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(Investigative Ophthalmology and Visual Science. 2005;46:1755-1764.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1039
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Human RPE Expression of Cell Survival Factors

Ping Yang,1 Jessica L. Wiser,1 James J. Peairs,1 Jessica N. Ebright,1 Zachary J. Zavodni,1 Catherine Bowes Rickman,1,2 and Glenn J. Jaffe1

1From the Departments of Ophthalmology and 2Cell Biology, Duke University Medical Center, Durham, North Carolina.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine basal and tumor necrosis factor (TNF)-{alpha}–regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-{kappa}B.

METHODS. Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-{kappa}B or ß-galactosidase and treated with TNF-{alpha} for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively.

RESULTS. Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1ß-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-{alpha} upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-{alpha} in a time-dependent manner, whereas c-IAP1, survivin, Bcl-xL, and TRAF-2 protein levels were not influenced by TNF-{alpha} treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-{alpha} treatment. Overexpression of mutant I{kappa}B blocked TNF-{alpha}–induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-xL proteins were localized diffusely in RPE cytoplasm.

CONCLUSIONS. Multiple RPE cell survival factors are expressed by human RPE cells. TNF-{alpha} regulates expression of some of these factors in an NF-{kappa}B–dependent manner, whereas others are not influenced by NF-{kappa}B. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR).

Retinal pigment epithelial (RPE) cells form a monolayer of cuboidal cells located between the photoreceptors of the neurosensory retina and the choroidal capillary bed. The RPE comprises an important blood–retinal barrier component and performs many important functions essential to the visual process. Normally, RPE cells remain in a quiescent state and survive over an individual’s lifetime.1 2

Age-related macular degeneration (AMD) is an idiopathic retinal degenerative disease that is the leading cause of irreversible vision loss in the Western world among persons older than 65 years.3 AMD is characterized by clinical signs, ranging from a few soft drusen and pigmentary changes in the macular RPE with normal visual acuity, to large areas of RPE atrophy or choroidal neovascular membranes (CNVMs) and associated blindness.4 RPE cell apoptosis is an important feature of the advanced forms of this disease.5 6 Although vision loss in AMD is caused by photoreceptor damage in the central retina, RPE atrophy is a prominent disease component.7 8

Proliferative vitreoretinopathy (PVR), the principal cause of retinal reattachment surgical failure, is a potentially blinding disease. PVR is characterized by uncontrolled cell proliferation and migration into the subretinal space and vitreous cavity and onto the retinal surface and undersurface.9 10 The RPE cell is thought to be a key cell-type in this disease. RPE cell migration and proliferation and associated collagen secretion contribute to membrane formation in PVR.9 10 Contraction of these membranes can lead to retinal detachment and subsequent loss of vision. The factors responsible for unwanted survival of migrating proliferating RPE cells in this condition have not been clearly defined.

Nuclear transcription factor (NF)-{kappa}B is a major regulator of cell life and death.11 12 In many cells, activation of NF-{kappa}B blocks apoptosis, induces cell proliferation, blocks differentiation, and promotes metastasis.12 NF-{kappa}B activation in cancer cells by chemotherapy can blunt the ability of the cancer therapy to induce cell death by producing antiapoptotic factors such as cellular inhibitor of apoptosis proteins (c-IAPs), cellular Fas-associated death domain (FADD)-like interleukin-1ß-converting enzyme-like inhibitory protein (c-FLIP), and tumor necrosis factor (TNF) receptor associated factor (TRAF).13 14 15 16 Other important survival factors are members of the B-cell leukemia/lymphoma (Bcl)-2 family. These proteins inhibit apoptotic cell death and include Bcl-2, Bcl-xL, and A1 (also designated Bfl-1).17

TNF-{alpha} is one of the most studied activators of NF-{kappa}B. It is widely expressed in epiretinal membranes, vitreous, and subretinal fluid of eyes with PVR18 19 and has also been identified in CNVMs of eyes with AMD.20 TNF-{alpha} is a major regulator of RPE cell activities, including cell attachment, spreading, chemotaxis, migration, and proliferation.21 22 Numerous studies indicate that specific NF-{kappa}B inhibition enhances TNF-{alpha}–induced apoptosis in a variety of cell types otherwise resistant to TNF-{alpha}–induced cell death.23 24 25 26 Furthermore, NF-{kappa}B is activated in eyes with PVR27 (Jaffe GJ, unpublished data, 2001) and AMD.28 However, RPE cells are resistant to TNF-{alpha}–induced apoptosis, even after specific NF-{kappa}B blockade.29

Despite the fact that RPE cells may remain intact over a person’s lifetime, and RPE cell survival and apoptosis play crucial roles in diseases such as PVR and AMD,5 6 7 8 9 10 there is very little information regarding RPE cell survival factor expression and mechanisms that control their expression. Because TNF-{alpha} is present in eyes with these disorders and activates NF-{kappa}B, it is important to determine whether survival factor expression is regulated by NF-{kappa}B, and whether there are survival factors that are expressed independently of this transcription factor. To better understand these mechanisms, we determined RPE cell survival factor expression and whether survival factor expression was regulated by TNF-{alpha} in an NF-{kappa}B–dependent manner.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
RPE Cell Culture and Adenoviral Infection
Human donor eyes were obtained from the North Carolina Eye Bank, Inc. (Winston- Salem, NC), in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. RPE cells for culture studies were harvested from eyes as previously described.30 Cells were grown in Eagle’s minimal essential medium (MEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) and 1x antibiotic-antimycotic (Invitrogen) at 37°C in a humidified environment containing 5% CO2. RPE cells (1 x 105) were seeded in six-well plates (Corning-Costar Inc., Corning, NY). Twenty-four hours later, cells were incubated with fresh medium for an additional 24 hours and then were left untreated or were infected with adenovirus encoding either mutant inhibitor (I)-{kappa}B (I{kappa}B)31 (University of North Carolina at Chapel Hill Gene Delivery Core, Chapel Hill, NC) or ß-galactosidase (LacZ) at a multiplicity of infection (MOI) of 10, as we have previously described.29 32 Cells were stimulated with or without TNF-{alpha} (1.1 x 103 U/mL; R&D Systems, Inc., Minneapolis, MN) for various times. In the remainder of the text "basal" expression refers to cells grown in MEM without TNF-{alpha} treatment as well as to freshly isolated cells. We chose TNF-{alpha} concentrations that we have shown previously to stimulate cytokine gene expression and cause I{kappa}B degradation and NF-{kappa}B activation in RPE cells.29 32

Preparation of Freshly Isolated Human RPE and RPE/Choroid Samples
Human eyes from six donors (45–66 years of age) were obtained from the North Carolina Eye Bank stored in preservative (RNAlater; Ambion, Austin, TX), with an average death-to-procurement time of 4 hours 15 minutes. To avoid introducing donor-related bias, one eye of each donor was used in the study. Eyes were stored at 4°C for up to 2 weeks or at –20°C until they were processed. For RPE isolation, the anterior segment, iris, lens, and vitreous of each eye were carefully extracted. Using a 4-mm trephine punch, we removed pieces of the peripheral retina and collected the underlying RPE/choroid tissue. Tissue punches were obtained from four of the six eyes and pooled for RNA isolation of the RPE/choroid sample. After removal of the tissue punches, posterior poles were cut into quadrants. Each quadrant was rinsed with sterile phosphate-buffered saline (PBS) and the neural retina was gently teased away from the RPE. After removal of the retina, RPE cells free of choroidal contamination were collected as follows: RPE cells were carefully brushed away from Bruch’s membrane and washed into a Petri dish with PBS. In the event that RPE cells were stuck to the retina, the same brush and washing techniques were used to separate the RPE from the neural retinal tissue. The composition of the resultant RPE cell suspension was analyzed with light microscopy and found to consist almost exclusively of RPE cells (Fig. 1D) . Cells from each eye were recovered into a 2-mL microcentrifuge tube with a wide-bore transfer pipette and then spun down at 5000 rpm for 5 minutes at 4°C. RPE cell–enriched pellets were pooled from all six donor eyes for RNA isolation of the RPE-enriched sample.



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  		FIGURE 1. Human RPE cells expressed multiple survival factors. Quantification of the relative levels of survival factor transcripts in cultured RPE cells grown in medium alone (A) or with TNF-{alpha} (1.1 x 103 U/mL) (B) for 4 hours. Logarithmic fluorescence history versus cycle number of survival factor genes and the reference gene GAPDH. The colors corresponding to each gene (duplicate reactions) are shown below the fluorescence tracing. (C). Quantification of the relative levels of multiple survival factors, in freshly isolated RPE-enriched cells (D) and fresh RPE/choroid samples. Expression of genes in RPE-enriched cells was calculated relative to expression of RPE/choroid samples: 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = [(CT, target – CT, GAPDH)RPE-enriched – (CT, target – CT, GAPDH)RPE/choriod]. (D) Light micrograph of human RPE cells obtained from donor eyes and used to generate RNA in the RPE-enriched sample. After careful removal of neural retina, RPE cells were brushed away from Bruch’s membrane into a PBS solution and collected with a wide-bore transfer pipette for RNA isolation. Cell suspension (20 µL) was plated on a gelatin-subbed slide for light microscopy and stained with Mayer hematoxylin. The sample was highly enriched in RPE cells, as demonstrated by lack of blood cells and limited photoreceptor–retina debris.

 
RNA Isolation, Purification, and cDNA Synthesis
Total RNA was isolated from cultured RPE cells (RNeasy; Qiagen Inc., Valencia, CA), according to the manufacturer’s protocols. The RNA was then DNase-digested, quantified, and used as a template to generate cDNAs as described below for the freshly isolated RPE samples.

RNA from both the RPE-enriched and RPE/choroid samples was purified immediately after tissue preparation (TRIzol; Invitrogen) and glycogen.33 After isolation and DNase treatment (DNA-free; Ambion) as described by the manufacturer, RPE/choroid and RPE-enriched RNA samples were further treated to remove visible melanin contamination. The size exclusion chromatography procedure34 was used to eliminate melanin pigment with the following adaptations: approximately 0.05 g of gel beads (Bio-Gel P-60 beads; Bio-Rad, Hercules, CA) was hydrated in 1.1 mL of 10 mM sodium acetate as described by the manufacturer. Four hundred microliters (400 µL) of hydrated beads/column was removed to a filter microcentrifuge tube, pelleted at 1000 rpm for 1 minute, and washed four times with 400 µL 10 mM sodium acetate. The melanin-contaminated RNA was then mixed with 20 µL of 10 mM sodium acetate and added to the washed beads. The column was incubated on ice for 10 minutes and then spun down at 1000 rpm for 1 minute. The flow-through was collected and an additional 100 µL of 10 mM sodium acetate was added to the beads. After another 10-minute incubation on ice, the sample was spun at 1000 rpm for 2 minutes, and the flow-through was collected. The purified RNA from both samples was reconcentrated by ethanol precipitation with glycogen and then quantified by fluorescence at 530 nm, using an RNA quantitation reagent (RiboGreen RNA; Molecular Probes, Eugene, OR), as described by the manufacturer. First-strand cDNAs were synthesized from equal amounts of total RNA (3 µg/reaction), with a cDNA synthesis kit (iScript; Bio-Rad).

Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) of survival factor mRNA was performed with a detection system (iCycler IQ; Bio-Rad).33 35 Briefly, duplicate reactions were prepared with 20 µL of PCR master mix consisting of 10 µL master mix (iQ SYBR Green Supermix; Bio-Rad), 1 µL cDNA template, 1 µL each of gene-specific primer pairs (20 nM; Table 1 ) and 7 µL RNase-free water. Reactions were denatured at 95°C for 2 minutes and amplified for 50 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. Real-time quantification of all the survival factor genes was normalized to the threshold cycle (CT) value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)36 where CT equals the PCR cycle number at which the amount of amplified product reached 100 relative fluorescence units (RFU). Expression of genes in TNF-{alpha} stimulated samples was calculated relative to expression of the untreated samples: 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = [(CT, target – CT, GAPDH)TNF-{alpha}-stimulated – (CT, target – CT, GAPDH)untreated].37 A melting curve for all products was obtained immediately after amplification by increasing temperature in 0.4°C increments from 65° for 85 cycles of 10 seconds each. The presence of a single melting-temperature peak per primer pair and 2% agarose gel analysis confirmed PCR products. Each real-time experiment was repeated three times.


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  		TABLE 1. Primers for Real-Time PCR

 
Cell Extracts and Western Blot
Cell medium was removed, and cells were washed twice with cold Hanks’ balanced salt solution and lysed with RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). Lysates were transferred to 1.5-mL tubes (Eppendorf, Fremont, CA) and cleared by centrifugation. Total protein in the supernatants was measured by Bradford assay (Bio-Rad) with bovine serum albumin (BSA) used to generate the curve, according to the manufacturer’s instructions. Protein (30 µg) was electrophoresed on a 12.5% SDS-polyacrylamide gel overlaid with a 3.6% polyacrylamide stacking gel. The proteins were transferred to nitrocellulose membrane (Bio-Rad) with a mini transblot apparatus (Bio-Rad), according to the manufacturer’s directions. Recombinant human Bcl-2-related protein, A1 (R&D Systems, Inc.), and human whole-cell lysate from HL-60 acute promelocytic leukemia cells (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as positive controls for A1 and Bcl-2 Western blot analysis, respectively. Transfers were performed overnight at room temperature (RT). Nonspecific binding sites were blocked by immersing the membrane in 5% fat-free milk powder (SACO Foods Inc., Middleton, WI) for 30 minutes at RT. The blocking step was repeated, and then membranes were washed three times (5 minutes per wash) in Tris-buffered saline (73 mM Tris, 40 mM NaCl [pH 7.45]) containing 0.1% Tween-20 (TBST). The membranes were incubated overnight at 4°C with the following antibodies (Santa Cruz Biotechnology, Inc.) diluted in 5% milk: rabbit polyclonal antibody directed against c-IAP1 (1:2000), c-IAP2 (1:2000), TRAF-1 (1:2000), TRAF-2 (1:1500), Bcl-xL (1:800), Bcl-2 (1:800), survivin (1:800), A1 (1:500), and mouse monoclonal antibody against c-FLIP NF6 (1:500 in 5% milk; Alexis, San Diego, CA). The blots were then washed three times (20 minutes per wash) in TBST and incubated with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (1:5000 in 5% milk; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 4°C for 60 minutes. Immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham, Piscataway, NJ). Data shown in Figures 2 , Figures 3 , Figures 4 , Figures 5 , Figures 6 to 7 are from a representative experiment. In all the figures with histograms of densitometry data, optical density (OD) refers to the integrated density. Experiments in duplicate were separately repeated twice with similar results.



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  		FIGURE 2. c-FLIP protein levels were increased by TNF-{alpha} in RPE cells. RPE cells in duplicate wells were treated with medium alone or TNF-{alpha} (1.1 x 103 U/mL) for different times. Cytoplasmic proteins (30 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis. (A) Western blot probed with antibody to c-FLIP. The relative quantities of c-FLIPL and c-FLIS short form, determined by densitometry, are shown separately below each lane. (B) Blot in (A) was stripped and reprobed with antibody to GAPDH, a control for gel loading. The relative quantity of GAPDH protein is shown below each lane.

 


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  		FIGURE 3. TRAF-1 protein levels are increased by TNF-{alpha} in RPE cells. Cytoplasmic proteins (30 µg from RPE cells in duplicate wells, as in Fig. 2 ) were probed with antibody to TRAF-1 (A), stripped, and reprobed with antibody to GAPDH (B). The relative quantity of TRAF-1 and GAPDH, determined by densitometry, is shown below each lane.

 


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  		FIGURE 4. Effect of mutant I{kappa}B overexpression on TNF-{alpha}–induced TRAF-1 expression. RPE cells in duplicate wells were left untreated (no virus) or infected with adenovirus expressing mutant I{kappa}B or LacZ. One day after infection, cells were treated with medium alone or medium with TNF-{alpha} (1.1 x 103 U/mL) for 6 hours, and cytoplasmic proteins (30 µg) were probed with antibody to TRAF-1 (A), stripped, and reprobed with antibody to ß-catenin (B). The relative quantity of TRAF-1 and ß-catenin, determined by densitometry, is shown below each lane.

 


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  		FIGURE 5. RPE cell survival factor protein levels after TNF-{alpha} stimulation. RPE cells in duplicate wells were treated with medium alone or TNF-{alpha} (1.1 x 103 U/mL) for different times. Cytoplasmic proteins (30 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis.

 


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  		FIGURE 6. NF-{kappa}B blockade did not affect RPE survival factor protein levels. RPE cells in duplicate wells were left untreated or infected with adenovirus expressing mutant I{kappa}B or LacZ. One day after infection, cells were treated with medium alone or TNF-{alpha} (1.1 x 103 U/mL) for 6 hours. Cytoplasmic proteins (30 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis.

 


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  		FIGURE 7. Bcl-2 and A1 proteins were not detected in RPE cells. (A) RPE cells in duplicate wells were treated with medium alone or TNF-{alpha} (1.1 x 103 U/mL) for 16 and 24 hours. RPE cytoplasmic proteins (30 µg), 25 and 50 ng recombinant truncated A1 protein (amino acids 1-152, 18 kDa) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibody to A1 for Western blot analysis. (B) Protein (30 µg) aliquoted from the same RPE cell lysates used in blot (A) and 25 µg of whole-cell lysate from HL-60 cells were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis. The lanes corresponding to lysates from untreated RPE cells were cut from the remaining membrane. The membranes were probed on the same day with antibody to Bcl-2 and Bcl-xL, and then both membranes were visualized simultaneously with ECL. (C, D) Blots in (A) and (B) respectively, were stripped and reprobed with antibody to GAPDH. The relative quantity of GAPDH protein is shown below each lane.

 
Localization of TRAF-1 and Bcl-xL
Cell medium was removed, and cells were fixed with 4% paraformaldehyde for 20 minutes at 4C°, blocked with 5% nonimmune goat serum (NGS; Jackson ImmunoResearch Laboratories, Inc.) for 1 hour, and then incubated for 2 hours with rabbit polyclonal antibody directed against TRAF-1 or mouse monoclonal antibody against Bcl-xL (1:50 in 0.2% normal goat serum (NGS; Santa Cruz Biotechnology, Inc.) separately at RT. Cells were washed with PBS and then incubated with indocarbocyanine (Cy3)-conjugated goat anti-rabbit IgG antibody (1:400 in 0.3% Triton X-100/PBS; Jackson ImmunoResearch Laboratories, Inc.) or fluorescein (FITC)-conjugated goat anti-mouse IgG antibody (1:100 in 0.3% Triton X-100/PBS; Jackson ImmunoResearch Laboratories, Inc.) for 1 hour at RT. Cells were washed with PBS and then incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO) in PBS for 5 minutes. TRAF-1 and Bcl-xL fluorescent staining was imaged by a fluorescence microscope. Data shown in Figures 8 and 9 are from a representative experiment. Experiments in duplicate were separately repeated twice with similar results.



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  		FIGURE 8. TRAF-1 localization within cultured RPE cells. RPE cells were treated with medium alone (–TNF-{alpha}) or TNF-{alpha} (+TNF-{alpha}; 1.1 x 103 U/mL) for 16 hours. The cells were immunostained to localize TRAF-1 (A, B, E, F). Control: PBS rather than primary antibody (I, J). Nuclei were stained with DAPI (C, D, G, H, K, L). Bar, 10 µm.

 


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  		FIGURE 9. Bcl-xL localization. RPE cells without any treatment (–TNF-{alpha}) were immunostained to localize Bcl-xL (A, B). Control: PBS rather than primary antibody (E, F). Nuclei were stained with DAPI (C, D, G, H). Bar, 10 µm.

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Survival Factors Expressed by Human RPE Cells
Multiple survival factor genes are expressed in RPE cells in culture and in situ under basal conditions. To determine the relative abundance of these factors, their transcripts were analyzed by real-time RT-PCR. In cultured RPE cells, Bcl-x was the most highly expressed survival factor, c-IAP1, c-FLIP, TRAF-2, and survivin were expressed at intermediate levels, TRAF-1 and c-IAP2 were expressed at low levels, and Bcl-2 and A1 were expressed at very low levels relative to expression of the highly expressed housekeeping gene, GAPDH (Fig. 1A) . To ensure that basal survival factor mRNA expression in cultured human RPE cells reflects gene expression in their normal monolayer in situ, we then examined survival factor mRNA expression in samples of RPE cells obtained from human donor eyes. As shown in Fig 1C , mRNA expression of survival factors readily detected in untreated cultured RPE cells, c-FLIP, TRAF-1, TRAF-2, and Bcl-xL were also expressed in situ. Expression of these survival factors was enhanced in RPE-enriched cells in situ, relative to RPE/choroid preparations in situ. c-IAP1 and -2 were also identified in RPE-enriched cells in situ, but were even more highly expressed in RPE/choroid samples in situ (Figs. 1C 1D) . In contrast, Bcl-2 and A1 were not detected in RPE-enriched cells in situ, and, as shown in Figure 1A , these survival factors were also nearly undetectable in untreated cultured RPE cells. Survivin was not expressed in situ, although it was present in cultured cells.

In many cells, TNF-{alpha} stimulation causes transcriptional activation of genes that suppress apoptosis.11 12 We found that in RPE cells, TNF-{alpha} markedly upregulated TRAF-1, c-IAP2, and A1, moderately upregulated c-IAP1 and c-FLIP, and slightly upregulated TRAF-2. In contrast, Bcl-2 was slightly downregulated by TNF-{alpha}, whereas survivin, and Bcl-x were minimally and inconsistently influenced by exposure to TNF-{alpha} (Fig. 1B , Tables 2 3 ).


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  		TABLE 2. TNF-{alpha}-Regulated Survival Factor Gene Expression

 

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  		TABLE 3. Effect of NF-{kappa}B Blockade on TNF-{alpha}-Regulated Survival Factor Gene Expression

 
NF-{kappa}B Blockade of TNF-{alpha}–Induced Survival Factor Gene Expression
To determine whether TNF-{alpha}–upregulated expression of survival factor genes was modulated by NF-{kappa}B, we infected RPE cells with adenovirus expressing mutant I{kappa}B to block NF-{kappa}B activation, or with adenovirus expressing LacZ, as a transgene and viral control. After TNF-{alpha} exposure, the pattern of TNF-{alpha}–upregulated survival factor gene expression after infection with adenovirus expressing LacZ was similar, although somewhat reduced compared with uninfected cells. In contrast, TNF-{alpha}–upregulated survival factor gene expression was completely abolished by mutant I{kappa}B (Table 3) .

Effect of TNF-{alpha} on c-FLIP and TRAF-1 Protein Expression in RPE Cells
c-FLIP is a protein that is thought to be an important determinant of cell survival. It is structurally similar to pro-caspase-8 and competes with caspase-8 binding to FADD and thereby blocks apoptosis at the apex of the caspase cascade.13 38 We have shown that TNF-{alpha} upregulates c-FLIP protein levels in an NF-{kappa}B–dependent manner.29 In the current study, we determined the kinetics of this response and compared these kinetics to steady state protein production of other RPE cell survival factors. We found that TNF-{alpha} upregulated the long (FLIPL) and short (FLIPS) forms of FLIP in a time-dependent manner (Fig. 2A) . In contrast to c-FLIP, GAPDH was not greatly affected by TNF-{alpha} stimulation and thus served as a useful protein to standardize protein loading (Fig. 2B) .

TRAF-1 is another important NF-{kappa}B-targeted survival factor that binds to several intracellular proteins, protein kinases, and NF-{kappa}B inhibitory proteins.39 As seen with c-FLIP, TNF-{alpha} upregulated RPE cell TRAF-1 levels in a time-dependent manner (Fig. 3A) . Again, GAPDH levels were not greatly affected by TNF-{alpha} stimulation (Fig. 3B) .

Effect of Overexpression of Mutant I{kappa}B on TNF-{alpha}–Induced TRAF-1 Protein Expression
We showed that TNF-{alpha} increases c-FLIP and TRAF-1 expression. We have reported that NF-{kappa}B blockade abolishes TNF-{alpha}–induced expression of the c-FLIP long and short forms.29 We next tested whether NF-{kappa}B inhibition affects TRAF-1 expression. Overexpression of mutant I{kappa}B completely blocked TRAF-1 protein expression (Fig. 4A) . As a control for gel loading and cytoplasmic phosphorylation, we used ß-catenin, a protein that is phosphorylated and degraded by the proteasome in response to an NF-{kappa}B–independent signal transduction pathway (Fig. 4B) .

c-FLIP and TRAF-1 protein levels were increased after TNF-{alpha} exposure, and NF-{kappa}B blockade suppressed the increased protein induced by TNF-{alpha}. We have previously found that RPE cells survive after TNF-{alpha} stimulation, despite NF-{kappa}B inhibition (which blocks NF-{kappa}B–dependent survival factors). Thus, we reasoned that other survival factors, in addition to c-FLIP and TRAF-1, may be present that would prevent RPE cell death after TNF-{alpha} stimulation and NF-{kappa}B blockade. We found that multiple survival factor proteins, including c-IAP1, c-IAP2, TRAF-2, Bcl-xL, and survivin were produced by RPE cells; c-IAP1, TRAF-2, Bcl-xL, and survivin levels were not influenced by TNF-{alpha} treatment at any time point tested, and TNF-{alpha} only slightly increased c-IAP2 protein expression at 16 and 24 hours after TNF-{alpha} treatment (Fig. 5) .

We next conducted additional experiments to confirm the results from these time course experiments and to determine whether survival factor levels was affected by NF-{kappa}B blockade. NF-{kappa}B blockade did not affect RPE cell c-IAP1, c-IAP2, TRAF-2, Bcl-xL, or survivin expression (Fig. 6) .

Bcl-2 family members play an important role in mitochondria-mediated apoptosis and antiapoptosis. A1, a Bcl-2 family member, also is an NF-{kappa}B–dependent survival factor.11 12 Low level Bcl-2 and A1 mRNA expression was observed by real-time RT-PCR, and A1 levels were upregulated by TNF-{alpha}. However, we were unable to detect A1 or Bcl-2 protein under conditions in which Bcl-xL was readily detected (Fig. 7) even when higher protein concentration (50 µg) were loaded (not shown).

Localization of TRAF-1 and Bcl-xL Protein within RPE Cells
We demonstrated that TNF-{alpha} greatly upregulated RPE cell expression of TRAF-1 protein and that Bcl-xL protein levels were not affected by TNF-{alpha} stimulation on Western blot. These proteins were further analyzed for their localization within RPE cells as representative examples of NF-{kappa}B–dependent and –independent RPE cell survival factors, respectively. TRAF-1 was diffusely localized throughout the RPE cytoplasm, lightly staining in the untreated group and qualitatively much brighter in the TNF-{alpha}–treated group (Fig. 8) . Bcl-xL was also localized within the RPE cytoplasm (Fig. 9) .


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It is thought that the relative balance between anti-and proapoptotic factors determines whether a cell lives or dies.40 RPE cells are critically important to maintain neural retinal function. Thus, one might expect that RPE cells that are exposed to oxidative and inflammatory insults on an ongoing basis throughout life, but have a very low turnover rate,1 2 would express many antiapoptotic proteins. The present studies confirm this hypothesis. We found that multiple RPE cell survival factor genes are expressed both in cultured RPE cells and in RPE cells freshly isolated from human donor eyes. The levels of at least two of these, TRAF-1 and c-FLIP, were upregulated by the inflammatory cytokine TNF-{alpha}, an effect that was blocked when NF-{kappa}B was inhibited.

For four survival factors, c-FLIP, TRAF-1, TRAF-2, and Bcl-xl, in situ gene expression was higher in RPE-enriched samples than RPE/choroid samples, indicating higher expression in RPE relative to choroid. c-IAP1 and -2 were also expressed in RPE-enriched samples; however, expression levels were higher in RPE/choroid than RPE-enriched cells, perhaps reflecting relatively higher expression in choroidal vasculature. Regardless, taken together, these data indicate that survival factors that are expressed by RPE cells in situ, are also expressed by cultured RPE cells.

In general, RPE cell survival factor protein expression under basal conditions mirrored mRNA expression, as determined by real-time RT-PCR. Similarly, increased c-FLIP, TRAF-1, and c-IAP2 protein levels paralleled increases in mRNA expression after TNF-{alpha} stimulation, whereas Bcl-xL, TRAF-2, and survivin protein levels and steady state mRNA expression were not affected greatly by TNF-{alpha}. In contrast, c-IAP1 mRNA expression was increased by TNF-{alpha} stimulation, but protein levels were not greatly affected by the treatment. It is possible that protein levels were slightly increased but were below our ability to detect differences. Alternatively, survival factor protein production may be regulated by post transcriptional mechanisms, as has been reported previously.41 42 43

In the present report, we found that cultured RPE cells produce TRAF-1 and -2 and c-IAP1 and -2 proteins. To our knowledge, these data represent the first demonstration of TRAF and c-IAP family member proteins in RPE cells. The pattern of TRAF and c-IAP expression under basal conditions and after exposure to inflammatory cytokine stimulus is very cell-type and tissue specific.44 45 46 47 48 In the present study, human RPE cells expressed high TRAF-2 and c-IAP1 and -2 protein levels under basal conditions and high TRAF-1 levels after TNF-{alpha} treatment, an expression pattern that is similar to that observed in salivary gland cell clones (ACMT-6 and -7).49 In some cells, expression of individual IAP family members is sufficient to suppress apoptosis, whereas in others, coexpression of multiple different c-IAPs is necessary.15 49 50 51 The relative roles of TRAF and IAP family members individually, or cooperatively, in RPE cell apoptosis remains to be determined.

Survivin, a c-IAP family member, was expressed by cultured RPE cells, albeit at relatively low levels, but not by freshly isolated RPE cells. Among the survival factors examined in the present report, survivin was the only one that was not expressed at similar levels in both cultured and freshly isolated cells. Survivin has a very restricted distribution and is usually detected in neoplastic cells.52 Lack of expression in situ probably reflects inherent differences between cultured cells and their in situ counterparts.

Members of the Bcl-2 protein family are crucial apoptosis regulators. We detected Bcl-2 and A1 mRNA expression by real-time RT-PCR, and A1 mRNA levels were regulated by TNF-{alpha}. However, measured Bcl-2 and A1 mRNA levels were very low, compared with Bcl-x and other survival factor mRNA, and we were unable to detect Bcl-2 and A1 protein, despite ready detection of positive control proteins. Bcl-2 transgene–mediated overexpression prevents RPE cell apoptosis in an oxidative stress model.53 However, in this same study, as in ours, endogenous Bcl-2 protein was not readily detected.53 Furthermore, Bcl-2 tends to be highly expressed in embryos and declines postnatally after differentiation and maturation.17 In contrast, we readily detected Bcl-xL protein in cultured and freshly isolated human RPE cells, both by Western blot and immunocytochemistry. Similarly, other investigators have identified Bcl-xL protein in bovine RPE, and in those cells it protects them from serum-deprivation–induced apoptotic cell death.54 Taken together, these data suggest that Bcl-xL, but not Bcl-2 or A1, is an important endogenous RPE cell survival factor. Further studies are needed to provide further support to this contention.

In pathologic conditions such as AMD and PVR, RPE cells are exposed to the inflammatory cytokine TNF-{alpha}.18 19 20 TNF-{alpha} activates the extrinsic apoptosis pathway when it binds to TNF receptor 1. In most cells, apoptotic cell death does not occur, because TNF-{alpha} also activates NF-{kappa}B which upregulates antiapoptotic genes. For example TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, survivin, and A1 have been identified as NF-{kappa}B–regulated survival factor genes.11 12 13 14 15 16 In certain retinal diseases such as PVR, there is unwanted proliferation of RPE cells that migrate into the subretinal space, onto the retinal surface, and into the vitreous cavity. Therefore, theoretically, downregulation of antiapoptotic proteins by suppression of NF-{kappa}B activity would be a useful treatment strategy to induce apoptosis in these RPE cells, as has been proposed for apoptosis induction in neoplastic cells.13 14 15 16 However, we have demonstrated that human RPE cells are resistant to TNF-{alpha}–mediated extrinsic pathway apoptotic cell death, despite NF-{kappa}B blockade.29 The persistent c-IAP1, c-IAP2, survivin, Bcl-xL, and TRAF-2 protein levels after NF-{kappa}B inhibition observed in the present report may help to explain RPE cell resistance to TNF-{alpha}–induced apoptosis even after NF-{kappa}B blockade. These results further highlight the cell–type specificity of therapeutic strategies to induce apoptotic cell death. We hypothesize that these survival factors may provide RPE cells with additional protection from TNF-{alpha}–induced cell death. Although it is beyond the scope of the present work, studies are currently under way in our laboratory to test this hypothesis.

RPE cells are exposed to continual oxidative stress throughout life. This oxidative stress arises from several sources, including photoreceptor outer segment phagocytosis, exposure to peroxidized lipid membranes, and photooxidative reactive oxygen intermediates.55 Oxidative stress–induced RPE cell apoptosis has been proposed as a major pathophysiological mechanism in AMD.4 Perhaps more surprising is that RPE cells in most individuals are able to withstand this oxidative onslaught.1 2 Oxidative stress activates the intrinsic apoptosis pathway, a process mediated by enhanced mitochondria membrane permeability. Survival factors, which include c-IAPs and Bcl-2 family members identified in the present study, are thought to inhibit the intrinsic pathway of apoptotic cell death by preventing increased mitochondria permeability and subsequent release of proapoptotic proteins or by blocking caspases activated by proteins released by leaky mitochondria.17 46 47 We speculate that, cumulatively, these survival factors, along with antioxidant enzymes such as catalase Mn-Zn superoxide dismutase, and glutathione peroxidase and antioxidant vitamins, protect RPE cells from oxidative stress–induced apoptotic cell death.


   Footnotes
 
Supported by National Eye Institute Grants NEI R01-EY9106 (GJJ) and P30EY05722 (Core Grant), R01-EY11286 (CBR), and a Career Development Award from Research to Prevent Blindness (CBR).

Submitted for publication August 30, 2004; revised November 9, 2004, and January 7, 2005; accepted January 11, 2005.

Disclosure: P. Yang, None; J.L.Wiser, None; J.J. Peairs, None; J.N. Ebright, None; Z.J. Zavodni, None; C.B. Rickman, None; G.J. Jaffe, 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.

Corresponding author: Glenn J. Jaffe, Department of Ophthalmology, Box 3802, Duke University Eye Center, Durham, NC 27710; jaffe001{at}mc.duke.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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