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Methylmercury, an Environmental Electrophile Capable of Activation and Disruption of the Akt/CREB/Bcl-2 Signal Transduction Pathway in SH-SY5Y Cells

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Methylmercury, an Environmental Electrophile Capable of Activation and Disruption of the Akt/CREB/Bcl-2 Signal Transduction Pathway in SH-SY5Y Cells

Takamitsu Unoki et al. Sci Rep.

Abstract

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

Figures

Figure 1
Figure 1. Translocation of phosphorylated Akt in the nucleus.
SH-SY5Y cells were exposed to 0.5 μM of MeHg for 6 h. Nuclear and cytoplasmic fractions were subjected to Western blotting with indicated antibodies. Nuclear Akt band intensity in control was defined as 100%. *p < 0.05 vs. control. Each value is the mean ± S.D. of three determinations (A). The localization of phosphorylated-Akt (p-Akt) (green) was detected along with F-actin (red) and the nucleus (blue). The regions enclosed by the white squares are magnified in the lower panels. Scale bars, 10 μm. (B).
Figure 2
Figure 2. Activation of Akt and CREB and induction of Bcl-2 by MeHg in SH-SY5Y cells.
The cells were exposed to 1 μM of MeHg for the indicated time and then were subjected to western blotting and the band intensities were quantified (A). The cells were exposed to the indicated concentrations of MeHg for 6 h. The whole lysates were subjected to western blotting and the band intensities were quantified (B). The cells, transfected with CRE-luciferase and the pRLTK expressed vector, were exposed to 1 μM of MeHg for the indicated time. The luciferase assay was performed as described in the materials and methods (C). *p < 0.05 vs. control. Each value is the mean ±  S.D. of three determinations.
Figure 3
Figure 3. Inhibition of the MeHg-mediated Akt/CREB/Bcl-2 signaling pathway by an Akt inhibitor in SH-SY5Y cells.
SH-SY5Y cells were pretreated with wortmannin (0.5 μM) for 30 min, and then the cells were exposed to MeHg for 12 h. The cells were also exposed to MeHg for 12 h in the presence or absence of LY294002 (5 μM). The whole cell lysates were subjected to western blotting and the band intensities were quantified (A). The cells were transfected with CRE-luciferase and then treated with wortmannin (0.5 μM) for 30 min following exposure of MeHg for 6 h. The luciferase assay was performed as described in the materials and methods (B). *p < 0.05 vs. control MeHg (0 μM). #p < 0.05 vs. control MeHg (0.5 μM or 1 μM). Each value is the mean ±  S.D. of three determinations.
Figure 4
Figure 4. Protective role of Akt/CREB/Bcl-2 signaling in MeHg-induced cell death in SH-SY5Y cells.
SH-SY5Y cells were pretreated with wortmannin (5 μM) for 30 min and then the cells were exposed to MeHg (1 μM) for 6 h. The cells were stained with annexin V and propidium iodide (PI). Scale bar, 50 μm (A). The cells were pretreated with wortmannin (0.5 μM) for 30 min following exposure of MeHg for 24 h, and then an MTT assay was performed (B). The cells were transfected with control, Bcl-2–1, or Bcl-2–2 siRNA for 48 h and the expression level of Bcl-2 was examined by western blotting (C, upper). The siRNA transfected cells were exposed to MeHg for 24 h, and then cell viability was measured by the MTT assay (C, lower). *p < 0.05 vs. control. Each value is the mean ±  S.D. of three determinations.
Figure 5
Figure 5. Disruption of Akt/CREB/Bcl-2 signaling by higher concentrations of MeHg in SH-SY5Y cells.
SH-SY5Y cells were exposed to MeHg (0, 2, 5, and 10 μM) for 6 h, and subjected to western blotting (A) and the band intensities were quantified (B). The cells were transfected with CRE-luciferase and exposed to MeHg (0, 2, and 5 μM) for 3 h. The luciferase assay was performed as described in the materials and methods (C). *p < 0.05 vs. control MeHg (0 μM). #p < 0.05 vs. control MeHg (2 μM). Each value is the mean ±  S.D. of three determinations.
Figure 6
Figure 6. S-Mercuration of Akt and CREB by MeHg in SH-SY5Y cells.
SH-SY5Y cells were treated with MeHg for 3 h and a BPM-precipitation assay was performed. The precipitated proteins were detected by western blotting with the indicated antibodies (A). The cell lysate before the separation was used to estimate the alteration of protein expression by MeHg stimulation. The band intensities were quantified (B). Recombinant CREB (0.5 mg/mL) was reacted with MeHg (20 μM) for 30 min at 37 °C, and then the proteins were subjected to SDS-PAGE following Coomassie brilliant blue staining (C, upper). The CREB protein on the gel was cut out using a gel-band cutter and the total Hg concentration in the CREB was analysed by AAS. The mercury content was expressed as Hg ng/ng of protein (C, lower). *p < 0.05 vs. control. Each value is the mean ±  S.D. of three determinations.
Figure 7
Figure 7. Biphasic regulation of Akt/CREB/Bcl-2 signaling mediated by MeHg.

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