Oxidant-induced cell death in retinal pigment epithelium cells mediated through the release of apoptosis-inducing factor

Congxiao Zhang1, Judit Baffi1, Scott W. Cousins2 and Karl G. Csaky1,*

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


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, the pathways involved in oxidant-induced cell death of a primary cell of the retina, ARPE-19, were investigated and compared with a leukemic cell, U937 cells. Both ARPE-19 and U937 cells exhibited similar viability when exposed to menadione. At lethal doses, both cell lines demonstrated extensive membrane blebbing. However, although U937 cells exhibited caspase-3, -9 PARP cleavage and 200 bp laddering, no such cleavage or laddering was noted in ARPE-19 cells. Furthermore, addition of exogenous cytochrome c and ATP to a cell-free system again resulted in cleavage of caspase-3 and -9 in extracts of U937 but not ARPE cells. Further studies in ARPE-19 cells undergoing menadione-induced cell death demonstrated mitochondrial membrane depolarization, release of cytochrome c, nuclear translocation of apoptosis-inducing factor and subsequent 50 kilo-base pair laddering, and nuclear shrinkage. All of these findings were abrogated by the pretreatment of ARPE-19 cells with hepatocyte growth factor/scatter factor. These findings demonstrate the complex nature of cell death in primary cells of the retina and highlight the role of caspase-independent signals, growth factors and intracellular survival factors in programmed cell death pathways.

Key words: Apoptosis, RPE cells, Apoptosis-inducing factor, Caspase, Cell-free


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The retinal pigment epithelium (RPE) is a differentiated non-neuronal neuroepithelium-derived cell lying between the photoreceptors and the choriocapillaris. It serves a supportive role in photoreceptor function. During adulthood, RPE cells are thought to be involved in several retinal degenerative diseases including age-related macular degeneration (AMD). Although the exact pathophysiology of AMD is not known, repetitive oxidative stress is thought to be involved. This injury results in diffuse morphological changes in the RPE, including depigmentation, shrinkage, loss of apical villae and deposition of extracellular material (Green, 1999Go). Widespread death of the RPE, termed geographic atrophy (GA), is a severe complication of this disease and is responsible for severe vision loss in AMD patients (Bressler et al., 1988Go). At present, it is not known how RPE die in AMD.

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, 1998Go). 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., 2001Go), apoptosis-inducing factor (AIF) (Susin et al., 1999Go) or AMID (Wu et al., 2002Go). 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., 2001Go; Susin et al., 1999Go), cells deficient in caspase-3 or the caspase accessory molecule Apaf-1 (Joza et al., 2001Go; Susin et al., 2000Go) or with exogenous caspase inhibition (Dumont et al., 2000Go; Yasugi et al., 2000Go). AIF also translocates to the nucleus in retinal photoreceptors following retinal detachment (Hisatomi et al., 2001Go).

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.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
All cell culture reagents were purchased through Gibco-BRL (Gaithersburg, MD) unless otherwise indicated. Antibodies against caspase 3 (Chemicon, Temecula, California), PARP (Roche, Indianapolis, IN), cytochrome c (BD-Pharmingen, Palo Alto, CA), AIF, Oct-1 (Santa Cruz Biotechnologies, Santa Cruz, CA) and cytochrome c oxidase II (Molecular probes, Eugene, CA) were used. Anti-caspase-9 antibody was a kind gift of Y. Lazebnik (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY) and X. Wang (University of Texas Southwestern, Dallas, TX). Trans-retinoic acid and menadione were obtained from Sigma (St. Louis, MO), Z-VAD.fmk was from Enzyme Systems Inc. (Dublin, CA) and recombinant HGF/SF was from R&D systems (Minneapolis, MN). Human leukemic U937 cells were obtained from ATCC (Manassas, VA), primary human RPE cells were generated as described previously (Sullivan et al., 1996Go) and human ARPE-19 cells, a human RPE cell line, were kindly provided by Leonard Hjelmeland (University of California, Davis, CA).

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., 2001Go) 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., 2001Go) 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., 1997Go). 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., 1995Go). 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., 1993Go). 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.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell viability and caspase activation
Viability of ARPE-19 cells, a human retinal pigment epithelial cell line, and U937 cells, a leukemic cell line, following exposure to menadione, (Vitamin K3; 2-methyl-1,4-naphthoquinone) was determined using a colormetric dye-XTT-based assay. The dose responsiveness of both cell types was comparable (Fig. 1A). The morphological characteristics of cell death, such as cell shrinkage and prominent cell membrane blebbing, were also similar in both cell types (Fig. 1B). The extensive membrane blebbing was confirmed in ARPE-GFP-rRas cells, an ARPE-19 cell line genetically engineered to express GFP in the inner leaflet of the plasma membrane (Fig. 1B) and showed protrusions and detachments of cell membrane blebs.



View larger version (95K):
[in this window]
[in a new window]
 
Fig. 1. Cytoxocity and morphology of cells exposed to menadione. (A) Dependence of U937 ({square}) and ARPE-19 ({diamond}) cell survival on menadione concentration. 1x104 cells were exposed to menadione (1 µM to 250 µM) for 4 hours and allowed to recover for 24 hours. Cell viability was determined by XTT assay as described in Materials and Methods. Data are represented as mean±s.d. (n=3). (B) Both U937 cells and ARPE-19 cells were exposed to 50 µM menadione for 4 hours and were observed immediately under inverted microscopy. (Inserts) Control ARPE-GFP cells in which GFP is localized to the membrane (left, arrowheads) under similar conditions were observed under confocal fluorescent microscopy and showed the presence of membrane blebs (right, arrows). Images represent three independent experiments. Bar, top, 50 µm; bottom and inserts, 30 µm.

 

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.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2. Detection of caspase cleavage in whole cells and cell-free extracts. (A) 50-100 µg of whole lysates of U937 and ARPE-19 cells were analyzed by western blotting using antibodies against both pro- and cleaved forms of caspase-9 (top), -3 (middle) and PARP (bottom) in control cells and 4 hours after exposure to 50 µM menadione. (B) Cytosolic extracts of 5x104 cells were prepared as described in Materials and Methods, incubated with 10 µM of cytochrome c and 1 mM ATP for 37°C for 2 hours, separated on a reducing gel and analyzed by western blotting using antibodies against caspase-9 (top) and -3 (bottom). (C) ARPE-19 cells were preincubated with Z-VAD.fmk (100 µM) for 1 hour and exposed to various concentrations of menadione (1 µM to 100 µM) for 2 hours. Cell viability was determined using the XTT assay as indicated in Fig.1A. ({square}), menadione alone; ({diamond}), menadione with Z-VAD.fmk.

 

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., 1997Go). 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., 2000bGo). 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).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3. Nuclear changes in apoptotic cells. (A) Following exposure to 50 µM menadione for 6 hours, cells were fixed in 4% paraformaldehyde for 5 minutes and put on a coverslip with DAPI-containing mounting media and observed under epi-flurorescent microscopy. Bar, 10 µm. (B) 10 µg genomic DNA from U937 and ARPE-19 cells exposed to 50 µM menadione for 6 hours was separated either by 1.0% agarose gel electrophoresis (left) or pulse field gel electrophoresis (right) and visualized under UV illuminescence.

 

AIF translocation
In control ARPE-19 cells, mitochondria maintain a transmembrane potential ({Delta}{psi}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.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 4. Mitochondrial permeability changes during apoptotic cell death in ARPE-19 cells. (A) ARPE-19 cells, treated with 50 µM menadione for 4 hours were incubated with CMXRos, fixed, immunolabeled with anti-AIF antibodies and put on a coverslip with DAPI-containing mounting media. In control cells (top), immunostaining of AIF colocalized with CMXRos and was excluded from the DAPI-labeled nuclei. In menadione-treated cells (bottom), AIF is colocalized with DAPI-labeled nuclei, and CMXRos staining is diminished. Bar, 10 µm. (B) Redistribution of AIF and cytochrome c during ARPE-19 cell death. Whole cells or subcellular extracts of control or ARPE-19 cells treated with 50 µM menadione for 4 hours were analyzed for AIF and cytochrome c translocation using western blot analysis. Actin was used to normalize whole cell and cytosolic loading. Cytochrome c oxidase II and Oct-1 were used for normalization of mitochondrial and nuclear fraction loading, respectively. (Data represent two independent experiments). (C) The values are the percentage of ARPE-19 cells treated as in A displaying nuclear AIF and loss of CMXRos staining obtained by averaging three fields per slide in which approximately 150 cells per slide were counted. The results shown are mean±s.d., which are representative of two independent experiments. (*, P<0.001, ANOVA/t-test, unpaired).

 

4 hours after a lethal dose of menadione (50 µM) there was a loss of {Delta}{psi}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 {Delta}{psi}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 {Delta}{psi}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).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5. HGF/SF treatment of apoptotic ARPE-19 cells. (A) ARPE-19 cells were incubated with HGF/SF (50 ng/ml for 48 hours) and subsequently exposed to menadione (50 µM for 2 hours). After 24 hours recovery, cell viability was determined using a XTT assay as described in Fig. 1A. (B) Cells either left untreated or treated with HGF, menadione alone or HGF/SF + menadione as described in (A). AIF immunohistochemistry was performed as in Materials and Methods. The value is the percentage of cells displaying nuclear AIF obtained by an average of three fields per slide in which approximately 150 cells per slide were counted. The results shown are mean +/– std, representative of two independent experiments. (*, P<0.001, ANOVA/t-test, unpaired). (C) Appearance of ARPE-19 cells treated with menadione and HGF/SF as in (A). Immunohistochemistry for AIF and counter-staining with CMXRos or DAPI was performed as in Fig. 4A.

 

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 {Delta}{psi}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.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6. HGF/SF effects on AIF nuclear distribution and large scale DNA fragmentation. Nuclear extracts were analyzed by western blotting for AIF (top) obtained 6 hours following either no treatment or treatment with HGF/SF (50 ng/ml for 48 hours) and menadione (50 µM for 2 hours). The blot was stripped and re-blotted with anti-Oct-1 antibody (middle) to normalize for protein loading. Pulse field gel electrophoresis was performed (as described in Materials and Methods) on 10 µg of genomic DNA from ARPE-19 cells exposed to similar conditions (bottom).

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell death, brought about by activation of the PCD pathway, appears to play a prominent role in chronic degenerative diseases, such as Alzheimer's and Parkinson's disease and age-related macular degeneration (Hinton et al., 1998Go; Thompson, 1995Go). Therefore, the understanding and control of PCD pathways may provide a way to delay or prevent the onset of these diseases. The PCD pathway that has been most studied involves the release of mitochondrial cytochrome c, which results in activation of the caspase cascade and DNA fragmentation. Although alternative PCD pathways exist, their control over cell death is poorly understood (Leist and Jaattela, 2001Go). In particular, AIF-mediated cell death appears to play a principal role only in genetically altered cells or in the setting of exogenous caspase inhibition (Dumont et al., 2000Go; Joza et al., 2001Go; Loeffler et al., 2001Go; Susin et al., 2000Go; Susin et al., 1999Go; Yasugi et al., 2000Go). The present study provides evidence that a primary line of RPE cells suppresses activation of caspases in the presence of cytoplasmic cytochrome c and utilizes, instead, an alternative mechanism of AIF release as a principle initiator of PCD.

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., 1997Go). 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, 2001Go). AIF, a 57kDa flavoprotein, is targeted to the mitochondria where it is found in the intermembrane space of the mitochondria (Daugas et al., 2000aGo). Recent evidence suggests that intact activated PARP may be required for AIF release from the mitochondria (Yu et al., 2002Go). 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., 1999Go). 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., 2000bGo; Loeffler et al., 2001Go; Susin et al., 1999Go). 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., 2000Go).

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., 2002Go), 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., 1997Go) or through the generation of high intracellular levels of ROS (Samali et al., 1999Go). 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., 2001Go), 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., 2001Go; Osborne et al., 1997Go). Other investigators have demonstrated the disappearance of total cellular caspase-3 (Cai et al., 1999Go), an observation that is an imprecise measure of caspase activation. Under the influence of complete zinc depletion (Wood and Osborne, 2001Go), 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., 1999Go; Hyun et al., 2001Go). A2E activation by blue light activates fluorescenic caspase-3 chromophorbes (Sparrow and Cai, 2001Go; Sparrow et al., 2002Go; Suter et al., 2000Go) 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., 2001Go; Sebbagh et al., 2001Go). 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., 2001Go).

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., 2001Go) and appears to interfere with processing of caspase-9 cleavage during apoptosome assembly (Bruey et al., 2000Go; Garrido et al., 1999Go). ATP depletion also inhibit caspase activation (Leist et al., 1997Go) 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., 2001Go). 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., 1999Go). 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., 1990Go). The results in this study confirm previous findings about the ability of HGF/SF to affect apoptosis (Gao et al., 2001Go; Mildner et al., 2002Go).

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.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Barak, A., Morse, L. S. and Goldkorn, T. (2001). Ceramide: a potential mediator of apoptosis in human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 42,247 -254.[Abstract/Free Full Text]

Bressler, N. M., Bressler, S. B. and Fine, S. L. (1988). Age-related macular degeneration. Surv. Ophthalmol. 32,375 -413.[Medline]

Bruey, J. M., Ducasse, C., Bonniaud, P., Ravagnan, L., Susin, S. A., Diaz-Latoud, C., Gurbuxani, S., Arrigo, A. P., Kroemer, G., Solary, E. et al. (2000). Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat. Cell Biol. 2, 645-652.[CrossRef][Medline]

Cai, J., Wu, M., Nelson, K. C., Sternberg, P., Jr and Jones, D. P. (1999). Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 40,959 -966.[Abstract]

Chai, F., Truong-Tran, A. Q., Ho, L. H. and Zalewski, P. D. (1999). Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review. Immunol. Cell Biol. 77,272 -278.[CrossRef][Medline]

Coleman, M. L., Sahai, E. A., Yeo, M., Bosch, M., Dewar, A. and Olson, M. F. (2001). Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat. Cell. Biol. 3,339 -345.[CrossRef][Medline]

Daugas, E., Nochy, D., Ravagnan, L., Loeffler, M., Susin, S. A., Zamzami, N. and Kroemer, G. (2000a). Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 476,118 -123.[CrossRef][Medline]

Daugas, E., Susin, S. A., Zamzami, N., Ferri, K. F., Irinopoulou, T., Larochette, N., Prevost, M. C., Leber, B., Andrews, D., Penninger, J. et al. (2000b). Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14,729 -739.[Abstract/Free Full Text]

Dumont, C., Durrbach, A., Bidere, N., Rouleau, M., Kroemer, G., Bernard, G., Hirsch, F., Charpentier, B., Susin, S. A. and Senik, A. (2000). Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult. Blood. 96,1030 -1038.[Abstract/Free Full Text]

Ellerby, H. M., Martin, S. J., Ellerby, L. M., Naiem, S. S., Rabizadeh, S., Salvesen, G. S., Casiano, C. A., Cashman, N. R., Green, D. R. and Bredesen, D. E. (1997). Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17,6165 -6178.[Abstract/Free Full Text]

Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M. and Jaattela, M. (2001). Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153,999 -1010.[Abstract/Free Full Text]

Gao, M., Fan, S., Goldberg, I. D., Laterra, J., Kitsis, R. N. and Rosen, E. M. (2001). Hepatocyte growth factor/scatter factor blocks the mitochondrial pathway of apoptosis signaling in breast cancer cells. J. Biol. Chem. 276,47257 -47265.[Abstract/Free Full Text]

Garrido, C., Bruey, J. M., Fromentin, A., Hammann, A., Arrigo, A. P. and Solary, E. (1999). HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J. 13,2061 -2070.[Abstract/Free Full Text]

Gerasimenko, J. V., Gerasimenko, O. V., Palejwala, A., Tepikin, A. V., Petersen, O. H. and Watson, A. J. (2002). Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore. J. Cell Sci. 115,485 -497.[Abstract/Free Full Text]

Green, W. R. (1999). Histopathology of age-related macular degeneration. Mol. Vis. 5, 27.[Medline]

Green, D. R. and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281,1309 -1312.[Abstract/Free Full Text]

Hinton, D. R., He, S. and Lopez, P. F. (1998). Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration. Arch. Ophthalmol. 116,203 -209.[Abstract/Free Full Text]

Hisatomi, T., Sakamoto, T., Murata, T., Yamanaka, I., Oshima, Y., Hata, Y., Ishibashi, T., Inomata, H., Susin, S. A. and Kroemer, G. (2001). Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo. Am. J. Pathol. 158,1271 -1278.[Abstract/Free Full Text]

Hyun, H. J., Sohn, J. H., Ha, D. W., Ahn, Y. H., Koh, J. Y. and Yoon, Y. H. (2001). Depletion of intracellular zinc and copper with TPEN results in apoptosis of cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 42,460 -465.[Abstract/Free Full Text]

Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki, T., Elia, A. J., Cheng, H. Y., Ravagnan, L. et al. (2001). Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410,549 -554.[CrossRef][Medline]

Killingsworth, M. C., Sarks, J. P. and Sarks, S. H. (1990). Macrophages related to Bruch's membrane in age-related macular degeneration. Eye 4, 613-621.[Medline]

Lashkari, K., Rahimi, N. and Kazlauskas, A. (1999). Hepatocyte growth factor receptor in human RPE cells: implications in proliferative vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 40,149 -156.[Abstract]

Lazebnik, Y. A., Cole, S., Cooke, C. A., Nelson, W. G. and Earnshaw, W. C. (1993). Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123, 7-22.[Abstract]

Leist, M. and Jaattela, M. (2001). Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell. Biol. 2,589 -598.[CrossRef][Medline]

Leist, M., Single, B., Castoldi, A. F., Kuhnle, S. and Nicotera, P. (1997). Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. 185,1481 -1486.[Abstract/Free Full Text]

Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S. and Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479 -489.[Medline]

Loeffler, M., Daugas, E., Susin, S. A., Zamzami, N., Metivier, D., Nieminen, A. L., Brothers, G., Penninger, J. M. and Kroemer, G. (2001). Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J. 15,758 -767.[Abstract/Free Full Text]

Martin, S. J., Newmeyer, D. D., Mathias, S., Farschon, D. M., Wang, H. G., Reed, J. C., Kolesnick, R. N. and Green, D. R. (1995). Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis. EMBO J. 14,5191 -5200.[Abstract]

Mildner, M., Eckhart, L., Lengauer, B. and Tschachler, E. (2002). Hepatocyte growth factor/scatter factor inhibits UVB-induced apoptosis of human keratinocytes but not of keratinocyte-derived cell lines via the phosphatidylinositol 3-kinase/AKT pathway. J. Biol. Chem. 277,14146 -14152.[Abstract/Free Full Text]

Osborne, N. N., Cazevieille, C., Pergande, G. and Wood, J. P. (1997). Induction of apoptosis in cultured human retinal pigment epithelial cells is counteracted by flupirtine. Invest. Ophthalmol. Vis. Sci. 38,1390 -1400.[Abstract]

Ravagnan, L., Gurbuxani, S., Susin, S. A., Maisse, C., Daugas, E., Zamzami, N., Mak, T., Jaattela, M., Penninger, J. M., Garrido, C. et al. (2001). Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat. Cell. Biol. 3, 839-843.[CrossRef][Medline]

Samali, A., Nordgren, H., Zhivotovsky, B., Peterson, E. and Orrenius, S. (1999). A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis. Biochem. Biophys. Res. Comm. 255, 6-11.[CrossRef][Medline]

Sebbagh, M., Renvoize, C., Hamelin, J., Riche, N., Bertoglio, J. and Breard, J. (2001). Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell. Biol. 3,346 -352.[CrossRef][Medline]

Sparrow, J. R. and Cai, B. (2001). Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2. Invest. Ophthalmol. Vis. Sci. 42,1356 -1362.[Abstract/Free Full Text]

Sparrow, J. R., Zhou, J., Ben-Shabat, S., Vollmer, H., Itagaki, Y. and Nakanishi, K. (2002). Involvement of oxidative mechanisms in blue-light-induced damage to A2E-laden RPE. Invest. Ophthalmol. Vis. Sci. 43,1222 -1227.[Abstract/Free Full Text]

Strunnikova, N., Baffi, J., Gonzalez, A., Silk, W., Cousins, S. W. and Csaky, K. G. (2001). Regulated heat shock protein 27 expression in human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 42,2130 -2138.[Abstract/Free Full Text]

Sullivan, D. M., Chung, D. C., Anglade, E., Nussenblatt, R. B. and Csaky, K. G. (1996). Adenovirus-mediated gene transfer of ornithine aminotransferase in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 37,766 -774.[Abstract]

Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M. et al. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397,441 -446.[CrossRef][Medline]

Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini, P., Ferri, K. F., Irinopoulou, T., Prevost, M. C. et al. (2000). Two distinct pathways leading to nuclear apoptosis. J. Exp. Med. 192,571 -580.[Abstract/Free Full Text]

Suter, M., Reme, C., Grimm, C., Wenzel, A., Jaattela, M., Esser, P., Kociok, N., Leist, M. and Richter, C. (2000). Age-related macular degeneration. The lipofusion component N-retinyl-N-retinylidene ethanolamine detaches proapoptotic proteins from mitochondria and induces apoptosis in mammalian retinal pigment epithelial cells. J. Biol. Chem. 275,39625 -39630.[Abstract/Free Full Text]

Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267,1456 -1462.[Medline]

Wood, J. P. and Osborne, N. N. (2001). The influence of zinc on caspase-3 and DNA breakdown in cultured human retinal pigment epithelial cells. Arch. Ophthalmol. 119, 81-88.[Abstract/Free Full Text]

Wu, M., Xu, L. G., Li, X., Zhai, Z. and Shu, H. B. (2002). AMID, an AIF homologous mitochondrion-associated protein, induces caspase-independent apoptosis. J. Biol. Chem. 277,25617 -25623.[Abstract/Free Full Text]

Yasugi, E., Kumagai, T., Nishikawa, Y., Okuma, E., Saeki, K., Oshima, M., Susin, S. A., Kroemer, G. and Yuo, A. (2000). Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells. FEBS Lett. 480,197 -200.[CrossRef][Medline]

Yu, S. W., Wang, H., Poitras, M. F., Coombs, C., Bowers, W. J., Federoff, H. J., Poirier, G. G., Dawson, T. M. and Dawson, V. L. (2002). Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297,259 -263.[Abstract/Free Full Text]


Related articles in JCS:

Caspase-independent apoptosis – the eyes have it

JCS 2003 116: 1002. [Full Text]