Endoplasmic reticulum stress preconditioning modifies intracellular mercury content by upregulating membrane transporters

Endoplasmic reticulum (ER) stress preconditioning protects cells against methylmercury (MeHg) cytotoxicity by inducing integrated stress responses such as eIF2α phosphorylation, ATF4 accumulation, and nonsense-mediated mRNA decay (NMD) suppression. Here we demonstrated that ER stress preconditioning results in the upregulation of membrane transporters, leading to a decrease in intracellular mercury content. Our analyses showed that ER stress preconditioning upregulated the expression of methionine transporters that affect the cellular influx of MeHg, LAT1, LAT3, and SNAT2; and a membrane transporter that affects the efflux of MeHg, ABCC4, in MeHg-susceptible myogenic cells. Among these, ABCC4 transporter expression exhibited the greatest elevation. The functional significance of ABCC4 transporter in the efflux of MeHg was shown by the ABCC4 inhibition study. Additionally, we identified the role of phospho-eIF2α/ATF4 pathway in the upregulation of LAT1, SNAT2, and ABCC4 and the role of NMD suppression in LAT3 upregulation. Further, we detected that ER stress preconditioning amplified membrane transporter expression most likely through the translation of the upregulated mRNAs caused by ATF4-dependent transcription and NMD suppression. Taken together, these results suggested that the phospho-eIF2α/ATF4 pathway activation and NMD suppression may represent therapeutic targets for the alleviation of MeHg cytotoxicity by enhancing mercury efflux besides inducing protective stress responses.

It has been reported that ISR regulates the expression of genes involved in amino acid import 12 . Notably, MeHg is readily incorporated into the sulfur-containing amino acid cysteine to form a MeHg-cysteine complex because MeHg has a high affinity for the sulfur atom in thiol groups. As the MeHg-cysteine complex conformationally mimics the essential amino acid methionine, the complex can be transported to the cells via amino acid transporters for methionine [13][14][15] . Therefore, ER stress preconditioning may affect intracellular Hg concentration through changes in the gene expression of methionine transporters. Additionally, the ATP-binding cassette (ABC) transporter cassette C subfamily 4 (ABCC4) protein, which functions in the efflux of glutathione conjugates, has been shown to be associated with the cellular efflux of MeHg as well 16,17 .
In turn, NMD represents an mRNA quality control mechanism that detects the premature termination codon (PTC) located 5′-upstream of the last exon-exon junction and degrades PTC-containing mRNAs 18 . NMD suppression study suggested that NMD regulates transcripts with amino acid metabolism and transport functions that include upstream open reading frame(s) (uORF), alternative splicing that introduces nonsense codons, or frame-shifts and introns in the 3′untranslated region 19 . Furthermore, Abcc4 has been reported to have alternatively spliced transcripts (Abcc4s) bearing nonsense codons. Together, these findings suggest that NMD suppression caused by ER stress preconditioning may lead to the change in such membrane transporters mRNA expression. Our previous study showed that NMD suppression upregulates ATF4 mRNA, suggesting that ER stress preconditioning amplifies the accumulation of ATF4, a key transcriptional activator involved in adaptation to stresses, through NMD suppression-mediated increase in ATF4 mRNA and eIF2α phosphorylation-mediated translation facilitation of ATF4 protein-coding ORF 11 . Therefore mild ER stress-induced ISR may play a role in changes in the gene expression of membrane transporters.
Here we investigated the effect of ER stress preconditioning on the expression of these membrane transporters and on intracellular Hg concentration following exposure to MeHg using MeHg-susceptible myogenic cells. The role of mild ER stress-induced ISR in modifying the expression of these membrane transporters was also determined.

Results
Effect of TPG pretreatment on intracellular Hg concentration and the expression of membrane transporters following MeHg exposure. Intracellular Hg concentration increased soon after the exposure to MeHg in mouse myogenic MeHg-susceptible C2C12-DMPK160 cells. The influx of MeHg was more rapid in cells pretreated with 0.3 μg/ml TPG for 16 h than that in non-preconditioned cells (Fig. 1A). The peak of Hg concentration occurred 3 h following MeHg exposure in non-preconditioned cells but at 1 h post-exposure in preconditioned cells. Furthermore, the efflux of MeHg was also more rapid in cells pretreated with TPG than in TPG-untreated cells. These data suggested that the efflux of MeHg was more highly activated than the influx following MeHg exposure and that ER stress preconditioning further enhanced this phenomenon thus alleviating the load of MeHg on cells.
Next we examined the mRNA expression of methionine transporters related to the influx of MeHg, L-type amino acid transporter (LAT) 1, LAT3, and sodium coupled amino acid transporter 2 (SNAT2) 14,[20][21][22] ; and ABCC4 related to the efflux of MeHg. As shown in Fig. 1B, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses demonstrated that the exposure to MeHg caused an upregulation of the mRNA expression of Lat1, Snat2, and Abcc4 in a dose-dependent manner, but not of Lat3. Among these, Abcc4 upregulation was markedly higher. ER stress preconditioning further upregulated the gene expression of all of these membrane transporters with Abcc4 mRNA levels being exceptionally high (Fig. 1C). An alternative splicing variant of ABCC4 (ABCC4s) bearing nonsense codons was also upregulated (Fig. 1D). The results of western blot analysis showed an increase in the expression of LAT1, SNAT2, and ABCC4 in preconditioned cells compared to non-preconditioned cells. Treatment with MeHg modified the expression of the membrane transporters, and especially enhanced ABCC4 proteins in non-preconditioned cells (Fig. 1E). Notably, the difference in intracellular Hg content between preconditioned and non-preconditioned cells corresponded to the changes in the expression of these transporters (Fig. 1A,C,E). mTOR expression in cells exposed to MeHg. The LAT family is critical for the control of protein translation and cell growth through mammalian target of rapamycin complex 1 (mTORC1) signaling, which plays a role in cap-dependent mRNA translation and is also associated with amino acid metabolism 23 . Therefore, we examined the effect of MeHg exposure and ER stress preconditioning on mTORC1 signaling. As shown in Fig. 2A, Mtor mRNA expression was downregulated following MeHg exposure whereas pretreatment with TPG upregulated Mtor expression. Consistent with this, western blot analysis demonstrated that phosphorylation of 4EBP1, a direct substrate of mTORC1, was down-regulated following exposure to MeHg and activated under ER stress preconditioning (Fig. 2B).
The role of methionine transporters and ABCC4 on intracellular Hg content following MeHg exposure. Next, we investigated the role of methionine transporters and ABCC4 on intracellular Hg content using synthetic siRNAs against LAT1, LAT3, or SNAT2 and the specific ABCC4 inhibitor Ceefourin 1 24 . Knockdown of Lat1, Lat3, or Snat2 in C2C12-DMPK160 cells was confirmed by RT-qPCR (Fig. 3A). As shown in Fig. 3B, the intracellular Hg content was significantly decreased in Lat1, Lat3, and Snat2 knockdown cells. A decrease in intracellular Hg content was corresponded to the previous data on MeHg uptake described in Lat1 knockdown cells 15 . In contrast, the intracellular Hg content in cells treated with 2 μM Ceefourin 1 was significantly higher than that in non-treated cells (Fig. 3C). These results suggest that methionine transporters and ABCC4 are related to intracellular mercury concentration via the influx and the efflux function of MeHg, respectively, beginning at the early stages of MeHg exposure. Effect of Perk knockdown on membrane transporter expression. Because ER stress preconditioning induces ISR but not ATF6 and Xbp1 pathways 11 , we next analyzed the effect of each of ISR on the expression of membrane transporters in order to determine the mechanism by which prior mild ER stress upregulates these proteins.
First, we investigated the effect of eIF2α phosphorylation on membrane transporter expression. For this, we depleted the eIF2α phosphorylation kinase PERK using siRNA knockdown methodology. Knockdown of Perk in C2C12-DMPK160 cells was confirmed by RT-qPCR and western blot analysis (Fig. 4A,B). As shown in Fig. 4C, pretreatment with TPG upregulated Lat1, Lat3, Snat2, and Abcc4 mRNAs in both non-silencing (NS) siRNA-and Perk siRNA-transfected cells. However, the mRNA levels of all these transporters were significantly lower in Perk knockdown cells compared to NS siRNA-transfectants. The results indicated that the upregulation of these transporter mRNAs by ER stress preconditioning involved PERK expression.
Next we investigated the effect of knockdown of Perk on the other ISR factors, NMD suppression and ATF4 accumulation. NMD suppression was estimated for the expression of non-protein coding small nucleolar RNA host gene 1 (Snhg1) mRNA, which harbors a PTC. Pretreatment with TPG upregulated Snhg1 mRNA in both NS siRNA-transfectants and Perk knockdown cells, suggesting that NMD was suppressed in these TPG-treated cells (Fig. 4D). However, the level of Snhg1 mRNA was significantly downregulated in Perk knockdown cells compared to that in NS siRNA-transfectants. The induction of Atf4 mRNA by ER stress preconditioning was also significantly suppressed in Perk knockdown cells compared to NS siRNA-transfectants (Fig. 4E).
Effect of eIF2α phosphorylation on the membrane transporter expression. The role of phosphorylation of eIF2α on membrane transporter expression was further investigated using mutant non-phosphorylatable eIF2α-transfected cell lines. For this, a human wild type (WT) EIF2A construct encoding serine at position 52 or a mutant EIF2A-SA construct encoding the non-phosphorylatable alanine at position 52 was transfected into C2C12-DMPK160 cells and selected by puromycin to establish stable cell lines. Endogenous eIf2α in both established cell lines was knocked down following transfection with a targeted siRNA as confirmed by western blotting (Fig. 5A). Western blot analyses confirmed the establishment of a WT cell line that expressed phospho-eIF2α under mild ER stress as well as a mutant SA cell line without phospho-eIF2α under the condition of endogenous eIf2α knockdown (Fig. 5B).
As shown in Fig. 5C, pretreatment with TPG upregulated Lat1, Lat3, Snat2, and Abcc4 mRNAs in both WT and mutant SA cell lines. However, the levels of all transporters mRNAs were significantly lower in the mutant SA cell line compared to the WT cell line. The results indicated that the upregulation of these transporter mRNAs by ER stress preconditioning was dependent upon the phosphorylation of eIF2α. In addition, significant downregulation of non-protein coding Snhg1 mRNA (Fig. 5D) and ATF4 (Fig. 5B,E) were observed in mutant SA cell line under mild ER stress as compared to the WT cell line. Effect of Atf4 knockdown on membrane transporter expression. Next, we investigated the effect of knockdown of Atf4, a transcriptional activator that modulates a wide spectrum of downstream genes involved in adaptation to stresses, on LAT1, LAT3, SNAT2, and ABCC4 expression in preconditioned cells. Knockdown of Atf4 in C2C12-DMPK160 cells was confirmed by RT-qPCR and western blotting (Fig. 6A,B). As shown in Fig. 6C, pretreatment with TPG upregulated Lat1, Lat3, Snat2, and Abcc4 mRNAs in both NS siRNA-and Atf4 siRNA-transfected cells. However, the levels of Lat1, Snat2, and Abcc4 mRNAs were significantly downregulated in Atf4 knockdown cells compared to those in NS siRNA-transfectants, whereas the level of Lat3 mRNA was significantly upregulated in Atf4 knockdown preconditioned cells compared to NS siRNA-transfected preconditioned cells. The results of western blot analysis showed a decrease in the expression of LAT1, SNAT2, and ABCC4 proteins and an increase in LAT3 in Atf4 knockdown preconditioned cells compared to NS siRNA-transfected preconditioned cells (Fig. 6D). The results suggested that the upregulation of LAT1, SNAT2, and ABCC4 caused by ER stress preconditioning involved ATF4 whereas LAT3 upregulation did not. Pretreatment with TPG upregulated Snhg1 mRNA in both NS siRNA-transfectants and Atf4 knockdown cells. However, the level of Snhg1 mRNA     (Fig. 6E), suggesting that ER stress preconditioning-mediated NMD suppression was not a downstream event of Atf4 accumulation.

Effect of NMD suppression on membrane transporter expression.
To investigate the effect of NMD suppression, another ISR factor, on the expression of membrane transporters, knockdown of the NMD components Smg-1 or Smg-7 was performed. Knockdown of Smg-1 or Smg-7 in each siRNA-transfected C2C12-DMPK160 cell type was confirmed by western blotting (Fig. 7A). NMD inhibition was supported by the decrease (following Smg-1 knockdown) or increase (following Smg-7 knockdown) in phospho (p)-UPF1, a central component of NMD, for which the UPF1 phosphorylation and dephosphorylation cycle is essential 25 . In both Smg-1 and Smg-7 knockdown cells, significant upregulation of Snhg1 mRNA was observed compared to NS siRNA-transfectants and the level was further upregulated by ER stress preconditioning (Fig. 7B). In addition, the induction of Atf4 mRNA by ER stress preconditioning was higher in NMD-suppressed cells compared to NS siRNA-transfected preconditioned cells and significantly higher in Smg-7 knockdown cells (Fig. 7C). Western blot analysis confirmed that phosphorylation of eIF2α and ATF4 accumulation in preconditioned NS-or NMD suppressed-cells (Fig. 7D).
As shown in Fig. 7E, pretreatment with TPG upregulated Lat1, Lat3, Snat2, and Abcc4 mRNAs in both NS siRNA-transfectants and NMD-suppressed cells. However, the levels of these transporter mRNAs were significantly higher in NMD suppressed cells compared to NS siRNA-transfectants. Western blot analyses showed an increase in the expression of LAT3, SNAT2, and ABCC4 in NMD-suppressed preconditioned cells compared to NS siRNA-transfected preconditioned cells (Fig. 7F). The results suggested that the upregulation of these membrane transporters by mild ER stress involved NMD suppression. A decrease in intracellular Hg content was confirmed in Smg-1 knockdown preconditioned cells whereas that was increased in Atf4 knockdown cells (Fig. 7G).

Discussion
In this study, we demonstrated that ER stress preconditioning increased both the cellular influx and the efflux of MeHg and modified intracellular Hg content. RT-qPCR and western blot analyses of the expression of membrane transporters that affect cellular influx (LAT1, LAT3, SNAT2), and efflux (ABCC4) of MeHg, revealed that all of these membrane transporters mRNAs and LAT1, SNAT2, and ABCC4 proteins were upregulated and that the upregulation of ABCC4 was exceptionally high (Fig. 1C,E). The results of siRNA study for methionine transporters and ABCC4 inhibition study suggested that methionine transporters and ABCC4 represented a critical factor for the influx and efflux of MeHg, respectively (Fig. 3). Together with the time course study of intracellular Hg content (Fig. 1A) and membrane transporter expression after the exposure to MeHg (Fig. 1B), the findings indicated that the efflux of MeHg was activated than the influx after the exposure to MeHg and that this phenomenon was further enhanced by ER stress preconditioning. The results thus suggested that ER stress preconditioning alleviated the load of MeHg on cells through the enhanced efflux of MeHg caused by highly upregulated expression of ABCC4.
It has been reported that the induction of LAT family proteins and SNAT2 is coincident with mTORC1 pathway activation. However, our study demonstrated that MeHg downregulated the mTORC1 pathway despite the upregulation of LAT1 and SNAT2 expression (Figs 1B,E and 2A,B). As intracellular amino acid availability is recognized as a major mTORC1 regulatory mechanism 26 , the MeHg-induced inactivation of mTORC1 signaling suggested the likelihood of poor amino acid metabolism in MeHg-exposed cells. Notably, MeHg is easily incorporated in sulfur-containing amino acid cysteine and can thereby be transported to cells as a MeHg-cysteine complex, which might disturb amino acid availability. In turn, a defect of amino acid availability might underlie in part the loss of body weight exhibited by MeHg-exposed rats 8,27 .
We also identified that ER stress preconditioning protected against MeHg-induced mTORC1 signaling inactivation ( Fig. 2A,B). Because the protective effect of ER stress preconditioning against MeHg cytotoxicity is due to the induction of ISR 11 , we investigated the role of ISR including eIF2α phosphorylation, accumulation of ATF4, and NMD suppression in the upregulation of membrane transporters in order to determine the mechanism by which prior ER stress upregulates membrane transporter expression. The results of siRNA-mediated knockdown study of Perk, Atf4, or NMD components and the study using mutant non-phosphorylatable eIF2α-transfected cells uncovered that NMD suppression was related to the upregulation of LAT3 expression and that ATF4 accumulation led to upregulated LAT1, SNAT2, and ABCC4 expression (Figs 4-7). Our results are consistent with a prior report that ATF4 regulates the expression of the amino acid transporters LAT1 and SNAT2 [28][29][30] .
transporter mRNAs caused by ATF4-dependent transcription and NMD suppression. We confirmed a decrease in intracellular Hg content in Smg-1 knockdown preconditioned cells (Fig. 7G). Our findings were summarized in Fig. 8. Notably, the identification of these mRNAs as targets of NMD is consistent with its role in regulating transcripts encoding proteins involved in amino acid metabolism and transport that carry various traits predisposing them to errors in translation 19 . In particular, among the transporters examined in this study, it has been reported that LAT1 contains uORFs and exon with PTC 19 and that alternatively spliced transcripts of Abcc4 (Abcc4s) bear nonsense codons 31 . Accordingly, we also found that ER stress preconditioning increased alternatively spliced transcripts of Abcc4 (Fig. 1D). Additionally, it is expected that Lat3 also contains uORF, 3′UTR exon and/or uncharacterized feature of NMD substrate 32 because Lat3 mRNA and LAT3 could only be upregulated through NMD suppression (Figs 6C,D and 7E,F).
Notably, the results of a microarray study comparing control cells to those with knockdown of the NMD component Upf1 suggested that almost of 10% of non-mutated mRNAs are regulated by NMD 19 . The diverse transcripts induced by NMD suppression included mRNAs that play an important part in mediating the unfolded protein response, integrated stress response, and in amino acid transport and metabolism, as well as proto-oncogene mRNAs. In the current study, we also demonstrated that NMD suppression under ER stress preconditioning upregulated membrane transporter mRNAs related to the influx and efflux of MeHg as well. Furthermore, NMD suppression under ER stress preconditioning upregulated ATF4 mRNA and amplified the accumulation of ATF4 through eIF2α phosphorylation-mediated translation facilitation of ATF4 protein-coding ORF, resulting in the transcription facilitation of the membrane transporter mRNAs (Fig. 8).
Finally, it has been reported that one-third of alternative transcripts examined contain a PTC that renders them targets for NMD 33 . It is considered therefore that NMD suppression induced by a variety of stresses should stabilize many of these NMD-candidate transcripts. The involvement of these NMD-candidate transcripts in proteome diversity consequent to varied stresses remains to be elucidated; however, the findings of the current study support the potential of NMD regulation as a tool to dynamically alter gene expression and protect cells against environmental stresses.
In conclusion, we have demonstrated that ER stress preconditioning resulted in the upregulation of membrane transporters through the activation of the phospho-eIF2α/ ATF4 pathway and NMD suppression, leading to a decrease in intracellular Hg content. ER stress preconditioning also improved intracellular amino acid metabolism. Together, the results indicate that intracellular Hg content is able to be modulated through membrane transporter upregulation consequent to promoting the activation of the phospho-eIF2α/ATF4 pathway and NMD suppression. Therefore, these factors may represent therapeutic targets for the alleviation of MeHg cytotoxicity by inducing not only protective stress responses but also enhanced efflux of elevated levels of Hg.

Methods
Cell culture and drug treatments. The mouse myogenic C2C12-DMPK160 cell line, which is susceptible to MeHg treatment 34 , was cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceuticals) supplemented with 10% fetal bovine serum (HyClone), 300 μg/ml glutamine (Nissui Pharmaceuticals) and 0.4 mg/ ml Geneticin (G418) (Thermo Fisher Scientific) and exposed to MeHg in serum-free Cosmedium (Cosmo Bio Co., Ltd) as described previously 35 . Thapsigargin (TPG) (Sigma-Aldrich) was prepared as described previously 11 . Ceefourin 1 (Abcam) stock was dissolved in dimethylsulfoxide (WAKO). Ceefourin 1 was added 5 min prior to MeHg treatment. For the preconditioning study, 0.3 µg/ml TPG was added to the cells for 16 h prior to MeHg treatment. After removal of TPG, cells were exposed to MeHg as described previously 35 .