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| The Signal Transduction Pathways for Antioxidant Response Element - Mediated Phase II Enzyme Expression 김상건*#8228;강건욱#8228;김윤균 서울대학교 약학대학 종합약학연구소 *Corresponding author Tel. 02-880-7840 Fax. 02-872-1795 E-mail. sgk@snu.ac.kr Phase II Enzyme Induction by Chemoprotectives and Toxicants Exposure of mammalian cells to a variety of chemoprotective compounds confers resistance against oxidative stress and a broad set of xenobiotics generating oxidative stress (e.g. pro-oxidants and carcinogens). The capacity of mammalian cells to maintain cellular functions during oxidative stress resides in rapid induction of protective enzymes, which decrease oxidative stress by reducing reactive oxygen species (ROS). Table 1 summarizes some biologically relevant ROS. Chemoprotective compounds exert their effects by inducing phase II detoxifying enzymes such as glutathione S-transferases (GST) and quinone reductase. The mechanistic basis for chemoprotective responses has been intensively studied since the discovery of carcinogen metabolism by chemical-induced cellular enzymes (Conney et al., 1956). Chemoprotective agents induce the enzymes that metabolize carcinogens to less reactive forms (Miller et al., 1958). In particular, the induction of phase II enzymes may represent a protective adaptive response to oxidative stress. Recent studies raised the notion that GST may also be involved in the intracellular signaling pathway responsible for cell survival. GST inhibits formation of Jun-c-Jun NH2-terminal kinase (JNK) complex and subsequently blocks mitogenic signaling induced by oncogenic ras-p21 (Villafania et al., 2000). Hence, the regulation of GST gene expression may be coupled with cell cycle control when cells are exposed to oxidative stress. Classical classification of the chemically-inducible enzymes consists of two category: one is a monofunctional inducer, which induce phase II detoxifying enzymes; and another is a bifunctional inducer which induces both phase II and phase I oxidizing enzymes (Prochaska et al., 1985; Talalay et al., 1988; Wattenberg, 1985). Coinduction of both phase I and II enzymes by bifunctional inducers can be mediated through unique receptors and translocators. For instance, the transcriptional activation of the CYP1A1 gene, a representative phase I enzyme, is mediated by the Ah-receptor and its nuclear translocator ARNT (Prochaska and Talalay, 1988). Phase I enzymes in general represent an activating system for many natural and synthetic carcinogens, while phase II enzymes often serve to detoxify harmful activated carcinogens. Thus, the selective induction of phase II enzymes, but not phase I enzymes, would be beneficial to prevent chemical-induced carcinogenesis. Several natural and synthetic compounds exert the protective activities against certain carcinogens. Among the natural compounds, a variety of compounds have protective effects through induction of phase II enzymes. These compounds include sulfides in garlic and polyphenols in green teas (Table 2). In addition, numerous studies provided evidence that the synthetic anti- and pro-oxidants [e.g. tert-butylhydroquinone (t-BHQ), butylhydroxyanisole (BHA)] as well as toxicants (e.g. thiazoles) are capable of inducing phase II enzymes (Table 2). Antioxidant Response Element As far as the induction of phase II enzymes is concerned, the discovery of antioxidant response element (ARE) may be a most important event among the scientific findings. The inducible expression of phase II enzymes (e.g. rGSTA2) by phenolic antioxidants has been extensively studied (Bergelson et al., 1994; Wasserman and Fahl, 1997; Venugopal and Jaiswal, 1998). Transcriptional activation of the antioxidant genes by these compounds has been traced to a cis-acting transcriptional enhancer called the antioxidant response element (ARE) or the electrophile response element (EpRE). ARE coordinately regulates the expression of a battery of antioxidant genes. The binding proteins to the ARE consensus sequence involve Nrf proteins and Maf family members (Venugopal and Jaiswal, 1998; Moinova and Mulcahy, 1999). Signals activated by oxidative stress stimulate transduction of Nrf activity and subsequent activation of ARE (Venugopal and Jaiswal, 1998; Moinova and Mulcahy, 1999). A series of studies have shown that endogenously generating ROS as well as external stimuli such as phenolic antioxidants induce the detoxifying enzymes including mEH and GSTs via activation of ARE consisting of Nrf2 and Maf (Kang et al., 2000; Kang et al., 2001a, 2001b). The initial step to elucidate molecular mechanistic basis of the phase II gene induction was identification of the cis-acting ARE. AREs have been found in the promoters of diverse detoxifying genes [e.g. rat and mouse GST-Ya (Rushmore et al., 1990; Friling et al., 1990), rat GST-P (Okuda et al., 1989), rat and human quinone reductase/DT-diaphorase (QR) (Favreau and Pickett, 1991; Li and Jaiswal, 1992)] (Fig. 1). Later, AREs were also found in the promoters of genes that encode cellular antioxidant enzymes such as γ-glutamylcysteine synthetase (Wild et al., 1999), heme oxygenase-1 (HO-1) (Prestera et al., 1995), and ferritin L and H chains (Wasserman and Fahl, 1997). Because the ARE core sequence (GTGACNNNGC) shows some similarity with the activating protein-1 (AP-1)-binding site (TGACTCA), the initial studies have been focused on the role of AP-1 such as c-Jun and c-Fos on the phase II enzyme expression (Friling et al., 1992; Jaiswal, 1994; Nguyen et al., 1994). Nonetheless, recent studies strongly supported the contention that a member of the basic leucine zipper transcription factor family, Nrf1/2 (NF-E2-related factor 2), is involved in the activation of ARE. As a matter of fact, Nrf2 can bind to the ARE core sequence in association with small molecular protein family, Maf protein such as Maf G or Maf K (Itoh et al., 1997). It seems clear that Nrf-2 plays a central role in the ARE-dependent gene transcription. A number of anti-oxidative genes could no longer be induced by electrophilic or ROS-generating agents in Nrf2-deficient macrophages (Hayes et al., 2000), and phenolic antioxidant BHA failed to induce GST in the Nrf2 knockout mice. Despite this enthusiastic hypothesis, some other evidence raised the possibility that distinct transcription factor(s) bind to ARE region and control the GST gene transcription. Chen and Ramos reported that CEBP-β and CBP300 might be involved in the regulation of the gene transcription using GST-Ya promoter CAT-assay and gel shift analysis (2000). A number of transcription factors are phosphorylated by distinct members of kinase family triggered in response to a variety of stimuli (Gupta et al., 1995; Tan et al., 1996). Among cytochrome P450 families, transcription of the CYP1A1 gene is mediated with AhR and ARNT. Phosphorylation processes by PKC and tyrosine kinase are included in the activation of AhR or ARNT (Backlund et al., 1997; Long et al., 1998). Because phosphorylation of ARE transcription factor(s) by cellular kinases could be a potential important regulatory step, we will focus on the mechanistic basis and signal pathway responsible for the Nrf2-mediated ARE-dependent phase II enzyme induction. In this communication, the current knowledge and future perspective of signal transduction pathways for the induction of phase II detoxifying enzymes will be reviewed. Tight Binding of Inactive Nrf2 to Cytosolic Keap1 A recent study provided evidence that Nrf2 activity is repressed by its tight binding with the cytoskeleton-associated protein Keap1 and the complex localization is limited in the cytoplasm (Itoh et al., 1999). In comparing the human and chicken Nrf2 amino acid sequences, Ioth et al found six highly conserved regions, and one of the regions, named Neh2, was required for the negative regulation of Nrf2 activity in HD3 erythroblasts using deletion mutant analysis. This led to the hypothesis that the Neh2 domain interacts with a cellular protein, Keap1. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. They also showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and to potentiate the ARE-mediated response. They postulated that Keap1 and Nrf2 may constitute a crucial cellular sensor for oxidative stress, and mediate a key step in transducing the signaling pathway that leads to transcriptional activation of phase II enzymes by the Nrf2 nuclear shuttling mechanism. Role of MAP Kinases in ARE-Mediated Phase II Enzymenduction Information is restricted as to how the signal is transduced from cytosolic Nrf-2 or other transcription factor(s) to nuclear target point of ARE. Many research groups have studied phosphorylation steps for ARE activation. A number of cellular stresses and lethal insults (e.g. cytotoxic chemicals) engage the mitogen-activated protein (MAP) kinases and concomitantly induce transactivation of the targeted genes (Amato et al., 1998; Fritz and Kaina, 1999). Three distinct mammalian MAP kinase modules including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase have been characterized (Cahill et al., 1996; Treisman, 1996). Stress-activated protein kinase cascade involves the activation of JNK, which consequently induces AP-1-mediated transactivation of the genes. An attempt to clarify the signaling pathway for the phase II enzyme induction was made by Yu et al. (1999, 2000). The role of ERK in the induction of phase II detoxifying enzymes has been studied. In their studies, treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of ERK. Inhibition of ERK with PD98059 or by transient transfection with a dominant negative mutant of MKK1 abolished ERK activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and the antioxidant response element (ARE)-linked reporter gene activity by the inducers. In addition, the expression of Ras and its mutants failed to affect the chemical-inducible activation of ARE. Based on the observations, they proposed the possibility that the induction of ARE-mediated phase II detoxifying enzymes be mediated by the MAP kinase through direct activation of Raf-1. They also proposed the hypothesis that ARE activation was negatively regulated by p38 kinase (Yu et al., 2000). Treatment of HepG2 and Hepa1c1c7 cells with t-BHQ stimulated the activity of p38 kinase. Inhibition of p38 kinase activation by SB203580 a specific inhibitor enhanced the induction of quinone reductase activity and the activation of ARE reporter gene by tBHQ. Consistent with this result, interruption of the p38 kinase pathway by overexpression of a dominant-negative mutant of p38 kinase or MKK3, an immediate upstream regulator of p38 kinase, potentiated the activation of the ARE reporter gene by t-BHQ. In addition, inhibition of p38 kinase activity augmented the induction of ARE reporter gene activity by the known chemical inducers of phase II enzymes. In contrast to their reports, we found out that PD98059 failed to inhibit the ARE binding activity and rather treatment of cells with PD98059 markedly increased rGSTA2 expression in H4IIE cells (Kang et al., 2001a). Hence, strong evidence was provided that ERK may be responsible for the negative regulation of rGSTA2 expression, and the induction of rGSTA2 by the inhibition of ERK might constitute another plausible mechanism for rGSTA2 induction. We found out that PD98059 failed to affect the ARE binding activity in H4IIE cells, which raised the possibility that other distinct pathway(s) may exist for rGSTA2 induction. The difference between quinone reductase and rGSTA2 expression may result from the distinct regulatory pathway for phase II enzyme expression. Alternatively, the difference may be due to cell type-specificity. These data highly support the conclusion that the MAP kinase including ERK and p38 kinase are not responsible for the phosphorylation of Nrf-2 (Kang et al., 2001a; Huang et al., 2000). This is consistent with the recent report by Huang et al (2000). In their study, neither ERK inhibitor nor p38 kinase inhibitor affected the phosphorylation of Nrf-2 in HepG2 cells. PKC-Mediated Phosphorylation of Nrf-2 The question as to what cellular kinase phosphorylates Nrf-2 was recently resolved by Pickett? research group (Huang et al., 2000). They showed the role of the PKC pathway in the ARE activation and proved that PKC-directed phosphorylation of Nrf2 is a critical event for its nuclear translocation in response to oxidative stress. Using a reporter gene assay, ARE-directed transcription was activated by phorbol 12-myristate 13-acetate (PMA) a representative PKC activator, but completely suppressed by staurosporine and Ro-32-0432, selective inhibitors of PKC. Immunocytochemistry and Western blot analysis revealed that both PMA and t-BHQ promoted the nuclear localization of Nrf2, which was blocked by staurosporine or Ro-32-0432. They demonstrated that PMA transiently activated Nrf2 phosphorylation, whereas t-BHQ or β-naphthoflavone led to persistent stimulation, which was abolished by staurosporine, but not by U0126 and SB203580, the respective inhibitors of MKK and p38 kinases. In addition, purified Nrf2 could be phosphorylated in vitro by the catalytic subunit of PKC, or by PKC immunoprecipitated from cell lysates. These findings demonstrated that Nrf-2 was phosphorylated by PKC (Fig. 2). However, it remains to be further clarified whether multiple phosphorylation sites exist in Nrf-2 and other cellular kinases phosphorylate Nrf-2. Essential Role of PI3-Kinase on the ARE-Dependent rGSTA2 Induction Although PKC plays a key role in the phosphorylation of Nrf-2, it is not obvious whether PKC activation is an initial triggering step detecting the intracellular redox change. PKC is a kind of downstream kinase to transduce signal into target molecules in response to extracellular stimuli (e.g. IP3-induced calcium release and Ras activation). Hence, the signal detection system (e.g. receptor or upstream kinase) may exist in close proximity to the plasma membrane. Phosphatidylinositol 3-kinase (PI3-kinase) is a lipid kinase that phosphorylates phosphatidylinositols at the 3 position of the inositol ring. This enzyme has been found to be associated with the activation of cellular survival signals in response to several growth factors and has been implicated in mitogenesis and cell transformation (Daulhac et al., 1999). In addition, the phosphorylated forms of phosphatidylinositol act as second messengers for several kinases, including the serine-threonine Akt kinase and ribosomal S6 kinase (Lin et al., 1999). PI3-kinase is also involved in the regulation of the small GTPase Rac by growth factors (e.g. platelet-derived growth factor) and Rac plays an important role in the activation of c-Jun NH2 terminal kinases (Hawkins et al., 1995; Fritz and Kaina, 1999). Our research group found out that PI3-kinase regulates the ARE activation inducible by oxidative stresses such as sulfur amino acids deprivation (SAAD) and t-BHQ and the subsequent transcriptional activation of rGSTA2. Depletion of hepatic GSH increases the susceptibility of cells and animals to free radical-induced damage because GSH plays a critical role in the detoxification of oxidative metabolites produced from endogenous and exogenous molecules. We have shown that deprivation of sulfur amino acids from culture medium leads to a decrease in the cellular GSH level, which subsequently elevates oxidative stress as evidenced by an increase in the fluorescence of DCF (Kang et al., 2000). The level of rGSTA2 mRNA was significantly increased after incubation of cells with t-BHQ or exposure to SAAD medium (Table 3). t-BHQ increased the rGSTA2 mRNA maximally at 12 h, followed by gradual returning toward that of control at 48 h. Whereas SAAD caused an increase in rGSTA2 mRNA at 24 - 48 h. Western blot analysis revealed that rGSTA1/2 subunit began to be induced 6 h after t-BHQ treatment, peaked at 12-24 h and extended up to 48 h. The rGSTA1/2 subunit in H4IIE cells was also induced 48 h after SAAD and extended up to 72 h (Table 3). To determine whether the PI3-kinase cascade was involved in activation of the ARE-binding transcription factors, cells were incubated with wortmannin and LY294002, PI3-kinase inhibitors, 30 min before t-BHQ treatment or exposure to SAAD medium. The activation of ARE by t-BHQ or SAAD was inhibited by the presence of wortmannin or LY294002 (Kang et al., 2000). To confirm that inhibition of ARE activation by the PI3-kinase inhibitors prevented rGSTA2 induction, rGSTA2 mRNA level was also monitored in cells incubated with each PI3-kinase inhibitor (Table 4). Either wortmannin or LY294002 significantly inhibited the increase in rGSTA2 mRNA by t-BHQ or SAAD. In addition, we showed that the expression of microsomal epoxide hydrolase, another phase II enzyme, was also controlled by the PI3-kinase pathway (Kang et al., 2001b). These results provided strong evidence that the activity of PI3-kinase was essential in the regulatory pathway leading to the activation of ARE and subsequent phase II enzyme induction (Fig. 3). Conclusion 1. Phase II enzyme can be induced by both chemoprotectives and toxicants. 2. The transcriptional activation of phase II enzymes is controlled by a cis-acting transcriptional enhancer called ARE in response to ROS. The binding proteins to the ARE consensus sequence involve Nrf proteins and Maf family members. 3. Nrf2 activity is repressed by its tight binding with the cytoskeleton-associated protein Keap1 and the complex localization is limited in the cytoplasm. 4. Phosphorylation of Nrf-2 by PKC is required for the nuclear translocation, which may serve as a component of ARE. 5. PI3-kinase activity is essential in the regulatory pathway leading to the activation of ARE and subsequent phase II enzyme induction. 6. 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