ToxSci Advance Access published online on January 6, 2009
Toxicological Sciences, 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, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC
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, College of Medicine, Taipei Medical University, Taipei 110 (Taiwan), ROC
¶ Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, ROC
1 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 (C.M. Shih)
Received November 1, 2008; revision received December 20, 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-ERK 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 (AVOs) and autophagosomes as well as on the processing of microtubule-associated protein 1 light chain 3 (LC3), 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 (siRNA) 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.
2 These authors made an equal contribution to this work.