| Summary: | 博士 === 臺北醫學大學 === 醫學科學研究所 === 97 === Cadmium (Cd), an environmental pollutant with high cytotoxicity, accumulates in the liver and kidney after exposure and may result in pulmonary disease, carcinogenicity, and hepato- and nephrotoxicities. It has been reported that Cd exerts its toxicity through caspase-dependent or -independent apoptosis. However, the role of autophagy in the Cd-mediated cell death is unclear. In this study, using MES-13 mesangial cells as a cell model, we investigated the detail molecular mechanism and the role of autophagy in the cadmium-induced cytotoxicity. After treatment with Cd, the MES-13 mesangial cells underwent autophagy and apoptosis in a dose- and time-dependent manner, as revealed by acridine orange and annexin V/PI staining methods on a flow cytometer, respectively. Of which, the autophagy was confirmed by the formation of autophagosomes and processing of LC-3, hallmarks of autophagy, using electron microscopy and immunoblot, respectively. To demonstrate the involvement of glycogen synthase kinase-3?? (GSK-3???w, we employed a selective inhibitor of GSK-3??, SB 216763, and the siRNA technique to knockdown the expression of GSK-3??, both of which resulting in decrease of the percentage of Cd-induced autophagy. Following this line, cells harboring the GSK-3?? gene by transient transfection were more sensitive to exposure to Cd, suggesting that GSK-3?? plays a crucial role in regulating the Cd-induced autophagy. Furthermore, the GSK-3?? activation was inhibited by treatment with N-acetylcysteine (NAC), a ROS scavenger, suggesting that the Cd-induced GSK-3?? activation may be mediated by ROS burst. Consistent with our previous reports, the intracellular hydrogen peroxide (H2O2) was increased 2.6-fold after 4 h exposure to Cd and dimished rapidly within 1 h as revealed by flow cytometry with 2′-7′-dichlorofluorescein diacetate (DCFH-DA) staining. The NAC was able to abolish the Cd-induced ROS burst and autophagy, which suggests that ROS-GSK-3?? signaling is involved in the Cd-induced autophagy. In addition, a parallel experiment using Fluo-3 AM staining indicated that Ca2+ was increased within 0.5 h after Cd treatment. To demonstrate the involvement of Ca2+, we employed a cytosolic Ca2+ chelator and an IP3R inhibitor, both of which effectively inhibited Cd-induced cell death. Moreover, a knockdown of calcineurin by small interfering RNA resulted in an elevation of [Ca2+]i and cell death, suggesting that the release of Ca2+ from the endoplasmic reticulum plays a crucial role in Cd-induced cell death. In addition, activation of ERK was observed after treatment with Cd, and this was eliminated by pretreatment with BAPTA-AM. Furthermore, cells pretreated with the MEK1/2 inhibitor PD 98059 or U0126 showed a decrease in the percentage of autophagy induced by Cd, indicating that Cd-induced autophagy occurs through the Ca2+-ERK signaling pathway. In addition, cells pretreated with general caspase inhibitor, Z-VAD-fmk, were resistant to Cd, suggesting that caspase activation is involved in the cytotoxicity of Cd. To investigate the relationship between ROS and Ca2+, mesangial cells were pretreated with NAC followed by Cd exposure, which resulted in decrease of Cd-induced elevation of Ca2+ levels, ERK phosphorylation and apoptosis, suggesting that Ca2+ signaling may be a downstream event of ROS. Finally, inhibition of autophagy and apoptosis by treatment with 3-MA and Z-VAD-fmk resulted in reverse of cytotoxicity of Cd. These findings demonstrate that Cd induced two types of cell death, including autophagy and apoptosis. Furthermore, Ca2+-ERK and ROS-GSK-3?? signaling play a crucial role in the Cd-induced autophagy. In addition, Ca2+-mitochondrion-caspase signaling pathway was found to involve in the Cd-mediated apoptosis.
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