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From the Department of Ophthalmology, Columbia University, New York.
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Abstract |
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METHODS. Human RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light. Caspase-3 activity was assayed by observing cleavage of a fluorogenic peptide substrate, and the effect of a peptide inhibitor of caspase-3 (Z-DEVD-fmk) on the quantity of apoptotic nuclei was determined. ARPE-19 cells were transfected with either a neomycin-selectable expression vector containing Bcl-2 cDNA or a control neomycin-selectable expression vector without Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein expression was confirmed by Western blot analysis. Cell viability was assayed by TdT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidino-2-phenylindole (DAPI) staining and by fluorescence staining of the nuclei of membrane-compromised cells.
RESULTS. In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of blue light exposure. The incidence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected with Bcl-2.
CONCLUSIONS. Blue light illumination of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a proteolytic caspase cascade and that is regulated by Bcl-2.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Analysis of extracts of human RPE has revealed that the major hydrophobic fluorophore of RPE lipofuscin is A2E,13 14 a quaternary pyridinium salt14 15 16 that is generated after hydrolytic cleavage of the all-trans-retinal-phosphatidylethanolamine conjugate, A2PE (phosphatidyl-pyridinium bisretinoid).17 It is also now clear that the accumulation of A2E has adverse consequences for the cell. Thus, as an amphiphilic detergent, A2E has been shown to exert a detergent-like perturbation of cell membranes,18 an effect that may explain the propensity for A2E to interfere with the adenosine triphosphatase (ATPase)–dependent acidification of lysosomes.19 A2E has also been shown to confer a susceptibility to photo-induced damage.20 21 In particular, the blue (480 nm) region of the spectrum was found to induce the death of A2E-containing cultured RPE cells in a manner that was directly dependent on the A2E content of the cells.20 Conversely, green light (540 nm) was considerably less effective. This wavelength dependence was consistent with the absorbance and excitation spectra of A2E.20
Although the photochemical events triggering apoptosis under conditions
of blue light exposure are not fully understood, the cell death program
is probably executed by caspases, a family of cysteine-dependent
proteases located in the cytoplasm.22
23
24
25
26
27
Several lines of
evidence indicate that the caspase-mediated cleavage of manifold
cellular substrates, including enzymes involved in DNA repair,
structural components of the cytoplasm and nucleus, and various protein
kinases, is directly responsible for the demise of the cell. Caspases
are synthesized as inactive zymogens (procaspases) whose activation
requires cleavage on the carboxyl side of aspartate residues to
liberate one large (
20 kDa) and one small (10 kDa) subunit. The
active enzyme is then formed as a tetramer consisting of two of each of
these subunits. Caspases cleave substrate proteins exclusively after
aspartate residues, and the sequence of the four amino acid
NH2-terminals to the cleavage site determines the
substrate specificity of the different caspases. Distinct members of
the caspase family are involved in both the initiation and execution
phases of apoptosis, with the initiator caspases coupling cellular
signaling pathways to caspase activation and the downstream effector
caspases being responsible for the cleavage of cellular substrates.
Although several human caspases have been identified, it is becoming increasingly clear that not only does the specific subset of caspases recruited and the sequence in which they are activated vary with the particular cell death paradigm, but the cascade may also exhibit cell-type specificity.24 28 29 30 31 Furthermore, the inhibition of caspases does not always prevent cell death elicited by proapoptotic signals.32 Perhaps the best studied of the cell death pathways are those that are triggered by binding of cognate ligand to one of a number of cell surface death receptors. Subsequent clustering of the receptor leads to physical association with an adaptor protein at the cytoplasmic face and ultimately to the clustering and activation of initiator caspases. Conversely, other cell death pathways are initiated by mitochondria, with cytochrome c and probably other proteins being released from the mitochondrial intermembrane space. On entering the cytosol, cytochrome c forms a complex with an adaptor protein (APAF-2), thereby recruiting and activating the initiator caspase. Upstream of this process, an additional level of regulation is provided by the Bcl-2 family of proteins, many but not all of which reside in the mitochondrial outer membrane and among other actions, control the release of mitochondrial apoptogenic factors, such as cytochrome c.30 33 34 35 36 37 Nevertheless, because one antiapoptotic member of this family, Bcl-2 protein, can inhibit some apoptotic paradigms but not others, it is possible that certain death stimuli can either circumvent or operate downstream of Bcl-2.
Although a number of exogenous photosensitizers—for instance, those used in photodynamic therapy38 39 —have been studied for their ability to initiate apoptosis after their activation by specific wavelengths of light, far less is understood of the mechanism by which an identified, naturally occurring fluorophore, such as A2E, induces apoptosis after exposure to visible light. By studying caspase-3 as an effector of the death process and Bcl-2 as a potential negative regulator, we addressed the molecular pathways involved in executing RPE cell death in the context of A2E and blue light.
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Methods |
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A2E Synthesis and Loading
A2E was synthesized from all-trans-retinal and
ethanolamine14
and stored as a stock solution in dimethyl
sulfoxide (DMSO). For loading of RPE cells, A2E was delivered in
100-µM concentrations in culture media, as previously
described.18
The autofluorescence of cell-associated A2E
was detected by epifluorescent illumination under a microscope
(Axiovert S100; Carl Zeiss, Thornwood, NY) and standard fluorescein
isothiocyanate (FITC) filters (460–500-nm excitation, 510–560-nm
emission).
Blue Light Illumination
Cells growing in eight-well plastic chamber slides were exposed,
either to a single spot of blue light delivered from a 100-W mercury
lamp (480 ± 20 nm; 35 mW/mm2, 60
seconds)20
or to a light line delivered from a tungsten
halogen source (470 ± 20 nm; 0.4 mW/mm2;
20-minute exposure), as indicated. These wavelengths are consistent
with the excitation spectrum of A2E.20
.
Caspase-3 Cleavage Activity
Caspase-3–like protease activity was studied by detecting the
cleavage of the cell-permeable fluorogenic peptide substrate GDEVDGI
(Gly-Asp-Glu-Val-Asp-Gly-Ile; PhiPhiLux-G2D2; Alexis, Laufelfingen,
Switzerland).40
41
42
43
Briefly, 5 hours after light exposure,
the cells were incubated for 1 hour at 37°C in the dark with 10 µM
substrate prepared in RPMI-1640 supplemented with 10% fetal calf serum
(FCS). Immediately after washing, the cleaved substrate was detected by
fluorescence microscopy using rhodamine-appropriate filters.
Inhibition of Caspase-3
Before exposure to blue light, A2E containing ARPE-19 cells was
preincubated at 37°C for 1 hour with 20 µM
Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-fmk; Alexis), a
cell-permeable peptide inhibitor whose tetrapeptide structure is based
on the optimal sequence recognized by caspase-3.44
Transfection of Bcl-2
ARPE-19 cells grown in 100-mm culture dishes to 70% to 80%
confluence were transfected with the neomycin-resistant
pSFFV/Bcl-2 plasmid (a generous gift from Ralph Buttyan,
Columbia University, the College of Physicians and Surgeons, New York,
NY).45
Control cells were transfected with the
neomycin-resistant expression vector (pCMV-Script; Stratagene, La
Jolla, CA) without insertion of the Bcl-2 cDNA. Transfection
complexes were prepared by preincubation of plasmid (4 µg) with
reagents (Plus-Lipofectamine; Gibco–Life Technologies, Grand Island,
NY) in serum-free/antibiotic-free Dulbecco’s modified Eagle’s medium
(DMEM; 0.75 ml), according to the manufacturer’s instructions.
Subsequently, 1.5 ml of the reagent complex was gently mixed with the 5
ml of DMEM in each culture plate. After 3 hours of incubation at 37°C
and 8.5% CO2, 6.5 ml of antibiotic-free medium
containing 20% FCS was added to each dish. Twenty-four hours after the
start of transfection, the cells were replated in DMEM with 10% FCS at
a density of 105 cells/100-mm dish, and after an
additional 24 hours, G418 sulfate (700 µg/ml; Gibco–Life
Technologies) was added to begin selection. Medium containing G418 was
renewed weekly, and after 3 weeks, individual colonies were isolated
with cloning rings and were subcultured and eventually expanded.
Seventeen pSFFV Bcl-2 and eight pCVM-Script-neo clones were screened
for Bcl-2 expression.
Probe Generation and In Situ Hybridization
To generate RNA probes for Bcl-2 in situ
hybridization, a fragment of a 630-bp Bcl-2 cDNA was
subcloned into the pBluescript II KS(±) vector.
BssHII/EcoRI (antisense) and
BssHII/BamHI (sense) linearized DNA templates
were purified by electrophoresis on agarose gel and transcribed using
the digoxigenin RNA labeling system (Boehringer–Mannheim,
Indianapolis, IN). Forward transcription from the T7 promoter generated
the antisense probe. Before hybridization, probe size was determined by
electrophoresis on a 1% agarose gel, and labeling efficiency was
detected by dot blot hybridization.
Cells grown in eight-well chambers were fixed with 4% paraformaldehyde for 20 minutes, washed in phosphate-buffered saline (PBS), and digested with 7.5 µg/ml proteinase K in 50 mM EDTA and 0.1 M Tris-HCl (pH 8.0) for 20 minutes at 37°C. After rinsing in 0.2% glycine to arrest digestion, the sections were acetylated in 0.25% acetic anhydride containing 0.1 M triethanolamine for 10 minutes. The slides were prehybridized for 2 hours at 37°C in a solution containing 50% formamide, 2x SSC, 1x Denhardt’s solution, 10% dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), 4 mM EDTA, 250 µg/ml yeast t-RNA, and denatured salmon testis DNA. Hybridization of Bcl-2 antisense digoxigenin UTP-labeled RNA probe (2.5 ng/µl) was then performed overnight at 42°C. Sense probe served as a negative control. RNase A was subsequently applied to the sections for 30 minutes to digest any unbound probe. The slides were then washed repeatedly with gentle agitation in declining concentrations of SSC (2x to 0.1x SSC) followed by digoxigenin buffer (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl). After blocking with 10% normal serum, the sections were incubated for 2 hours with alkaline phosphatase–conjugated anti-digoxigenin polyclonal sera (Boehringer–Mannheim), diluted 1:750 in 100 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 10% normal serum. The bound antibody was detected using nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate as substrate (Boehringer–Mannheim).
Detection of Bcl-2 by Western Blot Analysis
Cells were washed with PBS and lysed in 50 mM Tris-HCl (pH 7.5)
containing 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and
protease inhibitors. Lysates were centrifuged at 12,000g for
10 minutes, and the protein concentration of the supernatant was
determined using a protein assay system (Bio-Rad, Richmond, CA). Bcl-2
proteins were immunoprecipitated using monoclonal antibody to human
Bcl-2 (Dako, Glostrup, Denmark), and samples containing equal amounts
of immunoprecipitated protein (20 µg) were separated by 10% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membranes. Bcl-2 protein was detected
using the antibody to human Bcl-2, and binding of secondary antibody
was detected using the blot detection system (Hybond ECL; Amersham
Pharmacia Biotech, Piscataway, NJ).
Assays of Cell Viability
To count nonviable cells using assays based on the detection of
nuclear condensation and DNA fragmentation, terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) of apoptotic
nuclei was performed, together with 4'6'-diamidino-2-phenylindole
(DAPI) labeling of all nuclei, 6 hours after blue light exposure. For
staining, cultures were fixed in 2% paraformaldehyde for 30 minutes,
permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (2
minutes, 4°C), incubated in TdT together with dUTP-rhodamine (37°C,
60 minutes; Boehringer–Mannheim), and stained with DAPI (0.3 µM). To
determine the percentage of apoptotic nuclei, TUNEL- (rhodamine) and
DAPI-stained nuclei were visualized by fluorescence microscopy (x40
objective), and counting was performed from digital images after
contrast enhancement by computer (PhotoShop ver. 5; Adobe, San Jose,
CA).
Nonviable cells were also labeled by fluorescence-exclusion assays that allow for the labeling of apoptotic nuclei because of a loss of plasma membrane integrity during the latter stages of apoptosis. Accordingly, 12 and 18 hours after blue light exposure, the nuclei of dead cells were stained with the membrane-impermeable dyes propidium iodide (15 µM in medium, 15-minute incubation; Molecular Probes, Eugene, OR) or Dead Red (1:500 dilution, 15-minute incubation; Molecular Probes) alone or in combination with Hoechst 33342 (5 µg/ml) to stain all nuclei. In all experiments, replicates were assayed as indicated in the figure legends.
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Results |
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The role of caspase-3 in mediating the blue light–induced death of A2E-containing RPE cells was further assessed by exposing the cells to a spot of 480 nm illumination (60 seconds) in the presence of the caspase-3 inhibitor Z-DEVD-fmk. Using a fluorescence assay in which the nuclei of nonviable cells were labeled with the membrane-impermeable dye Dead Red 18 hours after light exposure, we observed that treatment with Z-DEVD-fmk reduced the numbers of nonviable cells in the 0.5-mm diameter zones corresponding to the areas of illumination (Figs. 3A 3B ). Counting of fluorescently labeled nuclei in the illuminated fields revealed that the addition of Z-DEVD-fmk decreased the numbers of apoptotic nuclei to an average of 55% of control numbers (three experiments with two-tailed P = 0.0006, 0.02, and 0.05, by Student’s t-test; Fig. 3C ). As previously reported,20 the frequency of apoptotic nuclei among cells that had not been loaded with A2E but that were exposed to blue light was not greater than background levels observed in nonilluminated regions of the cultures (not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Apoptosis induced in A2E-loaded RPE cells by blue light was inhibited by approximately 50% in the presence of the caspase-3 inhibitor, Z-DEVD-fmk. This level of inhibition is consistent with that achieved in other studies using Z-DEVD-fmk to block apoptosis in whole cells induced by a variety of agents.51 52 53 The failure of Z-DEVD-fmk to completely prevent apoptosis may be interpreted as evidence for a redundant caspase-3–independent pathway. On the other hand, because signal augmentation occurs along the caspase cascade, blockage of a single protease may reflect the kinetics of amplification along this enzyme pathway.51 If even a small amount of caspase is activated, it could be sufficient to induce the death program. In addition, although Z-DEVD-fmk is membrane permeable, some restriction on the penetrability of the tetrapeptide inhibitor is suggested by the observation that considerably higher concentrations of inhibitor are required to inhibit the death of intact cells than for inhibition of caspase-3 in cell-free systems.54 55
The Bcl-2 protein resides on the outer of the two mitochondrial membranes and is one member in a family of proteins that play a pivotal role in the regulation of cell death. Some of the members of the Bcl-2 family, such as Bcl-2, Bcl-XL, and Bcl-w, inhibit apoptosis, whereas others, for instance Bax, Bak, and Bad, are promoters.27 33 Ectopic expression of Bcl-2 in a transgenic approach has been shown to rescue photoreceptor cells in retinal degeneration slow (rds) mice,56 but has little or only temporary effects in other forms of retinal degeneration in mice, including a model of light damage.57 58 59 Similarly, enforced overexpression of Bcl-2 in cultured cells has been shown to confer a resistance to apoptosis induced by many,45 60 61 62 63 64 65 but not all,60 66 67 68 cell death stimuli. Under some circumstances, the ratio between the pro- and anti-apoptotic molecules is considered to be at least one of the determinants of the susceptibility of a cell to a death stimulus.69 In keeping with this, the extent to which overexpression of Bcl-2, after transfection into cultured cells, inhibits apoptosis has been shown to vary with the level of Bcl-2 protein expression.61
A number of mechanisms have been proposed to explain the ability of Bcl-2 to suppress apoptosis. For instance, the formation of heterodimers between antiapoptotic and proapoptotic proteins is thought to lead to the neutralization of activity.69 Apart from heterodimerization, Bcl-2 also can protect against release of the apoptogenic protein cytochrome c35 70 and avert a loss of mitochondrial membrane potential by inducing an H+ efflux from the mitochondria71 —measures that guard against downstream caspase activation.
Because the accumulation of lipofuscin by aging RPE cells is greatest in the macula,11 12 A2E-mediated blue light damage may contribute to the development of areas of RPE atrophy within the parafovea. In fundus photographs, RPE atrophy can initially appear as multiple small (150–200 µm) lesions that slowly enlarge and coalesce to form the large geographic areas of atrophy typical of non-neovascular AMD.72 73 It is interesting that laser scanning ophthalmoscopy reveals focal areas of increased autofluorescence at locations on the fundus that are otherwise unremarkable ophthalmoscopically.3 74 It has been suggested that these areas of increased autofluorescence may correspond to groups of RPE cells that contain higher quantities of lipofuscin than surrounding cells and that may be at risk for cell loss.3 74
Although epidemiologic studies concerned with a potential causal relationship between light exposure and AMD have been inconclusive,75 76 it is potentially relevant that the Chesapeake Bay Waterman Study found that individuals with advanced AMD, including geographic atrophy, reported the highest estimates of blue light exposure during the 20-year period leading up to the study.76 The propensity for blue light damage to the RPE may be particularly significant in the elderly aphakic or pseudophakic eye, wherein lipofuscin accumulation is substantial and the crystalline lens, which yellows with age and thus provides some protection from blue light, has been removed. Indeed, in an investigation analyzing associations between lens opacities and AMD, it was concluded that cataract extraction, without implantation of a UV-blue light–absorbing intraocular lens, leads to an increased risk of AMD.77 In another study, progression to AMD also occurred more frequently in eyes undergoing cataract extraction with intraocular lens implantation than in fellow eyes.78 The contribution of A2E to the pathogenesis of AMD under these and other conditions, deserves further study.
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Acknowledgements |
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Footnotes |
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Submitted for publication October 30, 2000; revised January 25, 2001; accepted February 7, 2001.
Commercial relationships policy: N.
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.
Corresponding author: Janet R. Sparrow, Department of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032. jrs88{at}columbia.edu
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J. R. Sparrow, J. Zhou, S. Ben-Shabat, H. Vollmer, Y. Itagaki, and K. Nakanishi Involvement of Oxidative Mechanisms in Blue-Light-Induced Damage to A2E-Laden RPE Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 1222 - 1227. [Abstract] [Full Text] [PDF]
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S. Ben-Shabat, C. A. Parish, H. R. Vollmer, Y. Itagaki, N. Fishkin, K. Nakanishi, and J. R. Sparrow Biosynthetic Studies of A2E, a Major Fluorophore of Retinal Pigment Epithelial Lipofuscin J. Biol. Chem., February 22, 2002; 277(9): 7183 - 7190. [Abstract] [Full Text] [PDF]
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J. K. McBee, J. P. Van Hooser, G.-F. Jang, and K. Palczewski Isomerization of 11-cis-Retinoids to All-trans-retinoids in Vitro and in Vivo J. Biol. Chem., December 14, 2001; 276(51): 48483 - 48493. [Abstract] [Full Text] [PDF]
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