PERSPECTIVE
The Saga of Leucine Zippers Continues
In Response to Oxidative Stress

Leo E. Otterbein and Augustine M. K. Choi

Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Oxidative stress results from either overproduction of reactive oxygen species (ROS), attenuation of endogenous enzymatic and nonenzymatic antioxidant defense systems, or both. The cellular and molecular adaptive responses to oxidative stress involve increased expression of antioxidant enzymes, phase II detoxification enzymes, and stress-inducible cytoprotective genes aimed at reversing the oxidant imbalance and achieving cellular homeostasis. The ability and efficacy of these adaptive responses to achieve and maintain redox homeostasis in large part dictate whether the cell or organ will survive the oxidant burden. Central to these adaptive responses are the activation of specific signal transduction pathways, which then regulate selective and specific induction of antioxidant and detoxifying enzymes, and cytoprotective genes to confer cytoprotective functions. A vital component of these signaling pathways are the critical role of transcription factors which regulate the coordinate induction of antioxidant enzymes and cytoprotective genes. Transcription factors represent the "key" to activating and regulating downstream effector genes which provide cytoprotection, and many of them are sensitive to changes in cellular redox state or oxidative stresses. The redox-sensitive transcription factors, nuclear factor (NF)-B and activator protein (AP)-1, are two of the most prominent regulators of cellular responses to oxidative stress.

The family of AP-1 transcription factors has attracted much attention for numerous investigators, as they play critical roles in regulating target genes involved not only in adaptive responses to oxidative stress, but also in the regulation of gene products involved in a plethora of biologic processes, including, but not limited to, inflammation, cell differentiation and proliferation, and cell death or apoptosis. AP-1 belongs to the family of basic region/leucine zipper (bZIP) transcription factors (1), which are characterized by a basic domain required for sequence-specific DNA binding activity and a region containing heptad repeats of leucine and hydrophobic residues required for dimerization to other bZIP protein family members. For instance, the "classic AP-1" family of transcriptional activators include Jun-Jun or Jun-Fos dimers, which bind to the 12-O tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE) consensus sequence TGAC/GTC/AA (see Table 1). The classification of bZIP family of proteins became more complex by the discovery of the antioxidant response element (ARE) in 1991 by Pickett and colleagues (2). The ARE is a unique cis-acting regulatory sequence found in the 5' regulatory region of genes encoding enzymes involved in the phase II metabolism of xenobiotics (2, 3). For example, phase II enzymes such as glutathione S-transferase (GST) Ya subunit genes and NAD(P)H:quinone oxidoreductase (NQO1) contain the ARE in the 5' upstream region, and induction of these enzymes resulting from cellular exposure to ROS is regulated by the activation of the ARE (2, 3). Pickett and colleagues suggested that the sequence 5'-GTGACNNNGC'3', where N is any nucleotide, represents the core sequence of the ARE, is required for transcriptional activation by ROS, and may represent a major signaling pathway by which cells sense and respond to oxidative stress (Table 1). This ARE core sequence is similar to the sequence recognized by the "classic AP-1" family members, and many investigators hypothesized that members of the Jun and Fos families of transcription factors may be binding to the ARE sequence genes such as the GST and NQO1 genes. This hypothesis was supported by studies illustrating the induction of Jun and Fos in response to oxidative stress (1, 4). However, recent data clearly show that transcription factors distinct from the "classic AP-1" family members of Jun and Fos regulate transcriptional activation of the ARE. For example, several studies suggest that the transcription factor Nrf2 plays an important role in regulating ARE-mediated gene transcription (5). Nrf2 is a member of the "cap `n' collar" family of bZIP transcription factors (CNC-bZIP), highly homologous to the NF-E2 transcription factors (10- 12). Transcription factor NF-E2 was originally identified as an erythroid cell-specific DNA binding protein which recognizes the consensus sequence TGCTGAG/CTCAT/C, which contains the TPA responsive element TGAG/CTCA, and exhibits high similarity to the consensus sequence of the ARE (12) (Table 1). Unlike the erythroid cell specific NF-E2, Nrf2 is expressed in a variety of tissues including the lung (10). Nrf1, another member of the CNC-bZIP family of transcription factors, is much less characterized to date. The complexity of the ARE transcriptional complex is further highlighted by recent studies demonstrating that Nrf2 can also heterodimerize with small Maf proteins forming the ARE binding complex (13). Maf family proteins (MafF, MafG, and MafK) recognize DNA sequence motif called MARE 5'-TGCTGAG/CTCAGCA) (Table 1). The function of Nrf2-maf protein dimers is not known, but it has been proposed that Nrf2-maf dimers act both as positive and negative regulators of gene transcription (16).

In this issue of AJRCMB, Cho and coworkers (20) demonstrate the physiologic function of Nrf2 using an in vivo model of oxidant-induced lung injury. Hyperoxia alone enhanced steady-state levels of Nrf2 mRNA expression in the lungs of mice exposed to hyperoxia, suggesting that Nrf2 can behave as a stress response gene in response to hyperoxic stress. Other bZIP proteins, such as Fos and Jun and activating transcription factor 4 (ATF4), have also been shown to be directly induced by cellular stresses (4, 5, 21). It is interesting to note that high basal levels of Nrf2 mRNA expression were observed in the Nrf2^(+/+) wild-type mice, confirming earlier observations of high levels of Nrf2 mRNA expression in the lung of developing mice embryo (10). In situ hybridization studies of lung tissue from mice embryo localized abundant Nrf2 mRNA expression in the bronchial and alveoli epithelium (10). Furthermore, Cho and coworkers show that hyperoxia can enhance not only the mRNA expression of Nrf2 but also the functional activity of Nrf2. Using oligonucleotide probes specific for NF-E2 and ARE, hyperoxia increases the protein-DNA complex formation in the lung after exposure to hyperoxia. Supershift analysis using Nrf2 antibodies was inconclusive. Most importantly, using Nrf2^(/) null mice, the authors clearly show that the Nrf2^(/) null mice were more susceptible to hyperoxic lung injury compared with Nrf2^(+/+) wild-type mice as assessed by epithelial cell injury markers, macrophage inflammation, and pulmonary permeability. The protective effects of Nrf2 against hyperoxic stress complements earlier observations made by Kan and colleagues that Nrf2^(/) null mice exposed to another oxidant such as BHT exhibited more severe acute lung injury and lower levels of mRNA expression for antioxidants, including NQO1, UGT, catalase, and SOD1, when compared with Nrf2^(+/+) wild-type mice (22). Ishii and colleagues have also shown that peritoneal macrophages isolated from Nrf2^(/) null mice exhibited decreased expression of oxidative stress-inducible genes such as heme oxygenase-1 (HO-1), stress protein A170, and peroxiredoxin MSP 23 (23). Cho and colleagues further showed that many of the ARE-regulated antioxidant enzymes and phase II enzymes were also modulated in Nrf2^(/) null mice after hyperoxia. Hyperoxia-induced mRNA levels of NQO1, GST, UGT, Gpx2, and HO-1 were significantly lower in the Nrf2^(/) null mice compared with Nrf2^(+/+) wild-type mice. This modulation of phase II enzymes by hyperoxia and Nrf2 provides insights to the functional role of phase II detoxifying enzymes in hyperoxic lung injury.

Activation of Nrf2 by ROS does not accompany its transcriptional induction; rather, ROS liberate Nrf2 from Keap1, a recently-identified cytoskeleton-associated factor which binds to the N-terminal of Nrf2 and negatively regulates Nrf2 activity (24). Under normal conditions, Nrf2 exists in an inactive state in the cytoplasm, in part or fully as a consequence of binding to Keap1. After exposure to electrophiles or ROS, Nrf2 is transported to the nucleus by an unknown mechanism (Figure 1), but may involve PKC mediated phosphorylation of Nrf2 (25). The mechanism by which Nrf2-bZIP dimer complex (bZIP proteins: e.g., Maf, Fos, Jun, ATF4) translocate into the nucleus after being released from the inhibitory subunit Keap1 is poorly understood (Figure 1). This Nrf2-Keap1 pathway is similar to the well-characterized NF-B-mediated pathway. Furthermore, the delineation of the critical upstream signaling pathways which regulate Nrf2- and ARE-mediated gene transcription of antioxidant enzymes and cytoprotective genes will provide insights to the specificity and redundancy of cellular stress signals and downstream gene activation. Recent studies have implicated the role of the mitogen-activated protein kinase and PI-3 kinase pathways (26).

View larger version (83K): [in this window] [in a new window]   Figure 1.   Schematic diagram of the antioxidant stress response through Nrf2. Reactive oxygen species liberate Nrf2 from Keap1 into the nucleus. Nrf2 heterodimers with bZIP proteins (e.g. Maf, Fos, Jun, ATF), and binds to the ARE found in the 5' regulatory region of genes encoding for cytoprotective and detoxification enzymes such as HO-1, GST and NQO1.

This study of Cho and coworkers further supports the emerging paradigm that Nrf2-mediated gene expression represents a central and critical regulatory system to coordinate cellular defense against oxidative stress (22, 23, 29, 30). The coordinated Nrf2 and other bZIP-regulated expression of cytoprotective genes will be important not only in various ROS-related disease processes such as aging, carcinogenesis, atherosclerosis, ischemia, and neurodegenerative disorders, but also in a variety of lung disorders such as asthma, emphysema, interstitial lung disease, and adult respiratory distress syndrome.

Many challenges lie ahead as we start to unravel the complex and delicate regulatory elements which mediate a host's responses to oxidative stress. The ever-expanding family of bZIP proteins, from the "classic AP-1" to ARE to MARE to NFE2 to Nrf2 proteins, all discovered in the last decade, strongly suggests that more subfamily members of bZIP proteins will be uncovered in the future. Indeed, a recent study identified ATF4, a member of the bZIP family of proteins, as an Nrf2-interacting protein which modulated HO-1 gene transcription in response to oxidative stress (21). With this increasing number of distinct but overlapping cis acting regulatory elements mediating gene expression and transcription in response to oxidative stress, the bigger hurdle is to unravel the critical trans acting elements, identifying the precise hetero or homo dimers of bZIP proteins binding to the cognate regulatory sequences. Using state of the art techniques such as yeast 2 hybrid systems and chromatin in vivo gel shift assays, we will undoubtedly not only unravel new bZIP proteins and new interactions between known bZIP proteins, but will also disprove some of the old "classical AP-1" bZIP proteins mediated gene transcription. With more rigorous experimentation, one should not be surprised if we learn in the future that some of the "classical" AP-1-mediated events may indeed be due to either ARE or Nrf2 or Maf-mediated events. To avoid unnecessary confusion in the literature, we must be cautious in hastily making conclusions on AP-1-mediated gene regulation, before rigorous examination of other bZIP proteins involved in the transcriptional response to oxidative stress. Importantly, we need to also identify the target genes of bZIP proteins, either individually or collectively, which confer the functional cytoprotection against oxidative stress. We are poised to tackle this challenge in that we can use approaches such as microarray, SAGE, or proteomics to identify target genes and gene products using in vitro and in vivo models of either overexpression or deletion of these specific bZIP transcription factors. The saga of leucine zippers marches on: will it ever be completely zipped?

	Footnotes

Address correspondence to: Augustine M. K. Choi, M.D., Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, MUH 628NW, 3459 5th Avenue, Pittsburgh, PA 15213. E-mail: choiam{at}msx.upmc.edu

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