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Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells

Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE and subsequent death of photoreceptors have been observed in age-related macular degeneration (AMD). Although the consequences of GSH...

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Autores principales: Sun, Yun, Zheng, Yingfeng, Wang, Chunxiao, Liu, Yizhi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037763/
https://www.ncbi.nlm.nih.gov/pubmed/29988039
http://dx.doi.org/10.1038/s41419-018-0794-4
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author Sun, Yun
Zheng, Yingfeng
Wang, Chunxiao
Liu, Yizhi
author_facet Sun, Yun
Zheng, Yingfeng
Wang, Chunxiao
Liu, Yizhi
author_sort Sun, Yun
collection PubMed
description Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE and subsequent death of photoreceptors have been observed in age-related macular degeneration (AMD). Although the consequences of GSH depletion have been described previously, questions remain regarding the molecular mechanisms. We herein examined the downstream effects of GSH depletion on stress-induced premature senescence (SIPS) and cell death in human RPE cells. Briefly, cultured ARPE-19 cells were depleted of GSH using: (1) incubation in cystine (Cys(2))-free culture medium; (2) treatment with buthionine sulphoximine (BSO, 1000 µM) to block de novo GSH synthesis for 24–48 h; or (3) treatment with erastin (10 µM for 12–24 h) to inhibit Cys(2)/glutamate antiporter (system x(c)(−)). These treatments decreased cell viability and increased both soluble and lipid reactive oxygen species (ROS) generation but did not affect mitochondrial ROS or mitochondrial mass. Western blot analysis revealed decreased expression of ferroptotic modulator glutathione peroxidase 4 (GPX4). Increased autophagy was apparent, as reflected by increased LC3 expression, autophagic vacuoles, and autophagic flux. In addition, GSH depletion induced SIPS, as evidenced by increased percentage of the senescence-associated β-galactosidase-positive cells, increased senescence-associated heterochromatin foci (SAHF), as well as cell cycle arrest at the G1 phase. GSH depletion-dependent cell death was prevented by selective ferroptosis inhibitors (8 μM Fer-1 and 600 nM Lip-1), iron chelator DFO (80 μM), as well as autophagic inhibitors Baf-A1 (75 nM) and 3-MA (10 mM). Inhibiting autophagy with Baf-A1 (75 nM) or 3-MA (10 mM) promoted SIPS. In contrast, inducing autophagy with rapamycin (100 nM) attenuated SIPS. Our findings suggest that GSH depletion induces ferroptosis, autophagy, and SIPS. In addition, we found that autophagy is activated in the process of ferroptosis and reduces SIPS, suggesting an essential role of autophagy in ferroptosis and SIPS.
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spelling pubmed-60377632018-07-11 Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells Sun, Yun Zheng, Yingfeng Wang, Chunxiao Liu, Yizhi Cell Death Dis Article Glutathione (GSH) protects against oxidative damage in many tissues, including retinal pigment epithelium (RPE). Oxidative stress-mediated senescence and death of RPE and subsequent death of photoreceptors have been observed in age-related macular degeneration (AMD). Although the consequences of GSH depletion have been described previously, questions remain regarding the molecular mechanisms. We herein examined the downstream effects of GSH depletion on stress-induced premature senescence (SIPS) and cell death in human RPE cells. Briefly, cultured ARPE-19 cells were depleted of GSH using: (1) incubation in cystine (Cys(2))-free culture medium; (2) treatment with buthionine sulphoximine (BSO, 1000 µM) to block de novo GSH synthesis for 24–48 h; or (3) treatment with erastin (10 µM for 12–24 h) to inhibit Cys(2)/glutamate antiporter (system x(c)(−)). These treatments decreased cell viability and increased both soluble and lipid reactive oxygen species (ROS) generation but did not affect mitochondrial ROS or mitochondrial mass. Western blot analysis revealed decreased expression of ferroptotic modulator glutathione peroxidase 4 (GPX4). Increased autophagy was apparent, as reflected by increased LC3 expression, autophagic vacuoles, and autophagic flux. In addition, GSH depletion induced SIPS, as evidenced by increased percentage of the senescence-associated β-galactosidase-positive cells, increased senescence-associated heterochromatin foci (SAHF), as well as cell cycle arrest at the G1 phase. GSH depletion-dependent cell death was prevented by selective ferroptosis inhibitors (8 μM Fer-1 and 600 nM Lip-1), iron chelator DFO (80 μM), as well as autophagic inhibitors Baf-A1 (75 nM) and 3-MA (10 mM). Inhibiting autophagy with Baf-A1 (75 nM) or 3-MA (10 mM) promoted SIPS. In contrast, inducing autophagy with rapamycin (100 nM) attenuated SIPS. Our findings suggest that GSH depletion induces ferroptosis, autophagy, and SIPS. In addition, we found that autophagy is activated in the process of ferroptosis and reduces SIPS, suggesting an essential role of autophagy in ferroptosis and SIPS. Nature Publishing Group UK 2018-07-09 /pmc/articles/PMC6037763/ /pubmed/29988039 http://dx.doi.org/10.1038/s41419-018-0794-4 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Sun, Yun
Zheng, Yingfeng
Wang, Chunxiao
Liu, Yizhi
Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title_full Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title_fullStr Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title_full_unstemmed Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title_short Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
title_sort glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037763/
https://www.ncbi.nlm.nih.gov/pubmed/29988039
http://dx.doi.org/10.1038/s41419-018-0794-4
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