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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Autores principales: 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
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Publicado: Public Library of Science (PLoS) 2012
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spelling oai:doaj.org-article:3074edb5378746eeb564cfacb5d478592021-11-18T08:13:54ZCytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.1932-620310.1371/journal.pone.0045754https://doaj.org/article/3074edb5378746eeb564cfacb5d478592012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23049853/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203In 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.Woo Jin JeongJee Hyun RhoYoung Geol YoonSeung Hee YooNa Young JeongWon Yeol RyuHee Bae AhnWoo Chan ParkSae Heun RhoHee Seong YoonYung Hyun ChoiYoung Hyun YooPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e45754 (2012)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
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.
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.
format article
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
title 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_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_sort cytoplasmic and nuclear anti-apoptotic roles of αb-crystallin in retinal pigment epithelial cells.
publisher Public Library of Science (PLoS)
publishDate 2012
url https://doaj.org/article/3074edb5378746eeb564cfacb5d47859
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