|Molecular Vision 2003;
Received 28 February 2003 | Accepted 31 July 2003 | Published 5 August 2003
Down-regulation of RPE65 protein expression and promoter activity by retinoic acid
Rosalie K. Crouch
Department of Ophthalmology, Medical University of South Carolina, Charleston SC, USA
Correspondence to: Rosalie K. Crouch, Ph.D., Department of
Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue,
Charleston, SC 29425; Tel: (843) 792-2814; FAX: (843) 792-1732; email:
Dr. Ma is now at the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: RPE65 is critical for the normal formation of 11-cis retinal and thus photoreceptor function. Opsin expressed in HEK293 cells has been reported to form rhodopsin on the addition of all-trans retinol, indicating that the machinery for retinoid isomerization is present. RPE65 has been previously identified in HEK293 cells at both the RNA and protein levels. To further understand retinoid metabolism in these cells and the control of RPE65 expression, HEK293 cells were used as a model to determine if retinoic acid (RA) affects RPE65 promoter activity.
Methods: RPE65 levels were determined by Western blots. RA regulation of RPE65 promoter activity was monitored using the luciferase reporter assay after transient transfection of HEK293 cells with the RPE65 promoter. Deletion and truncation promoter mutants were assessed for activity.
Results: RA down-regulates RPE65 protein expression and promoter activity. The RA receptors (RARs), RARα, -β, and -γ, and the retinoid X receptors (RXRs), RXRα, -β, and -γ, were all identified in these cells and shown to mediate the regulation of RPE65 mRNA expression. After deletion of the AP1, AP4 or NF1 transcription factor binding sites, the RA down-regulation was decreased, but the decrease was not associated with a single transcription factor. The truncation promoter constructs P60, P153 and P257 showed increases in promoter activity, indicating an inhibitory element had been removed, and the down-regulatory effect of RA was decreased.
Conclusions: The down-regulation of RPE65 by RA is occurring at the transcription level. Multiple elements in the RPE65 promoter may contribute to this regulation.
RPE65 is a major protein of the retinal pigment epithelium (RPE) . The protein is also present in cone photoreceptors [2,3] and has been identified in the iris pigment epithelium at the mRNA level . RPE65 is highly conserved among vertebrate species. In humans, mutations in the RPE65 gene have been found in severe retinal degenerations, accounting for 10-15% of Leber's congenital amaurosis, a severe form of autosomal recessive childhood onset retinal dystrophy [5-7], as well as some cases of retinitis pigmentosa . In spite of numerous studies on the protein itself and on animal models in which the protein has naturally mutated [9,10] or has been removed by genetic manipulation (i.e., the RPE65 knockout (Rpe-/-) mouse ), the mechanism of action of this protein is still not understood [12,13].
The expression of RPE65 has been reported to be related to the development of photoreceptors  and the differentiation of ARPE-19 cells . The accumulation of RPE65 mRNA, starting during late embryogenesis, parallels that of the rat and mouse opsin mRNAs, suggesting that common factors may control the activation of genes in photoreceptors and the RPE . The function of RPE65 is still not known, but studies on Rpe-/- mice have shown that 11-cis retinal, which is essential for the visual process, is not generated . Administration of 9- or 11-cis retinal to Rpe-/- mice results in the formation of the rod visual pigment, rhodopsin, and this pigment is functional as measured by electroretinograms [16,17]. A second consequence of the lack of RPE65 in the Rpe-/- mouse is the accumulation of retinyl esters in the RPE, as the usual processing of these esters to 11-cis retinal is interrupted.
Retinoic acid (RA) is not known to be directly involved in the visual cycle, but has been shown to be generated in the RPE as a result of the action of light on photoreceptors . However, RA can induce retinal precursor cells to develop into different cell types . It is well established that RA plays an important role in regulating growth and differentiation [20,21] through the regulation of gene expression [22,23] in many tissues. Two families of nuclear receptors mediate RA's biological effects: the RA receptors (RARα, -β, and -γ) and the retinoid X receptors (RXRα, -β, and -γ). These receptors form heterodimers that act as transcription activators or repressors via binding to the specific nucleotide sequences, retinoic acid response elements (RAREs), on the target genes .
RPE65 has not been previously identified at the protein level in primary cultured RPE cells, although its mRNA has been demonstrated in the RPE-derived cell line ARPE-19 . In the ARPE-19 cells, the transcription of RPE65 mRNA was delayed by 2-3 weeks in the presence of RA . Recently, the RA up-regulation of several cone specific genes, including RPE65, was reported in the Weri-Rb-1 retinoblastoma cell line .
Previously, we reported that RPE65 is expressed (unexpectedly) at both the mRNA and protein level in HEK293 cells . HEK293 cells are possible neuronal progenitors of retinal lineage  widely used in biological research including studies on visual function [29,30]. Using measurements of early receptor current as a monitor of rhodopsin, Brueggemann and Sullivan  observed that HEK293 cells expressing opsin have functional retinoid processing machinery, as rhodopsin regeneration occurred after application of not only 11-cis retinal but also all-trans retinal or all-trans retinol to these cells. The finding that the HEK293 cells have the ability to generate 11-cis retinal, and thus contain the illusive retinoid isomerase, emphasizes the importance of exploring retinoid metabolism in this system . We therefore have used this cell line to examine if RA regulates RPE65 expression. The studies reported here reveal that RA down-regulates both the protein expression and promoter activity of RPE65, the presence of which is critical for normal functioning of the visual cycle.
Cell culture and RA treatment
The HEK293 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and was grown in culture medium containing DMEM/F12 (1:1), 10% (v/v) fetal calf serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). All-trans RA, all-trans retinol, 9-cis RA, 4-([E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB), and N-(4-hydroxyphenyl)-retinamide (4HPR) were purchased from Sigma-Aldrich (St. Louis, MO). The RARα agonist Ro-40-6055 was a generous gift from Hoffmann-La Roche, Ltd (Basel, Switzerland). Cells (2x106) were plated on 100 mm tissue culture dishes and RA in ethanol (1 μL) was applied to the culture medium at final concentrations of 10-9-10-5 M. Cells were harvested at about 90% confluence after 72 h incubation. For the time course experiments, RA in ethanol (1 μL) was applied at a final concentration of 10-6 M after 0, 24 and 48 h and cells harvested at approximately 90% confluence after 72 h incubation. The same volume of vehicle, ethanol, was added to the control cells for all experiments.
Western blot analysis
The harvested cell pellets were treated for 30 min on ice with lysis buffer (50 mM Tris-base, pH 7.6, 150 mM NaCl, 1 M EDTA, 1% (v/v) NP40) to which the protease inhibitors (complete protease inhibitor 1 tablet/10 mL, pepstatin 10 μg/mL, pefabloc SC 1 mg/mL, and E64 10 μg/mL (Roche; Indianapolis, IN)) were added. The cells were centrifuged at 10,000x g for 15 min and the supernatant assayed for protein concentration (Bio-Rad Laboratories; Hercules, CA). Soluble proteins (100 μg) were resolved on 10% SDS polyacrylamide gels and electrotransferred onto nitrocellulose membranes. The membranes were probed with polyclonal anti-RPE65 rabbit antiserum (a gift from Dr. T. M. Redmond, National Eye Institute, Bethesda, MD; 1:1,000 dilution) overnight, followed by horse-radish peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (Amersham Biosciences Corp.; Piscataway, NJ). The immunoblots were reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa; Iowa City, IA; 1:10,000 dilution) followed by horseradish peroxidase-conjugated horse anti-mouse IgG secondary antibody (Amersham Biosciences, Corp.; Piscataway, NJ; 1:10,000 dilution).
Cell cycle analysis by flow cytometry
RA (10-6 M) was applied to the HEK293 cells, and the vehicle in the same volume was added to control cells. Cells were incubated in the dark at 37 °C in a 5% CO2 incubator for 24, 48, or 72 h, washed twice with PBS, spun at 500x g for 5 min, resuspended in 70% (v/v) ethanol in PBS and incubated at 4 °C for more than 4 h. Cells were again spun at 600x g for 10 min and washed with 5 mL PBS. The cell pellet was resuspended in 1 mL PBS with 50 μg/mL propidium iodide and 100 μg/mL RNase A (Sigma-Aldrich; St. Louis MO). After incubation at room temperature for 45 min, samples were analyzed by flow cytometry (MUSC facility).
HEK293 cells were pooled, resuspended in 50 M sodium phosphate buffer containing 0.32 M sucrose, and three cycles of freeze-thaw performed. The preparation was performed as described previously . In detail, cells were centrifuged at 600x g for 10 min to generate the cell nuclei (Fraction 1) as the pellet. The supernatant was centrifuged at 100,000x g for 1 h to separate the plasma membrane-microsomal fraction (Fraction 2) and soluble fraction (Fraction 3). The microsomal pellet was washed three times with 50 mM sodium phosphate buffer containing 0.32 M sucrose and centrifuged at 100,000x g for 15 min. To the microsomal fraction, 0.3% (v/v) of CHAPS in sodium phosphate buffer with complete protease inhibitors was added. The above mixture was incubated on ice overnight and recentrifuged at 100,000x g for 15 min (Fraction 2). These three fractions were used for Western blot analysis.
Immunofluoresence staining and confocal microscopy
HEK293 cells grown on coverglasses were fixed with methanol and permeabilized with 0.3% (v/v) Triton X-100. Cells were incubated with anti-RPE65 rabbit antiserum (1:1,000 dilution) and were further incubated with Texas Red labeled goat anti-rabbit secondary antibody (1:200 dilution; Vector Laboratories, Inc.; Burlingame, CA). Imaging was performed on a Bio-Rad MRC-1000 laser scanning confocal microscope, and the images were processed with Adobe Photoshop 5.0 software (Adobe Systems, Inc., San Jose, CA).
Deletion mutations of RPE65 promoter
The RPE65 promoter was a generous gift from Dr. Debra A. Thompson, University of Michigan Medical School . The 1345 bp fragment in pBluescript II sk (-) was digested with Sac I (5') and Hind III (3') for the 1345 bp, and Bgl II (5') and Hind III (3') for the 450 bp promoter sequence, and subcloned into pGL3 basic vector containing the firefly luciferase gene (Promega; Madison, WI). The 1345 bp promoter in pGL3 served as the template for all the mutations generated. Deletions of AP1, AP4 and NF1 transcription factor binding sites were performed by Quick-change Mutagenesis Kit (Stratagene; La Jolla, CA). For the truncation mutations of RPE65 promoter, 3' primer containing the Hind III restriction site and 5' primers containing the Mlu I, Nhe I, and Xho I restriction sites were designed. After PCR, the corresponding 257 bp, 153 bp, and 60 bp PCR products were passed through G25 columns, digested by the corresponding restriction enzymes, purified by agarose gel and ligated into the pGL3 vector containing the corresponding restriction enzyme sites. All the restriction enzymes were purchased from Promega (Madison, WI).
Transient transfection and RA treatment
The pGL3-basic vector, the pGL3-control vector (Promega; Madison, WI), and the RPE65 promoter-reporter construct (1 μg, each) were transfected into the HEK293 cells using LIPOFECTAMINETM 2000 (Invitrogen; Carlsbad, CA) reagent. Cells were moved into normal growth medium 24 h after transfection. RA in 1 μL ethanol was applied to the cells at final concentrations of 10-9 to 10-5 M. RA analogues were applied to the cells at final concentration of 10-6 M. Control cells received the same volume of the vehicle. Luciferase activity was measured after 24 h incubation. For time course experiments, cells were changed into normal growth medium 24 h after transfection, RA in 1 μL ethanol was applied at a final concentration of 10-6 M, and cells were incubated for 12, 24, and 48 h. Control cells received the same volume of the vehicle and were incubated for 48 h. To correct for differences in transfection efficiency, protein assays were performed following each luciferase assay. All experiments were performed in triplicate.
Luciferase activity assay
Cell lysate was prepared by scraping the HEK293 cells vigorously in 200 μL of 1x passive lysis buffer (PLB; Promega; Madison, WI) followed by 2 cycles of freeze/thaw. The cell lysate (20 μL) was transferred into a luminometer tube containing 100 μL of luciferase assay reagent (Promega; Madison, WI). Light emission was measured using an Auto Lumat LB 953 luminometer (EG&G, Wallac, Inc; Gaithersburg, MD), and luciferase activity was recorded . Luciferase activity of the pGL3-control vector was used to monitor the transfection efficiency and served as background correction, which is subtracted from the measured RPE65 promoter activity, for the activity of RPE65 promoter-reporter construct. A protein assay was performed using BioRad DC Protein Assay (Bio-Rad Laboratories; Hercules, CA) to normalize luciferase activity.
Reverse transcription (RT-) PCR and Southern blot analysis
Total RNA from HEK293 cells was isolated by the acid guanidinium isothiocyanate-phenol/chloroform extraction method . The RT analysis was performed as described previously . The mixture of 1 μL of the RT product, 10 pmol of the respective primer and 3 μL of 2 mM dNTP was subjected to PCR using the Expand High Fidelity PCR System (Roche Diagnostics Corp.; Indianapolis, IN). The PCR conditions for the RARs were 1 cycle at 95 °C for 5 min followed by 36 cycles of 95 °C of 1 min, 55 °C for 100 s, 72 °C for 80 s and 1 cycle at 72 °C for 3 min in a Robocycler (Stratagene; La Jolla, CA). The PCR conditions for the RXRs were the same except that the annealing temperature was 59 °C instead of 55 °C. All PCR products were probed with 32P-labelled nested primers in a Southern blot analysis as described previously . The PCR and Southern primers were designed according to GenBank sequences of human RARs and RXRs, and are listed in Table 1.
Localization of RPE65 to both the cytosolic and membrane-associated fractions in HEK293 cells
Confocal microscopy utilizing immunostaining with anti-RPE65 polyclonal antibody showed that the RPE65 protein is distributed throughout the cytoplasm except for the nucleus (Figure 1A). No staining was detected in the absence of primary or secondary antibody (data not shown). Figure 1B is a bright field image of the same cell shown in Figure 1A. The subcellular localization of RPE65 was confirmed by fractionation with differential centrifugation followed by Western blot analysis. RPE65 was detected in both cytoplasm and microsomal fractions (Figure 1C). The high specificity of the anti-RPE65 polyclonal antibody used in our experiments is demonstrated by the recognition of a single band in Western blot analysis using HEK293 cells. This band had an identical molecular weight to the purified recombinant human RPE65 protein  and bovine RPE homogenate (Figure 1D). These data show that RPE65 is both cytosolic and membrane associated in HEK293 cells.
Resistance of HEK293 cells to RA treatment
Cell cycle analysis was conducted using flow cytometry to determine the effect of RA treatment. The data show no changes of cell cycle when 10-6 M RA was applied to HEK293 cells (Figure 2A) compared with the control (Figure 2B) to which vehicle alone was applied. Treatment of RA for 24, 48, and 72 h did not cause G1 phase retention or changes in the cell number in M and G2 phase. These results confirm the previous data of van der Leede et al. , who demonstrated that RA does not affect cell cycle progression and hence differentiation of the HEK293 cells.
Identification of RA down-regulation of RPE65 protein expression
HEK293 cells were incubated with RA (10-9-10-5 M for 72 h) and the RPE65 levels assayed by Western blot analysis. With the increasing concentrations of RA, RPE65 expression decreased to approximately 50% of the non-treated cells as normalized to GAPDH (Figure 3A,B). HEK293 cells were treated with 10-6 M RA for 24, 48, and 72 h, respectively, to determine the time dependence of RA treatment. RPE65 expression decreased following prolonged incubation with RA to an almost steady level within 48-72 h (Figure 3C,D).
Down-regulation of RPE65 promoter activity by RA
We tested the hypothesis that the RA regulation of RPE65 expression is at the transcriptional level. Twenty-four h after HEK293 cells were transiently transfected with the 1345 bp RPE65 promoter-luciferase construct, RA was applied at the concentrations of 10-9 to 10-5 M, incubated for 24 h, and luciferase activity measured. Results showed that the RPE65 promoter activity is down-regulated by RA in a concentration dependent manner (Figure 4A). The time course of regulation was determined by luciferase activity measurements after incubating these cells with 10-6 M RA for 12, 24, and 48 h. The data demonstrated that the RA down-regulation of the RPE65 promoter activity is time dependent (Figure 4B).
Detection of RARs and RXRs in HEK293 cells
In order to investigate the mechanism of the RA regulation, we performed RT-PCR and Southern blot analysis using primers designed according to GenBank sequences of human RARs and RXRs as listed in Table 1. All the PCR products were confirmed with 32P-labelled nested primers in Southern blot analysis. Our experiments detected the PCR products of 947 bp for RARα, 1016 bp for RARβ, 1051 bp for RARγ, 933 bp for RXRα, 999 bp for RXRβ, and 1030 bp for RXRγ (Figure 5). Therefore, the RARs (RARα, RARβ, RARγ) and RXRs (RXRα, RXRβ, RXRγ) are present in HEK293 cells and may potentially be involved in the RA down-regulation of RPE65 expression in these cells.
Down-regulation of RPE65 promoter activity through the RARs and RXRs
Cells were treated with RA analogues 24 h after transient transfection of HEK293 cells with RPE65 promoter-reporter construct, to examine the involvement of RA receptors in the regulation of RPE65 promoter activity. The luciferase activity of the RPE65 promoter in HEK293 cells was measured after 24 h incubation with one of the following: all-trans retinol, the RAR and RXR pan-agonist all-trans and 9-cis RA , the synthetic RAR selective agonist TTNPB , the potent RARγ agonist and moderate RARβ activator 4-hydroxyphenyl retinamide (4HPR) , or the RARα agonist Ro-40-6055 . All-trans retinol had very little activity, as it presumably had to be metabolized into all-trans RA in order to perform its function. The above agents decreased the RPE65 promoter activity by 40-60% (Figure 6). In summary, the above results showed that the down-regulation of RPE65 transcription by RA is mediated by both RARs and RXRs.
Effect of promoter truncation and deletions on RA regulation of RPE65 promoter activity
In order to identify the promoter sequences that mediate the RA down-regulation of RPE65 expression, two sets of mutations were generated on RPE65 promoter sequences (Figure 7A) and luciferase activity measured (Figure 7B,D). Deletion of the AP1, AP4 and NF1 transcription factor binding elements slightly increased the activity of the promoters (Figure 7B). Each deletion decreased the response of that promoter to RA (p<0.05, Figure 7C). The basic promoter activity of the 450 bp truncated RPE65 promoter dramatically increased compared with that of the 1345 bp RPE65 promoter (Figure 7D), suggesting that strong inhibitory elements may exist in that portion of the sequence. The truncated RPE65 promoters P257, P153 and P60 showed increases in the activity of the respective promoter (Figure 7D). There is an effect of RA on each of these truncated promoters, but none are as dramatic as with the complete P1345 promoter (Figure 7E). These data suggest multiple sequence elements in the promoter may contribute to the complex mechanism of RA regulation of RPE65 expression.
The function of RPE65 is still unknown. In the RPE, this protein is quite abundant and studies on the Rpe-/- mouse have shown that this protein is essential for the generation of 11-cis retinal from all-trans retinol  in the visual cycle. Mutations of the protein have been associated with a number of retinal degenerations (e.g., [5,8]) suggesting that disruption of this step in the visual cycle is dehabilitating for retinal function. In the bovine RPE, two forms of the RPE65 protein have been observed: a cytosolic, as well as a membrane-associated form with different molecular weights . Interestingly, our results utilizing the HEK293 cells have shown that the protein has a similar subcellular localization, both in the cytoplasm and microsomal fractions.
Our experiments on HEK293 cells show that RA down-regulates RPE65 expression at the transcriptional level in both a concentration and time dependent manner. The RA suppression of RPE65 expression is reversible since the expression level of RPE65 recovered after removal of RA (data not shown). Previous studies using [3H]-thymidine incorporation showed that RA does not change the rate of DNA synthesis in the HEK293 cells . Our flow cytometry data showing no changes of cell cycle also indicate that the HEK293 line is RA resistant, and that RA does not influence the proliferation of HEK293 cells. These results exclude the possibility that RA-induced down-regulation of RPE65 expression is the consequence of global suppression of gene expression due to the inhibition of HEK293 cell proliferation. We also observed that the degree of cell confluence does not influence the levels of RPE65 expression and promoter activity (data not shown). These results demonstrate that the down-regulation of RPE65 by RA in HEK293 cells is not a general response to cell growth and proliferation.
We and others  have failed to find RPE65 protein expression in cultured primary RPE cells. The identification of RPE65 protein in HEK293 cells was an unexpected finding, as previously the protein had been found only in ocular tissues. In HEK293 cells the down-regulation of RPE65 expression by RA treatment is effective within 72 h. Recent studies reported the down-regulation of RPE65 transcription in ARPE19 cells. However, these experiments employed a period of about 20 days for each treatment .
Interestingly, RA has also been found to up-regulate the expression of certain genes involved in photoreceptor function in cell lines. RA is known to up-regulate the expression of Rh1 (the predominant rhodopsin) gene expression in Drosophila . In Weri-Rb-1 and Y79 retinoblastoma cell lines, RA has been reported to up-regulate human cone arrestin mRNA expression . Whereas RA has no effect on interphotoreceptor retinoid binding protein (IRBP), it increases cone rod homeobox (CRX) mRNA significantly in Weri-Rb-1 cells . In a recent microarray study in Weri-Rb-1 cells , RA was found to up-regulate RPE65 transcription after 7 days incubation, along with other cone specific genes. RPE65 has been identified in cone but not rod photoreceptors [2,3]. The function of this protein in cones is still unknown.
In general, RA regulates gene expression by binding to RA receptors and forming RAR and RXR heterodimers. The heterodimers interact with RARE on the target genes. Using RT-PCR and Southern blot analysis, we identified all types of the RARs and RXRs in the HEK293 cell line. Experiments were performed to investigate whether RA analogues regulate RPE65 expression. All-trans and 9-cis RA are pan-agonists of RARs and RXRs . Our data show that 9-cis RA down-regulates RPE65 promoter activity to the same extent as all-trans RA. TTNPB (RAR selective activator), Ro-40-6055 (a RARα agonist) and 4HPR (a strong RARγ and moderate RARβ transcriptional activator) also down-regulate RPE65 promoter activity. Therefore, RA down-regulation of RPE65 protein expression is through the retinoid receptors and both RARs and RXRs are responsible for this down-regulation.
To further investigate the mechanism of RA regulation of RPE65 expression, two series of mutations of the RPE65 promoter were generated. Only changes in promoter activity were found on deletion of AP1, AP4, or NF1 transcription factor binding sites. The effect of RA on each of these mutants was less than the effect of RA on the full promoter, but there was not a dramatic association with one site. The results on the luciferase activity of the truncated RPE65 promoters indicate that no RARE exists in any single truncated region of the promoter, but rather that the regulation mechanism involves multiple elements. It is possible that the RARE may exist in the RPE65 promoter region, but arranged in a unique form (e.g., the response element may split apart and be separated by a number of nucleotides) . The TATA and CAAT box motifs of the RPE65 promoter may also possess RA-inducible promoter activity . Also, the possibility cannot be excluded that the RARE may exist in the intron sequences or the 3' untranslated region of the RPE65 gene.
In summary, using HEK293 cells as a model, these studies confirm that RA regulates the expression of RPE65. We have also shown that the RPE65 promoter is down-regulated by RA and that multiple retinoid receptors may be responsible for this down-regulation. As RPE65 is critical for the normal function of the visual cycle, these data suggest that the RA levels are of importance in the developing eye to ensure the appropriate levels of expression of this protein.
The authors thank Dr. Michael Redmond (NEI, NIH) for the anti-RPE65 antibody and Dr. Debra Thompson (University of Michigan Medical School) for the RPE65 promoter construct. We thank Drs. Donald Menick and Yoshi Hiro (Cardiology Division, MUSC) for use of the luminometer and assisting on the confocal microscope; Drs. Baerbel Rohrer, Mas Kono and Steve Kubalak for critical reading of the manuscript, and Candace Enockson, MT (ASCP), for assistance with the flow cytometry analyses. This work was supported in part by a fellowship from the National Science Foundation/EPSCoR (Y.C.); grants from the National Institutes of Health EY04939 (R.K.C.) and EY12231 (J.x.M.); the Foundation for Fighting Blindness, Owings Mills, MD; and an unrestricted grant to the Department of Ophthalmology at MUSC from Research to Prevent Blindness (RPB), Inc., New York, NY. R.K.C. is a RBP Senior Scientific Investigator.
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