We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, and protection against 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;4HPR), 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 4HPR are mediated. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, may be involved in 4HPR-induced effects on RPE cells. SCD 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. In addition, given the likely importance of microRNAs (miRNAs) as post-transcriptional regulators of gene expression 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. In the past year we have made progress in the following areas: 1) Stearoyl-CoA desaturase (SCD) regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in ARPE-19 cells. Increased expression of BiP/GRP78, ATF4 and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR-41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases such as AMD. A manuscript describing this work is currently under revision for publication. 2) We continued our work on the role of miRNAs in regulating the inflammatory response of the retinal pigment epithelium (RPE), implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB (NF- κB) pathway. We investigated the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines. We treated confluent cultures of RPE cells established from adult human donor eyes with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to the proinflammatory cytokines by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic. Our results clearly showed that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines. The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p was dependent on IFN-γ. Thus, miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying retinal degenerative diseases such as AMD through by negatively regulating the NF-κB pathway by targeting the expression of IRAK1. A manuscript describing these findings was published during this reporting period. We are currently conducting experiments to modulate the effect of proinflammatory cytokines on the inflammatory response using anti-inflammatory agents such as resveratrol. 3) We have continued 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, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. 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? Do mechanisms like epithelial-mesenchyme transition play a role in this process? 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 have developed methods for establishing bovine RPE cells in primary culture. Using these we are analyzing expression of visual cycle and other genes and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. The study is still ongoing. 4) We continued analysis of post-transcriptional modifications of IRBP. In this reporting period we have concentrated on establishing whether phosphorylation of IRBP occurs in vivo. An HPLC method to purify phosphopeptides has been established and these are being analyzed by mass spectrometry. We continue to collaborate within the LRCMB and with other laboratories and sections (LI, Molecular Structure and Functional Genomics), as well as with extramural labs in the analysis of retinoid and other compounds.
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