The Antioxidant Responsive Element A Site for Nrf2 Transcription Factor Binding

As mentioned above, common to several phase II detoxification enzymes is their transcriptional regulation by a conserved element located in the promoter region designated as antioxidant responsive element (ARE). This cis-acting regulatory element has been identified in the 5' flanking region of GST Ya and NQO1 genes in mammals and may also be present in other phase II enzymes. The ARE shows sequence similarity to activator protein (AP-1), and has been demonstrated to be a binding site for c-Jun, jun-B, jun-D, Fra1, and c-fos. More recent investigations have also identified a small basic leucine zipper protein (bZIP), designated Nrf-2, that can interact with the ARE, and is the prime candidate for mediating the induction of detoxification gene expression (Fig. 4). The important role of Nrf2 has been demonstrated by the work of Bloom et al. Using site-directed mutagenesis in the DNA-binding region of Nrf2, Bloom and colleagues demonstrated a decreases in Nrf2's ability to interact with the ARE (148). The overall outcome was a reduction in expression of the marker enzyme NQO1. Nrf2 can function alone or interact with c-jun, jun-B, and jun-D by formation of hetermodimers positively regulating ARE-mediated gene expression in mammalian cells (149). In contrast, interaction of Nrf2 with the small Maf proteins c-Maf, MafG,

Figure 4 The proposed mechanism of phase II detoxification enzyme induction by oxidative stress and electrophilic agents such as isothiocyanates. PKC, protein kinase C; ERK1/2, extracellular-signaling kinases; JNK1, c-jun-n-ter-minal-kinase; Nrf2-, NF-E2-related factor; Keapl, Kelch-like ECH-associated protein; NQO1, quinone reductase; GST, glutathione-S-transferase. (Adapted from Refs. 148,154-156.)

Figure 4 The proposed mechanism of phase II detoxification enzyme induction by oxidative stress and electrophilic agents such as isothiocyanates. PKC, protein kinase C; ERK1/2, extracellular-signaling kinases; JNK1, c-jun-n-ter-minal-kinase; Nrf2-, NF-E2-related factor; Keapl, Kelch-like ECH-associated protein; NQO1, quinone reductase; GST, glutathione-S-transferase. (Adapted from Refs. 148,154-156.)

and MafK in addition to c-fos negatively regulates ARE-mediated gene expression (150,151). Indeed, overexpression of MafG, MafK, and c-fos proteins in HepG2 cells suppresses the expression of ARE-related gene transcription such as the marker enzymes NQO1 and GST Ya (152).

C. Nrf2 Regulation: The Mechanism of Nrf2: Keap1 Dissociation

Nrf2 is sequested in the cytoplasm by the formation of a complex with the actin-binding protein Keap1. During insult by enzyme inducers such as ITCs, Nrf2 is released from Keap1. This allows Nrf2 to translocate to the nucleus and mediate the transcriptional activation of phase II detoxification enzymes via interaction with the ARE (153). To elucidate how Nrf2 causes enzyme induction Huang et al. focused on the mechanisms by which Keap1 dissociates from Nrf-2. Their results suggest that the involvement of protein kinase C (PKC) mediates phosphorylation of Nrf2 during oxidative stress. Once again, mutational studies were used to generate Nrf2 mutants that had an impaired ability to induce ARE-mediated transcription (154,155). Dinkova-Kostova et al. proposed an alternative mechanism by which interactions with thiol groups present in the binding domain of Keap1 are disrupted by enzyme inducers. When Keap1 was exposed to sulforaphane and bis(2-hydroxyben-zylidene)acetone, the number of free thiol residues was decreased, suggesting a direct interaction with electrophiles at key sites on Keap1. The authors propose that the thiol-containing domains present on Keap1 are the sensors that allow for the dissociation of Keap1 from Nrf2, which potentiates the induction of phase II detoxification enzymes (156).

D. The In Vivo Function of Nrf2

Mice null for Nrf2(—/—) show reduced expression of several key enzymes such as GSTs, at both the mRNA and protein level, when compared to wild-type mice (Nrf2+/+). Moreover, the characteristic loss of GST expression increased the sensitivity of Nrf2 (—/—) mice to xenobiotics such as butylated hydroxyanisole and acetaminophen (157,158,160). Thimmulappa et al. using an oligonucleotide microarray approach, have identified Nrf2-regulated genes in Nrf2 (—/—) and Nrf2 (+/+) mice exposed to sulforaphane (159). Comparative analysis of treatment groups identified several genes previously showed to be induced by sulforaphane; among these were NQO1, GST, GCS, and UGT. In addition, these investigators identified several groups of genes involved in xenobiotic detoxification, antioxidant function, and biosynthetic enzymes involved in GSH and glucuronic acid pathways. Moreover, many of these enzymes were express only in the Nrf2 (+/+) mice.

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