Elsevier

Toxicology Letters

Volume 262, 16 November 2016, Pages 135-141
Toxicology Letters

Atg5-dependent autophagy plays a protective role against methylmercury-induced cytotoxicity

https://doi.org/10.1016/j.toxlet.2016.09.007Get rights and content

Abstract

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. However, molecular mechanisms underlying MeHg toxicity remain elusive. We show that MeHg reduced mouse embryonic fibroblast (MEF) viability in a dose-dependent manner. Furthermore, MeHg treatment increased levels of autophagy markers LC3-II and p62, possibly by acting on the MAPKs signaling pathway in several cell types. MeHg exposure elevated the number of LC3 puncta in stable GFP-LC3 MEFs and the number of autophagic vacuoles. The accumulation of LC3-II and p62 increased further when complementing MeHg with autophagy inhibitor, chloroquine. Moreover, we found that autophagy-related gene 5-deficient (Atg5−/−) MEFs exhibited higher sensitivity and higher levels of p62 compared to their wild-type counterparts following MeHg exposure. This suggested that p62 was upregulated at the transcription level by MeHg and degraded by Atg5-dependent autophagy. Our data demonstrate that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity.

Introduction

Methylmercury (MeHg) is a global, highly lipophilic environmental pollutant (Driscoll et al., 2013). MeHg causes serious damage to various organs in humans and experimental animals. Consumption of contaminated fish and other aquatic seafood is reported to be the primary environmental source of MeHg exposure (Kim and Zoh, 2012, Li et al., 2010). MeHg is absorbed in the intestine and is readily distributed to various tissues in the body, including the most sensitive of targets—the brain—and the developing fetus. However, despite extensive research over the last half century, the molecular mechanisms underlying MeHg toxicity are not fully understood. As previously demonstrated, MeHg induces oxidative stress through an increase in intracellular reactive oxygen species (ROS), such as peroxide and superoxide anions (Shanker and Aschner, 2003). MeHg also displays unique affinity for thiol groups of proteins, such as Mn-superoxide dismutase, transporters, and tubulin (Kanda et al., 2014). Such MeHg-induced protein modification and/or ROS generation may lead to accumulation of damaged proteins and disruption of various biological processes, thus explaining the onset of MeHg toxicity.

Macroautophagy (hereafter referred to as autophagy), a major and highly conserved degradation pathway in eukaryotes, prevents the accumulation of misfolded or damaged proteins, protein aggregates, and damaged organelles in the cytoplasm (Mizushima, 2007). Cytoplasmic material is engulfed by double-membrane-bound structures (autophagosomes) and delivered to lysosomes for degradation. Autophagy gene (ATG)-related proteins coordinate specific steps in autophagy and sequestration (Suzuki and Ohsumi, 2007). Among known Atg proteins, microtubule-associated protein 1 light chain 3 (LC3), a mammalian ortholog of yeast Atg8, is necessary for autophagosome formation (Kabeya et al., 2000). During autophagy, cytosolic LC3-I is conjugated to phosphatidylethanolamine (PE) to generate LC3-II, which is then recruited to the autophagosomal membrane. Thus, LC3-I to LC3-II conversion reflects the progression of autophagy, and detecting LC3-II by immunoblot analysis is often used to monitor autophagy activity (Mizushima, 2004, Mizushima et al., 2010).

Substrate selectivity in autophagy is key to achieving proper degradation of damaged material. Another fundamental protein for autophagy, SQSTM1/p62, interacts specifically with ubiquitinated proteins (Katsuragi et al., 2015) and LC3-II. p62 serves as an adaptor that brings ubiquitinated damaged proteins into the autophagosome for degradation (Weidberg et al., 2011). p62 has been also extensively studied as a scaffold regulator for atypical protein kinase C, extracellular signal-regulated kinases (ERK1), NF-kappaB, and caspase-8 (Bitto et al., 2014, Katsuragi et al., 2015). Various functions of p62 have been suggested; however, the role of p62 in regulating cell signaling pathways and its detailed molecular actions under stress conditions have received little attention.

Recent studies have shown that MeHg induces autophagy in human neural stem cells (Chang et al., 2013) and rat primary astrocytes (Yuntao et al., 2014). These observations indicate that autophagy may help counter MeHg toxicity. Given the impact of MeHg as a pollutant, a link between autophagy and MeHg toxicity would shed new light on the role of autophagy in stress responses. In this report, we demonstrate autophagy activation and LC3-II and p62 upregulation by MeHg exposure in several cell types. This was further confirmed by using Atg5−/− mouse embryonic fibroblasts (MEFs). These exhibited higher sensitivity than Atg5+/+ MEFs following MeHg exposure. Our results suggest that Atg5-dependent autophagy is a major cell survival mechanism involved in responding to MeHg-induced cytotoxicity. We also discuss possible signaling pathways involved in autophagy induction following MeHg stress.

Section snippets

Cell culture and methylmercury treatment

Immortalized Atg5−/−and Atg5+/+ MEFs (Dr. Noboru Mizushima, University of Tokyo), and human neuroblastoma SH-SY5Y cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 292 μg/mL L-glutamine. Human colon carcinoma Caco-2 cells (ATCC) were cultured in MEM supplemented with 10% FCS, 0.1 mM non-essential amino acids, 100 U/mL penicillin, 100 μg/mL

Cytotoxic effect of MeHg

The effect of MeHg on MEFs was investigated using the WST assay. Fig. 1A and B show a concentration–response study in which MEFs were exposed to different amounts of MeHg for 24 h. The IC50 was approximately 3.5 μM after treatment with MeHg (Fig. 1A). When cells were treated with 2 μM MeHg, cell viability was significantly reduced compared to the control (p < 0.05), whereas a 1 μM MeHg dose did not have any significant effect. To further examine the cytotoxicity of MeHg on MEFs, lactate dehydrogenase

Discussion

Autophagy is one of the main pathways responsible for eliminating damaged, nonfunctional, or aggregated proteins and organelles. Thereby, autophagy is vital for ensuring proper cell functioning. However, the impact of MeHg, an environmental pollutant, on autophagy has been little understood in spite of its various adverse effects on cell function and viability. In this study, we first assessed MeHg cytotoxicity in MEFs and found that 1 μM MeHg had little effect on cell viability, whereas 2–10 μM

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

Atg5+/+, Atg5−/−, and stable GFP-LC3 MEFs were generously provided by Dr. Noboru Mizushima (University of Tokyo). We thank Miss. M. Adachi, A. Kobayashi, S. Minami, Mr. N. Ibaraki, T. Yasuda, and R. Harada for technical assistance.

Funding: This work was supported in part by Grants-in-Aid for Research Activity start-up (Grant Number 26893043) and Challenging Exploratory Research (Grant Number 16K15381).

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