Cadmium toxicity toward autophagy through ROS-activated GSK-3beta in mesangial cells.
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-3beta (GSK-3beta) 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-3beta inhibitor SB 216763 or the small interfering RNA technique to knockdown the expression of GSK-3beta resulted in a decrease of Cd-induced autophagy. In contrast, overexpression of GSK-3beta by transient transfection potentiated Cd toxicity toward the mesangial cells, suggesting that GSK-3beta plays a crucial role in regulating Cd-induced autophagy. Moreover, a decrease of the phosphorylated level at Ser9 of GSK-3beta was observed by immunoblot after treatment with Cd, indicating GSK-3beta was activated by Cd. This phenomenon was reversed by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), demonstrated that ROS might activate GSK-3beta. In fact, intracellular hydrogen peroxide (H(2)O(2)) 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-3beta signaling pathway.
Wang
Sheng-Hao
SH
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan, ROC.
Shih
Yung-Luen
YL
Kuo
Tai-Chin
TC
Ko
Wun-Chang
WC
Shih
Chwen-Ming
CM
D000111:Acetylcysteine / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002104:Cadmium / Q000633:toxicity*; D002460:Cell Line; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D050527:Mesangial Cells / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D007211:Indoles; D008301:Maleimides; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C417521:SB 216763; D002104:Cadmium; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D000111:Acetylcysteine