Tributyltin activates the Keap1–Nrf2 pathway via a macroautophagy-independent reduction in Keap1

Abstract

Tributyltin (TBT) is an environmental chemical, which was used as an antifouling agent for ships. Although its use has been banned, it is still persistently present in ocean sediments. Although TBT reportedly causes various toxicity in mammals, few studies on the mechanisms of biological response against TBT toxicity exist. The well-established Keap1–Nrf2 pathway is activated as a cytoprotective mechanism under stressful conditions. The relationship between TBT and the Keap1–Nrf2 pathway remains unclear. In the present study, we evaluated the effect of TBT on the Keap1–Nrf2 pathway. TBT reduced Keap1 protein expression in Neuro2a cells, a mouse neuroblastoma cell line, after 6 hr without altering mRNA expression levels. TBT also promoted the nuclear translocation of Nrf2, a transcription factor for antioxidant proteins, after 12 hr and augmented the expression of heme oxygenase 1, a downstream protein of Nrf2. Furthermore, TBT decreased Keap1 levels in mouse embryonic fibroblast (MEF) cells, with the knockout of Atg5, which is essential for macroautophagy, as well as in wild-type MEF cells. These results suggest that TBT activates the Keap1–Nrf2 pathway via the reduction in the Keap1 protein level in a macroautophagy-independent manner. The Keap1–Nrf2 pathway is activated by conformational changes in Keap1 induced by reactive oxygen species or electrophiles. Furthermore, any unutilized Keap1 protein is degraded by macroautophagy. Understanding the novel mechanism governing the macroautophagy-independent reduction in Keap1 by TBT may provide insights into the unresolved biological response mechanism against TBT toxicity and the activation mechanism of the Keap1–Nrf2 pathway.

INTRODUCTION

Tributyltin (TBT) compounds were previously used in antifouling paint, which prevents barnacles and other marine organisms from adhering to the bottom of ships. The use of TBT was subsequently banned because it causes imposex in snail species, including Thais clavigera (Kotake, 2012). Even though the use of TBT has been banned, it persists in ocean sediments because of its long half-life. TBT can enter the human food chain via the consumption of marine organisms that have taken up TBT. In addition to inducing reproductive organ abnormalities in snails, TBT is reportedly immunotoxic (Li and Li, 2020) and obesogenic (Chen et al., 2021; da Costa et al., 2019). There are also prior studies on the neurotoxicity of TBT in rats, including inducing behavioral abnormalities (Ema et al., 1991). Studies have reported that concentration of TBT in human blood varies from tens to hundreds nM (Whalen et al., 1999; Kannan et al., 1999). Therefore, there are concerns regarding its plausible effects on mammals, including humans, as well as marine organisms.

Several studies have been conducted to understand the mechanisms of TBT toxicity. TBT exerts potent agonist action on the nuclear receptors peroxisome proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), causing endocrine disruption by their activation (le Maire et al., 2009; Nakanishi, 2008; Hiromori et al., 2009). TBT causes thymic atrophy by starving cortical thymocytes and T-cells (Raffray and Cohen, 1993; Ueno et al., 2009). Furthermore, we have shown previously that TBT reduces the expression of ionotropic glutamate receptor 2 (GluA2), one of the subunits of the α-amino-3-hydroxy-5-mesoxazole-4-propionic acid (AMPA)-type glutamate receptor, which in turn increases intracellular Ca²⁺ influx, promoting neuronal fragility (Nakatsu et al., 2006, 2009). Furthermore, we have previously reported that TBT induces lysosomal dysfunction and reduces cell viability in human neuroblastoma SH-SY5Y cells (Hatamiya et al., 2022).

When cells are exposed to various stressors, multiple defense mechanisms are activated. The activation of Keap1–Nrf2 pathway protects cells from various stimuli such as electrophiles and oxidative stress. NF-E2-related factor 2 (Nrf2) is a pZip transcription factor that belongs to the Cap 'n' Collar family (Motohashi et al., 2002). Nrf2 has seven different functional domains and promotes the transcription of various downstream factors via nuclear transfer (Canning et al., 2015). Kelch-like ECH-associated protein 1 (Keap1) is a homodimeric protein containing three major domains with different roles. Keap1 and Nrf2 have domains that bind to each other, with the Kelch domain of Keap1 and Neh2 domain of Nrf2 (Baird and Dinkova-Kostova, 2011). Keap1 also forms a complex with the Cullin scaffold protein Cullin3 (Cul3) and the Ring protein Rbx1 to form the E3 ubiquitin ligase. Under normal conditions, Keap1 binds to Nrf2 and negatively regulates Nrf2 expression by proteasomal degradation (Baird and Dinkova-Kostova, 2011; Baird and Yamamoto, 2020). However, in the presence of electrophilic substances and oxidative stress, many reactive cysteines of the Keap1 protein react to stress-inducing substances and lead to a disruption of the Keap1 structure. Consequently, the bond between Keap1 and Nrf2 is broken and Nrf2 is translocated into the nucleus. This in turn promotes the transcription of various antioxidant factors, resulting in a stress response (Baird and Yamamoto, 2020; Yamamoto et al., 2008; Takaya et al., 2012).

No prior studies have been conducted to evaluate the effects of TBT on the Keap1–Nrf2 pathway. Evaluating these effects will provide further information on mechanisms of TBT toxicity and biological response to TBT toxicity, which have not been elucidated. In the present study, the effects of TBT on the Keap1–Nrf2 pathway in mouse neuroblastoma Neuro2a cells were investigated.

MATERIALS AND METHODS

Reagents

TBT (T0363) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethyl sulfoxide (DMSO) (043-07216) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Cell Counting Kit-8 (CK04) and cytotoxicity LDH assay kit-WST (CK12) were purchased from Dojindo Laboratories (Kumamoto, Japan). Bullet Blocking One for western blotting (13779-14), Chemi-Lumi One L (07880-54), One super (02230-14), and One ultra (11644-24) were purchased from Nacalai Tesque Ltd. (Kyoto, Japan). Skimmed milk was purchased from Meiji Co., Ltd. (Tokyo, Japan). Can Get Signal Solution 1 (NKB-201), Solution 2 (NKB-301), SuperPrep II Cell Lysis & RT Kit for qPCR (SCQ-401), and KOD SYBR qPCR Mix (QKD-201) were obtained from TOYOBO Co., Ltd. (Osaka, Japan).

Cell culture

Wild-type (WT), autophagy-related 5 (Atg5) knockout (KO) mouse embryonic fibroblast (MEF) cells (Kuma et al., 2004), and mouse neuroblastoma Neuro2a cell line were purchased from RIKEN BioResource Research Center (Ibaraki, Japan). Neuro2a cells, WT MEF cells, and Atg5 KO MEF cells were cultured following a previously published protocol, which describes the methods for culturing HeLa cells (Yabuki et al., 2021), with slight modifications. Neuro2a cells were seeded at a density of 3.5 × 10⁴ cells/cm² on uncoated 100 mm dishes in all experiments.

Drug treatment

TBT was dissolved in DMSO and adjusted to a concentration of 0 and 700 nM. Cells were exposed to TBT for 3, 6, 12, and 24 hr. The final concentration of DMSO in the culture medium was adjusted to 0.1%.

Cell-viability and cell-toxicity assay

Cell viability and cytotoxicity studies were performed following previous studies (Yabuki et al., 2021) using CCK8 and LDH assay kits.

Antibodies

Rabbit anti-Nrf2 (sc-13032), mouse anti-LaminB1 (sc-377000), mouse anti-vinculin (sc-73614), and mouse anti-heme oxygenase 1 (sc-390991) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Mouse anti-Keap1 (M224-3) and mouse anti-Atg5 (PM050) were purchased from Medical & Biological Laboratories Co., Ltd. (Tokyo, Japan). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A9169) and HRP-conjugated anti-mouse IgG (A9044) were purchased from Sigma-Aldrich Co. LLC. (St Louis, MO, USA).

Whole-cell protein extraction

Neuro2a cells, WT, and Atg5 KO MEF cells were seeded on 100 mm dishes. Whole-cell protein extraction was performed using the method based on a prior study (Miyara et al., 2016b) with a few modifications.

Nucleocytoplasmic fractionation

Nucleocytoplasmic fractionation was performed based on prior studies (Yabuki et al 2021; Ishida et al., 2017) with a few modifications.

Western blotting

Western blotting was performed according to prior studies (Yabuki et al., 2021; Miyara et al., 2016a) with a few modifications. Membranes were blocked at room temperature for 1 hr or 5 min with tris-buffered saline containing 0.1% tween 20 (TBS-T) + 5% skimmed milk (blocking buffer) or Bullet Blocking One. Thereafter, they were washed with TBST and incubated overnight at 4°C with primary antibodies diluted in blocking buffer or Can Get Signal Solution 1. The membrane-bound primary antibodies were detected by incubating the membranes for 1 hr at room temperature with secondary antibodies diluted in blocking buffer or Can Get Signal Solution 2.

RT-qPCR analysis

Neuro2a cells were seeded in 96-well microplates. Cell lysis, reverse transcription, and real-time PCR were performed following a previous study (Yabuki et al., 2021) with slight modifications. The sequences of the primers used in the present study are as follows: mouse keap1 forward 5'-CAGGTACACACTAGAGGATCACA-3', mouse keap1 reverse 5'-GTGGATGCCTTCGATGGACA-3', mouse GAPDH forward 5'-ACTAACATCAAATGGGGTGAC-3', mouse GAPDH reverse 5'-ATTGCTGACAATCTTGAGTGA-3'.

Statistical analysis

All results are expressed as mean ± standard deviation (S.D., n = 3 or 4, independent experiments). Statistical significance was analyzed using Tukey’s post-hoc test, utilizing Mini-StatMate software (ATMS, Chiba, Japan). Statistical significance was set at p < 0.05.

RESULTS

TBT decreased cell viability in Neuro2a cells

In this study, we used TBT (700 nM), as it has been previously reported that this concentration causes cell death and impairs lysosomal function in SH-SY5Y cells within 24 hr of exposure. To determine the toxicity of TBT to Neuro2a cells, the cells were exposed to a culture medium containing 0 and 700 nM TBT for 6 and 24 hr, and cell viability and lactose dehydrogenase (LDH) release in culture were measured. TBT (700 nM) did not alter cell viability after 6 hr of treatment; however, after 24 hr of treatment, cell viability was significantly reduced compared with that of the control cells (Fig. 1a). TBT treatment also significantly increased the amount of LDH released into the culture medium after 24 hr compared with those released by the control cells (Fig. 1b). These results suggested that treatment with 700 nM TBT for 24 hr caused cytotoxicity in Neuro2a cells.

Fig. 1

TBT reduces cell viability in Neuro2a cells. a.b) Neuro2a cells were exposed to 0 and 700 nM TBT for 6 and 24 hr. Cell viability was measured using the Cell Counting Kit-8 (CCK8) assay kit. Cytotoxicity was determined by measuring the activity level of LDH, which is released from the cells upon cell membrane damage. Data are represented as mean ± S.D. from three independent experiments. ***p < 0.001 compared to control.

TBT promoted nuclear translocation of Nrf2 and increased the expression of an antioxidant protein in Neuro2a cells

To examine whether TBT affects the Keap1–Nrf2 pathway, we first evaluated changes in the nuclear translocation of Nrf2 following TBT treatment. Neuro2a cells were exposed to 0 and 700 nM TBT for 3, 6, 12, and 24 hr; thereafter, the nuclear accumulation of Nrf2 protein was measured using western blotting. Nrf2 protein could not be detected in the cytoplasmic fraction, probably because of continuous degradation by the proteasome (Sekhar et al., 2002). A significant increase in the nuclear migration of Nrf2 was observed in the nuclear fraction at 12 and 24 hr after TBT exposure (Fig. 2a, b). Next, we examined whether TBT affects the expression of proteins transcriptionally regulated by Nrf2. Heme oxygenase 1 (HO-1), one of the downstream factors of Nrf2, is upregulated by various stimuli, including nitric oxide, heavy metals, and cytokines (Loboda et al., 2016). Neuro2a cells were exposed to 0 and 700 nM TBT for 3, 6, 12, and 24 hr; thereafter, the protein expression level of HO-1 was evaluated using western blotting. The results showed a significant increase in HO-1 protein expression at 12 hr after TBT exposure (Fig. 2c, d). These results suggested that TBT activates the Keap1–Nrf2 pathway in Neuro2a cells.

Fig. 2

TBT promotes nuclear translocation of Nrf2 and increases the expression of antioxidant proteins in Neuro2a cells. a.b) Neuro2a cells were treated with 0 and 700 nM TBT for 3, 6, 12, and 24 hr. Subsequently, Neuro2a cells were collected separately from the nucleus and cytoplasm. The protein expression level of Nrf2 was assessed using western blotting. Vinculin was used as a loading control for the cytoplasmic fraction and Lamin B1 as a loading control for the nuclear fraction. Data are represented as the mean ± S.D. from three independent experiments. ***p < 0.001 compared to the control. c.d) Neuro2a cells were treated with 0 and 700 nM TBT for 3, 6, 12, and 24 hr. The protein expression level of HO-1 was assessed using western blotting. The protein expression of vinculin was measured as a loading control. Data are represented as the mean ± S.D. from three independent experiments. ***p < 0.001 compared to the control.

TBT rapidly decreased Keap1 protein expression in a Keap1 mRNA-independent manner

Keap1 negatively regulates Nrf2 expression by binding to Nrf2 under normal conditions. Under conditions of stress, the binding between Keap1 and Nrf2 is disrupted, which promotes the nuclear translocation of Nrf2 (Taguchi et al., 2011). Herein, we examined the effect of TBT on Keap1 expression. After exposure of Neuro2a cells to 0 and 700 nM TBT for 3, 6, 12, and 24 hr, the expression level of Keap1 protein was assessed using western blotting. TBT significantly reduced the expression of Keap1 protein at 6, 12, and 24 hr after TBT exposure (Fig. 3a, b). Next, we tested the changes in Keap1 mRNA expression after TBT exposure to examine whether the reduction in Keap1 protein expression after exposure was dependent on Keap1 mRNA expression levels. Neuro2a cells were exposed to 0 and 700 nM TBT for 3, 6, 12, and 24 hr and Keap1 mRNA was analyzed using RT-qPCR. The results showed that TBT did not alter Keap1 mRNA expression levels (Fig. 3c). These results indicated that TBT promotes the loss of Keap1 protein without affecting the expression level of Keap1 mRNA in Neuro2a cells.

Fig. 3

TBT rapidly reduces Keap1 protein expression in a Keap1 mRNA-independent manner. a.b) Neuro2a cells were treated with 0 and 700 nM TBT for 3, 6, 12, and 24 hr. The protein expression level of Keap1 was assessed using western blotting. Vinculin was used as a loading control. Data are represented as the mean ± S.D. from three independent experiments. *p < 0.05, **p < 0.01 compared to control. c) Neuro2a cells were treated with 0 and 700 nM TBT for 3, 6, 12, and 24 hr. The mRNA expression of Keap1 was assessed using RT-qPCR. GAPDH mRNA level was used as an internal standard. Data are represented as the mean ± S.D. from three independent experiments.

The reduction in Keap1 protein expression following TBT exposure is independent of macroautophagy

Keap1 contains a DC domain that binds to the macroautophagy substrate p62 (SQSTM1) and is widely reported to be degraded by macroautophagy (Zhang et al., 2005; Ichimura et al., 2013; Taguchi et al., 2012). Therefore, we investigated whether the loss of Keap1 induced by TBT exposure is because of macroautophagy degradation. Atg5 plays a crucial role in the formation of autophagosomes, which are essential for macroautophagy (Matsushita et al., 2007). Atg5 KO and WT MEF cells were treated with 0 and 700 nM TBT-containing medium for 6 and 24 hr respectively, and the expression levels of Keap1 protein were evaluated using western blotting. No difference in the level of reduction in Keap1 protein was found in Atg5 KO MEF cells compared with those in WT MEF cells at 6 and 24 hr after TBT exposure (Fig. 4a, b). These results suggested that the TBT-induced Keap1 protein loss is independent of macroautophagy.

Fig. 4

The reduction in Keap1 protein expression following TBT exposure was independent of macroautophagy. a.b) Atg5 KO MEF cells and WT MEF cells were treated with 0 and 700 nM TBT for 6 and 24 hr. The expression level of Keap1 protein was assessed using western blotting. Vinculin was used as a loading control. Data are represented as the mean ± S.D. from three independent experiments. *p < 0.05, **p < 0.01 compared to control.

DISCUSSION

Various chemicals, including methyl mercury (MeHg), activate the Keap1–Nrf2 pathway. However, whether organotins, such as TBT, activate the Keap1–Nrf2 pathway has not been studied earlier. In the present study, we investigated the effect of TBT on the Keap1–Nrf2 pathway in Neuro2a and MEF cells. In Neuro2a cells, 700 nM TBT activated the Keap1–Nrf2 pathway before it caused cell death. Furthermore, TBT rapidly promoted the decrease in Keap1 protein level before the translocation of Nrf2 to the nucleus. We also demonstrated that the reduction in Keap1 protein expression was independent of macroautophagy because TBT exposure also reduced Keap1 protein expression in Atg5 KO MEF cells.

Herein, we demonstrated that TBT facilitates the nuclear transfer of Nrf2 (Fig. 2). In the presence of reactive oxygen species (ROS), Keap1 forms intramolecular disulfide bonds around Cys226, 613, and 622/624, and the conformational change in Keap1 leads to a disruption of Keap1–Nrf2 binding (Suzuki et al., 2019). In the presence of electrophiles, the binding of electrophiles to various sensor cysteines, mainly Cys151, 273, and 288 of Keap1, promotes the disruption of binding between Keap1 and Nrf2 (Baird and Yamamoto, 2020; Sekhar et al., 2010; Kumagai et al., 2013). It is reported that TBT does not generate ROS under exposure concentration conditions similar to those in the present study (Nakatsu et al., 2007). There are no previous studies on TBT covalently binding to Cys residues. TBT reduces the Keap1 protein level before it promotes the nuclear translocation of Nrf2 (Fig. 2, 3); therefore, TBT may activate Nrf2 by degrading the Keap1 protein. This would be irrespective of the conventional activation mechanism elucidated, which states that the Keap1 protein changes its conformation and dissociates its bond from Nrf2.

In the present study, it was demonstrated that TBT decreases the Keap1 protein level irrespective of macroautophagy, widely known as the Keap1 protein degradation pathway (Fig. 4). When Hepa1c1c7 cells are exposed to fenofibrate, Keap1 is degraded via macroautophagy approximately 18 hr after exposure (Park et al., 2015). It has also been reported that ROS are generated in the liver of mice 16 hr after fasting and refeeding and that Keap1 is degraded by p62-mediated macroautophagy (Bae et al., 2013). Therefore, most previous studies indicate that the degradation of the Keap1 protein starts relatively later. In the present study, the degradation of Keap1 started much earlier after exposure to TBT in comparison to the findings of prior studies, which suggests that the mechanism of degradation of Keap1 by TBT is different from that established previously. Keap1 is ubiquitinated when exposed to oxidative stress; however, it is not degraded by the proteasome (Takaya et al., 2012; Zhang et al., 2005). It was also reported that Keap1 is degraded by chaperone-mediated autophagy (CMA) under long-term oxidative stress conditions and serum deprivation (Zhu et al., 2022). In macroautophagy, autophagy-associated proteins form autophagosomes, which transport waste proteins to lysosomes where they are degraded. In CMA, the heat shock cognate chaperone of 70 kDa (hsc70) and lysosome-associated membrane protein type 2A (LAMP-2A), lysosomal membrane protein receptor, transfer unutilized proteins directly into lysosomes for their degradation (Wu et al., 2015). Our previous studies have shown that TBT disrupts lysosomal function (Hatamiya et al., 2022), suggesting that CMA may not contribute to the degradation of Keap1 by TBT. Therefore, TBT might have lowered the Keap1 protein level through a new pathway of disappearance, which is different from that described in prior studies.

In the present study, we found that TBT activates the Keap1–Nrf2 pathway before causing cell death (Fig. 1–3). In our study, Nrf2 significantly increased the expression of HO-1 protein after nuclear translocation. HO-1 is an important cytoprotective protein that not only releases potent antioxidants but also produces factors that regulate cell growth and apoptosis (Loboda et al., 2016). Several prior studies state that activation of the Keap1–Nrf2 pathway protects neurons from MeHg-induced toxicity in SH-SY5Y cells and in vivo animal models (Kumagai et al., 2013; Yoshida et al., 2014). Furthermore, naphthoquinone activates the Keap1–Nrf2 pathway, which acts as a biological response in HepG2 cells and primary mouse hepatocytes (Miura et al., 2011; Abiko et al., 2021). Therefore, activation of the Keap1–Nrf2 pathway may have been instrumental in protecting cells against TBT toxicity. In contrast, mutations in Keap1 and aberrant activation of Nrf2 hrave also been reported to promote oncogenesis and cause toxicity (Taguchi et al., 2011; Ohta et al., 2008). Therefore, we need to examine the relationship between TBT-induced activation of the Keap1–Nrf2 pathway and cytotoxicity more minutely.

In the present study, we demonstrated that TBT activates the Keap1–Nrf2 pathway before causing cell death. We also found that TBT reduced Keap1 protein expression by macroautophagy-independent pathways considerably faster than promoting the nuclear translocation of Nrf2. This raises the possibility that TBT rapidly degrades Keap1 protein through a novel mechanism, thereby activating the Keap1–Nrf2 pathway. There have been few studies on the biological response mechanisms to TBT toxicity and/or detailed time-course studies on the reduction in the Keap1 level and nuclear transfer of Nrf2. TBT may cause severe toxicity if exposed to mammals for long-term exposure. Research on the mechanisms of biological response to TBT toxicity is essential. Further clarification of the degradation mechanism of Keap1 protein will provide an innovative perspective on the mechanisms of biological response to TBT toxicity and also delineate a novel mechanism of Nrf2 activation.

ACKNOWLEDGMENTS

We thank Prof. Noboru Mizushima for providing WT and Atg5−/− MEF cells. We would like to thank Editage (www.editage.com) for English language editing. The present study was funded by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research(B) 20H04342 (to Y.K).

Conflict of interest

The authors declare that there is no conflict of interest.

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