1 National Eye Institute, Building 10 Room 10N119, 9000 Rockville Pike,
Bethesda, Maryland 20892-1857, USA
2 Department of Ophthalmology, University of Miami, Miami, Florida
* Author for correspondence (e-mail: kcsaky{at}helix.nih.gov)
Accepted 27 January 2003
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Summary |
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Key words: Apoptosis, RPE cells, Apoptosis-inducing factor, Caspase, Cell-free
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Introduction |
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The classic programmed cell death (PCD) pathway involves activation of
pro-caspases with concomitant changes in cellular structure and 200 bp DNA
fragmentation (Green and Reed,
1998). Both the loss of mitochondrial transmembrane potential and
subsequent release of cytochrome c are thought to be early steps in this
process. Multiple caspase-independent pathways of cell death also exist. These
include mechanisms involving cathepsins
(Foghsgaard et al., 2001
),
apoptosis-inducing factor (AIF) (Susin et
al., 1999
) or AMID (Wu et al.,
2002
). AIF, a 57 kDa flavoprotein that is normally found in the
intermembrane space of mitochondria, induces apoptosis but results in
large-scale DNA fragmentation. AIF has been identified as a primary mechanism
for apoptosis in cells genetically engineered to overexpress AIF
(Loeffler et al., 2001
;
Susin et al., 1999
), cells
deficient in caspase-3 or the caspase accessory molecule Apaf-1
(Joza et al., 2001
;
Susin et al., 2000
) or with
exogenous caspase inhibition (Dumont et
al., 2000
; Yasugi et al.,
2000
). AIF also translocates to the nucleus in retinal
photoreceptors following retinal detachment
(Hisatomi et al., 2001
).
In this report, we demonstrate that primary RPE cells under conditions of oxidant-induced cell death undergo mitochondrial depolarization with subsequent release of cytochrome c but without subsequent caspase-9, -3, PARP cleavage or 200 bp DNA fragmentation. However, under this lethal condition, cells exhibit membrane blebbing and AIF release, which results in large-scale DNA fragmentation. Furthermore, AIF release can be inhibited by pre-treatment with hepatocyte growth factor/scatter factor (HGF/SF), with a resulting increase in cell survival. These data indicate that under certain conditions, primary cells use AIF release as a principle mechanism for cell death, and highlights the potential limitation of caspase-inhibitory therapies for degenerative disorders.
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Materials and Methods |
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Cell culture
ARPE-19 and primary human RPE cells were maintained in Dulbecco's modified
Eagle's medium Ham's F12 1:1 (DMEM/F12) supplemented with 10% fetal
bovine serum (FBS) with 100 u/ml of penicillin and streptomycin. For all
experiments ARPE-19 and human RPE cells were plated at confluence and allowed
to differentiate in DMEM/F12 media supplemented with 1% FBS and 25 nM
transretinoic acid without antibiotics for a minimum of 15 days. The human
leukemic U937 cells were grown in suspension in RPMI 1640 medium supplemented
with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 4.5g/l glucose and 1.0 mM sodium
pyruvate. For all treatments, cells were used at a density of 0.5 million
cells/ml.
RPE membrane blebbing
2x104 ARPE-19 cells stably expressing green fluorescent
protein (GFP) targeted to the inner leaflet of the plasma membrane
(ARPE-GFP-c'-rRas) were generated as described previously
(Strunnikova et al., 2001) and
seeded onto collagen-IV-coated eight-well chamber slides (LAB-TEK, Nalge Nunc
International, Naperville, IL). After exposure to menadione, cells were fixed
in 4% paraformaldehyde and examined under a dual-channel laser scanning
confocal microscope (Leica, Exton, PA).
Cell viability assay (XTT assay)
U937 cells or ARPE-19 cells (1x104) were seeded in 96-well
micro-culture plates and treated with various doses of menadione. The number
of surviving cells was measured by the XTT-{sodium
3'-[(1-phenylaminocarbonil)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene
sulfonic acid hydrate} assay (Cell Proliferation Kit II; Roche Molecular
Biochemicals, Indianapolis, IN) following a 24 hour recovery period.
Western blot analysis
Crude cell extract was obtained as previously described
(Strunnikova et al., 2001)
with the following minor modification. Briefly, cells were lysed in 33.3 mM
glucose, 33.3 mM Tris-HCl, pH 8, 6.7 mM EDTA, 2 M urea, 2% 2-mercaptoethanal,
1% SDS, supplemented with protease inhibitors (Complete-Mini; Roche Molecular
Biochemical, Indianapolis, IN) by repetitive disruption through a 20-gauge
needle. The lysate was centrifuged at 12,000 g for 15 minutes
at 4°C, and the protein concentration in the supernatant was determined
using a protein assay kit (Protein Assay Reagent kit; Pierce, Rockford, IL).
Proteins were separated on a 10% SDS-polyacrylamide gel (Nupage; Invitrogen,
Carlsbad, CA), transferred to a nitrocellulose filter (Millipore, Bedford,
MA), stained with an appropriate antibody and developed by chemiluminescence
(Western blot analysis kit, Roche Molecular Biochemica, Indianapolis, IN). The
blots were exposed to Xray film (X-OMAT AR; Eastman Kodak, Rochester, NY) and
the image was processed using Adobe Photoshop (Adobe System, Mountain View,
CA).
Isolation of subcellular fractions
Nuclei-free, mitochondria-free cytosolic extracts were prepared as
described previously (Ellerby et al.,
1997). Briefly, cells were swelled in buffer A (50 mM PIPES, pH
7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT plus of protease
inhibitors) and gently disrupted with a Dounce homogenizer. The cell
lysate was centrifuged for 30 minutes at 16,000 g at 4°C
and the clarified supernatant was collected. Mitochondial fraction was
prepared as described elsewhere (Martin et
al., 1995
). Cells were suspended in buffer B [250 mM sucrose, 20
mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1
mM DTT, cocktail of protease inhibitors] and gently disrupted by passing
through an ice-cold cell homogenizer. Unlysed cells and nuclei were pelleted
(750 g) for 10 minutes. The supernatant was further
centrifuged (22,000 g) for 15 minutes and the pellets
containing mitochrondria were collected and lysed. The cellular debris was
removed by centrifugation (22,000 g) for 15 minutes. The
supernatants containing mitochondrial proteins were collected. Nuclei were
prepared as reported previously (Lazebnik
et al., 1993
). Briefly, the cells were allowed to swell and lysed
on ice in nuclear buffer (NB) (1 mM PIPES, pH 7.4, 10 mM KCl, 2 mM
MgCl2, 1 mM DTT, 10 µM cytochalasin B and cocktail of protease
inhibitors). The resulting homogenate was layered over 30% sucrose in NB. The
nuclei were pelleted by centrifugation (800 g) for 10 minutes.
For caspase activation, the 16,000 g cytosolic extract was
incubated with 10 µM cytochrome c (Sigma, St. Louis, MO) and 1 mM dATP for
2 hours at 37°C.
Immunocytochemistry
Differentiated ARPE cells were grown on eight-well chamber slides (Lab-Tek)
and treated as previously described. Cells were fixed in methanol:acetone
50:50 (vol/vol) for 10 minutes at 20°C and blocked in 10% normal
donkey serum containing 0.6% Triton X-100 in phosphate buffer (PBS) (pH 7.4)
for 1 hour at room temperature. Slides were incubated with anti-AIF antibody
and were then treated with Cy2-conjugated secondary antibody (Jackson
ImmunoResearch). The cells were mounted with DAPI containing anti-fade
mounting medium (Vectashield, Vector Laboratories, Inc. Burlingame, CA) and
viewed with an epi-fluorescence microscope (Olympus Optical Co., LTD,
Melville, NY) equipped with a cooled charge-coupled device camera. Images were
digitally captured using NIH Image 1.52 software and recompiled in Adobe
Photoshop (Adobe System, San Jose, CA). For negative controls, the slides were
incubated with 10% normal donkey serum containing 0.6% Triton X-100 instead of
the primary antibody and showed a lack of specific immunoreactivity. The
mitochrondria of ARPE-19 cells were labeled using 200 nM MitoTracker Red
CMXRos (Molecular Probe, Eugene, OR) following the manufacturer's
recommendation.
DNA gel electrophoresis
Genomic DNA was extracted according to procedures recommended by the
manufacturer (Genomic DNA preparation Kit, Promega, Madison WI). For detecting
the oligonucleosomal DNA, the DNA was fractionated on a 1.2% agarose/EtBr gel
in Bio-Rad powerPAC/1000 (constant voltage 100V, 1.5 hours). For the detection
of large-scale DNA fragments, the DNA sample was analyzed in a Bio-Rad CHEF-DR
III (1% agarose; 0.5xTBE; 200 V; 15 hours; pulse wave 60 seconds;
120° angle) (Bio-Rad Laboratories, Richmond, CA) using the low range PFG
marker (New England Biolab, Beverly, MA) as the molecular weight standard.
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Results |
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To elucidate the biochemical pathways of RPE cell death, cells were exposed to a lethal dose (50 µM) of menadione and comparisons were made to U937 cells, a cell line that uses a caspase-3-dependent pathway for cell death. Both ARPE and U937 cells express caspases-3 and -9 as 32 kDa and 48 kDa proforms, respectively, and the 112 kDa effector molecule, poly-ADP-ribose polymerase (PARP) (Fig. 2A). Induction of cell death in U937 cells resulted in the cleavage, at 4 hours, of caspase-3 to its reactive fragments of 19 kDa and 17 kDa and caspase-9 to its reactive fragments of 37 kDa and 35 kDa (Fig. 2A). Similarly, PARP cleavage to an 85 kDa fragment was also detected.
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However, under similar conditions, in ARPE-19 cells no cleavage of caspase-3 or -9 was detected (Fig. 2A). Additionally, PARP remained intact.
When applying a broad-spectrum caspase inhibitor, Z-VAD.fmk, to ARPE-19 cells and exposing them to increasing doses of menadione, cell death was not attenuated as determined by the XTT assay (Fig. 2C), reinforcing the concept that caspases are not involved in decisive steps of ARPE-19 cell death induced by menadione.
Cell free system
To further explore the state of caspase activation in ARPE-19 cell death
and to ensure that depletion of ATP or excessive generation of ROS was not
responsible for the inhibition of the caspases, an in-vitro cell system, using
cystolic fractions of ARPE-19 and U937 cells, was studied. Previous studies
have shown that caspases can be activated when cytosol from non-apoptotic
cells is incubated with cytochrome c and ATP at 37°C
(Ellerby et al., 1997).
Initial experiments (Fig. 2B)
were performed to characterize this process in cytosol prepared from U937
cells. Incubation of purified cytosol with 1 mM ATP and 10 µM cytochrome c
led to both caspase-3 and -9 activation. Processing of endogenous procaspase-3
was demonstrated by immunoblotting, which showed the appearance of a 17 kDa
fragment (Fig. 2B). Similarly,
procaspase-9 was detected and in the presence of cytochrome c, it was
processed into 35 and 37 kDa forms (Fig.
2B).
Cytosol from ARPE-19 cells was prepared in a similar fashion to U937 cytosol. Western blot analysis revealed the presence of both caspase-3 and -9 in the fraction (Fig. 2B). However, following addition of cytochrome c and ATP no cleavage of these caspases could be detected (Fig. 2B) regardless of the amount of cytochrome c added or the time of incubation (data not shown), indicating that cytochrome-c-dependent programmed cell death pathways are not active in ARPE-19 cells. Results obtained from a primary cell line of human retinal pigment epithelial cells indicated a similar pattern with the absence of caspase-3 cleavage noted in the presence of cytochrome c (Fig. 2B).
DNA fragmentation
Detection of nuclei changes by using the DNA-binding dye DAPI in U937 and
ARPE-19 cells 6 hours after exposure of a minimally lethal dose of menadione
(50 µM) revealed marked differences between these two cell types.
Typically, several stages of nuclear change can be seen during programmed cell
death, with the formation of nuclear bodies as the end result. This finding
was evident in apoptotic U937 cells (Fig.
3A) where the nuclei showed typical condensed chromatin. However,
ARPE-19 cells demonstrated only nuclear shrinkage
(Fig. 3A), similar to that seen
in stage I nuclear changes. This type of change can also be seen in nuclei
exposed to AIF (Daugas et al.,
2000b). Upon further examination, nuclear DNA from apoptotic U937
cells 6 hours after 50 µM menadione treatment showed internucleosomal 200
bp fragment laddering typical of programmed cell death
(Fig. 3B). In contrast, nuclear
DNA from ARPE-19 cells under similar conditions did not demonstrate this 200
bp laddering (Fig. 3B);
instead, cleavage to 50 kbp fragments, detected only by pulse-field
electrophoresis (Fig. 3C), was
observed. This phenomenon is also typical of that seen in cells undergoing
AIF-dependent DNA fragmentation. 200 bp fragment laddering was not seen in
ARPE-19 cells at later time points (24 hours) or with higher doses of
menadione (data not shown).
|
AIF translocation
In control ARPE-19 cells, mitochondria maintain a transmembrane potential
(m) that can be visualized by the membrane potential
sensitive dye, CMXRos (Fig.
4A). Immunofluorescence detection of AIF, found in >90% of
cells, yielded a punctate cytoplasmic staining pattern with some preference
for the perinuclear area, which is typical for mitochondrial localization with
sparing of the nucleus. Merged images confirmed this localization.
|
4 hours after a lethal dose of menadione (50 µM) there was a loss of
m verified by the absence of CMXRos staining
(Fig. 4A), with subsequent
translocation of AIF into the nucleus as demonstrated by colocalization of AIF
immunostaining with DAPI, resulting in nuclear staining in >90% of cells.
The frequency of cells demonstrating both loss of
m and
AIF translocation at 4 hours (Fig.
4C) indicated a close correlation between these two events.
Western blot analysis of whole cell lysate, nuclear and mitochondrial
fractions was used to track the intracellular movement of both AIF and
cytochrome c in ARPE-19 cells 4 hours after exposure to 50 µM menadione.
Both the 57 kDa AIF and the 15 kDa cytochrome c were detected in control
ARPE-19 cells (Fig. 4B). After
the onset of programmed cell death, the 57 kDa AIF was found at a low level in
mitochondria but appeared in the nuclear fractions. Cytochrome c was also
translocated, as is typically seen with loss of m, from
the mitochondria into the cytosol (Fig.
4B).
Inhibition of AIF translocation
HGF/SF protects epithelial cells and tumor cells against programmed cell
death by inhibiting the loss of mitochondria-permeability transition and
preventing the subsequent release of AIF and cytochrome c. The XTT assay of
cell viability was used to measure the effect of HGF/SF pretreatment on
menadione-induced toxicity of ARPE-19 cells. HGF/SF pretreatment increased
significantly the LD90 concentration from 25 µM to 75 µM
menadione (P<0.001) (Fig.
5A).
|
Redistribution of AIF in ARPE-19 cells treated with 50 µM menadione
alone or with pre-treatment with HGF/SF was studied by using AIF-specific
immunofluorescense. Compared with cells exposed to 50 µM menadione alone
(Fig. 4A), pre-treatment with
HGF/SF prevented m depolarization, as demonstrated by
maintenance of CMXRos staining (Fig.
5C). Additionally, AIF remained within the mitochondria of cells
and no changes in nuclear appearance could be detected. The frequency of cells
demonstrating AIF nuclear distribution was also determined: whereas >90% of
cells exposed to menadione (50 µM) alone exhibited nuclear AIF,
pretreatment with HGF/SF completed abrogated this phenomenon
(Fig. 5B).
Prevention of AIF redistribution as a possible mechanism of HGF/SF survival in ARPE-19 cells exposed to menadione was further confirmed by examining AIF nuclear translocation using western blot analysis (Fig. 6). Nuclear fractions of ARPE-19 cells pretreated with HGF/SF and exposed to 50 µM menadione showed an absence of AIF in the nuclear fractions compared to cells treated with menadione alone. Detection of the nuclear protein, Oct-1, confirmed the presence of equivalent amounts of protein in the fractions. Additionally, 50 kbp sized DNA fragments were also not seen in nuclear DNA from cells pretreated with HGF/SF. However, this fragmentation was observed only in cells treated with menadione alone (Fig. 6). These results further reinforce the concept that prevention of AIF nuclear translocation and subsequent DNA damage are keys steps in HGF/SF protection in ARPE-19 cells exposed to lethal doses of menadione.
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Discussion |
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PCD has both cellular and biochemical features. Cells undergoing PCD
typically shows signs of cell shrinkage, membrane blebbing and nuclear
condensation. Classic PCD biochemical pathways typically revolve around
mitochondrial membrane depolarization, with subsequent release of cytochrome c
from the mitochondria. Cytoplasmic cytochrome c forms a complex, termed the
apoptosome, with caspase-9 and Apaf-1, which results in the cleavage of
caspase-9 into an active 35 kDa active fragment, which subsequently cleaves
caspase-3 (Li et al., 1997).
The active 17 kDa fragment of caspase-3 further activates effector molecules
leading to DNA fragmentation. The prominence of this PCD pathway in various
cell types supports the use of caspase inhibitors to delay cell death in
several disease states. The role of alternative PCD pathways, such as release
of AIF, AMID or activation of cathepsins, is still unclear
(Leist and Jaattela, 2001
).
AIF, a 57kDa flavoprotein, is targeted to the mitochondria where it is found
in the intermembrane space of the mitochondria
(Daugas et al., 2000a
). Recent
evidence suggests that intact activated PARP may be required for AIF release
from the mitochondria (Yu et al.,
2002
). Although PARP activation was not directly measured in the
present study, the absence of PARP cleavage in ARPE-19 cells following lethal
injury assures its availability to induce AIF release. AIF subsequently
translocates to the nucleus where it results in DNA fragmentation producing
large fragments (50 kbp) through an unknown pathway
(Susin et al., 1999
). This
form of DNA fragmentation can also result in TUNEL-positive cell staining.
Although it appears that AIF can be co-released with other mitochondria
proteins in cells undergoing PCD, no evidence exists of its utility as a
primary mechanism of cell death in adult cells. The majority of studies have
implicated AIF involvement in the early stages of apoptosis, where it is
responsible for stage 1 chromatin condensation. AIF release also propagates
apoptosis by affecting the barrier function of mitochondria, resulting in the
release of cytochrome c and AIF from other mitochondria
(Daugas et al., 2000b
;
Loeffler et al., 2001
;
Susin et al., 1999
). In the
present study, 4 hours after a lethal injury, essentially all ARPE-19 cells
demonstrating AIF nuclear translocation had lost their mitochondrial membrane
potential. Interestingly in lymphocytes AIF release in the absence of caspase
activation does not lead to cell death
(Dumont et al., 2000
).
Menadione is a quinone that generates intracellular reactive oxygen species
(ROS) that result in mitochondrial membrane depolarization with subsequent
activation of caspase-dependent PCD
(Gerasimenko et al., 2002), as
was seen in this study in U937 cells. Menadione at high doses (250 µM) can
indirectly inhibit caspase activity through a reduction in ATP levels
(Leist et al., 1997
) or
through the generation of high intracellular levels of ROS
(Samali et al., 1999
).
However, in both U937 and ARPE cells, menadione at 50 µM induced features
of apoptosis, including membrane blebbing and DNA fragmentation. Interesting,
AIF-knockout cells (aif-/Y ES) survived in the
presence of 150 µM menadione but only when Z-VAD.fmk was added
(Joza et al., 2001
),
indicating that AIF is one of the pathways in menadione-induced cell death and
further suggesting that menadione acts in a redundant fashion through both the
caspases and AIF.
Several previous studies of oxidant-induced RPE cell death have focused on
indirect evidence of caspase-3 activation through the use of TUNEL staining
(Barak et al., 2001;
Osborne et al., 1997
). Other
investigators have demonstrated the disappearance of total cellular caspase-3
(Cai et al., 1999
), an
observation that is an imprecise measure of caspase activation. Under the
influence of complete zinc depletion (Wood
and Osborne, 2001
), direct evidence of caspase-3 fragmentation has
been shown in the RPE cells, but this is in stringent conditions that may have
little relevance to the normal state of these cells
(Chai et al., 1999
;
Hyun et al., 2001
). A2E
activation by blue light activates fluorescenic caspase-3 chromophorbes
(Sparrow and Cai, 2001
;
Sparrow et al., 2002
;
Suter et al., 2000
) but direct
detection of caspase-3 fragments has not been presented.
The molecular pathways connecting the biochemical and cellular changes
observed in PCD are still under investigation. Under typical conditions of
caspase-3 activation, membrane blebbing occurs through the cleavage of ROCK1,
leading to phosphorylation of myosin light chains
(Coleman et al., 2001;
Sebbagh et al., 2001
). In our
study, AIF release without caspase activation still produced blebbing. Whether
these PCD and cell membrane blebbing pathways can be activated independently
is still controversial, but clearly the present data confirm previous reports
that caspase activation is not required for some of the cellular changes
associated with PCD (Foghsgaard et al.,
2001
).
The present study provides evidence that RPE cells possess protective
mechanisms to guard against inadvertent caspase activation even in the
presence of cytochrome c release. Heat shock protein 27 (Hsp27) is abundant in
RPE cells (Strunnikova et al.,
2001) and appears to interfere with processing of caspase-9
cleavage during apoptosome assembly (Bruey
et al., 2000
; Garrido et al.,
1999
). ATP depletion also inhibit caspase activation
(Leist et al., 1997
) but in
the present study, using a cell free assay, ATP was not rate limiting. Of
interest is recent work that has indicated that Hsp70 binds to AIF and
prevents its activation (Ravagnan et al.,
2001
). Future work on PCD in RPE cells will focus on the use of
this protein as a possible protective factor in oxidant-induced cell
death.
Growth factor control of PCD is an emerging field of study. RPE cells
possess c-Met, the receptor for HGF-SF
(Lashkari et al., 1999). A
prominent source of HGF-SF in vivo in the retina may be macrophages normally
found in the tissue surrounding the RPE and recruited in AMD
(Killingsworth et al., 1990
).
The results in this study confirm previous findings about the ability of
HGF/SF to affect apoptosis (Gao et al.,
2001
; Mildner et al.,
2002
).
In the activation of oxidant-induced RPE cell death, a process which has been postulated to play a role in the terminal events of atrophic AMD, it is becoming clear that a complex interaction of pro-PCD signals and the presence of anti-PCD proteins determines the fate of the cell. Discovering what controls the balance of these two opposing forces is the focus of ongoing work.
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