by AMPK enhances the activity. In addition, S6K1 directly phosphorylates mTOR at Ser2448, and enhances its activity (Chiang and Abraham, 2005).
It was observed that insulin stimulates the Nrf2 activity and induces GSTA2 (Kang et al., 2002). Insulin activates Akt and RSK1 downstream of PI3K, which contributes to the cell viability. Therefore, GSTA2 induction by insulin might be mediated by the activation of the mTOR-related complex. In contrast, ceramide inhibits GSTA2 expression by degrading HNF1. The hypothesis that a decrease in mTOR activity is coupled with GSTA2 repression is supported by the observation that ceramide decreases the activity of S6K1 and protein synthesis through a downstream pathway of mTOR (Hyde et al., 2005). Therefore, it is highly likely that mTOR signaling is an important cell signal pathway for regulating GST expression as well as cell viability.
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PHYSIOLOGICAL IMPLICATIONS |
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TOPABSTRACTGST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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Antioxidants are substances that either directly or indirectly protect cells from the harmful effects of toxic free radicals. Antioxidants and antioxidant enzymes include ascorbic acid,
-tocopherol, ß-carotene, polyphenol, flavonoids, GSH, superoxide dismutase, catalase, and GSH peroxidase. Antioxidant agents can either scavenge ROS or stimulate the detoxification mechanism within the cells, resulting in the removal of ROS. Mammals have novel antioxidant defense systems to protect against the cellular damage caused by oxidative stress. The capacity of cells to maintain cellular homeostasis during oxidative stress resides in their ability to induce protective enzymes, which decrease the level of oxidative stress caused by ROS. ARE was identified as a cis-acting element that is responsible for inducing the phase II enzymes. AREs are also found in the promoter regions of a wide variety of antioxidant enzymes (e.g., -glutamylcysteine synthetase, heme oxygenase-1 and ferritin L and H chains) and of the genes encoding detoxifying genes (e.g., rat and mouse GSTA2, rat GSTP, rat, and human quinone reductase) (Kang et al., 2003a).
The phase II enzymes, GST, microsomal epoxide hydrolase, and uridine diphosphate-glucuronyl transferase, are generally responsible for metabolic detoxification (Goldstein and Faletto, 1993). Chemoprotective agents induce phase II enzymes that metabolize toxicants and carcinogens to less reactive forms. These conjugation reactions are catalyzed by phase II detoxifying enzymes, which protect cells. Similarly, GSH conjugations catalyzed by GST allow highly reactive carcinogens or radical intermediates to be eliminated through the excretion machinery (Salinas and Wong, 1999). Obviously, the induction of the phase II enzymes contributes to cytoprotection and potentially to the self-repair of cells exposed to oxidative stress. Cytoprotective agents induce GSTA2 and concomitantly activate the PI3K-Akt/ERK-RSK1-mTOR pathways that activate the transcription factors favoring cell viability. Therefore, the induction of GSTA2 is an adaptive response that protects against oxidative stress. Besides cytoprotective agents, prooxidants, and toxicants (e.g., t-BHQ, butylhydroxyanisole, and thiazoles) induce the phase II enzymes, which may reflect the adaptive responses to harmful challenges.
These results support the concept that the adaptive induction of the phase II enzymes in response to external stimuli appears to be regulated by different signaling pathways and the differential activation of distinct transcription factors. This hypothesis is strengthened by the observations that oxidative stress and electrophilic compounds antagonize the insulin-induced activation of mTOR-dependent signaling and S6K1 activity despite their strong induction of the antioxidant genes (Patel et al., 2002). For example, hydrogen peroxide decreases the level of 4E-BP1 phosphorylation, which is a substrate of mTOR (O'Loghlen et al., 2006). Oxidative stress strongly activated ERK but failed to activate Akt (Patel et al., 2002). It is possible that the induction of GSTA2 by oxidative stress results from the activation of the transcription factors regulated by a different signaling pathway. Overall, the mechanistic basis and cell signaling for the induction of GSTA2 by electrophilic chemicals may be different from that by cytoprotective agents.
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ACKNOWLEDGMENTS |
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This study was supported by The Korea Research Foundation Grant (KRF-2004-015-E00096), Ministry of Education, Republic of Korea.
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REFERENCES |
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TOPABSTRACTGST EXPRESSION AS A...TRANSCRIPTION FACTORS FOR GST...PI3K-RSK-mTOR SIGNALING AND...PHYSIOLOGICAL IMPLICATIONSREFERENCES |
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