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Cytoplasmic and Nuclear Anti-Apoptotic Roles of aB-Crystallin in Retinal Pigment Epithelial Cells
Published: Wednesday, September 26, 2012
Author: Woo Jin Jeong et al.

by 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

In addition to its well-characterized role in the lens, aB-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 aB-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 aB-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 aB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that aB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that aB-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 aB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of aB-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 aB-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 aB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.