TThe 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. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMD). Retinoic acid, derived from oxidation of vitamin A, affects many cellular functions including cell growth, differentiation, and apoptosis. This effect is mediated through transcriptional regulation by the nuclear hormone receptors RAR and RXR for which retinoic acids are ligands. Synthetic analogs of retinoic acid also have significant effects on cellular function. One such analog, fenretinide, N-(4-hydoxyphenyl)retinamide (4HPR), has long been used as a cancer preventive agent. Recently, it has been proposed as a therapeutic agent for lipofuscin-based retinal diseases. At low doses, we have shown that 4HPR induces neuronal differentiation of cultured ARPE-19 human retinal pigment epithelial cells. At higher doses it causes apoptosis. We are interested in how these effects of 4HPR are mediated. In another avenue of regulation, microRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. The extent of their importance is just beginning to be realized. 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 ARPE19 cells due to agents with which they are treated in our experiments. 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. The role of IRBP in regulation of visual cycle may require receptors for transfer of retinoids. 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 previously found that 4HPR-induced neuronal differentiation of ARPE-19 cells is mediated through an MAPK/ERK1/2 signal transduction pathway. In addition, we identified a number of genes that are differentially expressed in 4HPR-induced neuronal type differentiation of RPE cells as well as in apoptosis. IGFBP5, one such gene down regulated in 4HPR induced differentiation is known to regulate the signal transduction pathway mediated by IGF-1, which is involved in cell growth, differentiation and apoptosis. In the past year we found that the neuronal differentiation of cultured human RPE cells induced by 4HPR is associated with down regulation of IGFBP5. This down regulation of IGFBP5 is inhibited by U0126, an inhibitor of MEK1/2, indicating the involvement of MAPK pathway. The ectopic addition of recombinant IGFBP5 is unable to block both the decrease in IGFBP5 expression as well as the neuronal differentiation, indicating that IGFBP5 may not be a direct mediator of the neuronal differentiation. On the other hand, overexpression of C/EBPβ(CCAAT/enhancer binding proteins) not only blocks the appearance of neuron-like morphology but also the observed decrease in the expression of IGFBP5. The activity of the human IGFBP5 promoter that contains the C/EBP element was decreased by 4HPR by approximately 2.5-fold as compared to basal conditions. Deleting C/EBP element from the IGFBP5 promoter abolished the activity in both basal and differentiating conditions. These data suggest that C/EBP regulates IGFBP5 expression at transcriptional level. 2) Inflammatory processes are known to be involved in the development of age-related macular degeneration (AMD). Human retinal pigment epithelial (HRPE) cells in cultures respond to inflammatory cytokines by increasing the expression of many chemokines and cytokines. Therefore, we investigated the role of miRNA in this process in collaboration with Chandra Nagineni and John Hooks (NEI-LI). By microarray analysis, miR-155 was identified as the predominant miRNA induced by cytokine mix (IFN-γ, TNF-αand IL-1β) in HRPE cells. Real-time PCR analysis showed that the miR-155 expression increased 10-fold under this condition. The observed alteration in miR-155 expression by the cytokine mix was associated with marked increases in the expression of CCL2, CCL5, CXCL9, CXCL10 and IL-6 transcripts as well as corresponding proteins. Individual treatment with IFN-γ, TNF-αor IL-1βshowed noticeable but small increase in miR-155 expression, but combination of any two exaggerated this effect. These results show that inflammatory cytokines can modulate miR-155 expression in human RPE cells. Thus, miR-155, an miRNA implicated in immunity, infection and oncogenesis, could play an important role in inflammatory processes leading to AMD or other retinal degenerative diseases. 3) We have hypothesized that multiple variants or isoforms of IRBP may play different roles in the function of IRBP, and two forms of IRBP have been identified since its earliest characterization (IRBP I and II). It is possible that this is due to differential glycosylation of different populations and may have a functional relevance. In the past year we extended our studies of this modification. While no differences were noted in the monosaccharide composition of IRBP I and II, and IRBP I and II had similar asparagine-linked glycans, the proportion of each glycan differed between IRBP I and II. That is, the main asparagine-linked component of IRBP I was a complex type glycan, whereas, that of IRBP II was a hybrid type structure. The glycan structures of IRBP I and II may be important in the function of IRBP in the IPM. Another possible mechanism for IRBP post-translational modification is its phosphorylation. We are trying to identify which potential phosphorylation sites are utilized. The size of IRBP is making this task more difficult than anticipated. The relationship of phosphorylation to the differential glycosylation is also being investigated.
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