ToxSci Advance Access originally published online on January 6, 2009
Toxicological Sciences 2009 108(1):124-131; doi:10.1093/toxsci/kfn266
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Cadmium Toxicity toward Autophagy through ROS-Activated GSK-3β in Mesangial Cells
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* Graduate Institute of Medical Sciences
Department of Biochemistry, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC
Department of Pathology and Laboratory Medicine, Shin Kong Wu Ho-Su Memorial, Hospital, Taipei 111 (Taiwan), ROC
School of Medical Laboratory Science and Biotechnology
¶ Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC
|| Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan, ROC
2 To whom correspondence should be addressed at Department of Biochemistry, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110, ROC. Fax: +886-2-86421158. E-mail: cmshih{at}tmu.edu.tw.
Received November 1, 2008; accepted December 22, 2008
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Abstract |
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We previously demonstrated that cadmium (Cd) is able to induce autophagic cell death through a calcium-extracellular signal-regulated kinase pathway. Here, the object of this study is to investigate the role of glycogen synthase kinase-3β (GSK-3β) in the induction of autophagy. After treatment with Cd, MES-13 mesangial cells were determined to have undergone autophagy based on the formation of acidic vesicular organelles and autophagosomes as well as on the processing of microtubule-associated protein 1 light chain 3, using flow cytometry with acridine orange staining, electron microscopy, and immunoblot, respectively. Use of the GSK-3β inhibitor SB 216763 or the small interfering RNA technique to knockdown the expression of GSK-3β resulted in a decrease of Cd-induced autophagy. In contrast, overexpression of GSK-3β by transient transfection potentiated Cd toxicity toward the mesangial cells, suggesting that GSK-3β plays a crucial role in regulating Cd-induced autophagy. Moreover, a decrease of the phosphorylated level at Ser9 of GSK-3β was observed by immunoblot after treatment with Cd, indicating GSK-3β was activated by Cd. This phenomenon was reversed by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), demonstrated that ROS might activate GSK-3β. In fact, intracellular hydrogen peroxide (H2O2) was 2.6-fold elevated after 3 h of exposure to Cd. Both Cd-induced ROS bursts and autophagy were reduced by NAC and vitamin E. In summary, this study demonstrated that, in MES-13 mesangial cells, Cd-induced autophagy was mediated through the ROS-GSK-3β signaling pathway.
Key Words: cadmium; autophagy; GSK-3β; ROS; mesangial cells.
1 These authors made an equal contribution to this work.