Validation of the multiple sensor mechanism of the Keap1-Nrf2 system

Abstract

The Keap1-Nrf2 system plays a critical role in cellular defense against electrophiles and reactive oxygen species. Keap1 possesses a number of cysteine residues, some of which are highly reactive and serves as sensors for these insults. Indeed, point mutation of Cys151 abrogates the response to certain electrophiles. However, this mutation does not affect the other set of electrophiles, suggesting that multiple sensor systems reside within the cysteine residues of Keap1. The precise contribution of each reactive cysteine to the sensor function of Keap1 remains to be clarified. To elucidate the contribution of Cys151 in vivo, in this study we adopted transgenic complementation rescue assays. Embryonic fibroblasts and primary peritoneal macrophages were prepared from mice expressing the Keap1-C151S mutant. These cells were challenged with various Nrf2 inducers. We found that some of the inducers triggered only marginal responses in Keap1-C151S-expressing cells, while others evoked responses in a comparable magnitude to those observed in the wild-type cells. We found that tert-butyl hydroquinone, diethylmaleate, sulforaphane, and dimethylfumarate were Cys151 preferable, whereas 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PG-J₂), 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im), ebselen, nitro-oleic acid, and cadmium chloride were Cys151 independent. Experiments with embryonic fibroblasts and primary macrophages yielded consistent results. Experiments testing protective effects against the cytotoxicity of 1-chloro-2,4-dinitrobenzene of sulforaphane and 15d-PG-J₂ in Keap1-C151S-expressing macrophages revealed that the former inducer was effective, while the latter was not. These results thus indicate that there exists distinct utilization of Keap1 cysteine residues by different chemicals that trigger the response of the Keap1-Nrf2 system, and further substantiate the notion that there are multiple sensing mechanisms within Keap1 cysteine residues.

Introduction

The transcription factor Nrf2 (NF-E2-related factor 2) plays an important role in cellular defense against electrophilic and oxidative insults [1], [2]. Under unstressed conditions, the cellular level of Nrf2 is maintained at a low level, as Keap1 (kelch-like ECH-associated protein 1), an adaptor component of Cul3 (Cullin 3)-based ubiquitin E3 ligase complex, promotes ubiquitination and proteasomal degradation of Nrf2. On exposure to electrophiles or reactive oxygen species, the ubiquitination of Nrf2 ceases, leading to the stabilization and nuclear translocation/accumulation of Nrf2 and the subsequent induction of Nrf2 target genes [1]. A variety of Nrf2 inducers have been reported, most of which are electrophilic and readily react with cysteine thiols [3]. Keap1 is a cysteine-rich protein possessing 27 and 25 cysteine residues in the human and mouse proteins, respectively. A number of studies detected covalent modifications of Keap1 cysteine residues following exposure to electrophiles using in vitro labeling and mass spectrometry [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The functional significance of these cysteine residues has been examined in several experimental systems exploiting site-directed mutagenesis of Keap1. The function of mutant Keap1 molecules has been tested by transfection into culture cell lines (in transfecto) or by ectopic overexpression in zebrafish embryos [8], [9], [11], [14], [15], [16], [17]. We also established the significance of three cysteine residues, i.e., Cys151, 273, and 288, for the regulation of Nrf2 activity using a transgenic approach [18].

Of the Nrf2-inducing chemicals, tert-butylhydroquinone (tBHQ) is readily autooxidized to the electrophilic metabolite tert-butylbenzoquinone and modifies the cysteine residues of Keap1 [9], [13]. Because cells and mice exclusively expressing the Keap1-C151S mutant showed decreased accumulation of Nrf2 in response to tBHQ [8], [14], [18], Cys151 has been assumed critical for the Keap1 sensor activity against tBHQ. It has also been suggested that diethylmaleate (DEM), ebselen, dimethylfumarate (DMF), sulforaphane (SFN), and 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im) require Cys151 to trigger the Keap1-Nrf2 response in vitro or in transfecto [8], [14], [17], [19], [20], although several conflicting results have also been reported [5], [12].

On the other hand, the presence of Cys151-independent Nrf2 inducers has also been recognized. For instance, the cells or zebrafish embryos expressing the Keap1-C151S mutant responded to 15-deoxy-△^12,14-prostaglandin J₂ (15d-PGJ₂) at levels comparable to those of wild-type cells or zebrafish embryos. Furthermore, exposure of Keap1 to 15d-PGJ₂ resulted in covalent modification of Cys273 but not Cys151 [8]. Similarly, heavy metals such as cadmium chloride (CdCl₂) and arsenic compounds activated Nrf2 signaling in a Cys151-independent manner [9], [16]. These observations suggest that the modification of distinct cysteine residues results in a common downstream consequence; namely Keap1 halts to ubiquitinate Nrf2. Thus, we surmise that there are multiple sensor mechanisms within the cysteine residues of Keap1. However, critical target cysteine residues have not been identified for each compound in vivo, so that the precise contribution of each reactive cysteine residue to the sensor function of Keap1 remains to be clarified.

One of the critical parameters affecting the responsiveness of the Keap1-Nrf2 system is the expression ratio of Keap1 and Nrf2 proteins. Different ratios produced in overexpression experiments in transfecto gives rise to different outcomes. Indeed, as for the necessity of the BTB (broad complex-tramtrack-bric a brac) domain of Keap1, it has been difficult to draw a conclusion based on overexpression experiments. In this regard we recently developed a transgenic complementation rescue assay in which the expression levels of Keap1 and Nrf2 were regulated to within a range comparable to that in wild-type mice, and demonstrated the necessity of the BTB domain in vivo [18].

In order to rigorously verify the validity of the multiple cysteine sensor system for the Keap1-Nrf2 system, in this study we systematically and comprehensively examined the responsiveness of the C151S mutant of Keap1 against a variety of Nrf2 inducers exploiting the transgenic complementation rescue approach. The results demonstrated the presence of Cys151-preferable and Cys151-independent inducers of Nrf2 in vivo, indicating that various chemical inducers utilize reactive Keap1 cysteine residues distinctly. This study has established a basis for the analysis of in vivo function of Keap1 and Nrf2 inducers.