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. ? ? NORPEG (novel retinal pigment epithelial cell gene, RAI14), another retinoic acid regulated gene that we originally characterized from the human retinal pigment epithelial (RPE) cell line ARPE-19, encodes a protein consisting of 980 amino acid residues. The presence of ankyrin repeats region and coiled-coil domains, structural domains implicated in protein-protein interactions, suggests that NORPEG associates with multiple binding partners. It may be an important organizing protein in this regard.? ? 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 induces apoptosis in ARPE-19 cells in a dose-and time-dependent manner through activation of caspases 2 and 3, and generation of reactive oxygen species (ROS). In addition, we found that the expression of heme oxygenase-1 (HO-1), a stress response protein, and the growth arrest and DNA damage-inducible transcription factor 153 (Gadd153) were increased in response to the ROS generation. Furthermore, RAR antagonists were able to block the 4HPR-induced ROS generation, the expression of its downstream mediator Gadd153, and apoptosis. These results clearly demonstrate that 4HPR induces apoptosis in human retinal pigment epithelial cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO-1.? ? 2) 4HPR-induced neuronal differentiation of ARPE-19 cells is mediated through an MAPK/ERK1/2 signal transduction pathway. 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 neuronall differentiation indicating that IGFBP5 may not be a direct mediator of the neuronal differentiation.? ? 3) Protein-protein interaction involving NORPEG protein was investigated using the yeast-two hybrid (Y2H) system. The N-terminal 334 amino acid residues when used as a bait was found to interact with only one protein, the kinesin family member 1A (KIF1A). However, bait containing the C-terminal 105 amino acid residues interacted with more than 40 different proteins. The Y2H results are being verified using tandem affinity purification and co-immunoprecipitatation techniques. A novel protein domain was identified by sequence analysis, defining a family of genes that are structurally related to NORPEG. This domain was used as one of the baits for yeast-two-hybrid experiments. Results from the Y2H experiments were compared with the mass spec analysis of proteins co-precipitated with NORPEG. Binding partners for NORPEG were found among cytoskeletal components, plasma membrane adherence junctions, and the nuclear splicesomal machinery, all of which are consistent with the varied subcellular localizations observed for NORPEG by confocal microscopy. Confocal immunofluorescence analysis of native bovine retinal pigment epithelium with an anti-NORPEG antibody showed staining of adherens junctions, specialized structures by which epithelial cells connect with each other. We developed a rabbit monoclonal antibody against the human NORPEG protein. The antibody preparation specifically reacts with the 110 kDa protein band corresponding to the NORPEG on western blot. This antibody also shows immunoreactivity towards murine and bovine orthologs of NORPEG and promises be a useful reagent to study the function of this protein. ? ? 4) In 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. This provides futher 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 chain composition.
