Nrf2 Activation and Its Coordination with the Protective Defense Systems in Response to Electrophilic Stress

Molecular responses mediated by sensor proteins are important for biological defense against electrophilic stresses, such as xenobiotic electrophile exposure. NF-E2-related factor 2 (Nrf2) has an essential function as a master regulator of such cytoprotective molecular responses along with sensor protein Kelch-like ECH-associated protein 1. This review focuses on Nrf2 activation and its involvement with the protective defense systems under electrophilic stresses integrated with our recent findings that reactive sulfur species (RSS) mediate detoxification of electrophiles. The Nrf2 pathway does not function redundantly with the RSS-generating cystathionine γ-lyase pathway, and vice versa.


Nrf2 Activation under Electrophilic Stress
Nrf2 is a key regulator of cellular defenses against oxidative, electrophilic and environmental stresses. In the nucleus, Nrf2 forms a dimer with the small Maf protein (sMaf) and binds to the CNC-sMaf binding element (CsMBE) (formerly described as the antioxidant/electrophile response element (ARE/EpRE)) located in the regulatory regions of many genes responsible for cellular defense [21,22]. Nrf2 cooperatively regulates antioxidant proteins; such as heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL), to synthesize glutathione (GSH); phase-II xenobiotic detoxifying enzymes, such as GSH S-transferase (GST) and UDP-glucuronosyltransferase (UGT); and phase-III xenobiotic transporters, such as multidrug resistance-associated protein (MRP) [21,[23][24][25][26][27][28]. GSH is an abundant sulfhydryl found in various tissues that plays an important role in detoxification of electrophiles via GSH adduct formation. Although the pKa value of GSH is relatively high (9.12), indicating that about 2% of GSH exists as GS -, its deprotonated form that interacts with electrophiles, GST is able to lower this pKa value, thereby facilitating GSH adduct formation with electrophiles [29,30]. Such a polar metabolite is associated with detoxification of electrophiles because the GSH adduct is then excreted into the extracellular space through MRP [31]. These findings suggest that Nrf2 is essential for repression of environmental electrophile-mediated toxicity through GSH adduct formation and its excretion into the extracellular space.
Nrf2 is regulated by Kelch-like ECH-associated protein 1 (Keap1), an adaptor subunit of Cullin 3-based E3 ubiquitin ligase (Cul3). Under normal conditions, Keap1 binds to Nrf2 in the cytoplasm and promotes the ubiquitination and proteasomal degradation of Nrf2 and, thus, acts as a negative regulator of Nrf2 [32][33][34]. Keap1 is also a sensor protein for oxidative and electrophilic insults through the modification of its highly reactive cysteine residues. Thus, when the interaction between Nrf2 and Keap1 is disrupted, proteasomal degradation of Nrf2 decreases, causing de novo Nrf2 to build up within the cell, leading to increased translocation of Nrf2 into the nucleus (Figure 1). Under steady state conditions, Nrf2 is captured by Keap1 and constantly ubiquitinated by Cul3 in the cytosol leading to degradation through the ubiquitin-proteasome system and resulting in the inhibition of Nrf2 translocation from the cytoplasm to the nucleus (inset). Keap1 is modified through its highly reactive cysteine residues by electrophiles that are exposed exogenously or generated endogenously as second messengers, thereby diminishing its activity to hold Nrf2. As a result, Nrf2 newly synthesized translocates into the nucleus and interacts with a partner protein sMaf resulting in formation of a heterodimer that binds to the CsMBE (formerly known as ARE or EpRE), thereby upregulating its downstream genes that are involved in cellular defense (e.g., MRP, HO-1 and GCL). Autophagosomal degradation of Keap1 is also upregulated through its electrophilic modifications. Dotted gray arrows indicate processes disrupted by electrophilic stresses.
The cysteine code is an attractive point to understand the nature of Keap1 function as the sensor for electrophilic compounds. But the sensing mechanism is complicated. Saito et al. analyzed the specificity of Keap1 cysteine residues against chemical inducers of Nrf2 using mutants of three major cysteine residues, Cys 151, Cys273 and Cys288 of Keap1 [54]. They classified Nrf2 inducers into four classes: class I (Cys151 preferring), class II (Cys288 preferring), class III (Cys151/Cys273/Cys288 collaboration preferring) and class IV (Cys151/Cys273/Cys288 independent). However, it seems difficult to find the chemical consistency of each class. For example, both 15-deoxy-prostaglandin J 2 (15d-PGJ 2 ) and prostaglandin A 2 (PGA 2 ) belong to the prostaglandin family and share similar structural characteristics; 15d-PGJ 2 and PGA 2 belong to class II and class IV, respectively. Diethyl maleate and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole, which differ greatly in structure and size, both belong to class I. Therefore, the complex properties of electrophiles may be involved in the determination of the Keap1 modification site(s). Keap1 may sense various electrophilic substances through the combination of various cysteine residues.

Autophagosomal Degradation of Keap1
Analysis of liver-specific Atg7 (autophagy related 7)-deficient mice revealed that p62 protein is abnormally accumulated and Nrf2 is constitutively activated when the function of autophagy is disrupted. In this case the Keap1 protein is upregulated, suggesting that the Keap1 protein is constitutively degraded through the autophagy pathway [62]. Treatment of cells with TBQ, diethyl maleate (DEM) or 1,2-NQ accelerated the Keap1 degradation, indicating that Nrf2 accumulation is the dominant cause to provoke liver damage in autophagy-deficient mice. The autophagy pathway maintains the integrity of the Keap1-Nrf2 system for normal liver function by governing Keap1 turnover [63] (Figure 1).

Keap1-Independent Pathway
Recent findings have also revealed that Keap1-independent signal pathways might contribute to electrophile-induced Nrf2 activation and cytoprotective responses against electrophile exposure. Culbreth et al. found a Keap1-independent Nrf2 activation pathway in MeHg-exposed rat primary astrocytes, possibly involving the regulation of a member of the src family kinase named Fyn [64,65]. It has been reported that Fyn can phosphorylate Nrf2 favoring its export from the cell nucleus [66,67]. MeHg exposure in vitro increased gene expression of HO-1 and NQO1 as well as nuclear Nrf2 localization.
Oncogenic signal pathways are involved in Nrf2 expression at the transcription level. Oncogenic alleles of KRAS (kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1) and cMYC (avian myelocytomatosis virus oncogene cellular homolog) increase Nrf2 mRNA level through binding to TPA response element located in the Nrf2 promoter region [68][69][70]. Such an oncogene-induced Nrf2 transcription upregulates antioxidant and drug-resistant capacity in tumorigenic cells. In this sense, the application of Nrf2 inhibitors to anti-cancer treatment is of interest [68].

The Nrf2 and CSE Pathways: Parallel Contribution to the Repression of Electrophilic Stress
Reactive sulfur species (RSS) containing mobilized sulfur atom(s) bound to the thiol groups of CysSH and GSH or hydrogen sulfide (H 2 S) (e.g., CysSSH/CysSSSH, GSSH/GSSSH and HSSH/HSSSH) have been shown to be highly nucleophilic and antioxidative molecules [13,18,71]. The pKa value of RSS is lower than that of monosulfides, such as GSH and CysSH; e.g., CysSSH has a pKa value of 4.34 as predicted by chemical calculations [72]. This implies that RSS predominantly exists as the deprotonated form of SH (thiolate ion, S -) at physiological pH. Further, RSS such as GSSH and CysSSH, but not monosulfides such as GSH and CysSH, contain mobilized sulfur capable of capturing electrophiles. This leads to the formation of sulfur adducts [19,73,74].
While cystathionine γ-lyase (CSE) has been known as the final trans-sulfuration enzyme essential for cysteine biosynthesis from cystathionine [81], we reported that CSE can also catalyze the production of cysteine persulfide (CysSSH) from cystine (CysSSCys) as a substrate [18] (Figure 3). In turn, CysSSH spontaneously produces other reactive persulfide/polysulfide species, such as GSSH, and also H 2 S as a derivative [18,19]; thus, CSE act as an RSS generating enzyme (Figure 3). Mitochondrial cysteine-tRNA synthetase 2 (CARS2) also catalyzes formation of CysSSH from CysSH [82] (Figure 3). The metabolite analysis of wild-type (WT) and CSE knockout (KO) mice exposed to MeHg showed that (MeHg) 2 S was present at significant levels in various WT mouse tissues, but not at appreciable levels in CSE KO mouse tissues [79], suggesting that CSE is a critical enzyme involved in capturing xenobiotic electrophiles through formation of sulfur adducts in vivo. Furthermore, knockdown of CSE enhanced Cd-induced cytotoxicity, whereas overexpression of CSE protected cells from this electrophile-induced cytotoxicity [83]. In vivo studies have shown that CSE deletion in mice causes increased sensitivity to acetaminophen-induced hepatotoxicity, Cd-induced hepatotoxicity and MeHg-induced toxicity [20,77,84]. Overall, this suggests that CSE is a crucial factor engaged in the protection against electrophiles because this enzyme is involved in the production of RSS, thereby capturing electrophiles to yield their sulfur adducts [5,85] (Figure 3). Xenobiotic electrophiles selectively modify sensor proteins through thiolate ions at low doses and thus inhibit their activities. As a result, activation of effector molecules leads to a cellular adaptive response. However, a higher dose of xenobiotic electrophiles causes nonspecific protein modification, disrupting these signals, leading to cell death. Our previous studies confirmed such a dose-dependent transition, focusing on some sensor proteins that mediate cellular redox signaling pathways during exposure to xenobiotic electrophiles; phosphatase and tensin homologue (PTEN) modification by MeHg [11] and 1,4-NQ [75] as well as heat shock protein 90 (HSP90) by 1,4-NQ [12] and cadmium [77]. The fact that these xenobiotic electrophile-mediated redox signaling pathways and cellular toxicity are negatively regulated by RSS through the formation of their sulfur adducts, indicate that RSS act as the initial defense system for xenobiotic electrophile exposure [12,75,77] (Figure 3).

The Nrf2 and CSE Pathways Contribute to the Elimination of Electrophilic Stress in a Parallel Manner
Nrf2 plays a key role in the detoxification of electrophiles via formation of GSH adducts and subsequent excretion into extracellular spaces, whereas the CSE-mediated defense mechanism is facilitated through the sulfur adduct formation by RSS. These findings suggest that there is a non-canonical pathway of detoxification of environmental electrophiles conducted by CSE in addition to the canonical pathway regulated by Nrf2. In fact, CSE/Nrf2 double-KO mice and their hepatocytes were more sensitive to various electrophiles, such as MeHg, Cd, 1,4-NQ, crotonaldehyde and acrylamide, than their single-KO counterparts [20], indicating that both factors are involved in the repression of electrophile-induced toxicity in parallel pathways. Thus, not only does GSH adduct formation regulated by Nrf2 play a critical role in the protection against electrophiles but so does sulfur adduct formation regulated by CSE-producing RSS. Collectively, critical and parallel participation of Nrf2 and CSE in the protection against environmental electrophiles is like two sides of the same coin.
Incidentally, our study also revealed that CSE deletion did not substantially affect the cytotoxicity elicited by MeHg, 1,4-NQ, crotonaldehyde and acrylamide in primary mouse hepatocytes, in contrast to Nrf2 deletion [20]. A possible cause of this observation is that alternative defense pathway(s) may compensate for the lack of CSE and protect cells from the electrophilic damage. We found that CSE deletion enhanced Cd-mediated activation of Nrf2 and its downstream protein HO-1 in primary mouse hepatocytes [20]. Such a phenomenon may be also seen in MeHg and 1,4-NQ because of exposure of cultured cells to MeHg [46,65,86] and the 1,4-NQ-activated [87] Nrf2 signaling pathway. These findings suggest that the function of Nrf2 is potentiated in CSE deficient hepatocytes, thus protecting cells from xenobiotic electrophiles. Figure 4 shows the integration of our knowledge of the Nrf2 and CSE pathways. Upon electrophilic stresses, numerous studies confirmed that Nrf2 acts as a master regulator for cytoprotective molecules along with its activation mechanism through Keap1 as a sensor protein. CSE is another key factor implicated in the protection against electrophilic stresses. CSE is involved in RSS production, and works to capture electrophiles to yield their sulfur adducts. RSS-mediated protection against electrophiles appears to be a primary defense system compared with the Nrf2-dependent detoxification system with GSH because RSS are constitutively produced by CSE, whereas the gene expression of GCL, GST and MRP associated with conjugation with GSH and subsequent excretion of GSH adducts into the extracellular space are cooperatively regulated by Nrf2 [21,23]. As discussed above, the Nrf2 and CSE pathways do not function redundantly because CSE/Nrf2 double-KO mice and their hepatocytes were more sensitive to various electrophiles than their single-KO counterparts [20], though it is reported that the expression of CSE is, at least in part, transcriptionally regulated by ATF4 (activating transcription factor-4) [88,89]. Some studies suggested that Nrf2 is a positive regulator of ATF4 under the condition of electrophilic stress [90,91]. Thus, cross-talk between Nrf2 signaling and CSE expression might be mediated by ATF4 in response to electrophilic stresses. It is reported that RSS/ROS mediates 1,2-NQH 2 -SOH generation as sulfenic acid from 1,2-NQ, and 1,2-NQH 2 -SOH modifies Keap1 through Cys171, thereby activating Nrf2 [92]. Thus, RSS are able to affect Nrf2 activation through electrophile metabolic fate, and further studies are required to clarify the crosstalk between the Nrf2 and CSE pathways. . The Nrf2 and CSE pathways contribute to the elimination of electrophilic stress in a parallel manner. Nrf2 is a central coordinator for the induction of detoxifying genes that in turn are translated into proteins that are associated with GSH adduct formation and subsequent excretion into the extracellular space (e.g., GCL, GST and MRP). Aside from Nrf2, RSS generation mediated by CSE contributes to detoxifying electrophiles through sulfur adduct formation. For various electrophiles, upregulated genes and sulfur adducts have been uniquely identified (gray arrows).

Discussion
There are numerous examples of the beneficial effects elicited by pretreatment with Nrf2 activators as potential preventive therapeutics [93]. In fact, isothiocyanate and sulforaphane activated Nrf2 and upregulated downstream proteins associated with MeHg excretion, and suppressed mercury accumulation and intoxication in mice exposed to MeHg [94]. We speculate that directly trapping electrophiles by RSS would also be an effective strategy for electrophile detoxification. Therefore, the intake of foods and/or supplements containing not only Nrf2 activators but also sulfane sulfur-dependent chemicals may help to diminish the health risks of electrophilic stresses.
Funding: This work was supported by JSPS KAKENHI Grant Numbers JP18H05293 to Y.K., JP19K16368 to T.U., and JP18K14895 to M.A.

Acknowledgments:
We thank Renee Mosi (Canada), from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.