Cadmium Toxicity toward Autophagy through ROS-Activated GSK-3β 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-3 beta (GSK-3 beta) 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-3 beta inhibitor SB 216763 or the small interfering RNA technique to knockdown the expression of GSK-3 beta resulted in a decrease of Cd-induced autophagy. In contrast, overexpression of GSK-3 beta by transient transfection potentiated Cd toxicity toward the mesangial cells, suggesting that GSK-3 beta plays a crucial role in regulating Cd-induced autophagy. Moreover, a decrease of the phosphorylated level at Ser9 of GSK-3 beta was observed by immunoblot after treatment with Cd, indicating GSK-3 beta was activated by Cd. This phenomenon was reversed by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), demonstrated that ROS might activate GSK-3 beta. 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-3 beta signaling pathway.