Nstrated in HeLa and CHO cells that aAcrystallin inhibited Caspase-9 and

Nstrated in HeLa and CHO cells that aAcrystallin inhibited Caspase-9 and Caspase-3 activity and prevented chemically-triggered apoptosis as well as apoptosis induced by over-expression of pro-apoptotic Bim and Bax. In these cells, aA-crystallin-mediated anti-apoptotic function was directly related to its chaperone activity by enhancing PI3K/Akt survival pathway and reducing phosphatase tensin homologue (PTEN) activity [10]. During ER stress-induced retinal pigment epithelial (RPE) cell apoptosis, aB-crystallin has been shown to protect cells against mitochondrial dysfunction by inhibiting Bax and CHOP upregulation, attenuating caspase activation and restoring mitochondrial permeability transition [47]. McGreal and colleagues also reported that aB-crystallin interacted with cytochrome C and preserved mitochondrial membrane potential under oxidative stress [48]. Additionally, in mouse retinal explants exposed to oxidative stress, RPE-secreted aB-crystallin was able to provide neuroprotection toa-Crystallin Cytoprotective ActionFigure 7. Generation and expression of aA-crystallin deletion mutants. (A) Schematic representation of the various deletion mutants of aAcrystallin. (B) Western blot and (C) immunofluorescence analyses in transiently transfected 293T cells showing expression of wt and mutant aAcrystallin proteins fused to luciferase using luciferase antibody. doi:10.1371/MedChemExpress 69-25-0 journal.pone.0055372.ga-Crystallin Cytoprotective ActionFigure 8. The C-terminal extension domain of aA-crystallin was sufficient to provide protection against Bax-induced apoptosis. Twenty-four hours post-transfection, 293T cells transfected with Bax (Bax/pRluc) or with Bax and aA-crystallin wt or mutants were assayed in TUNEL assay. Counting of TUNEL-positive apoptotic cells showed that aA_wt (Bax/aA_wt), aA_90-143 (Bax/aA_90-143) and aA_144-173 (Bax/aA_144-173) significantly inhibited Bax-induced apoptosis, whereas N-terminal aA_1-89 (Bax/aA_1-89) and aA_1-116 (Bax/aA_1-116), along with aA_64-143 (Bax/ aA_64-143) containing the a-crystallin domain, did not prevent apoptosis. (* p,0.05, ** p,0.01, *** p,0.005 by t-test versus Bax/aA_wt). Data are the mean 6 SE of two to three independent experiments. doi:10.1371/journal.pone.0055372.gadjacent photoreceptor cells through inhibition of Caspase-3 and PARP activation [49]. Several studies reported on altered expression of a-crystallins in inherited retinal diseases [28,30], light-induced retinal degeneration [32] as well as early- and late-stage ARMD [33,50?2]. It is thus tempting to speculate that a-crystallins may be involved in the development of these degenerative diseases. However, their role in inherited retinal degeneration has not been studied yet and the molecular mechanisms that may regulate a-crystallin-mediated protection against photoreceptor apoptosis remain KDM5A-IN-1 price unknown. This prompted us to assess the cytoprotective 24786787 role of a-crystallins in 1317923 the survival of photoreceptor-like 661W cells. We reported a dosedependent decrease in cellular viability following STS treatment in lentiviral-mediated 661W cells stably expressing aA- or aBcrystallin, as reflected by increased TUNEL-positive apoptotic cells and decreased cellular ATP content. Moreover, we showed that a-crystallins prevented apoptosis through the inhibition of Caspase-3/-7 activity. It has been shown in vivo in aA2/2crystallin and aB2/2-crystallin knock-out mice that RPE lacking a-crystallins was more susceptible to apoptosis when subjected to H2O2-induc.Nstrated in HeLa and CHO cells that aAcrystallin inhibited Caspase-9 and Caspase-3 activity and prevented chemically-triggered apoptosis as well as apoptosis induced by over-expression of pro-apoptotic Bim and Bax. In these cells, aA-crystallin-mediated anti-apoptotic function was directly related to its chaperone activity by enhancing PI3K/Akt survival pathway and reducing phosphatase tensin homologue (PTEN) activity [10]. During ER stress-induced retinal pigment epithelial (RPE) cell apoptosis, aB-crystallin has been shown to protect cells against mitochondrial dysfunction by inhibiting Bax and CHOP upregulation, attenuating caspase activation and restoring mitochondrial permeability transition [47]. McGreal and colleagues also reported that aB-crystallin interacted with cytochrome C and preserved mitochondrial membrane potential under oxidative stress [48]. Additionally, in mouse retinal explants exposed to oxidative stress, RPE-secreted aB-crystallin was able to provide neuroprotection toa-Crystallin Cytoprotective ActionFigure 7. Generation and expression of aA-crystallin deletion mutants. (A) Schematic representation of the various deletion mutants of aAcrystallin. (B) Western blot and (C) immunofluorescence analyses in transiently transfected 293T cells showing expression of wt and mutant aAcrystallin proteins fused to luciferase using luciferase antibody. doi:10.1371/journal.pone.0055372.ga-Crystallin Cytoprotective ActionFigure 8. The C-terminal extension domain of aA-crystallin was sufficient to provide protection against Bax-induced apoptosis. Twenty-four hours post-transfection, 293T cells transfected with Bax (Bax/pRluc) or with Bax and aA-crystallin wt or mutants were assayed in TUNEL assay. Counting of TUNEL-positive apoptotic cells showed that aA_wt (Bax/aA_wt), aA_90-143 (Bax/aA_90-143) and aA_144-173 (Bax/aA_144-173) significantly inhibited Bax-induced apoptosis, whereas N-terminal aA_1-89 (Bax/aA_1-89) and aA_1-116 (Bax/aA_1-116), along with aA_64-143 (Bax/ aA_64-143) containing the a-crystallin domain, did not prevent apoptosis. (* p,0.05, ** p,0.01, *** p,0.005 by t-test versus Bax/aA_wt). Data are the mean 6 SE of two to three independent experiments. doi:10.1371/journal.pone.0055372.gadjacent photoreceptor cells through inhibition of Caspase-3 and PARP activation [49]. Several studies reported on altered expression of a-crystallins in inherited retinal diseases [28,30], light-induced retinal degeneration [32] as well as early- and late-stage ARMD [33,50?2]. It is thus tempting to speculate that a-crystallins may be involved in the development of these degenerative diseases. However, their role in inherited retinal degeneration has not been studied yet and the molecular mechanisms that may regulate a-crystallin-mediated protection against photoreceptor apoptosis remain unknown. This prompted us to assess the cytoprotective 24786787 role of a-crystallins in 1317923 the survival of photoreceptor-like 661W cells. We reported a dosedependent decrease in cellular viability following STS treatment in lentiviral-mediated 661W cells stably expressing aA- or aBcrystallin, as reflected by increased TUNEL-positive apoptotic cells and decreased cellular ATP content. Moreover, we showed that a-crystallins prevented apoptosis through the inhibition of Caspase-3/-7 activity. It has been shown in vivo in aA2/2crystallin and aB2/2-crystallin knock-out mice that RPE lacking a-crystallins was more susceptible to apoptosis when subjected to H2O2-induc.