Cross-talk between Transcription Factors AhR and Nrf2: Lessons for Cancer Chemoprevention from Dioxin

Throughout life, humans are subjected episodically to numerous stressors, including ultraviolet irradiation, products of combustion, pesticides, herbicides, heavy metals, and other environmental pollutants, as well as various toxicants ingested in food, such as phytochemicals in edible plants; pyrolysis products in cooked meat; and mycotoxin contaminants in cereals, nuts, and maize. To ensure survival in the face of such challenges, mammalian cells have evolved a variety of inducible genetic programs that enable them to adapt to the presence of harmful xenobiotics. These programs entail upregulation of discrete batteries of genes for drug-metabolizing enzymes, drug transporters, and various cytoprotective proteins that allow an increased rate of xenobiotic elimination from the body, restoration of normal homeostasis, and removal of damaged macromolecules. Among transcription factors that mediate adaptation to foreign compounds, the aryl hydrocarbon receptor (AhR) and nuclear factor-erythroid 2–related factor 2 (Nrf2) have been widely studied. It is well established that the AhR is responsible for induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) of genes that contain a xenobiotic response element (XRE, also sometimes called a dioxin response element) in their promoter regions. It is also clear that Nrf2 is responsible for induction by structurally diverse electrophiles and quinones of genes that contain an antioxidant response element (ARE, also sometimes called an electrophile response element) in their promoter regions. For many years, the pathways leading to induction of XRE-driven genes and induction of ARE-driven genes were thought to be entirely separate. In particular, TCDD was assumed to activate transcription essentially only through the XRE. In the highlighted paper, however, Klaassen and colleagues have presented strong in vivo evidence that TCDD can stimulate cross-talk between AhR and Nrf2, insofar that they have demonstrated induction by dioxin of numerous mouse glutathione S-transferase (Gst) genes that are not known to contain functional XREs in their upstream regulatory regions (Yeager et al., 2009). The ability of compounds such as TCDD and PAHs to induce the expression of members of the cytochrome P450 (CYP)1A family with aryl hydrocarbon hydroxylase activity, and thereby increase the rate of metabolism of aromatic xenobiotics, has been known for at least 40 years. Activation of CYP genes by TCDD or PAH is mediated by AhR, a ligand-activated transcription factor that is a member of the basic helix-loop-helix/Per-Arnt-Sim family (Gu et al., 2000). The AhR is normally retained in the cytoplasm in a complex with heat-shock protein 90, ARA9, and p23, and upon ligand binding, it translocates to the nucleus where it is recruited to XRE sequences in the promoters of target genes as a heterodimer with AhR nuclear translocator, another basic helix-loop-helix/Per-Arnt-Sim family member (Gu et al., 2000). Figure 1 shows the structures of TCDD along with the PAHs benzoapyrene and 3-methylcholanthrene, the flavonoids β-naphthoflavone and quercetin, and the planar phytochemical indolo3,2-bcarbazole, all of which serve as ligands for the AhR (Nguyen and Bradfield, 2008). In the mouse, the classic AhR target genes are Cyp1a1, Cyp1a2, Cyp1b1, aldehyde dehydrogenase 3a1 (Aldh3a1), Gst alpha 1 (Gsta1), NAD(P)H:quinone oxidoreductase 1 (Nqo1), and UDP-glucuronosyl transferase 1a6 (Ugt1a6). Structures of xenobiotics that induce XRE- and/or ARE-driven gene expression. Recognition that oxidizable polyphenols and a diverse range of thiol-reactive soft electrophiles induce drug-metabolizing enzymes has its origins in the cancer chemoprevention field. From the mid-1970’s onwards, agents that inhibit chemical carcinogenesis, such as butylated hydroxyanisole (BHA), ethoxyquin, oltipraz, and sulforaphane, were found in the mouse to upregulate microsomal epoxide hydrolase, Gsta1, Gsta2, Gsta3, Gsta4, Gstm1, Gstm2, Gstm3, Gstm4, Gstp1, Gstp2, and Nqo1 (reviewed by Hayes et al., 2005; Holtzclaw et al., 2004). As some of the genes that are induced by BHA are also induced by TCDD, it was initially a matter of some controversy as to whether they are regulated in an AhR-independent manner. However, it is now accepted that Nrf2, a cap‘n’collar (CNC) basic region leucine zipper (bZIP) protein, mediates induction of these genes by BHA and other chemopreventive agents. In this case, the CNC-bZIP factor is recruited to ARE sequences in the promoters of target genes as a heterodimer with small MAF proteins. Unlike AhR, transcription factor Nrf2 is not ligand activated, rather it responds to xenobiotics and endogenous compounds that are thiol reactive and thus generate a redox signal. Inducing agents increase the nuclear abundance of Nrf2 by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a substrate adaptor protein for the cullin-3 ubiquitin ligase. Under normal homeostatic conditions, Keap1 represses Nrf2 by facilitating its proteasomal degradation. Inhibition of Keap1 involves modification of critical Cys residues in the protein that prevent it from serving as a ubiquitin ligase substrate adaptor (McMahon et al., 2006). Besides mediating induction of drug-metabolizing enzymes, an important function of Nrf2 is to regulate antioxidant proteins, such as those involved in glutathione biosynthesis (i.e., glutamate-cysteine ligase, glutathione reductase, and the cystine transporter), glutathione peroxidase, thioredoxin and thioredoxin reductase, sulfiredoxin, the stress-protein heme oxygenase 1, and 26S proteasomal subunits (reviewed by Hayes and McMahon, 2009). Early studies on the mechanisms by which xenobiotics induce drug-metabolizing enzymes categorized TCDD, PAHs, and β-naphthoflavone as bifunctional inducers because they increased both Cyp1a1 and Nqo1 enzyme activities. By contrast, the BHA metabolite tert-butyl-hydroquinone, along with oltipraz and sulforaphane (all shown in Fig. 1) were classed as monofunctional inducers because they increased only Nqo1 enzyme activity and not Cyp1a1 activity (Holtzclaw et al., 2004). It is now generally accepted that bifunctional inducers activate both XRE- and ARE-driven genes, while monofunctional inducers activate only ARE-driven genes. Given the fact that PAHs and β-naphthoflavone can be oxidized by CYP isoenzymes to generate metabolites that are electrophilic or can redox cycle, it is possibly not surprising that they could stimulate both XRE- and ARE-driven gene expression; in such instances, it was thought that the parental compound would stimulate transcription through the XRE, while the metabolite, or oxidative stress produced as a consequence of CYP enzyme activity, would stimulate transcription through the ARE. However, the conclusion that TCDD can activate ARE-driven gene expression demands further explanation because being relatively inert it will not produce electrophilic metabolites. Upon reflection, it seems likely that TCDD can induce ARE-driven genes by at least two indirect mechanisms. First, bidirectional cross-talk appears to exist at a genetic level between AhR and Nrf2; the gene promoter of Nrf2 contains at least one functional XRE (Miao et al., 2005), and the gene promoter of AhR contains several AREs (Shin et al., 2007). Using mouse liver Hepa 1c1c7 cells, Miao et al. (2005) showed that TCDD can increase the level of messenger RNA for Nrf2 and its protein level several fold. This is a surprising result because in the absence of stress, Keap1 ought to be capable of targeting newly translated Nrf2 for ubiquitylation and proteasomal degradation. Clearly, the reason why TCDD antagonizes repression of Nrf2 requires further investigation. A second possible mechanism through which TCDD might induce ARE-driven gene expression is by stimulation of a protein-protein interaction between AhR and Nrf2 that increases the stability of the CNC-bZIP factor. Recent evidence suggests that ligand-activated AhR can interact with various transcription factors, such as estrogen or androgen receptors, and modulate their activities (Ohtake et al., 2009). Possibly, a similar relationship exists between AhR and Nrf2, but evidence that the two transcription factors can physically interact is lacking at present. However, it was reported many years ago that treatment of Hepa 1c1c7 cells with TCDD resulted in an increase in a nuclear complex that binds specifically to a synthetic ARE sequence in an electrophoretic mobility shift assay and that antibodies against AhR can prevent formation of the ARE-binding complex (Vasiliou et al., 1995). It therefore seems possible that the AhR may associate with protein complexes bound to the ARE, but the functional significance of this association is unclear. Several research groups have examined induction of Nqo1 by TCDD in the mouse. Both Prochaska and Talalay (1988) and Ma et al. (2004) demonstrated that TCDD induced Nqo1 in murine Hepa 1c1c7 cells but not in Hepa 1c1c7-derived cells lacking a functional AhR. Furthermore, Ma et al. (2004) also demonstrated that while TCDD could induce Nqo1 in wild-type mouse embryonic fibroblasts, it was unable to do so in equivalent cells from Nrf2-null mice. In the highlighted paper, Yeager et al. (2009) have demonstrated clearly that induction of Nqo1 by TCDD in the livers of wild-type C57BL/6 mice is accompanied by an increase in the amount of nuclear Nrf2 protein and a corresponding increase in ARE-binding activity. Using knockout mice, these workers have also provided the first in vivo evidence that induction of Nqo1 requires both AhR and Nrf2. An important aspect of the work described by Yeager et al. (2009) is that they have demonstrated that Gstm1, Gstm2, Gstm3, Gstm6, Gstp2, Gstt2, and MGst1 are induced by TCDD and that this is dependent on Nrf2. As none of these class Mu, Pi, Theta, and microsomal transferase genes are known to contain an XRE, it appears that their induction by TCDD occurs via another mechanism. The fact that induction of these Gst genes requires Nrf2 implicates AREs in the process, but further work is required to confirm this hypothesis. The paper by Yeager et al. (2009) opens the way for many other lines of investigation. For example, it will be necessary to establish how TCDD stimulates accumulation of Nrf2 protein: is it merely due to induction of the Nrf2 gene or does it also entail a protein-protein interaction between AhR and either Nrf2 or Keap1 or does it also involve redox-dependent inactivation of Keap1? If AhR and Nrf2 proteins are capable of physically interacting, is the interaction influenced by ligand binding or redox stress? Assuming AhR and Nrf2 interact, can the receptor be recruited to ARE sequences in gene promoters, and if so, which genes? In particular, does TCDD activate all ARE-driven genes or just a subset of such genes? Do other AhR ligands besides TCDD induce ARE-driven genes? Lastly, it may be worth revisiting the issue of whether monofunctional inducers, such as tert-butyl-hydroquinone and sulforaphane, that activate Nrf2 might attenuate the activity of AhR and influence the expression of XRE-driven genes. The discovery of functional cooperation between AhR and Nrf2 has significant biological implications. Historically, the AhR has been associated with carcinogenesis, whereas Nrf2 is associated with cytoprotection against degenerative diseases. The future challenge is to determine whether cross-talk between these two transcription factors can be exploited to therapeutic advantage or to improve chemopreventive strategies.