Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 μM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1β and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1β mRNA showed an increase in choroid–RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.     

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.     

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.     

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.     

A serum-free defined medium for retinal pigment epithelial cells

Human and bovine RPE cells underwent changes in morphology and culture doubling times when passaged in serum-supplemented medium (CM). Furthermore, late passage human RPE cells subcultured in CM medium increased synthesis of three acidic, 43 000–63 000 D proteins. In order to provide a controlled environment for the study of RPE cells in vitro, we have devloped a method for growing human and bovine RPE in a serum-free defined medium (DM). RPE cells grown in DM required a 24 h pretreatment with CM to allow the cells to attach and spread on the substrate. Cells grown in DM retained an epithelioid morphology, a stable culture doubling time, and similar 2-D PAGE patterns through several subculturings.     

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.     

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by homogenates of human retinal epithelial cells

The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and all-trans-isomers of retinyl palmitate. Retinyl ester hydrolysis was time-, protein-, and pH-dependent. The 11-cis isomer was hydrolyzed at a rate which was approximately 20 times greater than that of the all-trans isomer. The 11-cis-retinyl palmitate hydrolase activity did not require detergents, unlike the all-trans-retinyl palmitate hydrolase activity, which required detergents for activity. The 11-cis-retinyl palmitate hydrolase activity was maximally active with the addition of 1.0% sodium taurocholate at about pH 8.5, was abolished by incubation at 50 degrees C for 10 min, and was quantitatively recovered in the pellet after centrifugation at 100,000 X g for 1 h. The rate of hydrolysis of 11-cis-retinyl palmitate became saturated with increasing concentrations of 11-cis-retinyl palmitate; under the assay conditions employed, the hydrolase activity had an apparent Km of 19 microM toward 11-cis-retinyl palmitate. All-trans-retinol and 11-cis-retinyl did not affect the rate of hydrolysis of 11-cis-retinyl palmitate, and addition of all-trans-retinyl palmitate only weakly inhibited the 11-cis-retinyl palmitate hydrolytic activities. These data indicate that the human retinal epithelium possesses distinct activities for the hydrolysis of 11-cis- and all-trans-retinyl esters and raise the possibility that these activities may provide a means of distinguishing the stereoisomers of retinol in this tissue.     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Proteome analysis of lipofuscin in human retinal pigment epithelial cells

Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age-related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.     

Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells.

Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.     

The cytoskeleton of chick retinal pigment epithelial cells in situ

Summary Gelatin-coated slides were used to obtain en face preparations of retinal pigment epithelium (RPE) from 6-to 21-day-old chick embryos in order to study the distribution of F-actin in microfilaments (MF) and the MF-associated proteins, myosin, tropomyosin,-actinin and vinculin in situ at different stages of development by fluorescence microscopy. The epithelial sheets were fixed in formaldehyde and then extracted in a solution containing 0.1% Triton X-100. NBD-Phallacidin was used to visualize the F-actin in MF, and antisera against myosin, tropomyosin,-actinin and vinculin were used to determine the distribution of these four MF-associated proteins. F-actin, myosin, tropomyosin,-actinin and vinculin were present in cortical rings around the apical ends of the RPE cells throughout this period of development. Of these proteins, only F-actin was identified in the apical processes of RPE cells. The increase in the amount of F-actin could be followed as the length and the number of apical processes increased with age and maturation of RPE cells. F-actin was first detected in numerous short apical processes on the surface of each RPE cell on day 12. From day 12 to day 17, they were at an intermediate stage of elongation and from day 17 onward all of the RPE cells had long F-actin-containing apical processes. These results indicate that the F-actin-containing MF assemble much later in the apical processes than in the cortical rings. Also the cortical rings and apical processes of RPE cells resemble those in absorptive intestinal cells in that the cortical rings in both cell types contain MF associated with myosin, tropomyosin,-actinin and vinculin while the MF in the apical processes and microvilli lack these MF associated proteins, and both of these structures lack talin. In addition to apical processes and cortical rings, stained fibers were also observed at a level below the cortical rings. The simple and highly reproducible en face method described is useful for determining changes in the organization of cytoskeletal components and other macromolecules in RPE cells and other epithelial cells in situ.     

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H₂O₂)-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H₂O₂-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H₂O₂ stimulation, and H₂O₂-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H₂O₂ elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H₂O₂-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H₂O₂ is Rac1. This study is the first to demonstrate that H₂O₂ induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H₂O₂-induced apoptosis as well as AIF/Bax translocation of RPE cells. Y.-C. Yang and T.-C. Ho contributed equally to the work described herein.     

The unusual microtubule polarity in teleost retinal pigment epithelial cells

In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule-dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types.     

On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells: 11-fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle

The synthesis of 11-fluoro-all-trans-retinol (11-F-tROL), which is shown to be an excellent substrate for processing by visual cycle enzymes, is described. It is isomerized to its 11-cis congener subsequent to its esterification by lecithin retinol acyl transferase (LRAT) approximately as well as is vitamin A itself. The enzymatic turnover of 11-F-tROL is unaccompanied by enzyme inhibition. The previously reported lack of isomerization of this substrate had been suggested as evidence for a carbonium mechanism in the critical enzymatic isomerization pathway in vision. The mechanism of this process remains unknown.     

Rat retinal pigment epithelial cells show specificity of phagocytosis in vitro

The retinal pigment epithelial (RPE) cell of the eye normally phagocytozes only retinal rod outer segments (ROS). The specificity of this phagocytic process was examined by incubating RPE cells with a variety of particle types. Confluent RPE cell cultures were incubated for 3 h at 37 degrees C in the presence of rat ROS, rat red blood cells (RBC), algae, bacteria, or yeast. Other cell cultures were incubated with equal numbers of ROS and one other particle type. Quantitative scanning electron microscopy was used to determine the numbers and morphology of particles bound to RPE cells, while double immunofluorescence labeling (Chaitin, M. H., and M. O. Hall, 1983, Invest. Ophthalmol. Vis. Sci., 24:812-820) was used to quantitate particle binding and ingestion. Both assays demonstrated phagocytosis to be a highly specific process. RPE cells bound 40-250 X more ROS than RBC, 30 X more ROS than algae, and 5 X more ROS than bacteria or yeast. Ingestion was more specific than binding; RPE cells ingested 970 X more ROS than RBC, 140 X more ROS than bacteria, and 35 X more ROS than yeast. The phagocytic preference for ROS was maintained in competition experiments with other particle types. Serum was found to be essential for phagocytosis. This study demonstrates that both the binding and ingestion phases of phagocytosis are highly specific processes.     

Release of iron by human retinal pigment epithelial cells.

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, take up iron in association with transferrin by a typical receptor-mediated mechanism (Hunt et al., 1989. J. Cell Sci. 92:655-666). This iron is dissociated from transferrin in a low pH environment and uptake is sensitive to agents that inhibit endosomal acidification. The dissociated iron enters the cytoplasm as a low molecular weight (less than 10 kD) component and subsequently binds to ferritin. No evidence for recycling of iron in association with transferrin was found. Nevertheless, much of the iron that is taken up is recycled to the extracellular medium, primarily from the low molecular weight pool. This release of iron is not sensitive to inhibitors of energy production or of vesicular acidification but is increased up to a maximum of about 40% of the total 55Fe incorporated when cells are incubated with serum or the medium is changed. When a short loading time for 55Fe from 55Fe-transferrin is used (i.e., when the low molecular weight pool is proportionately larger), a much larger fraction of the cell-associated radiolabel is released than when longer loading times are used. The data suggest that a releasable intracellular iron pool is in equilibrium with the externalized material. The released iron may be separated into a high and a low molecular weight component. The former is similar on polyacrylamide gel electrophoresis to ferritin although it cannot be immune precipitated by anti-ferritin antibodies. The low molecular weight 55Fe which is heterogeneous in nature can be bound by external apo-transferrin and may represent a form that can be taken up by cells beyond the blood-retinal barrier.