Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.
In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In...
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Public Library of Science (PLoS)
2012-01-01
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Series: | PLoS ONE |
Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23049853/pdf/?tool=EBI |
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author | Woo Jin Jeong Jee Hyun Rho Young Geol Yoon Seung Hee Yoo Na Young Jeong Won Yeol Ryu Hee Bae Ahn Woo Chan Park Sae Heun Rho Hee Seong Yoon Yung Hyun Choi Young Hyun Yoo |
author_facet | Woo Jin Jeong Jee Hyun Rho Young Geol Yoon Seung Hee Yoo Na Young Jeong Won Yeol Ryu Hee Bae Ahn Woo Chan Park Sae Heun Rho Hee Seong Yoon Yung Hyun Choi Young Hyun Yoo |
author_sort | Woo Jin Jeong |
collection | DOAJ |
description | In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis. |
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spelling | doaj.art-3074edb5378746eeb564cfacb5d478592022-12-21T21:32:49ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0179e4575410.1371/journal.pone.0045754Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.Woo Jin JeongJee Hyun RhoYoung Geol YoonSeung Hee YooNa Young JeongWon Yeol RyuHee Bae AhnWoo Chan ParkSae Heun RhoHee Seong YoonYung Hyun ChoiYoung Hyun YooIn addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23049853/pdf/?tool=EBI |
spellingShingle | Woo Jin Jeong Jee Hyun Rho Young Geol Yoon Seung Hee Yoo Na Young Jeong Won Yeol Ryu Hee Bae Ahn Woo Chan Park Sae Heun Rho Hee Seong Yoon Yung Hyun Choi Young Hyun Yoo Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. PLoS ONE |
title | Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. |
title_full | Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. |
title_fullStr | Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. |
title_full_unstemmed | Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. |
title_short | Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells. |
title_sort | cytoplasmic and nuclear anti apoptotic roles of αb crystallin in retinal pigment epithelial cells |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23049853/pdf/?tool=EBI |
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