The retinal pigment epithelium (RPE), a monolayer of highly differentiated epithelial cells located between the photoreceptors and choriocapillaris, is exposed to a variety of stress, including exposure to light, inflammatory mediators, and reactive oxygen species (ROS). The integrity of the RPE layer, especially that of the macular region, is critical for the retention of normal visual function during the aging process. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration. In the adult retina, the RPE is the major site of retinoic acid production from all-trans retinol generated during the visual process. Fenretinide, N-(4-hydroxyphenyl)retinamide, a retinoic acid derivative and a potential cancer preventive agent, exerts its chemotherapeutic effect in cancer cells through the induction of apoptosis. Fenretinide induced apoptosis in human RPE cells (ARPE-19) in culture in a dose ? and time ? dependent manner as indicated by the generation of mono- and oligonucleosomes. ROS generation was also increased in a dose- and time- dependent manner in fenretinide treated ARPE-19 cells, and pyrrolidinedithiocarbamate, a free radical scavenger, inhibited fenretinide induced apoptosis. AGN194301, an RARalpha receptor specific antagonist, effectively blocked the generation of ROS as well as apoptosis in fenretinide treated cells. These results suggest that in the RPE the potential exists for the formation of toxic levels of ROS via the retinoid signaling pathway. We have shown that at low concentrations fenretinide induces differentiation of ARPE-19 cells into a neuronal phenotype. Total RNA for RT-PCR analysis was extracted from control cells and cells treated with fenretinide in order to examine the potential role of the retinoic receptor pathway in neuronal differentiation of ARPE-19 cells. RARgamma and RXRbeta mRNA were up-regulated after 3 hours of fenretinide treatment, suggesting that fenretinide-induced neuronal differentiation of ARPE-19 cells is mediated by RAR and RXR receptor pathways. Studies on NORPEG, a retinoic acid regulated gene that is also developmentally regulated and that we originally characterized from ARPE-19 cells, revealed that the pat7 nuclear localization signal present in the NORPEG protein is able to target a GFP-fusion protein to the nucleus. The potential nuclear localization of the protein encoded by NORPEG which is regulated during development and by retinoic acid suggests that this protein may play an important role in RPE cell structure and function. We have found that in washes of the bovine interphotoreceptor matrix, interphotoreceptor retinoid-binding protein (IRBP) consists of two fractions that are separable based on their differential lectin binding. These two fractions were purified to homogeneity via ion ?exchange and size-exclusion HPLC and analyzed for phosphorylation. Both IRBP I and IRBP II were stained with phosphoprotein specific stain. Mass Spectrometric analysis of the phosphopeptides isolated from proteolytic digests revealed that the sites of phosphorylation were different for IRBP I and II. In the case of IRBP I, the phosphorylated peptides were localized toward the C-terminus whereas the phosphorylated peptides isolated from IRBP II were localized closer to the N-terminus. The differential phosphorylation of these two IRBP fractions could be of functional significance since protein phosphorylation / dephosphorylation is a common and important regulatory modification of proteins.
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