5 Transcriptional Mediators of Cellular Hormesis

Transcriptional Mediators of Cellular Hormesis Tae Gen Son, Roy G. Cutler, Mark P. Mattson, and Simonetta Camandola Abstract Hormesis is the beneficial adaptive response of cells and organisms to acute subtoxic doses of certain types of environmental stressors (e.g., heat, oxida- tion, environmental toxins). Repetitive hormesis through routine exercise, calorie restriction, or ingestion of low levels of phytotoxins with the diet can stimulate cel- lular catabolic turnover of damaged molecules and increase protective mechanisms. The net result is an improved ability of the organism to better cope with noxious insults (i.e., preconditioning). Key to the benefits of hormesis are (1) the intensity of the stress/toxin, which needs to be enough to stimulate an effective response without causing permanent damage (i.e., subtoxic) and (2) the duration of the expo- sure, which needs to be limited (acute) to allow repair and recovery. Fundamental to the hormetic adaptive response is gene expression regulation. Although different stressors elicit unique signature responses, the comparison of prototypical hormetic inducers has highlighted the role played by a few transcription factor families. The periodic pulsatile activation of Nrf2, NF-κB, HSF, and FOXO has been found to be essential to obtaining the beneficial effects of various hormetic stimuli in different biological models. This chapter discusses molecular mechanisms and gene targets for these transcription factor families in the hormetic adaptive context. Keywords Transcription factors · Hormesis · Exercise · Calorie restriction · Phytochemicals · HSF · Nrf2 · FOXO · NF-κB Introduction Exposing organisms and cells to brief periods of mild stress renders them more resistant to the potential challenges of a subsequent, even greater stress. This phe- nomenon, known as hormesis, depends on the ability of cells and organism to upregulate their stress response–induced gene expression and the related pathways T.G. Son (B) Laboratory of Neurosciences, National Institutes of Health, National Institute on Aging, Baltimore, MD 21224, USA e-mail: sont2@grc.nia.nih.gov 69M.P. Mattson, E.J. Calabrese, Hormesis, DOI 10.1007/978-1-60761-495-1_4, C© Springer Science+Business Media, LLC 2010 70 T.G. Son et al. of maintenance and repair. The validity of the hormetic concept has been provided by experiments with various biological systems and by using various chemical, physical, and biological stressors. Paradigms of hormetic stressors are exercise, calorie restriction, temperature shock, irradiation, and pro-oxidants (Minois, 2000; Rattan, 2004). Exercise represents an oxidative and metabolic stressor for the skele- tal muscle and cardiovascular systems by generating free radicals, acids, and other damaging intermediates (McArdle et al., 2002). However, or rather because of it, moderate daily exercise clearly improves the quality of life and prevents dis- ease (Rattan, 2004). In a similar way, organisms perceive nutrient limitation as a metabolic stress, yet calorie restriction has been shown to extend the lifespan and decrease morbidity in organisms as evolutionarily distant as Caenorhabditis elegans and primates (Kirkwood and Shanley, 2005; Bishop and Guarente, 2007a). The ability of cells and organisms to adapt to short- and long-term environmental changes by modifying their gene expression is achieved through several transcrip- tional and posttranscriptional mechanisms, including chromatin remodeling and transcription; cell signaling; mRNA splicing, polyadenylation, and localization; and mechanisms of protein localization, modification, and degradation. Because tran- scription is a pivotal event in most adaptive stress responses, much emphasis and effort have been placed on understanding transcriptional regulation mechanisms. Different stressors elicit unique expression patterns according to the nature, mag- nitude, and duration of the insult. As the signature mechanisms responsible for their effects have been unraveled, it has become increasingly clear that most, if not all, stress conditions share a conserved core response. Categories of stress-induced genes include those encoding heat-shock proteins and antioxidant enzymes and proteins involved in energy metabolism. Genes for which expression is repressed by stress include those involved in growth-related functions, thus reflecting a redirection of resources toward stress protection. In this chapter we review the basic concept of transcriptional regulation and sum- marize recent knowledge on the transcription factor families that are emerging as key players in the adaptive hormetic response. Nature of Transcriptional Regulation Eukaryotic DNA resides in the nucleus in 23 pairs of chromosomes containing 6 × 109 base pairs and interacts with thousands of specific DNA-binding transcription factors. The basic chromatin structure is the nucleosome unit. Each nucleosome consists of a core histone octamer formed by a central heterotetramer of histones H3 and H4 sandwiched between a pair of H2A and H2B heterodimers. Each histone octamer has approximately 147 base pairs of supercoiled DNA wrapped around it, and the nucleosomes are separated by variable, species-specific DNA linkers of 28 to 43 base pairs. The chromatin fiber is folded into a more condensed 30-nm struc- ture by a single molecule of histone H1 bridging together nucleosomal units by binding at the beginning, center, and end of the nucleosomal DNA. Multiple orders Transcriptional Mediators of Cellular Hormesis 71 of coiling then give rise to the compact 100- to 400-nm “coiled-coil” structure of interphase chromatin called heterochromatin. Both the coiled-coil and nucleoso- mal structures are stable at the physiological ionic strength and prevent unwanted gene transcription. The transcriptional activation of gene expression is achieved through decondensation and chromatin remodeling. The less compact structure of the transcriptionally active chromatin—euchromatin—allows access of appro- priate transcription factors to localize sequence-specific regions on target genes (Fig. 1). Once bound to their cognate DNA sequences, the transcription factors recruit coactivator proteins with histone acetylase activity to further loosen the chromatin structure and modify the nucleosome structure, allowing sliding of the chromosomal DNA. The additional recruitment of several basal transcription fac- tors then gives rise to the formation of the preinitiation complex at the transcription start site and assists the RNA polymerase II during the synthesis of the hnRNA (Fig. 1). Control of chromatin organization and binding to cis-regulatory sequences represent the major check points in transcriptional regulation. h1 h1 h1 h1 h1 AAAAAA A B DNA Nucleosome TF gTF RNApol PIC Fig. 1 Schematic representation of transcriptional activation of gene expression. A. Chromatin remodeling enzymes and histone acetylases cause decondensation of the DNA structure at the level of nucleosomes. B. Recruitment of transcription factors (TF) to specific accessible DNA-binding sites attracts coactivator and general transcription factors (gTF) to assist in the formation of the preinitiation complex (PIC) and transcription by RNA polymerase II (RNApol) Hormetic Signaling Pathways The application of genome-wide approaches has begun to provide a global view of gene expression responses to many different stress conditions (Murray et al., 2004; Kultz, 2005; Bahn et al., 2007). Signaling pathways that have began to emerge as 72 T.G. Son et al. common denominators between various insults include the Nrf2/ARE, FOXO, HSF, and NF-κB pathways. Nuclear Factor–Erythroid 2p45 (NF-E2)–Related Factor (Nrf2)/Antioxidant Response Element (ARE) Signaling Pathway Over several hundred million years of evolution, cells and organisms have developed a system of antioxidants and phase II detoxifying enzymes to protect them against the nefarious effects of electrophiles and omnipresent reactive oxygen species. Several studies have led to the identification of a common cis-acting enhancer ele- ment, 5′-GTGACnnnGC-3′, known as the antioxidant responsive element (ARE) or electrophile responsive element (EpRE), within the 5′ flanking regulatory regions of antioxidant and detoxifying genes (Surh, 2003; Shen et al., 2005). Among the ARE-responsive genes are glutathione-S-transferase (GST), glutathione reductase, epoxide hydrolase, aldehyde reductase, UDP-glucuronyl transferase (UGT), heme oxygenase-1 (HO1), NADP(H):quinone oxidoreductase (NQO1), thioredoxin, and γ-glutamylcysteine synthetase (γ-GCS) (Surh, 2003). The transcription factor that binds to the ARE consensus sequence and regulates the activation of ARE-target genes is Nrf2. Nrf2, Keap1, and Regulation of the ARE Pathway Nrf2 was isolated in 1994 as a factor binding to the NF-E2 sequence of the β-globin locus promoter region (Moi et al. 1994). It belongs to the “cap ‘n’ collar” (CNC) subfamily of the basic leucine zipper transcription factors (Zhang, 2006; Kensler et al., 2007). The family includes the closely related proteins p45 NF-F2, Nrf1, Nrf2, and Nrf3, plus two distantly related transcriptional repressors, Bach1 and Bach2 (Zhang, 2006; Kensler et al., 2007). Like other leucine zipper proteins, Nrf2 can- not bind to the ARE consensus sequence as a monomer or homodimer and must heterodimerize with members of the small Maf protein family for efficient DNA binding and transactivation activity (Itoh et al., 1997). Beside sMaf proteins, in vitro studies have shown that Nrf2 may form heterodimers with c-Jun and ATF4 (Venugopal and Jaiswal, 1998; He et al., 2001). Although the interaction with these proteins may be important in certain conditions (Venugopal and Jaiswal, 1998; He et al., 2001), their significance in vivo is unclear. Very little is known about the mechanisms regulating Nrf2 dimerization, the binding to the DNA, and the trans- activation activity. The interaction with Bach 1 and Bach 2 has been shown to downregulate the expression of HO-1 and ARE-dependent reporter gene expres- sion (Sun et al., 2002; Muto et al., 2002), leading to the assumption that they are Nrf2 transcriptional repressors. On the other hand, Nrf2 is able to bind the coac- tivator CREB-binding protein (CBP or p300), which in turn strongly enhances its transactivation activity (Katob et al., 2001). The best-understood Nrf2 regulatory Transcriptional Mediators of Cellular Hormesis 73 mechanism is the interaction with the negative regulator Kelch-like ECH-associated protein 1 (Keap1) (Zhang, 2006; Kensler et al., 2007). Keap1 contains two major domains, an N-terminal broad complex/tramtrack/bric-a-brac (BTB) domain, and a C-terminal double-glycine repeat domain (DGR), separated by an intervening region. The DGR domain is essential for binding to the actin cytoskeleton and to the N-terminal of Nrf2 (Zhang, 2006; Kensler et al., 2007). The BTB domain is required for Keap1 homodimerization and for interaction with the Cullin3/Rbx1 E3 ubiquitin ligase (Kobayashi et al., 2004). Based on structural data, it is believed that in the absence of activators, Keap1 homodimers bind Nrf2 and actin, caus- ing the retention of Nrf2 in the cytoplasm (Zhang, 2006; Kensler et al., 2007). Through the binding to Cul3/Rbx1 E3 ligase, Nrf2 is targeted for ubiquitination and subsequent proteasomal degradation (Kobayashi et al., 2004) (Fig. 2). On expo- sure to electrophilic compounds or reactive oxygen species, Nrf2 dissociates from Keap1, thus eluding degradation, and translocates in the nucleus, where it medi- ates the activation of ARE-target genes (Zhang, 2006; Kensler et al., 2007) (Fig. 2). Cytoplasm Nucleus Electrophiles, ROS, Phytochemicals ARE Target genes sMaf Nrf2 P CBP Nrf2 Keap1 Keap1 SH SH SH SH cul3 Nrf2 Keap1 Keap1 SHSH SHSH SHSH SHSH cul3 Nrf2 Ub Ub Ub Ub Nrf2 Ub Ub Ub Ub Proteasome Degradation SR SR Keap1 SRSR SRSR Keap1 S-----S Keap1 S-----S Keap1 Nrf2 Nrf2 Nrf2 Nrf2 Nrf2 Fig. 2 Mechanism of induction of Nrf2-dependent genes. Under basal conditions, Nrf2 is bound to Keap1, retained in the cytosol, and targeted for proteasomal degradation via association with Cullin3/Rbx1–E3 ubiquitin ligase (Cul3). Inducers cause the release of Nrf2 from Keap1, allowing escape from degradation and nuclear relocalization. In the nucleus, Nrf2 binds to its cognate ARE site in association with small Maf protein members (sMaf), together with the coactivator protein (CBP), and regulates the expression of target genes 74 T.G. Son et al. Because Keap1 is highly enriched in cysteine residues, it is believed to be an oxida- tive/electrophilic sensor. Many of the 27 cysteine residues are reactive and potential target sites for a direct interaction with electrophilic compounds (Nguyen et al., 2004; Kobayashi and Yamamoto, 2005). Cell-free experiments demonstrated that four cysteine residues located in the intervening region—Cys257, Cys273, Cys288, and Cys297—are extremely reactive and a direct target of certain phase II induc- ers, leading to Keap1/Nrf2 dissociation (Dinkova-Kostova et al., 2002). In addition, Cys273 and Cys288 are required for ubiquitylation and subsequent degradation by the proteasome (Zhang and Hannink, 2003). The importance of Keap1 in the reg- ulation of Nrf2 stability is substantiated by observations in vivo. Keap1 knockout mice survive only 3 weeks after birth (Wakabayashi et al., 2003). Of interest, liver and embryonic fibroblasts from these mice exhibit increased expression of phase II detoxifying enzymes and constitutively higher levels of nuclear Nrf2 (Wakabayashi et al., 2003). Hormetic Inducers of the Nrf2/ARE Pathway Together with genes for antioxidants and phase II detoxification enzymes, Nrf2 has been shown to regulate genes involved in cell growth and apoptosis, inflam- mation, and the ubiquitin-mediated degradation pathway (Kwak et al., 2003; Lee et al., 2003a; Cho et al., 2005). Several in vitro and in vivo studies had demon- strated that Nrf2 is fundamental for the defense against reactive oxygen species and the pathogenesis of lung, hepatic, and neurodegenerative diseases. A better understanding of the functions, targets, and inducers of Nrf2/ARE-mediated gene expression has been achieved with the generation of Nrf2-deficient mice (Ramos- Gomes et al., 2001). Loss of Nrf2 decreases constitutive and inducible target gene expression, enhancing the sensitivity of Nrf2-deficient mice to carcinogenesis and oxidative stress (Chan et al., 2001; Ramos-Gomez et al., 2001; Cho et al., 2002). Nrf2-deficient mice have a significantly higher risk of benzo[a]pyrene-induced gas- tric neoplasia than do wild-type mice, due to the reduction of constitutive hepatic and gastric activities of GST and NQO1 (Ramos-Gomes et al., 2001). Hyperoxia- induced expression of NQO1, GST, UGT, glutathione peroxidase-2 (GPx2), and HO1 is significantly lower in Nrf2-deficient mice (Cho et al., 2002). Lack of Nrf2 sensitizes neurons and astrocytes to oxidative stress by decreasing constitutive and inducible protective genes (Lee et al., 2003a; Lee et al., 2003b). In Caenorhabditis elegans the homolog of Nrf2, SKN1, integrates stress tol- erance responses with energy metabolism homeostasis, regulating lifespan (Tullet et al., 2008). Recently Tullet et al. (2008) showed that the insulin/IGF1 path- way directly regulates SKN1, with unique functions in different tissues. Restricted expression of SKN1 in ASI neurons (putative neuroendocrine cells) mediates calorie restriction–induced extension of lifespan, suggesting that its role in these neurons is to tune the systemic responses to nutrition (Bishop and Guarente, 2007b). It is intriguing that intestinal SKN1 responds to environmental stress by promoting the expression of phase II genes, and its prolongevity effect is distinct from its ASI- mediated role in calorie restriction (An and Blackwell, 2003; Bishop and Guarente, Transcriptional Mediators of Cellular Hormesis 75 2007). It is possible that the stress and tissue specific sensitivity and differential function of SKN1/Nrf2 observed in worms are conserved in higher vertebrates. Notably, we recently observed that in ARE-hPAP reporter mice, starvation causes a significant upregulation of hPAP Nrf2-driven expression in liver but not in the cerebral cortex (Fig. 3). In contrast to the situation in worms, in mice Nrf2 seems to mediate calorie restriction anticarcinogenic properties, but apparently it is dispens- able for its prolongevity benefits (Pearson et al., 2008). The discrepancy observed between worms and mice on how calorie restriction extends lifespan could be due to different mechanisms of IGF1/insuling-signaling regulation of Nrf2. -actin hPAP Liver hPAP -tubulin Cortex Ba lb/C AR E-h PA P AR E-h PA P AR E-h PA P ST Fig. 3 Tissue-specific effects of starvation on Nrf2 activation. ARE-driven human placental alkaline-phosphatase mice (ARE-hPAP) were subjected to 2 days of starvation (ST). Levels of hPAP were measured by Western blot analysis in extracts from liver and cerebral cortex using actin and tubulin as loading controls Several phytochemicals with beneficial health effects have been shown to medi- ate Nrf2 activity. Sulforaphane is an isothiocyanate present in high amounts in broccoli sprouts and cruciferous vegetables (Myzak and Dashwood, 2006). Sulforaphane’s anticarcinogenic and protective effects are, at least partially, due to its ability to activate the Nrf2/ARE pathway. Indeed, in vivo gene-expression analysis comparing wild-type mice and Nrf2-null mice led to the identification of specific sulforaphane upregulated ARE-dependent target genes (Thimmulappa et al., 2002). Sulforaphane protects cultured neurons against oxidative stress (Kraft et al., 2004) and dopaminergic neurons against mitochondrial toxins (Han et al., 2007). Administration of sulforaphane to mice can protect photoreceptors against degeneration in a retinal degeneration model (Kong et al., 2007). From a mech- anistic point of view, sulforaphane acts at different levels of the Nrf2 pathway. It suppresses Nrf2 proteasomal degradation, leading to a prolonged half-life and transcriptional activity (Jeong et al., 2005), and directly covalently binds the thiol groups in the inhibitor Keap1, thus causing the release of Nrf2 and its subsequent nuclear relocalization (Dinkova-Kostova et al., 2002). The antioxidant carnosol 76 T.G. Son et al. induces HO1 expression and activates ARE- reporter activity in PC12 cells in a phosphatidylinositol 3 kinase (PI3K)– and AKT-dependent fashion (Martin et al., 2004). Treatment of endothelial cells with epigallocatechin-3-gallate (EGCG), the major constituent of green tea, increases Nrf2 nuclear levels and upregulates HO1 expression (Wu et al., 2006). Resveratrol increases the activities of catalase, super- oxide dismutase, glutathione peroxidase, NQO1, and GST and upregulates Nrf2 and induces its translocation to the nucleus (Hsieh et al., 2006; Rubiolo et al., 2008). In addition, to the phytochemicals cited previously, several other natural compounds have been identified as inducers of the Nrf2/ARE pathway, including quercetin (Tanigawa et al., 2007), curcumin (Kang et al., 2007), phenethyl isothiocyanate (Son et al., 2008), and chalcone (Son et al., 2008). Forkhead Box O (FOXO) Transcription Factors The mammalian orthologs of C. elegans DAF-16 [a forkhead/winged-helix tran- scription factor, box O (FOXO)] transcription factors FOXO1, FOXO3a, FOXO4, and FOXO6 function as tumor suppressors and energy metabolism rheostats and are essential proteins for animals to reach their genetic maximum lifespan (Libina et al., 2003). The expression of FOXO-regulated genes can be controlled by any of the transcription factors, and specificity is achieved either by expression-pattern or by isoform-specific regulation. We will therefore