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. 2013:3:2346.
doi: 10.1038/srep02346.

Endoplasmic reticulum stress preconditioning attenuates methylmercury-induced cellular damage by inducing favorable stress responses

Fusako Usuki  1 , Masatake Fujimura, Akio Yamashita
Affiliations

Endoplasmic reticulum stress preconditioning attenuates methylmercury-induced cellular damage by inducing favorable stress responses

Fusako Usuki et al. Sci Rep. 2013.

Abstract

We demonstrate that methylmercury (MeHg)-susceptible cells preconditioned with an inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPase, thapsigargin, showed resistance to MeHg cytotoxicity through favorable stress responses, which included phosphorylation of eukaryotic initiation factor 2 alpha (Eif2α), accumulation of activating transcription factor 4 (Atf4), upregulation of stress-related proteins, and activation of extracellular signal regulated kinase pathway. In addition, ER stress preconditioning induced suppression of nonsense-mediated mRNA decay (NMD) mainly through the phospho-Eif2α-mediated general suppression of translation initiation and possible combined effects of decreased several NMD components expression. Atf4 accumulation was not mediated by NMD inhibition but translation inhibition of its upstream open reading frame (uORF) and translation facilitation of its protein-coding ORF by the phospho-Eif2α. These results suggested that ER stress plays an important role in MeHg cytotoxicity and that the modulation of ER stress has therapeutic potential to attenuate MeHg cytotoxicity, the underlying mechanism being the induction of integrated stress responses.

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Figures

Figure 1
Figure 1. Effect of pretreatment with TPG on MeHg cytotoxicity.
(A) Cell viability of C2C12-DMPK 160 cells pretreated with TPG 16 h before exposure to 0.4 or 0.5 μM MeHg was determined. Pretreatment with TPG (100–300 ng/ml) attenuated MeHg cytotoxicity. The viability of untreated cells was regarded as 100%. Values represent means ± SE (n = 6). *, **Significantly different from TPG-untreated and MeHg-treated cells by a one-way ANOVA (*p < 0.05, **p < 0.01). (B) Apoptosis analysis. The upper panel shows flow cytometry analysis of C2C12-DMPK160 stained with propidium iodide (PI) and FITC-Annexin V. The vertical axis indicates PI fluorescence intensity and horizontal axis Annexin V fluorescence. Exposure to 0.4 μM MeHg for 16 h increased the number of cells undergoing apoptosis (Annexin V-FITC-positive and PI-negative). A minor population of cells was observed to be Annexin V-FITC- and PI-positive, indicating that they were in end-stage apoptosis or already dead. The lower panel shows the profile of frequency of viable cells (Annexin V-FITC- and PI-negative) and cells undergoing apoptosis (Annexin V-FITC-positive and PI-negative). Pretreatment with TPG decreased the frequency of cells undergoing apoptosis.
Figure 2
Figure 2
(A) Flow cytometry analysis of C2C12-DMPK160 cells labeled with CM-H2DCFDA for investigation of intracellular ROS after 0.4 μM MeHg exposure for 4 or 7 h. Data shown are representative of 3 separate experiments. (B) Effect of pretreatment with TPG on SAPK/JNK, or ERK signaling pathways in C2C12-DMPK160 cells exposed to MeHg. Cells were pretreated with TPG (0.1 μg/ml) for 16 h and exposed to 0.4 μM MeHg. Total cell lysates prepared at the times indicated were analyzed by western blot using the indicated antibody probes. The images for each indicated probe were cropped from the same blot.
Figure 3
Figure 3. Effect of pretreatment with TPG on the expression of antioxidants in C2C12-DMPK160 cells.
Expression of Gpx1 (A), Txnrd1 (B), and Mn-Sod (C) mRNA was analyzed by quantitative real-time PCR. Total RNA was extracted from cells treated with 0.1 or 0.3 μg/ml TPG for 16 h. The histogram depicts the indicated mRNA normalized to β-actin. Values shown are means ± SE of 4 separate experiments.**Significantly different from TPG-untreated cells by a one-way ANOVA (**p < 0.01). (D) Western blot analyses of Nrf2, Txnrd1, and Mn-Sod. Western blots of C2C12-DMPK160 cells pretreated with 0.1 or 0.3 μg/ml TPG for 16 h were analyzed with the indicated antibody probes. The densitometric quantification of each protein was normalized to α-tubulin and is represented as fold increase over the non-pretreated cells. Although cropped blots were used, the gels were run under the same experimental conditions. Representative images of 3 samples are shown with quantitative data (means ± SE). *, **Significantly different from TPG-untreated cells by a one-way ANOVA (*p < 0.05, **p < 0.01).
Figure 4
Figure 4. Effect of pretreatment with TPG on the expression of Grp78 (A), Mt1 (B), or Atf4 mRNA (C) in C2C12-DMPK160 cells.
Total RNA was extracted from cells treated with 0.1 or 0.3 μg/ml TPG for 16 h, or cells exposed to 0.4 μM MeHg with or without 100 μM Trolox. Expression of Grp78, Mt1, or Atf4 mRNA was analyzed by quantitative real-time PCR. The histogram depicts the indicated mRNA normalized to β-actin. Values shown are means ± SE of 4 separate experiments. *, **Significantly different from TPG- and MeHg-untreated cells by a one-way ANOVA (*p < 0.05, **p < 0.01). #, ##Significantly different from Trolox-untreated cells by a one-way Welch's t-test (#p < 0.05, ##p < 0.01).
Figure 5
Figure 5. Changes in stress-related proteins after pretreatment with TPG.
(A) Effect of pretreatment with TPG on the expression of stress-related proteins after MeHg exposure analyzed by western blotting. Total cell lysates prepared at the times indicated were analyzed with the indicated antibody probes. Although cropped blots were used, the gels were run under the same experimental conditions. (B) Effect of pretreatment with TPG on the expression of Snhg1 mRNA. The histogram depicts Snhg1 mRNA normalized to β-actin analyzed by quantitative real-time PCR. Values are represented as fold increase over that of non-pretreated controls and are means ± SE of 4 separate experiments. **Significantly different from TPG- and MeHg-untreated cells by a one-way ANOVA (**p < 0.01). ##Significantly different from TPG-untreated cells by a one-way Welch's t-test (##p < 0.01). (C) Effect of pretreatment with TPG on the expression of NMD-related factors analyzed by western blotting. Total cell lysates prepared at the times indicated were analyzed with the indicated antibody probes. Although cropped blots were used in this figure, the gels were run under the same experimental conditions. (D) Effect of NMD suppression on the expression of Atf4 and Grp78. Western blots of C2C12-DMPK160 transfected with the indicated synthetic siRNAs were analyzed with the indicated antibody probes. Although cropped blots were used in this figure, the gels were run under the same experimental conditions. NMD inhibition was supported by the decrease (in the case of Smg-1 knockdown) or increase (in the case of Smg-7 knockdown) in Upf1 phosphorylation, a central component of NMD (NS, non-silencing). (E) Effect of NMD suppression on the expression of Atf4 mRNA. The histogram depicts Atf4 mRNA normalized to β-actin analyzed by quantitative real-time PCR. Values are represented as fold increase over that of NS-transfectants and are means ± SE of 4 separate experiments. #, ##Significantly different from NS-transfectants by a one-way Welch's t-test (#p < 0.05, ##p < 0.01).
Figure 6
Figure 6. Effect of knockdown of Grp78 or Mt1 on MeHg-induced cytotoxicity.
NS, non-silencing. (A) Synthetic siRNA-mediated knockdown of Grp78 or Mt1. Western blots of C2C12-DMPK160 transfected with the indicated synthetic siRNAs were analyzed with the indicated antibody probes. Although cropped blots were used in this figure, the gels were run under the same experimental conditions. (B) Cell viability study. Viability was determined 48 h after transfection of each siRNA. Values represent means ± SD (n = 6). #, ##Significantly different from Grp78-knockdown cells by a one-way Welch's t-test (#p < 0.05, ##p < 0.01). (C) γ-H2ax immunostaining. C2C12-DMPK160 cells transfected with the indicated siRNA were pretreated with 0.1 μg/ml TPG 16 h before MeHg exposure. Cells were fixed 11 h after MeHg exposure and stained with anti-γ-H2ax antibody. Representative photographs are shown. Bar = 50 μm. (D) Percentage of γ-H2ax-positive nuclei in Mt1- and Grp78-knockdown, and NS siRNA-transfected cells (means ± SE). Cells were detected by counterstaining of cell nuclei with Hoechst 33342. Three hundred cells in 3–5 fields were analyzed in 3 separate experiments. ##Significantly different from TPG-untreated and MeHg-treated cells by a one-way Welch's t-test (##p < 0.01).
Figure 7
Figure 7. Effect of pretreatment with TPG on the expression of antioxidants and Grp78 mRNAs in NS siRNA-transfectants and Mt1-knocked down cells.
The histogram depicts each mRNA normalized to β-actin, represented as fold increase over non-pretreated controls. Values shown are means ± SE of 4 separate experiments. **Significantly different from TPG-untreated cells by a one-way ANOVA (**p < 0.01). ##Significantly different from NS- transfectants by a one-way Welch's t-test (##p < 0.01).
Figure 8
Figure 8. Summarized stress responses induced by ER stress preconditioning.
ER stress preconditioning induces Eif2α phosphorylation. Translation of Atf4 coding region is enhanced by phospho-Eif2α-induced facilitation of the bypass of inhibitory uORF. NMD activity is suppressed by the combined effects of decreased expression of several NMD components, in addition to suppression of general translation initiation mediated by phospho-Eif2α, leading to Atf4 mRNA up-regulation.
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References

    1. Murry C. E., Jennings R. B. & Reimer K. A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136 (1986). - PubMed
    1. Yellon D. M. & Downey J. M. Spotlight on preconditioning. Cardiovasc. Res. 55, 425–428 (2002). - PubMed
    1. Hayashi T., Saito A., Okuno S., Ferrand-Drake M. & Chan P. H. Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death. J. Cereb. Blood Flow Metab. 23, 949–961 (2003). - PubMed
    1. Hung C. C., Ichimura T., Stevens J. L. & Bonventre J. V. Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J. Biol. Chem. 278, 29317–29326 (2003). - PubMed
    1. Zhang P. L. et al. Preinduced molecular chaperones in the endoplasmic reticulum protect cardiomyocytes from lethal injury. An. Clin. Lab. Sci. 34, 449–457 (2004). - PubMed

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