Choroidal neovascularization remains the leading cause of severe vision loss in patients with the exudative form of age-related macular degeneration (ARMD). Choroidal vessels grow through breaks in Bruch's membrane and proliferate under the retinal pigment epithelium (RPE) and the sensory retina. The RPE as well as the choriocapillaris are morphologically altered before neovascularization occurs.

In the vertebrate retina, the retinal pigment epithelium (RPE) performs specific functions critical to the normal process of vision. Although some of these functions are well documented, molecular data are still scarce. Using the RPE-specific monoclonal antibody RPE9, raised against human RPE cells, we have identified a novel 65 kD protein, conserved in mammals, birds, and frogs. This RPE-specific protein was found to be nonglycosylated. It was most effectively solubilized in the presence of detergent suggesting that it is associated with the RPE cell membranes. Its partitioning in the detergent phase of Triton X-114 and its solubilization in 0.75 M and 1.0 M KCl suggest that it interacts with the membrane either through a polypeptide anchor or charged amino acids. Cell fractionation by differential solubilization and differential centrifugation demonstrated that the protein was preferentially associated with the microsomal membrane fraction, where it is the major protein. Developmental expression of this 65 kD protein was examined in neonatal rats. Morphologically well-differentiated RPE cells did not express the 65 kD protein at birth. However, expression was detectable at postnatal day 4, that is, one to two days before the photoreceptors develop their outer segments, suggesting that the expression of the 65 kD protein may be coordinated with other developmental events in the intact retina. This is further supported by the fact that RPE cells in confluent culture lose the expression of this protein within two weeks, while they maintain their characteristic epithelial morphology. Because of its specificity, its evolutionary conservation, and its timing of expression, it is possible that this protein may be involved in one of the key roles of RPE and as such is an important molecular marker for RPE differentiation.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific for the retina and pineal gland, induces inflammatory changes in these two organs in immunized animals. We report here on the identification of an immunodominant determinant of bovine IRBP that is highly immunogenic and immunopathogenic in the Lewis rat. The peptide, which comprises the sequence 1169-1191 of bovine IRBP, was shown to be immunodominant by its capacity to stimulate lymphocytes sensitized against whole IRBP. A comparison was made between peptide 1169-1191 and another peptide, 1158-1180, which is nondominant but is immunogenic and immunopathogenic in the Lewis rat. Peptide 1169-1191 was found to be superior in its immunological capacities; the minimal dose of 1169-1191 needed to induce cellular immune response or disease in Lewis rats (0.02-0.1 nmol/rat) is congruent to 1,000 times smaller than that of 1158-1180. In addition, unlike the ocular disease induced by 1158-1180, the disease produced by 1169-1191 resembled that induced by whole IRBP in its kinetics and histopathological features. The immunological activity of 1169-1191 in the Lewis rat was localized to the 10 residues at the COOH terminus; no such activity was exhibited by the truncated peptide 1169-1188, which comprises the 20 residues at the NH2 terminus of the full peptide. The usefulness of this unique experimental system in analyzing the role of immunodominance in peptide immunogenicity and immunopathogenicity is underscored.

Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.

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RPE65 is essential for all-trans-to 11-cis-retinoid isomerization, the hallmark reaction of the retinal pigment epithelium (RPE). Here, we identify regulatory elements in the Rpe65 gene and demonstrate their functional relevance to Rpe65 gene expression. We show that the 5 flanking region of the mouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consensus GC and CAAT boxes. The mouse and human genes do share several cis-acting elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promoter/␤-galactosidase transgene containing bases -655 to ؉52 (TR4) of the mouse 5 flanking region was sufficient to direct high RPE-specific expression in transgenic mice, whereas shorter fragments (-297 to ؉52 or -188 to ؉52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activity in the human RPE cell line D407 but weak activity in the non-RPE cell lines HeLa, HepG2, and HS27. Functional binding of potential transcription factors to the octamer sequence, AP-4, and NFI sites was demonstrated by directed mutagenesis, electrophoretic mobility shift assay, and cross-linking. Mutations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synergistically regulate the RPE65 promoter function. Thus, we have identified the regulatory region in the Rpe65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the Rpe65 gene.All-trans-to 11-cis-isomerization of vitamin A is an obligate and tissue-specific enzymatic step in the renewal of 11-cisretinal, the universal chromophore of rhodopsin and other visual pigment proteins, in the visual cycle (1) of the retinal pigment epithelium (RPE).1 Several components, including 11-cis-retinol dehydrogenase (2), cellular 11-cis-retinaldehydebinding protein (CRALBP) (3, 4) and lecithin:retinol acyltransferase (5, 6), all essential to the visual cycle activity, are found highly expressed, but not exclusively expressed, in the RPE. However, the retinol isomerase activity (7-9), central to 11-cischromophore synthesis, is expected, mechanistically, to be highly tissue-specific. A tissue-specific component of the RPE, RPE65 (10 -12), which copurifies with 11-cis-retinol dehydrogenase (2), appears to play a crucial role in retinoid isomerization. Thus, in the Rpe65-deficient mouse (13), rod photoreceptor function is abolished due to lack of the 11-cis-retinal chromophore. Furthermore, mutations in the human RPE65 gene cause several forms of severe early onset blindness (14 -17). Clearly, RPE65 is essential to the visual cycle in general and to all-trans-to 11-cis-retinoid isomerization in particular. RPE65 is the major protein of the RPE microsomal membrane fraction. The bovine (10), human (18), dog (19), rat (20), a...

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