The 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 (AMRD). 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. ? ? 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. ? ? 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) 4HPR-induced neuronal differentiation of ARPE-19 cells is mediated through an MAPK/ERK1/2 signal transduction pathway. By blocking the upstream kinase MEK1/2 with specific inhibitor U0126 we abrogated the 4HPR-induced phosphorylation of ERK1/2 and SAPK/JNK, indicating that the neuronal differentiation occurs through a positive cross-talk between the ERK and the SAPK/JNK pathways. Both U0126 and the suppression of ERK1/2 expression with small interfering RNA effectively blocked the 4HPR-induced neuronal differentiation of RPE cells and the expression of calretinin. The activated ERK1/2 then induced a sequential activation of p90RSK, and increase in phosphorylation of transcription factors c-fos and c-jun leading to transcriptional activation of AP-1. Taken together, our results clearly demonstrate that c-Raf/MEK1/2 signaling cascade involving ERK1/2 plays a central role in mediating the 4HPR-induced neuronal differentiation and calretinin expression in the human ARPE-19 retinal pigment epithelial cell line. In addition, we have 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. AGN194301, a RAR antagonist, blocked both the down and up regulations of IGFBP5 and 6 indicating the involvement of retinoid signaling. Exogenous IGFBP5 was 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.? ? 2) We began a study on microRNA (miRNA) expression in the human RPE cell line, ARPE-19. miRNAs could play an important role in RPE pathophysiology due to their ability to exert profound effects on gene expression. Our results clearly demonstrate that a large number of miRNAs are expressed in ARPE19 cells in culture and that the mir-9 expression is regulated during the N-(4-Hydroxyphenyl)retinamide (4HPR)-induced apoptosis. A 2-fold increase in mir-9 expression is associated with a marked increase in the expression of HO-1 and GADD153 transcripts. We have also studied the expression of miRNA in retinoblastoma Y-79 cells. 4HPR treatment did not affect mir-9 expression in these cells, but mir-223 expression increased over 10-fold. These approaches will be expanded to cover the effect on miRNA expression in ARPE19 cells exposed to other agents.? ? 3) IRBP is again attracting attention as an important regulator and transporter of retinoids in the visual cycle. For example, in a recent collaboration with Koutalos and coworkers we showed that the rate of all-trans retinol removal from photoreceptors is dependent on concentration of IRBP, confirming that IRBP is the physiologically relevant carrier for this process, providing further evidence for the existence of a receptor on photoreceptors for IRBP. In other work to further explore role of IRBP in retinoid transport to and from the retina, we hypothesized that multiple variants or isoforms of IRBP may also play different roles. Two forms of IRBP have been identified since its earliest characterization. We have purified both forms and submitted them to glycan chain analysis. We found that they have different glycan chain composition. It is possible that this is due to differential glycosylation of different populations. It may have a functional relevance. This is being explored. Another possible mechanism for IRBP post-translational modification is its phosphorylation. We are making progress in methods development to identify which potential phosphorylation sites are utilized. The relationship of phosphorylation to the differential glycosylation is also being investigated.
