p62/sequestosome 1 attenuates methylmercury-induced endoplasmic reticulum stress in mouse embryonic fibroblasts

Highlights

• Methylmercury induces endoplasmic reticulum stress in mouse embryonic fibroblasts.

• MeHg-induced ER stress response is higher in p62 knockout MEFs than in wild-type MEFs.

• Knock-in of GFP-p62 MEFs attenuates ER stress response after MeHg exposure.

Abstract

Methylmercury (MeHg) is a hazardous environmental pollutant that causes serious toxicity in humans and animals, as well as proteotoxic stress. In our previous study, we found that MeHg induces the expression of p62/sequestosome 1 (p62) that selectively targets ubiquitinated proteins for degradation via autophagy, and that p62 might protect cells against MeHg toxicity. To further investigate the role of p62 in MeHg-induced stress responses, we evaluated the role of p62 in MeHg-induced endoplasmic reticulum (ER) stress in p62 knockout (p62KO) mouse embryonic fibroblasts (MEFs). Treatment of wild-type (WT) MEFs were treated with MeHg (1 μM) increased mRNA levels of Chop encoding C/EBP homologous protein, Trib3 encoding Tribbles homolog 3, and Dnajb9 encoding DnaJ heat-shock protein family (Hsp40) member B9 increased, suggesting that ER stress is elicited by MeHg stress. Additionally, p62KO MEFs treated with MeHg showed a higher mRNA expression of Chop and Trib3 relative to that in WT MEFs. Furthermore, knock-in of GFP-p62 to p62KO cells diminished the Chop and Trib3 induction responses to MeHg stress and resulted in a higher cell viability than that of p62KO MEFs. These results suggest that the protective role of p62 against MeHg toxicity is partly mediated by suppressing the ER stress response.

Introduction

Mercury is a highly toxic element that is ubiquitous in nature. Once mercury is released into the aquatic environment, it is methylated by sulfate-reducing bacteria to form methylmercury (MeHg) (Nogara et al., 2019). MeHg is biomagnified through the food chain and present in high concentrations in predatory fish (Groth III, 2010), with humans are exposed to MeHg almost directly by consuming contaminated fish and other animal products (Kuntz et al., 2009; Driscoll et al., 2013). MeHg can easily pass through the cellular membrane and cause cell damage, and the high affinity of MeHg for sulfhydryl groups in cellular proteins can lead to the accumulation of damaged proteins, resulting in reduced resistance to oxidative stress damage, inhibition of Na+/K + pump activity, impairment of protein synthesis, and eventually causes proteotoxic stress (Toyama et al., 2007; Zhang et al., 2013; Kanda et al., 2014; Unoki et al., 2018). Various experimental models indicate that endoplasmic reticulum (ER) stress (Rudgalvyte et al., 2013; Usuki et al., 2017), the ubiquitin–proteasome system and related proteins (Hwang et al., 2002), and autophagy (Takanezawa et al., 2016) play a crucial role in the suppressing MeHg toxicity.

We previously found that p62/sequestosome 1 (p62) is involved in the MeHg detoxification process (Takanezawa et al., 2017). p62 plays diverse biological roles in inflammation, oxidative stress, apoptosis, tumorigenesis, and ubiquitinated-protein degradation (Moscat and Diaz-Meco, 2012), and recently, several studies reported that the p62 is associated with the autophagy processes (Komatsu et al., 2010; Ichimura et al., 2013). p62 acts as a cargo receptor for inducing the degradation of ubiquitinated substrates via its ubiquitin-associated domain. Additionally, it directly interacts with the autophagosomal marker protein LC3 via an LC3-interacting region motif, and promotes the formation of autophagosomes (Itakura and Mizushima, 2011). We previously demonstrated autophagy induction via MeHg stress, and that induction mediated by the protein autophagy-related gene 5 is essential for cell survival under MeHg-induced stress conditions (Takanezawa et al., 2016). Moreover, we found that p62 expression is induced by exposure to MeHg in several cell lines, and that p62 contributes to the removal of MeHg-induced ubiquitinated proteins and attenuation of MeHg toxicity (Takanezawa et al., 2017). However, the involvement of p62 in pathways other than autophagy under MeHg exposure has not been investigated.

The ER is an essential intracellular organelle that serves several specialized functions, including synthesis and maturation of secretory and transmembrane proteins, calcium storage, and the production of phospholipids and sterols. A delay in or impairment of protein folding and processing results in the accumulation of misfolded proteins and development of ER stress (Hotamisligil and Davis, 2016). Accumulation of misfolded proteins in the ER and their aggregation disrupt normal cellular function and homeostasis, leading to the development of cellular toxicity (Schröder, 2008; Mollereau et al., 2014). To resolve the accumulation of unfolded or misfolded proteins, the ER induces a cascade of reactions called the unfolded protein response (UPR) to restore normal ER functions (Sicari et al., 2020). The UPR regulates cellular adaption by enhancing the protein-folding capacity of the ER and simultaneously reduces the synthetic load (Battson et al., 2017). However, if the UPR fails to restore normal ER function, cells undergo apoptosis in response to prolonged or overwhelming ER stress. The UPR is closely associated with the development of various human diseases, including metabolic diseases, neurodegenerative diseases, inflammatory diseases, and cancer (Hetz et al., 2013; Oakes and Papa, 2015; Mou et al., 2020).

Previous studies report a close interaction between MeHg and ER stress. The expression of glucose-regulated protein (GRP)78, an ER-resident molecular chaperone that prevents the aggregation of unfolded or misfolded proteins, is induced by MeHg in vivo (Zhang et al., 2013) and in vitro (Usuki et al., 2008). Additionally, Hiraoka et al. reported that exposure of mouse embryonic fibroblasts (MEFs) to GSK2606414, a specific protein kinase RNA-like ER kinase inhibitor, inhibits MeHg-induced cell death (Hiraoka et al., 2017). However, the molecular mechanisms associated with the attenuation of MeHg-induced ER stress and cellular protection remain largely unknown.

In our previous study, we used MEFs to investigate the role of p62 against MeHg toxicity. To further understand the role of p62 under MeHg exposure conditions, we investigated the potential role of p62 in MeHg-induced ER stress in MEFs in order to consider the relationship between our previous findings and ER stress response. Furthermore, MEFs are well suited for investigating the molecular mechanisms associated with ER stress response and evaluating the effects of p62 knockout (p62KO) using in vivo models. The results confirm a crucial role for p62 in MeHg-induced ER stress and help validate animal studies.