Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

Methylmercury (MeHg) is a widely distributed environmental pollutant that causes a series of cytotoxic effects. However, molecular mechanisms underlying MeHg toxicity are not fully understood. Here, we report that sequestosome1/p62 protects mouse embryonic fibroblasts (MEFs) against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. p62 mRNA and protein expression in MEFs were temporally induced by MeHg exposure p62-deficient MEFs exhibited higher sensitivity to MeHg exposure compared to their wild-type (WT) counterparts. An earlier and higher level of accumulation of ubiquitinated proteins was detected in p62-deficient cells compared with WT MEFs. Confocal microscopy revealed that p62 and ubiquitinated proteins co-localized in the perinuclear region of MEFs following MeHg treatment. Further analysis of MEFs revealed that ubiquitinated proteins co-localized with LC3-positive puncta upon co-treatment with MeHg and chloroquine, an autophagy inhibitor. In contrast, there was minimal co-localization in p62-deficient MEFs. The present study, for the first time, examined the expression and distribution of p62 and ubiquitinated proteins in cells exposed to low-dose MeHg. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity and is required for MeHg-induced ubiquitinated protein clearance.


Results
MeHg induces SQSTM1/p62 protein levels and the formation of p62 aggregates. SQSTM1/p62 interacts with LC3 and is a substrate for autophagy. Stimulation of autophagy typically decreases cellular p62 levels 20 . In our previous study, however, we showed that MeHg stimulated autophagy in MEF cells, but increased the p62 protein levels in several types of cells such as MEFs, Caco-2, and SH SY-5Y 9 . Hence, we first assessed the effect of MeHg on p62 mRNA expression. Induction of p62 mRNA was observed at 4 h after 1 µM MeHg treatment and the levels were maintained for 24 h (Fig. 1A). p62 protein levels increased in a time-dependent manner with the peak occurring at 16 h (Fig. 1B). Consistent with this finding, confocal microscopic analysis of MeHg-treated MEFs also showed high p62 protein levels at 24 h (Fig. 1C).
To better understand the effects of autophagy on p62 expression, we then investigated whether p62 induction after 1 µM MeHg exposure correlated with the accumulation of p62 aggregates. This study involved separating the soluble cytosolic proteins from the insoluble aggregate fraction ( Fig. 2A). MeHg-treated cells displayed a clear increase in p62 in both the cytosolic and insoluble aggregate fractions. Concurrent with the increase in p62, MeHg also increased LC3-II protein levels in both fractions.
Subsequently, we examined whether the expression pattern of p62 was associated with that of LC3-II (Fig. 2B). Expression of p62 increased following MeHg treatment in both the cytosolic (peak 12 h) and aggregate (peak 16 h) fractions. Importantly, the expression pattern of the LC3-II protein differed from that of p62 in the aggregate fraction.
Further, we tested p62 expression in autophagy-deficient cells, Atg5KO MEFs. Treatment with MeHg increased p62 levels temporally, peaking at 16 h in wild-type MEFs. Similarly, in Atg5KO MEFs, p62 protein levels increased upon MeHg exposure. However, the accumulation was much greater in Atg5KO MEFs than in wild-type MEFs (Fig. 3). LC3-I levels were low and unchanged, and LC3-II levels were very low in Atg5KO MEFs, with or without MeHg treatment (Fig. 3). These findings indicate that upregulation of p62 by MeHg is independent of autophagic activity. However, as LC3-II levels increased, p62 levels decreased subsequently in WT cells, compared to Atg5 KO MEFs, suggesting that MeHg activates autophagy and degrades p62 at later time points.
Clearance of ubiquitinated proteins is impaired in the absence of p62. p62 binds ubiquitin non-covalently both in vitro and in vivo 21,22 . To assess the function of p62 under MeHg exposure, we examined whether MeHg facilitated protein ubiquitination in MEFs via immunoblot analysis. Treatment of wild-type MEFs with 1 µM MeHg increased the quantity of ubiquitinated proteins temporally (Fig. 4). The extent of this accumulation in p62 knockout (p62KO) MEFs occurred earlier than that observed in wild-type MEFs.
Co-localization of p62 and ubiquitinated proteins. Because p62 contains a ubiquitin-associated domain that recognizes substrates targeted for degradation, we next investigated whether ubiquitinated proteins were co-localized with p62 after MeHg treatment. Immunofluorescence staining revealed that p62 was present in numerous round bodies in the perinuclear area while ubiquitinated proteins were located in the cytoplasm, with weak staining, in wild-type MEFs (Fig. 5A). In wild-type MEFs treated with 1 µM MeHg, we observed an increased number of p62-positive puncta in the perinuclear area, and a substantial proportion of p62 appeared to co-localize with the ubiquitinated proteins. Similar immunofluorescence staining of ubiquitinated proteins was seen in p62KO MEFs. Notably, robust accumulation of ubiquitinated proteins was seen upon 2 µM MeHg treatment (Fig. 5A).
To confirm the interaction between p62 and ubiquitinated proteins, cell lysates from MEFs with or without MeHg treatment were immunoprecipitated using an antibody against p62. MeHg treatment increased the interaction of p62 with ubiquitinated proteins (Fig. 5B). To further investigate the relationship between p62 and ubiquitinated proteins, cells were treated with chloroquine (CQ), a lysosomotropic agent inhibiting the autophagic flux, leading to an accumulation of ubiquitinated proteins. In cells treated with CQ, there was an extensive accumulation of p62 and ubiquitinated proteins in the perinuclear area. Following co-treatment of CQ with MeHg, ubiquitinated proteins were strongly co-localized with p62 (Fig. 5C).
The loss of p62 impairs co-localization of ubiquitinated proteins with LC3 puncta. p62 is believed to mediate the clearance of ubiquitinated proteins by autophagy via direct interaction with LC3 on the membrane through the LC3-interacting region. To confirm the involvement of p62 in MeHg-induced autophagy, we examined the distribution of ubiquitinated proteins and LC3 puncta in wild-type and p62KO MEFs. To clarify the distribution of LC3 and ubiquitinated proteins, cells were treated with the autophagy inhibitor, CQ. CQ elevated levels of ubiquitinated proteins that were mainly located in the perinuclear region of the cell, and mostly co-localized with LC3 in wild-type MEFs (Fig. 6A). The co-localization of ubiquitinated proteins and LC3 was enhanced in wild-type MEFs treated with 1 µM MeHg and CQ (Fig. 6B). In contrast, the number of ubiquitinated proteins overlapping with LC3 was substantially decreased in p62KO cells (Fig. 6C). Upon MeHg treatment, despite the abundant accumulation of ubiquitinated proteins, p62KO MEFs showed limited co-localization of these proteins with LC3 (Fig. 6D).

Involvement of p62 in protection against MeHg-induced toxicity.
To determine whether p62 had a protective role, we assessed the sensitivity of wild-type and p62KO MEFs to MeHg. Numerous rounded and/or shrunken cells were observed in MeHg-exposed p62KO MEFs. These are typical features of apoptotic cell death. Wild-type MEFs showed fewer cells with such morphological features (Fig. 7A). In addition, the cytotoxicity of MeHg in wild-type and p62KO MEFs was examined using the CCK-8 viability assay (Fig. 7B). Consistent with the Hoechest 33342/PI data, at each concentration (0.5-3 µM), p62KO MEFs had a lower survival rate than wild-type MEFs following 24 h of MeHg treatment. Furthermore, the GFP-p62 was transfected into p62KO MEFs using a retroviral expression system, and cell viability was assessed after MeHg exposure. As expected, GFP-p62 most probably fully compensated for p62 (Fig. 7C). These results suggest that p62 protects cells from MeHg-induced cell death.

Discussion
Autophagy is a conserved cellular process that generally protects cells by eliminating damaged, nonfunctional, or aggregated proteins and organelles. Autophagy plays a critical role in maintaining cellular homeostasis and protects cells from various environmental stressors. Among these, the role of autophagy against heavy metal stress has been little understood despite their adverse effects on cell function and viability. Recently, we reported that low-dose MeHg exposure promoted autophagy, and Atg5-dependent autophagy served to protect cells from MeHg cytotoxicity 9 . SQSTM1/p62 is an autophagic receptor and delivers ubiquitinated cargoe for autophagic degradation 11,23 , implying that p62 is involved in MeHg-induced autophagy. In the current study, we showed that p62KO MEFs exhibited an enhanced sensitivity to low-dose MeHg toxicity, demonstrating our hypothesis that p62 is another key molecule in MeHg-induced cytotoxicity.  Wild-type and p62-defecient mouse embryonic fibroblasts were seeded in 6-cm dishes for treatments. After treatment with 1 µM MeHg for 0-24 h, whole cell lysates were immunoblotted with anti-ubiquitin and p62 antibodies. GAPDH was used as the loading control.
To understand how p62 contributes to cell survival against MeHg toxicity, we first examined response of p62 to MeHg in wild-type MEFs. Levels of p62 mRNA (Fig. 1A) and protein (Fig. 1B-D) were upregulated temporally after exposure to 1 µM MeHg. These results indicated that p62 was not only a substrate of autophagy, but also an inducible protein, and that upregulation occurred through a transcriptional event in response to low-dose MeHg exposure. We observed in Atg5KO MEFs that p62 protein levels increased after MeHg exposure (Fig. 3), indicating that increased p62 levels upon MeHg exposure were not derived from an inhibition of autophagic activity. Moreover, we showed in wild-type MEFs treated with MeHg that similar levels of p62 protein were observed in both the 1% Triton X-100 soluble and insoluble fractions ( Fig. 2A,B). We found that extensive accumulation of LC3-II occurred in the insoluble fraction upon treatment with MeHg. These results indicate that MeHg-induced p62 forms protein aggregates immediately with LC3-II. p62 directly binds to ubiquitinated proteins and plays a role in the formation and clearance of protein aggregates. The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway are the main degradative pathways for intracellular ubiquitinated proteins. p62 is involved in both processes, wherein it mediates the delivery of ubiquitinated proteins 24,25 . This present results show that MeHg induced ubiquitinated proteins temporally and suggests that p62 is involved in their clearance. The accumulation of ubiquitinated proteins occurred earlier and to a greater extent (Fig. 4) and robust accumulation of ubiquitinated proteins was observed in p62KO MEFs rather than in WT cells (Fig. 5A), suggesting that p62 is involved in the clearance of ubiquitinated proteins induced by MeHg exposure. Furthermore, ubiquitinated proteins were co-localized with LC3 in wild-type MEFs ( Fig. 6A and B), while the extent of overlap between ubiquitinated proteins and LC3 was decreased in p62KO MEFs ( Fig. 6C and D). Therefore, p62 is probably involved in the degradation of MeHg-induced ubiquitinated proteins, via autophagy. To assess the contribution of UPS, we investigated the effect of a proteasome inhibitor, MG132. Co-treatment with MeHg and MG-132 resulted in increased accumulation of ubiquitinated proteins in insoluble fractions, rather than treatment with only MG132 (Fig. S1). Moreover, we detected insoluble ubiquitinated proteins of approximately 260 kD prominently in p62KO MEFs, suggesting that p62 is involved in the clearance of MeHg-induced ubiquitinated proteins via the UPS. Together, our findings suggest that not only autophagy but also the UPS contributes to clearance of MeHg-induced ubiquitinated proteins, and p62 plays important roles in their clearance via autophagic and the proteasomeal pathways.
Herein, p62KO MEFs exhibited an enhanced sensitivity to MeHg exposure compared to their wild-type counterparts ( Fig. 7A and B) and exogenous GFP-p62 was able to compensate for their loss of p62 (Fig. 7C), thereby strongly suggesting the significance of p62 in protecting cells from MeHg toxicity. However, mechanisms underlying the enhanced sensitivity of p62 MEFs to MeHg remain unclear. Since p62KO MEFs showed excess accumulation of ubiquitinated proteins by MeHg treatment, at least in part, they may have undergone MeHg-induced cell death. Furthermore, other possibilities of enhanced MeHg sensitivity of p62KO MEFs cannot be excluded, as p62 has multiple functions. p62 was shown to interact with Kelch-ECH-associated protein 1 (KEAP1) at the transcription factor NF-E2-related factor 2 (NRF2) binding site, thereby promoting NRF2 release from KEAP1 [26][27][28] . NRF2 activation induced the expression of many antioxidant genes 29 . p62 also interacts with tumor necrosis factor receptor-associated factor (TRAF6) and activates NF-κB 30 . It has been known that NF-κB promotes tumor cell survival through its ability to reduce reactive oxygen species (ROS) during transformation in cancer tissues 31 . In addition to accumulation of ubiquitinated proteins, as reported previously, MeHg induces oxidative stress through an increase in intracellular ROS, such as peroxide and superoxide anions 32 . Further studies are needed to investigate the molecular mechanisms underlying the role of p62 in regulating the NRF2 and TRAF6 pathways and in ROS production by MeHg exposure.
The present results show that increased levels of p62 interact with ubiquitinated proteins and play an essential role in their turnover following MeHg exposure. Neuronal inclusions contain aggregates of p62 and ubiquitinated proteins in patients with various neurodegenerative disorders such as Parkinson's disease and amyotrophic lateral sclerosis 33 . Similar neuronal changes have been observed in various animal models of MeHg exposure 34,35 , and MeHg was suggested to be a risk factor for other neuronal disorders such as Alzheimer's disease 36 and ALS 37 . However, the relation between p62 and ubiquitinated proteins induced by MeHg, and these neurodegenerative diseases, has not been established. To further define the effect of MeHg on neurodegenerative diseases, it is necessary to investigate the role of p62 during low-dose and long periods of MeHg exposure, especially through daily food intake, and understand the coordination between the development of neurodegenerative diseases and the p62-dependent degradation of ubiquitinated proteins.
In conclusion, this study reported, for the first time, the protective role of the selective autophagy receptor p62 against low-dose MeHg exposure, and its role in the clearance of MeHg-induced ubiquitinated proteins. In future, we aim to further characterize the mechanisms underlying p62-mediated ubiquitinated proteins are degraded to protect against MeHg toxicity. Understanding the roles and mechanisms underlying the regulation of low-dose MeHg-induced ubiquitinated proteins could offer interesting novel therapeutic approaches for neurodegenerative diseases.