The retinal pigment epithelium (RPE), a monolayer of highly differentiated epithelial cells located between the photoreceptors and choriocapillaries, is exposed to variety of stress, including exposure to light, inflammatory mediators, and reactive oxygen species. Derangements in signaling are predicted to participate in pathophysiology of the RPE. Included among these pathologies are retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and oxidative stress. In addition, signaling networks are crucial for normal homeostasis of retina/RPE-specific pathways. However, such networks are often poorly understood. We wish to develop an understanding of signaling networks with special emphasis on retinoid metabolism pathways and protection against light damage and oxidative stress. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMD) and may be regulated by retinoic acid (RA). RA affects many cellular functions including cell growth, differentiation, and apoptosis. Synthetic analogs of retinoic acid also have significant effects on cellular function. One such analog, fenretinide (N-(4-hydoxyphenyl)retinamide;4-HPR), has been used as a cancer preventive agent and has been proposed as a therapeutic agent for lipofuscin-based retinal diseases, and we are interested in how these effects of 4-HPR are mediated. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is involved in 4HPR-induced effects on RPE cells. In another avenue of regulation, microRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. Given the likely importance of this level of regulation in the response of RPE cells to various signals we are interested in determining changes in miRNA expression in RPE cells due to agents with which they are treated in our experiments. Bioinformatics approaches are used to collate gene expression data and to generate models for further testing. Lastly, while most, if not all, enzymatic or binding protein components of the visual cycle have been identified, signaling events in the visual cycle have received less attention. It is anticipated that visual cycle retinoid flux is regulated by such external stimuli as day/night status, ambient light level, as well as by relative levels of retinoid isomers. Receptor mediated uptake of all-trans retinol as well as secretion of 11-cis retinal, both perhaps involving interphotoreceptor retinoid binding protein (IRBP), are also not fully understood. A long-term goal is to identify such receptors for IRBP on the RPE and photoreceptor membrane surfaces. In the past year we have made progress in the following areas: 1) We continued efforts to identify the role of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, in 4HPR-induced apoptosis of RPE cells. SCD by its ability to regulate the ratio of saturated fatty acids to monounsaturated fatty acids is thought to control the structural integrity and fluidity of cell membranes, and thereby plays an important role in cell growth, differentiation and apoptosis. SCD also plays an important early role in the synthesis of sphingolipids and ceramides, important effectors in cellular biology. A close association between the production of ceramide and the onset of programmed cell death has been well established. We observed that SCD protein expression and enzyme activity is decreased in human RPE cells during 4-HPR-induced apoptosis. Interestingly, SCD protein expression was also decreased when cells were treated with tunicamycin and thapsigargin, known inducers of endoplasmic stress. We are investigating inhibition of 4HPR-induced apoptosis in human RPE cells by inhibitors of SCD by biochemical assays and microarray analysis of cultured RPE cells. We have established non-radioactive HPLC/mass spectrometric assays for several sphingolipids and ceramides to assist in this analysis. 2) Small RNAs such as microRNAs (miRNAs) play an increasingly important role in the post-transcriptional regulation of expression of many genes. miRNAs have been implicated in a wide variety of cellular processes, including inflammation, cell proliferation, tumorigenesis, etc., and could play an important role in regulating the inflammatory processes leading to AMD or other retinal degenerative diseases. We have continued our work on the role of miRNAs in regulating the inflammatory response of adult human retinal pigment epithelial (HRPE) cells in collaboration with Chandra Nagineni and John Hooks (NEI-LI). Our study of the role of the JAK/STAT pathway in mediating the regulation of miR-155 expression in HRPE cells by the inflammatory cytokines was published in this reporting period. However, two other microRNAs, miR-146a and miR-146b-5p, are thought to regulate the inflammatory process by their ability to target IRAK1 and TRAF6, two important genes involved in cytokine signaling, for translational repression. We investigated the role of mir-146a and miR-146b in the response to inflammatory cytokines. Both were induced but appear to be mediated by different mechanisms. Genomatix promoter analysis software for identifying canonical transcription factor binding sites was used to search for evolutionarily conserved patterns in the promoters of miR-146a and miR-146b. 3) We began a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. By rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. The various immortalized cell lines, such as the commonly used ARPE-19, have lost most native phenotypic features. What are the mechanisms regulating such gain or loss? We are particularly interested in the long-known but poorly understood loss by immortalized and primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells used for human transplant are fully competent to fulfill their intended role in restoring RPE function in treated eyes. Our experimental paradigm focuses on the loss of visual cycle competence by adult bovine RPE cells explanted into primary culture. We are developing methods for establishing bovine RPE cells in primary culture. We are analyzing expression of visual cycle genes and will correlate this to changes in gene regulation. 4) Analysis of genomic intervals mapped for hereditary retinal diseases was performed in order to prioritize candidate genes. Latent semantic indexing software was used to extract information from PubMed, utilizing word usage patterns that suggest associations with the pertinent diseases. In parallel with this, a repository of expression data for genes in each of the mapped retinal disease loci was compiled. This was done by extracting ESTs from all available libraries from human retina and RPE/choroid.
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