Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells

PLoS One. 2012;7(9):e45754. doi: 10.1371/journal.pone.0045754. Epub 2012 Sep 26.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis*
  • Caspases / metabolism
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cell Survival
  • Cells, Cultured / cytology
  • Cytoplasm / metabolism*
  • DNA / genetics
  • DNA / metabolism
  • Flow Cytometry / methods
  • Humans
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods
  • Mutagenesis, Site-Directed
  • Ploidies
  • Pyruvaldehyde / pharmacology
  • RNA, Small Interfering / metabolism
  • Retinal Pigment Epithelium / cytology*
  • Sequence Analysis, DNA
  • Serine / chemistry
  • Subcellular Fractions / chemistry
  • alpha-Crystallin B Chain / chemistry
  • alpha-Crystallin B Chain / physiology*

Substances

  • RNA, Small Interfering
  • alpha-Crystallin B Chain
  • Serine
  • Pyruvaldehyde
  • DNA
  • Caspases

Grant support

This work was supported by the National Research Foundation of Korea grant funded by the Korean government (2011-0001262). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.