Sensitization of RPE Cells by αB-Crystallin siRNA to SAHA-Induced Stage 1 Apoptosis through Abolishing the Association of αB-Crystallin with HDAC1 in SC35 Speckles

Abstract

purpose. To better understand the mechanism underlying the anti-apoptotic activity of αB-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 αB-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 αB-crystallin by siRNA alone did not effectively induce apoptosis in ARPE-19 cells. Silencing of αB-crystallin likely abolishes the anti-apoptotic activity of αB-crystallin. The data indicated that silencing of αB-crystallin sensitizes ARPE19 cells to SAHA-induced apoptosis and leads them to stage 1 apoptosis. αB-Crystallin associates with HDAC1 on SC35 speckles, and silencing of αB-crystallin abolishes this association, resulting in the induction of apoptosis. The data indicated that the association between αB-crystallin and HDAC1 on SC35 speckles plays a pivotal role in anti-apoptotic activity.

conclusions. Knockout of αB-crystallin may be a promising new approach to enhance therapeutic potency for proliferative vitreoretinopathy without compromising efficacy.

Crystallins are major structural proteins in the lens. However, since they were first found outside the vertebrate eye lens, ¹ their distributions in retinal pigment epithelial (RPE) cells, optic nerve, extraocular muscle, iris, ciliary body, cornea, and nonocular tissue have also been observed. ^{2 3 4} Thus, entirely different nonlens roles of α-crystallins have been suggested. To date, different functions for αB-crystallin have been described. αB-Crystallin shows in vitro chaperonelike activity, which is reduced on phosphorylation. ⁵ In vivo, αB-crystallin is important for the maintenance and control of the cytoskeleton. ⁶ It can interact in a phosphorylation-independent manner with type 3 intermediate filaments and probably protects the cytoskeleton during stress. ⁷ Ample evidence indicates the involvement of αB-crystallin in the ubiquitin proteasome system. ⁸ Although α-crystallins are observed mainly on the cytoplasm, they were observed on SC speckles, which is a nuclear compartment involved in the storage and recycling of splicing factors ^{9 10}  

The expression of αB-crystallin was also demonstrated on human RPE cells under in vivo and in vitro conditions. ¹¹ In the retina, a major function of crystallins appears to protect retinal neurons from damage by metabolic or environmental stress in light-damaged photoreceptors and in models of retinal degeneration. ^{12 13} αB-Crystallin on RPE cells may function as an anti-apoptotic protein. A previous study showed that RPE cells that had been stably transfected with αB-crystallin were more resistant to H₂O₂-induced cellular injury. ¹¹  

The prevention of apoptosis in RPE cells by αB-crystallin has significance in two pathologic ocular conditions. Because RPE cells may be lost by apoptosis in age-related macular degeneration (AMD), αB-crystallin may play a role in preventing the onset of AMD. Conversely, αB-crystallin could inhibit the efficacy of therapeutics in inducing apoptosis in epiretinal membranes in patients with proliferative vitreoretinopathy (PVR). 

PVR, the principal cause of failed retinal reattachment surgery, is a pathologic wound-healing process, with the migration and uncontrolled proliferation of cells forming an epiretinal membrane. ¹⁴ RPE cells contribute significantly to epiretinal membrane formation in PVR. ^{15 16} Although in some cases surgery can provide a suitable means of treatment for PVR, often it proves futile. Therefore, it would be beneficial to pharmacologically inhibit the proliferation of RPE cells. A variety of anticancer agents have been tested for their therapeutic potential. Unfortunately, however, most of the drugs have found limited clinical application because of toxicity, a relative lack of efficacy, and the need for multiple injections or for vitrectomy with vitreous infusion. ¹⁷  

Histone deacetylase inhibitors (HDACIs) are known to selectively act on proliferative cells in low concentrations and to induce apoptosis. ^{18 19} They target a family of enzymes that catalyzes histone acetylation modifications, particularly for histones H2A, H2B, H3, and H4. The balance of histone acetylation is maintained by histone acetyl transferase and HDACs, which play an important role in gene transcription. ²⁰ HDACs are classified into several groups based on homology to yeast proteins. The class 1 HDACs (1–3 and 8) are located in the nucleus and show ubiquitous expression in various human cell lines and tissues. Class 2 HDACs (4–7, 9, 10) can shuttle between the nucleus and the cytoplasm. Class 3 HDACs (SIRT1–7) need NAD⁺ for their activity to regulate gene expression. ²¹ In the past decade, numerous HDACIs have been identified. SAHA is an HDACI considered a therapeutic agent for various cell-proliferating conditions. ²²  

The present study was undertaken to examine the mechanism underlying the anti-apoptotic activity exerted by αB-crystallin on human RPE cell line ARPE-19 cells as it pertains to the treatment of PVR. As will be shown, silencing of αB-crystallin sensitizes ARPE19 cells to SAHA-induced stage 1 apoptosis, and abolishing the association of αB-crystallin with HDAC1 on SC35 speckles corresponds to the induction of apoptosis. 

Methods

Reagents

The following reagents were obtained commercially: rabbit polyclonal anti–human antibodies; caspase-3 and -6; cytochrome c; apoptosis-inducing factor (AIF); promyelocytic leukemia (PML); Bcl-2; HDAC1, HDAC3, and HDAC6; mouse monoclonal anti–human PML antibody; and heat shock protein 70 (Hsp70) antibody from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti–human αB-crystallin antibody from Stressgen (Ann Arbor, MI); mouse monoclonal anti–human poly (ADP-ribose) polymerase (PARP) antibody from Oncogene (Cambridge, MA); rabbit polyclonal anti–human acetylated histone H2A, H2B, H3, and H4 antibodies from Cell Signaling (Danvers, MA); mouse monoclonal anti–HDAC1 antibody from Upstate (Temecula, CA); FITC-conjugated goat anti–rabbit and horse anti–mouse IgGs from Vector (Burlingame, CA); horseradish peroxidase-conjugated donkey anti–rabbit and sheep anti–mouse IgGs from Amersham Pharmacia Biotech (Piscataway, NJ); Dulbecco modified Eagle medium (DMEM) and fetal bovine serum (FBS) from Gibco BRL (Gaithersburg, MD); mouse monoclonal anti–human SC35 antibody, Hoechst 33342, dimethyl sulfoxide, RNase A, proteinase K, aprotinin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) from Sigma (St. Louis, MO); SAHA from Alexis Biochemicals (San Diego, CA); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanine iodide (JC-1) from Molecular Probes (Eugene, OR); and enhanced chemiluminescence Western blotting detection reagent from Pierce (SuperSignal WestPico; Pierce, Rockford, IL). 

Cell Culture

ARPE-19 cells purchased from the American Type Culture Collection (Rockville, MD) were maintained at 37°C with 5% CO₂ in air atmosphere, with 1:1 mixture of DMEM and Ham F12 medium with 10% FBS. 

αB-Crystallin siRNA

Twenty-one nucleotide RNA with 3′-dTdT overhangs was synthesized by Dharmacon Research through the ready-to-use option. The AA-N19 mRNA targets were αB-crystallin target sequence (5′-AAUUGACCAGUUCUUCGGAGA-3′). As a negative control, the same nucleotides were scrambled to form a nongenomic combination. 

αB-Crystallin siRNA Transfection or Combination Treatment with SAHA

Transfection of siRNA was performed with the use of siPORT Amine and Opti-MEM media. Cells grown to a confluence of 40% to 50% in six-well plates were transfected with 100 nM final siRNA concentration per well. Transfection mixture was added to each well, and incubation occurred for 4 hours. Then 2 mL growth medium was added, and cells were incubated for another 20 hours. After siRNA transfection medium was removed, each well was washed in PBS solution. Cells were treated with various concentrations of SAHA for 48 hours. 

Assessment of Cell Viability

Cells were counted on a cell counter (Vi-Cell; Beckman Coulter, CA) with the use of an automated trypan blue exclusion assay. 

Hoechst Staining

Cell suspension was centrifuged onto a clean, fat-free slide glass with a cytocentrifuge. Samples were stained in 4 μg/mL Hoechst 33342 for 30 minutes at 37°C and were fixed for 10 minutes in 4% paraformaldehyde. 

Quantification of DNA Hypoploidy and Cell Cycle Phase Analysis by Flow Cytometry

Ice-cold 95% ethanol with 0.5% Tween 20 was added to the cell suspension to a final concentration of 70% ethanol. Fixed cells were pelleted and washed in 1% BSA-PBS solution. Cells were resuspended in 1 mL PBS containing 11 Kunitz U/mL RNase, incubated at 4°C for 30 minutes, washed once with BSA-PBS, and resuspended in propidium iodide solution (50 μg/mL). After cells had been incubated at 4°C for 30 minutes in the dark and washed with PBS, DNA content was measured on a flow meter (Epics XL; Beckman Coulter, Hialeah, FL), and data were analyzed using flow cytometry software (Multicycle; Phoenix Flow Systems, San Diego, CA), which allowed a simultaneous estimation of cell cycle parameters and apoptosis. 

DNA Electrophoresis

Cells (10⁶) were resuspended in 1.5 mL lysis buffer (10 mM Tris [pH 7.5], 10 mM EDTA [pH 8.0], 10 mM NaCl, and 0.5% SDS) into which proteinase K (200 μg/mL) was added. After samples were incubated overnight at 48°C, 200 μL ice-cold 5 M NaCl was added, and the supernatant containing fragmented DNA was collected after centrifugation. The DNA was then precipitated overnight at 20°C in 50% isopropanol and was RNase A–treated for 1 hour at 37°C. The DNA from 10⁶ cells (15 μL) was equally loaded on each lane of 2% agarose gels in Tris-acetic acid/EDTA buffer containing 0.5 μg/mL ethidium bromide at 50 mA for 1.5 hours. DNA fragments were separated by 1.8% agarose gel electrophoresis and visualized under ultraviolet light. 

Pulse Field Gel Electrophoresis

Pulse field gel electrophoresis (PFGE) was carried out in 0.5× TBE maintained at 14°C by circulating cool water for 16 hours (CHEF Mapper XA System; Bio-Rad, Hercules, CA). DNA in the gel was stained with EtBr and detected with photographic film (LAS-3000 Plus; Fuji, Kanagawa, Japan). Chromosomal DNA from Saccharomyces cerevisiae and a mixture of λ DNA, its concatemers, and HindIII-digested λ DNA were used as DNA size markers. 

Western Blot Analysis

Cells were washed twice with ice-cold PBS, resuspended in 200 μL ice-cold solubilizing buffer (300 mM NaCl, 50 mM Tris-HCl [pH 7.6], 0.5% Triton X-100, 2 mM PMSF, 2 μL/mL aprotinin, and 2 μL/mL leupeptin), and incubated at 4°C for 30 minutes. Lysates were centrifuged at 14,000 rpm for 15 minutes at 4°C. Protein concentrations of cell lysates were determined with the Bradford protein assay kit (Bio-Rad, Hercules, CA) and equivalent amounts were loaded onto 7.5% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) and were reacted with each antibody. Immunostaining with antibodies was performed using enhanced chemiluminescence substrate (SuperSignal WestPico; Pierce) and was detected with photographic film (LAS-3000 Plus; Fuji). Equivalent protein loading was confirmed by Ponceau S staining. 

Immunofluorescence Staining and Confocal Microscopy

Cells were cytocentrifuged and fixed for 10 minutes in 4% paraformaldehyde, incubated with each primary antibody for 1 hour, washed three times each for 5 minutes, and then incubated with FITC-conjugated secondary antibody for 1 hour at 37°C. Cells were mounted with PBS, and fluorescent images were observed under a laser-scanning confocal microscope (LSM 510; Zeiss, Göttingen, Germany). 

Assay of Mitochondrial Membrane Potential

Disruption of mitochondrial membrane potential (MMP) was measured using a specific fluorescent probe, JC-1, that was added directly to the cell culture medium (5 μg/mL final concentration) and incubated for 15 minutes at 37°C. Cells were stained with JC-1, and flow cytometry to measure MMP was performed (an Epics XL; Beckman Coulter). Data were acquired and analyzed using EXPO32 ADC XL 4 color software. 

Nuclear Fractionation

Cells (5 × 10⁷) swollen in ice-cold hypotonic CaRSB buffer were Dounce-homogenized. Cell homogenates were then spun down (2 × 3000 rpm for 15 minutes). 

Coimmunoprecipitation

Cell extracts were incubated with appropriate antibodies in extraction buffer at 4°C overnight. Immunocomplexes were precipitated with protein A-Sepharose beads for 2 hours and washed five times with extraction buffer before boiling in SDS sample buffer. Immunoprecipitated proteins or aliquots containing 40 μg protein were separated on SDS-polyacrylamide gels, and Western blot analysis was performed as described. Each coimmunoprecipitation experiment was confirmed through reciprocal immunoprecipitation (data not shown). 

Statistical Analysis

Four independent experiments were carried out. Mean values were calculated from data obtained from triplicates of each independent experiment. Results of the experimental and control groups were tested for statistical significance by the Kruskal-Wallis nonparametric test. 

Results

Sensitization of ARPE-19 Cells by αB-Crystallin siRNA to SAHA-Induced Cell Death

SAHA was reported to induce cell death in most cultured cells, with IC₅₀ values in the range of 1 to 50 μM as determined by viability assay on cells 48 hours after treatment. However, SAHA treatment alone did not reduce the viability of ARPE-19 cells at those concentrations. Notably, silencing of αB-crystallin by siRNA significantly sensitized ARPE-19 cells to SAHA-induced cell death (Fig. 1) . Given that the viability of ARPE-19 cells treated with 10 μM SAHA in combination with αB-crystallin siRNA was approximately 50%, we selected this single combination treatment for further exploration. 

Sensitization of ARPE-19 Cells by αB-Crystallin siRNA to SAHA-Induced Apoptosis

Although SAHA with αB-crystallin siRNA did not demonstrate ladderlike DNA fragments on agarose gel, disintegration of nuclear DNA into giant fragments of 1 to 2 Mbp and high molecular weight fragments of 200 to 800 kbp was recognized by PFGE (Fig. 2A) . Hoechst staining showed nuclear condensation (Fig. 2B) , and flow cytometry indicated accumulation of subdiploid apoptotic cells (Fig. 2C) . SAHA with αB-crystallin siRNA induced the degradation of procaspase-3 and -6 and PARP and the production of their cleaved products; it also downregulated Bcl-2 (Fig. 2D) . SAHA with αB-crystallin siRNA induced the release of cytochrome c and AIF from mitochondria (Fig. 2E)and reduced MMP (Fig. 2F) . 

Sensitization of ARPE-19 Cells by αB-Crystallin siRNA to SAHA-Induced Hyperacetylation of Histones and Downregulation of HDAC1

Although 10 μM SAHA alone did not induce histone hyperacetylation in ARPE-19 cells, silencing of αB-crystallin sensitized ARPE-19 cells to the ability of SAHA to induce histone acetylation (Fig. 3A) . We next examined whether the anti-apoptotic effect of αB-crystallin is associated with its modulation of SAHA-sensitive HDAC subtypes. Neither SAHA alone nor SAHA with αB-crystallin siRNA altered the expression levels of HDAC3 or HDAC6. Notably, SAHA at 10 μM slightly downregulated HDAC1, and this downregulation was substantially augmented by αB-crystallin siRNA (Fig. 3B) . 

Localization of αB-Crystallin to SC35 Speckles and Its Association with HDAC1

Confocal microscopy showed that αB-crystallin was distributed not only in the cytoplasm but also in the nuclei of control ARPE-19 cells. On the nucleus, αB-crystallin formed nuclear body–like structures and localized to SC35 speckles. Neither nuclear nor cytosolic αB-crystallins were observed in cells treated with αB-crystallin siRNA alone or with SAHA with αB-crystallin siRNA, indicating that silencing of αB-crystallin by siRNA efficiently eliminates αB-crystallin. SAHA alone did not alter the subcellular distribution of αB-crystallin (Fig. 4A) . αB-Crystallin did not localize to another nuclear body, the PML body (Supplementary Fig. S1A). 

We observed HDAC1 in the whole nucleus of ARPE-19 cells. It is notable that HDAC1 in the nucleus formed large, bright, specklelike structures. Confocal microscopy showed that HDAC1 colocalizes with SC35. αB-Crystallin siRNA alone did not alter the nuclear distribution of HDAC1. In contrast, SAHA alone treatment substantially evacuated nuclear HDAC1. However, its distribution in SC35 speckles was, in general, maintained in ARPE-19 cells. Notably, HDAC1 disappeared in SC35 speckles of ARPE-19 cells treated with SAHA with αB-crystallin siRNA (Fig. 4B) . Coimmunoprecipitation assay conducted on the nuclear fraction of ARPE-19 cells also indicated that αB-crystallin and HDAC1 associate in SC35 speckles and that SAHA with αB-crystallin siRNA abolishes this association, although SAHA alone did not alter this association (Fig. 4C) . HDAC1 did not localize to another nuclear body, the PML body (Supplementary Fig. S1B). Next, we examined whether HDAC1 stabilized by αB-crystallin prevents hyperacetylation of αB-crystallin by SAHA in SC35 speckles. Our data show that SAHA upregulated acetylated Hsp70 but that it did not alter the acetylation state of αB-crystallin (Fig. 4D) . Two other HDAC subtypes, HDAC3 and HDAC6, did not show evident localization in SC35 speckles. None of the three treatments above altered the subcellular location or expression level of HDAC3 and HDAC6 (Figs. 5A 5B) . Coimmunoprecipitation assay also demonstrated no interaction of HDAC3 or HDAC6 with αB-crystallin (Fig. 5C) . 

Discussion

Our novel observations add new insight into the role of αB-crystallin in RPE cells. Our data show for the first time that silencing of αB-crystallin by siRNA sensitizes ARPE-19 cells to SAHA-induced stage 1 apoptosis though SAHA usually does not effectively induce apoptosis in RPE cells. 

Early in the apoptotic process, DNA is cleaved into HMW fragments (stage 1), which degrade, in the next step, into oligonucleosomal size (stage 2). Although two stages of DNA degradation are observed in most apoptotic cells, some cells exhibit stage 1 DNA degradation but never reach stage 2. AIF is known to be the main factor responsible for HMW DNA fragmentation, indicating stage 1 apoptosis. ²³ Numerous previous studies related to apoptosis in ARPE-19 cells have demonstrated DNA degradation by a PFGE instead of by conventional gel electrophoresis, similar to the present study. Lack of data demonstrating the DNA ladder in ARPE-19 cells may explain the high chemoresistance of this cell. ²⁴ Considering that elucidating the mechanism of chemoresistance in RPE cells is an important and challenging task, future studies are needed. 

This study also shows for the first time that αB-crystallin in RPE cells may play an anti-apoptotic role in association with SC35 speckles. 

Although α-crystallins are soluble cytoplasmic proteins, they have been described in association with the nucleus. The nucleus contains several types of subnuclear structures such as nucleoli, SC35 speckles, Cajar bodies, and PML bodies, all of which have different nuclear activities. ^{10 25} As previous studies have shown, αB-crystallin localizes to SC35 speckles, ^{26 27} which are interchromatin granule clusters that contain snRNPs and other splicing components and that may function as sites for storage or recycling of splicing factors. ¹⁰  

To date, functional roles of αB-crystallin in the SC35 speckles have not been fully delineated. Previous data suggest that several chaperones, such as heat shock protein 27, have been demonstrated to localize in the nuclear compartment and to function in the recovery from nuclear protein aggregation. ²⁸ The SC35 speckle compartment constitutes a dynamic protein population, and there are presumably many opportunities for interaction between those proteins and αB-crystallin. ^{25 29} Thus, the specific association of αB-crystallin with SC35 speckles may indicate a chaperoning role of αB-crystallin in this compartment. In addition, αB-crystallin, like other chaperones, may be involved in the regulation of transcription. ³⁰  

HDACs can downregulate gene expression by core histone deacetylation. HDACs can also deacetylate nonhistone protein. Like other HDAC subtypes, HDAC1 is known to modulate numerous apoptosis-related factors. ³¹ Our data showing that HDAC1 associates with αB-crystallin on SC35 speckles raise an important issue on the functional role of the association of αB-crystallin and HDAC1 on SC35 speckles of ARPE-19 cells. Different HDACs are associated with different chaperone proteins. ³² Here we observed that HDAC1 is associated with αB-crystallin in SC35 speckles. This finding raises the interesting possibility that this association plays a role in the anti-apoptotic function of αB-crystallin. Given that ATP-dependent chaperone proteins are known to orient the histone substrate to the catalytic pocket of HDAC, we propose that the presence of αB-crystallin may enhance deacetylation of histones by HDAC1 in SC35 speckles. We also observed that the distribution of HDAC1 in SC35 speckles was maintained in cells treated with SAHA alone. This finding suggests that αB-crystallin may function in stabilizing its client protein, HDAC1, in SC35 speckles of ARPE-19 cells. A previous study showed that HDACI exerts its activity through the hyperacetylation of chaperones. ³³ Thus, we examined whether HDAC1 stabilized by αB-crystallin prevents hyperacetylation of αB-crystallin by SAHA in SC35 speckles. However, our data show that unlike hsp70, SAHA did not alter the acetylation state of αB-crystallin. 

Although the mechanism by which αB-crystallin exerts anti-apoptotic function in SC35 speckles remains speculative, our in vitro study provides a clue that silencing of αB-crystallin is likely to sensitize RPE cells to the apoptosis-inducing efficacy of SAHA by abolishing the anti-apoptotic activity exerted by αB-crystallin in SC35 speckles. There may be a significant gap in the knowledge of what substrates αB-crystallin interacts with in vivo, but our in vitro study suggests that the association of αB-crystallin and HDAC1 in SC35 speckles is implicated in pathologic ocular conditions related to apoptosis. 

It is unfortunate that a favorable therapeutic response to many drugs for PVR is often associated with severe toxicity in RPE cells. In this regard, knockout of αB-crystallin by siRNA could be a promising new approach to enhance the therapeutic potency in PVR so that lower doses may be used without compromising efficacy. 

²  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.

 Supported by Korea Research Foundation Grant (KRF-2006-J03501).

 Submitted for publication April 14, 2008; revised May 21, 2008; accepted August 18, 2008.

 Disclosure: S.J. Noh, None; W.J. Jeong, None; J.H. Rho, None; D.M. Shin, None; H.B. Ahn, None; W.C. Park, None; S.H. Rho, None; Y.H. Soung, None; T.H. Kim, None; B.S. Park, None; Y.H. Yoo, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Silencing of αB-crystallin by siRNA sensitizes ARPE-19 cells to SAHA-induced cell death. Ctrl, control; Si, small interfering RNA directed against αB-crystallin; Sc, scramble control. Assays were undertaken on ARPE-19 cells obtained 48 hours after treatment with SAHA with or without αB-crystallin siRNA or scrambled RNA. αB-Crystallin siRNA significantly sensitizes ARPE-19 cells to SAHA-induced cell death. *P < 0.05; **P < 0.01.

Figure 2.

 

Silencing of αB-crystallin by siRNA sensitizes ARPE-19 cells to SAHA-induced apoptosis. Ctrl, control; SiRNA, small interfering RNA directed against αB-crystallin; ScRNA, scrambled control siRNA; SAHA, 10 μM SAHA. Scale bar, 20 μm. Assays were undertaken in ARPE-19 cells obtained 48 hours after treatment with SAHA with or without αB-crystallin siRNA or scrambled RNA. (A) DNA electrophoresis (left) and PFGE (right). (B) Nuclear morphology after Hoechst staining. (C) Representative histograms showing cell cycle progression and induction of apoptosis. (D) Western blot assay on apoptosis-related proteins. β-Actin was used for the loading control. (E) Confocal microscopy showing the subcellular localization of cytochrome c and AIF. (F) Flow cytometry showing mitochondrial membrane potential.

Figure 3.

 

Silencing of αB-crystallin by siRNA augments histone acetylation and downregulation of HDAC1 induced by SAHA in ARPE-19 cells. Ctrl, control; SiRNA, small interfering RNA directed against αB-crystallin; ScRNA, scramble control siRNA; SAHA, 10 μM SAHA. Assays were undertaken on ARPE-19 cells obtained 48 hours after treatment with SAHA with or without αB-crystallin siRNA or scrambled RNA. (A) Western blot assay on acetylated histones. β-Actin was used for the loading control. (B) Western blot assay on HDAC1, HDAC3, and HDAC6. β-Actin was used for the loading control.

Figure 4.

 

Association of αB-crystallin and HDAC1 in SC35 speckles. Ctrl, control; SiRNA, small interfering RNA directed against αB-crystallin; SAHA, 10 μM SAHA. Scale bar, 20 μm. (A) Confocal microscopy showing the association of αB-crystallin with SC35. αB-Crystallin is distributed not only in the cytoplasm (arrowheads) but also in the nuclei of control ARPE-19 cells. In the nuclei of the control cells, αB-crystallin colocalizes with SC35 speckles (arrows). Neither nuclear nor cytosolic αB-crystallins were observed in cells treated with αB-crystallin siRNA alone (SiRNA) or SAHA with αB-crystallin siRNA (siRNA+SAHA), indicating that silencing of αB-crystallin by siRNA efficiently eliminates αB-crystallin. (B) Confocal microscopy showing the association of HDAC1 with SC35. HDAC1 is observed in the whole nuclei of ARPE-19 cells. Notably, HDAC1 formed a large, bright, specklelike structure. Confocal microscopy showed that HDAC1 colocalizes with SC35 (Ctrl). αB-Crystallin siRNA alone did not alter the nuclear distribution of HDAC1 (siRNA). SAHA treatment alone substantially evacuated nuclear HDAC1. However, its distribution in SC35 speckles was maintained in ARPE-19 cells (SAHA) in general. HDAC1 disappeared in SC35 speckles in ARPE-19 cells treated with SAHA and αB-crystallin siRNA (siRNA+SAHA). The profile of SC35 and HDAC1 fluorescence intensity is depicted (bottom). The intensity of SC35 is shown in red, and that of HDAC1 is shown in green. In contrast to the control cell (a), HDAC1 protein is concentrated within SC35 speckles in SAHA-treated cells (b). (C) Coimmunoprecipitation assay on the nuclear fraction of ARPE-19 cells shows αB-crystallin coimmunoprecipitated with HDAC1. (D) Coimmunoprecipitation assay on the nuclear fraction of ARPE-19 cells shows that unlike hsp70, SAHA with αB-crystallin siRNA did not alter the acetylation state of αB-crystallin.

Figure 5.

 

Confocal microscopy showing that treatment with neither SAHA alone nor SAHA with αB-crystallin siRNA alters the subcellular distribution of HDAC3 (A) or HDAC6 (B). Assays were undertaken on ARPE-19 cells obtained 48 hours after treatment with SAHA with or without αB-crystallin siRNA. Scale bar, 20 μm. (C) Coimmunoprecipitation assay on the nuclear fraction of ARPE-19 cells shows αB-crystallin is not coimmunoprecipitated with HDAC3 or HDAC6.

Supplementary Materials

Supplementary Figure S1 - (PDF)