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Human RPE Expression of Cell Survival Factors

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Human RPE Expression of Cell Survival Factors 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 PURPOSE. To determine basal and tumor necrosis factor (TNF)- etinal pigment epithelial (RPE) cells form a monolayer of ␣–regulated expression of retinal pigment epithelial (RPE) cell Rcuboidal cells located between the photoreceptors of the survival factors and whether regulation is dependent on nu- neurosensory retina and the choroidal capillary bed. The RPE clear transcription factor (NF)-␬B. comprises an important blood–retinal barrier component and performs many important functions essential to the visual pro- METHODS. Cultured human RPE cells were infected with ade- novirus encoding either mutant inhibitory (I)-␬Bor␤-galacto- cess. Normally, RPE cells remain in a quiescent state and survive over an individual’s lifetime.1,2 sidase and treated with TNF-␣ for various times. Freshly pre- Age-related macular degeneration (AMD) is an idiopathic pared RPE/choroid and RPE samples were isolated from human retinal degenerative disease that is the leading cause of irre- donor eyes. Real-time reverse transcription-polymerase chain versible vision loss in the Western world among persons older reaction, Western blot, and immunocytochemistry were used than 65 years.3 AMD is characterized by clinical signs, ranging to determine survival factor gene expression, cellular protein from a few soft drusen and pigmentary changes in the macular levels, and localization, respectively. RPE with normal visual acuity, to large areas of RPE atrophy or RESULTS. Multiple survival factor genes, including cellular inhib- choroidal neovascular membranes (CNVMs) and associated itor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-asso- blindness.4 RPE cell apoptosis is an important feature of the ciated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 advanced forms of this disease.5,6 Although vision loss in AMD (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain is caused by photoreceptor damage in the central retina, RPE (FADD)-like interleukin-1␤-converting enzyme-like inhibitory atrophy is a prominent disease component.7,8 protein (c-FLIP), were expressed in basal conditions in both Proliferative vitreoretinopathy (PVR), the principal cause of cultured RPE cells and RPE cells in situ, whereas survivin was retinal reattachment surgical failure, is a potentially blinding expressed only by cultured cells. TNF-␣ upregulated expres- disease. PVR is characterized by uncontrolled cell proliferation sion of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, and migration into the subretinal space and vitreous cavity and c-FLIP, and to a lesser extent c-IAP2 protein levels were in- onto the retinal surface and undersurface.9,10 The RPE cell is creased by TNF-␣ in a time-dependent manner, whereas thought to be a key cell-type in this disease. RPE cell migration c-IAP1, survivin, Bcl-x , and TRAF-2 protein levels were not and proliferation and associated collagen secretion contribute L 9,10 influenced by TNF-␣ treatment at any time point tested. In to membrane formation in PVR. Contraction of these mem- contrast, Bcl-2 and A1 proteins were not detected under basal branes can lead to retinal detachment and subsequent loss of conditions or after TNF-␣ treatment. Overexpression of mutant vision. The factors responsible for unwanted survival of migrat- I␬B blocked TNF-␣–induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, ing proliferating RPE cells in this condition have not been c-FLIP, and A1 gene expression and downregulated TRAF-1 clearly defined. ␬ protein levels. TRAF-1 and Bcl-x proteins were localized dif- Nuclear transcription factor (NF)- B is a major regulator of L 11,12 ␬ fusely in RPE cytoplasm. cell life and death. In many cells, activation of NF- B blocks apoptosis, induces cell proliferation, blocks differentia- CONCLUSIONS. Multiple RPE cell survival factors are expressed tion, and promotes metastasis.12 NF-␬B activation in cancer by human RPE cells. TNF-␣ regulates expression of some of cells by chemotherapy can blunt the ability of the cancer these factors in an NF-␬B–dependent manner, whereas others therapy to induce cell death by producing antiapoptotic factors are not influenced by NF-␬B. RPE cell survival factors may such as cellular inhibitor of apoptosis proteins (c-IAPs), cellular protect RPE cells from apoptosis normally and in diseases such Fas-associated death domain (FADD)-like interleukin-1␤-con- as age-related macular degeneration (AMD) and proliferative verting enzyme-like inhibitory protein (c-FLIP), and tumor ne- vitreoretinopathy (PVR). (Invest Ophthalmol Vis Sci. 2005;46: crosis factor (TNF) receptor associated factor (TRAF).13–16 1755–1764) DOI:10.1167/iovs.04-1039 Other important survival factors are members of the B-cell leukemia/lymphoma (Bcl)-2 family. These proteins inhibit ap- optotic cell death and include Bcl-2, Bcl-xL, and A1 (also des- 17 1 2 ignated Bfl-1). From the Departments of Ophthalmology and Cell Biology, TNF-␣ is one of the most studied activators of NF-␬B. It is Duke University Medical Center, Durham, North Carolina. widely expressed in epiretinal membranes, vitreous, and sub- Supported by National Eye Institute Grants NEI R01-EY9106 (GJJ) 18,19 and P30EY05722 (Core Grant), R01-EY11286 (CBR), and a Career retinal fluid of eyes with PVR and has also been identified 20 Development Award from Research to Prevent Blindness (CBR). in CNVMs of eyes with AMD. TNF-␣ is a major regulator of Submitted for publication August 30, 2004; revised November 9, RPE cell activities, including cell attachment, spreading, che- 2004, and January 7, 2005; accepted January 11, 2005. motaxis, migration, and proliferation.21,22 Numerous studies Disclosure: P. Yang, None; J.L.Wiser, None; J.J. Peairs, None; indicate that specific NF-␬B inhibition enhances TNF-␣–in- J.N. Ebright, None; Z.J. Zavodni, None; C.B. Rickman, None; G.J. duced apoptosis in a variety of cell types otherwise resistant to Jaffe, None TNF-␣–induced cell death.23–26 Furthermore, NF-␬B is acti- The publication costs of this article were defrayed in part by page 27 charge payment. This article must therefore be marked “advertise- vated in eyes with PVR (Jaffe GJ, unpublished data, 2001) and AMD.28 However, RPE cells are resistant to TNF-␣–induced ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 29 Corresponding author: Glenn J. Jaffe, Department of Ophthalmol- apoptosis, even after specific NF-␬B blockade. ogy, Box 3802, Duke University Eye Center, Durham, NC 27710; Despite the fact that RPE cells may remain intact over a [email protected]. person’s lifetime, and RPE cell survival and apoptosis play Investigative Ophthalmology & Visual Science, May 2005, Vol. 46, No. 5 Copyright © Association for Research in Vision and Ophthalmology 1755 Downloaded from iovs.arvojournals.org on 09/25/2021 1756 Yang et al. IOVS, May 2005, Vol. 46, No. 5 5–10 crucial roles in diseases such as PVR and AMD, there is very TABLE 1. Primers for Real-Time PCR little information regarding RPE cell survival factor expression ؅ ؅ and mechanisms that control their expression. Because TNF-␣ Gene Sequence (5 –3 ) is present in eyes with these disorders and activates NF-␬B, it is F CTG GCA TTG CCC TCA ACG ACC important to determine whether survival factor expression is GAPDH ␬ R CTT GCT GGG GCT GGT GGT CC regulated by NF- B, and whether there are survival factors that TRAF-1 F CCG GAA CAA GGT CAC CTT CAT GC are expressed independently of this transcription factor. To R TGG GCA TCC ACT GGC CAC G better understand these mechanisms, we determined RPE cell TRAF-2 F GGC CCT TCA ACC AGA AGG TGA CC survival factor expression and whether survival factor expres- R CGA TGT TCA TGT CGT TGA CTG GC sion was regulated by TNF-␣ in an NF-␬B–dependent manner. c-FLIP F ATT GCA TTG GCA ATG AGA CAG AGC R TCG GTG CTC GGG CAT ACA GG Bcl-x F GCC ACC CCG GGC TCT CTG C R CCG TCC AAT CTC CGG GCA CC MATERIALS AND METHODS Bcl-xL F GCA GGT ATT GGT GAG TCG GAT CGC R CAC AAA AGT ATC CCA GCC GCC G Bcl-2 F GAT GGG AAC ACT GGT GGA GGA TGG RPE Cell Culture and Adenoviral Infection R TCT GGA GGG CCC ACG GCA G Human donor eyes were obtained from the North Carolina Eye Bank, A1 F AAA TTG CCC CGG ATG TGG ATA CC Inc. (Winston- Salem, NC), in accordance with the provisions of the R TTT CCC AGC CTC CGT TTT GCC c-IAP1 F AGC CTG AGC AGC TTG CAA GTG C Declaration of Helsinki for research involving human tissue. RPE cells 30 R CCC ATG GAT CAT CTC CAG ATT CCC for culture studies were harvested from eyes as previously described. c-IAP2 F CCG TCA AGT TCA AGC CAG TTA CCC Cells were grown in Eagle’s minimal essential medium (MEM; Invitro- R AAG CCC ATT TCC ACG GCA GC gen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Hyclone Labo- Survivin F ATT CGT CCG GTT GCG CTT TCC ratories, Logan, UT) and 1ϫ antibiotic-antimycotic (Invitrogen) at 37°C R CAC GGC GCA CTT TCT TCG CAG ϫ 5 in a humidified environment containing 5% CO2. RPE cells (1 10 ) were seeded in six-well plates (Corning-Costar Inc., Corning, NY). F, forward; R, reverse. 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)-␬B(I␬B)31 (Uni- RNA Isolation, Purification, and cDNA Synthesis versity of North Carolina at Chapel Hill Gene Delivery Core, Chapel Total RNA was isolated from cultured RPE cells (RNeasy; Qiagen Inc., Hill, NC) or ␤-galactosidase (LacZ) at a multiplicity of infection (MOI) Valencia, CA), according to the manufacturer’s protocols.
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