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. 2015;11(7):1037-51.
doi: 10.1080/15548627.2015.1052208.

SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin

Huifeng Pi  1 , Shangcheng Xu, Russel J Reiter, Pan Guo, Lei Zhang, Yuming Li, Min Li, Zhenwang Cao, Li Tian, Jia Xie, Ruiqi Zhang, Mindi He, Yonghui Lu, Chuan Liu, Weixia Duan, Zhengping Yu, Zhou Zhou
Affiliations

SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin

Huifeng Pi et al. Autophagy. 2015.

Abstract

Cadmium is one of the most toxic metal compounds found in the environment. It is well established that Cd induces hepatotoxicity in humans and multiple animal models. Melatonin, a major secretory product of the pineal gland, has been reported to protect against Cd-induced hepatotoxicity. However, the mechanism behind this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd induced mitochondrial-derived superoxide anion-dependent autophagic cell death. Specifically, Cd decreased SIRT3 protein expression and activity and promoted the acetylation of SOD2, superoxide dismutase 2, mitochondrial, thus decreasing its activity, a key enzyme involved in mitochondrial ROS production, although Cd did not disrupt the interaction between SIRT3 and SOD2. These effects were ameliorated by overexpression of SIRT3. However, a catalytic mutant of SIRT3 (SIRT3(H248Y)) lacking deacetylase activity lost the capacity to suppress Cd-induced autophagy. Notably, melatonin treatment enhanced the activity but not the expression of SIRT3, decreased the acetylation of SOD2, inhibited mitochondrial-derived O2(•-) production and suppressed the autophagy induced by 10 μM Cd. Moreover, 3-(1H-1,2,3-triazol-4-yl)pyridine, a confirmed selective SIRT3 inhibitor, blocked the melatonin-mediated suppression of autophagy by inhibiting SIRT3-SOD2 signaling. Importantly, melatonin suppressed Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin exerts a hepatoprotective effect on mitochondrial-derived O2(•-)-stimulated autophagic cell death that is dependent on the SIRT3/SOD2 pathway.

Keywords: 3-MA, 3-methyladenine; 3-TYP, 3-(1H-1,2,3-triazol-4-yl)pyridine; ACTB, actin, β; Baf A1, bafilomycin A1; Cd, cadmium; CdCl2, cadmium chloride; GPT/ALT, glutamic-pyruvate transaminase (alanine aminotransferase); H2O2, hydrogen peroxide; LC3, microtubule-associated protein 1 light chain 3; O2•−, superoxide anion; SIRT1, sirtuin 1; SIRT3; SIRT3, sirtuin 3; SOD2; SOD2, superoxide dismutase 2, mitochondrial; SQSTM1/p62, sequestosome 1; autophagy; cadmium; hepatotoxicity; mROS, mitochondrial reactive oxygen species; mel, melatonin; melatonin; mitochondrial ROS; tf-LC3, tandem fluorescent mRFP-GFP-LC3B.

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Figures

Figure 1.
Figure 1.
For figure legend, see page 1040.
Figure 2.
Figure 2.
Mitochondrial-derived O2•− mediates Cd-induced autophagy in cultured HepG2 cells. (A) Quantification of mitochondrial-derived O2•− levels using a fluorescence spectrometer after HepG2 cells were treated with Cd at different concentrations for 12 h. HepG2 cells were preincubated with Mito-TEMPO (10 μM) for 2 h and then treated with 10 μM Cd, then the mitochondrial-derived O2•− levels (B), LC3 level (C) and cell viability (D) were determined. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 versus the control group, ##p < 0.01 vs. the Cd (10 μM) group. (n=6.)
Figure 3.
Figure 3.
Cd exposure increases acetylated-SOD2 expression in HepG2 cells in a dose-dependent manner. (A) Representative immunoblot of SOD2 protein levels in HepG2 cells. (B) SOD2 activity in HepG2 cells (C) Acetylation of SOD2 after Cd exposure was determined by immunoprecipitation with an anti-SOD2 antibody, followed by immunoblot analysis of acetylated-lysine. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 versus the control group. (n = 4.)
Figure 4.
Figure 4.
Cd exposure decreases SIRT3 protein expression and activity in a dose-dependent manner. (A) Quantitative real-time PCR analysis was applied to detect SIRT3 mRNA levels. (B) Representative immunoblot of SIRT3 protein levels in HepG2 cells. (C) SIRT3 activity was measured based on an enzymatic reaction using a SIRT3 assay kit. (D) Cell lysates were mixed with various concentrations Cd for 12 h. Antibodies against SIRT3 were added to immunoprecipitate the SIRT3-containing complexes, and then immunoblotted for SOD2 or SIRT3. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, *p < 0.05, **p < 0.01 vs. the control group. (n = 4.)
Figure 5.
Figure 5.
SIRT3-SOD2 modulates Cd-induced mitochondrial-derived O2•− accumulation and autophagy in cultured HepG2 cells. (A) SIRT3 overexpression induced deacetylation of SOD2 after 10 μM Cd treatment. (B) SOD2 activity in HepG2 cells. (C) Mitochondrial-derived O2•− production in HepG2 cells. (D) Representative immunoblot of LC3 protein levels in HepG2 cells. (E) Cell viability. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 versus control group, #p < 0.05, ##p < 0.01 vs. the Cd (10 μM) group. (n = 6.)
Figure 6.
Figure 6.
SIRT3 deacetylase deficiency does not affect mitochondrial-derived O2•− accumulation and autophagy in Cd-treated HepG2 cells. (A) SIRT3H248Y overexpression did not induce deacetylation of SOD2 after 10 μM Cd treatment. (B) SOD2 activity in HepG2 cells. (C) Mitochondrial-derived O2•− production. (D) Representative immunoblot of LC3 protein levels in HepG2 cells. (E) Cell viability. The results are expressed as a percentage of the control, which is set at 100%. The values are presented as the means ± SEM, **p < 0.01 versus control group, #p < 0.05 vs. the Cd (10 μM) group. (n = 6.)
Figure 7.
Figure 7.
Melatonin suppresses Cd-induced autophagic cell death. (A) Mitochondrial-derived O2•− production. (B) A representative immunoblot and quantification analysis of LC3. (C) Cell viability. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 vs. the control group, and #p < 0.05, ##p < 0.01 vs. the Cd (10 μM) group. (n = 6.)
Figure 8.
Figure 8.
Melatonin increases SIRT3 activity but not expression, and it inhibits acetylated-SOD2 expression after Cd treatment in vitro. Melatonin enhances SIRT3 activity (A) but not the expression of SIRT3 (B) at 12 h after exposure to 10 μM Cd. Melatonin induced deacetylation of SOD2 (C) and increased SOD2 activity (D) after Cd treatment. The results are expressed as a percentage of the control, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 versus the control group, and #p < 0.05 vs. the Cd (10 μM) group. (n = 4.)
Figure 9.
Figure 9.
3-TYP pretreatment abolishes the melatonin-suppressed autophagy in Cd-injured HepG2 cells. The effects of melatonin and 3-TYP pretreatment on mitochondrial-derived O2•− production (A), (B) A representative immunoblot and quantification analysis of LC3, (C) cell viability, (D) acetylated-SOD2 expression and (E) SOD2 activity. The results are expressed as a percentage of the Cd group, which is set at 100%. The values are presented as the means ± SEM, *p < 0.05, **p < 0.01 versus the Cd (10 μM) group, and #p < 0.05, ##p < 0.01 vs. the Mel + Cd group. (n = 6.)
Figure 10.
Figure 10.
Melatonin suppresses Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. (A) Serum GPT activity was measured. (B) Immunohistochemical analysis of LC3B and SOD2 (acetyl K68) expression in liver tissue. (C) SOD2 activity in liver tissue. The results are expressed as a percentage of the control group, which is set at 100 %. The values are presented as the means ± SEM, **p < 0.01 versus the Cd group, and #p < 0.05, ##p < 0.01 vs. the Mel + Cd group. (n = 8.)
Figure 11.
Figure 11.
Melatonin inhibits autophagic cell death by promoting SIRT3-SOD2-mROS pathway in cadmium-induced hepatotoxicity.
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