Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells
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 c...
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ndltd-TW-097TMC056590442016-05-04T04:31:29Z http://ndltd.ncl.edu.tw/handle/74919571758166773269 Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells 鎘細胞毒性之分子訊息探討 Sheng-Hao Wang 王聖豪 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. 施純明 2009 學位論文 ; thesis 94 zh-TW |
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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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author2 |
施純明 |
author_facet |
施純明 Sheng-Hao Wang 王聖豪 |
author |
Sheng-Hao Wang 王聖豪 |
spellingShingle |
Sheng-Hao Wang 王聖豪 Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
author_sort |
Sheng-Hao Wang |
title |
Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
title_short |
Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
title_full |
Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
title_fullStr |
Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
title_full_unstemmed |
Molecular Signals of Cadmium-Induced Cytotoxicity to Mesangial Cells |
title_sort |
molecular signals of cadmium-induced cytotoxicity to mesangial cells |
publishDate |
2009 |
url |
http://ndltd.ncl.edu.tw/handle/74919571758166773269 |
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