The MARCKS protein amount is differently regulated by calpain during toxic effects of methylmercury between SH-SY5Y and EA.hy926 cells

Abstract

Methylmercury (MeHg) is an environmental pollutant that shows severe toxicity to humans and animals. However, the molecular mechanisms mediating MeHg toxicity are not completely understood. We have previously reported that the MARCKS protein is involved in the MeHg toxicity to SH-SY5Y neuroblastoma and EA.hy926 vascular endothelial cell lines. In addition, calpain, a Ca²⁺-dependent protease, is suggested to be associated with the MeHg toxicity. Because MARCKS is known as a substrate of calpain, we studied the relation between calpain activation and cleavage of MARCKS and its role in MeHg toxicity. In SH-SY5Y cells, MeHg decreased cell viability along with increased calcium mobilization, calpain activation and a decrease in MARCKS amounts. However, pretreatment with calpain inhibitors attenuated the decrease in cell viability and MARCKS amount induced only by 1 µM but not by 3 µM MeHg. In cells with a MARCKS knockdown, calpain inhibitors failed to attenuate the decrease in cell viability caused by MeHg. In EA.hy926 cells, although MeHg caused calcium mobilization and a decrease in MARCKS levels, calpain activation was not observed. These results indicate that the participation of calpain in the regulation of MARCKS amounts is dependent on the cell type and concentration of MeHg. In SH-SY5Y cells, calpain-mediated proteolysis of MARCKS is involved in cytotoxicity induced by a low concentration of MeHg.

Keywords: MARCKS; calpain; methylmercury.

Fig. 1.

Attenuation of the MeHg-induced decrease in cell viability by calpain inhibitors. MeHg induced a dose-dependent decrease in the viability of SH-SY5Y and EA.hy926 cells (A, n=9 to 10). Effects of calpain inhibitors (0.3 µM MDL-28170 or calpeptin) on the decrease in cell viability induced by 1 µM (B, n=9) or 3 µM (C, n=9) MeHg in SH-SY5Y cells. Effects of calpain inhibitors (0.3 µM MDL-28170 or calpeptin) on the decrease in cell viability induced by 3 µM (D, n=5) or 10 µM (E, n=5) MeHg in EA.hy926 cells. Data are expressed as a percentage of vehicle-treated cells (control). Results are shown as mean ± SEM. *P<0.05 as compared with control (A) or as indicated (B–E). N.S.: not significant.

Fig. 2.

Calcium mobilization induced by MeHg. MeHg induced a dose-dependent increase in the fluo-4 F/F₀ ratio in SH-SY5Y cells (A, n=7) and in EA.hy926 cells (B, n=6). Data are expressed as a percentage of vehicle-treated cells (control). Results are shown as mean ± SEM. *P<0.05, as compared with control.

Fig. 3.

Calpain activation induced by MeHg, and effects of calpain inhibitors. The MeHg-induced change in 150/145 kDa SBDP was investigated in SH-SY5Y cells (A–C, n=5) and EA.hy926 cells (D–F, n=6). Representative immunoblots of 150/145 and 120 kDa SBDP and β-actin with specific antibodies are shown (A and D). Changes in 150/145 kDa SBDP were determined by densitometric analysis (B, C, E and F). Data are expressed as a percentage of vehicle-treated cells (control). Results are shown as mean ± SEM; *P<0.05.

Fig. 4.

Attenuation of the MeHg-induced decrease in MARCKS amount by calpain inhibitors. The MeHg-induced decrease in full-length MARCKS amounts and effects of calpain inhibitors were studied in SH-SY5Y cells (A–C, n=5) and EA.hy926 cells (D–F, n=6). Representative immunoblots of MARCKS and β-actin with specific antibodies are shown (A and D). Changes in MARCKS content were quantified by densitometric analysis (B, C, E and F). Data are expressed as a percentage of vehicle-treated cells (control). Results are shown as mean ± SEM; *P<0.05. N.S.: not significant.

Fig. 5.

Effects of calpain inhibitors on the MeHg-induced decrease in viability and on MARCKS content in SH-SY5Y cells with a MARCKS knockdown. Representative immunoblots of MARCKS and β-actin with specific antibodies are shown (A). Effects of MeHg and calpain inhibitors on full-length MARCKS amounts (B, n=5) and viability (C, n=8) in control and MARCKS knockdown cells. Data are expressed as a percentage of vehicle-treated cells. Results are shown as mean ± SEM. *P<0.05. N.S.: not significant.

Fig. 6.

Schematic representation of the regulation of full-length MARCKS amounts by MeHg in SH-SY5Y and EA.hy926 cells. The involvement of calpain in the regulation of MARCKS protein levels is dependent on cell types and the concentration of MeHg. In SH-SY5Y cells, MARCKS proteolysis by calpain mediates the cytotoxicity of the low concentration of MeHg.