Sensitization of RPE cells by alphaB-crystallin siRNA to SAHA-induced stage 1 apoptosis through abolishing the association of alphaB-crystallin with HDAC1 in SC35 speckles.
OBJECTIVE
To better understand the mechanism underlying the anti-apoptotic activity of alphaB-crystallin in RPE cells.


METHODS
Cells of the human retinal pigment epithelial line ARPE-19 were treated with a histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), with or without alphaB-crystallin siRNA. To examine the mechanism underlying the cell death induced in ARPE-19 cells, nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, Western blot analysis, confocal microscopy, and coimmunoprecipitation assay were undertaken.


RESULTS
The present study demonstrated that an HDACI, SAHA, at the usual doses or the silencing of alphaB-crystallin by siRNA alone did not effectively induce apoptosis in ARPE-19 cells. Silencing of alphaB-crystallin likely abolishes the anti-apoptotic activity of alphaB-crystallin. The data indicated that silencing of alphaB-crystallin sensitizes ARPE19 cells to SAHA-induced apoptosis and leads them to stage 1 apoptosis. alphaB-Crystallin associates with HDAC1 on SC35 speckles, and silencing of alphaB-crystallin abolishes this association, resulting in the induction of apoptosis. The data indicated that the association between alphaB-crystallin and HDAC1 on SC35 speckles plays a pivotal role in anti-apoptotic activity.


CONCLUSIONS
Knockout of alphaB-crystallin may be a promising new approach to enhance therapeutic potency for proliferative vitreoretinopathy without compromising efficacy.
Department of Anatomy and Cell Biology, Dong-A University College of Medicine and Medical Science Research Center, Busan, South Korea.
Seung Jin
SJ
Noh
Woo Jin
WJ
Jeong
Jee Hyun
JH
Rho
Dong Min
DM
Shin
Hee Bae
HB
Ahn
Woo Chan
WC
Park
Sae Heun
SH
Rho
Young Hwa
YH
Soung
Tae Hyun
TH
Kim
Bong Soo
BS
Park
Young Hyun
YH
Yoo
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002452:Cell Count; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D015536:Down-Regulation; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D056284:Histone Deacetylase 1; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D034741:RNA, Small Interfering; D018630:Vitreoretinopathy, Proliferative / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D000077337:Vorinostat; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D034741:RNA, Small Interfering; D038203:alpha-Crystallin B Chain; D000077337:Vorinostat; D004247:DNA; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases