Expression and differential polarization of the reduced-folate transporter-1 and the folate receptor alpha in mammalian retinal pigment epithelium

The differential polarized distribution of the reduced- folate transporter (RFT-1) and folate receptor alpha (FRalpha), the two proteins involved in the transport of folate, has been characterized in normal mouse retinal pigment epithelium (RPE) and in cultured human RPE cells. RPE cells mediate the vectorial transfer of nutrients from choroidal blood to neural retina. Whereas FRalpha is known to be present in many cell types of the neural retina, in situ hybridization analysis in the present study demonstrated that RFT-1 is present only in RPE. Laser-scanning confocal microscopy using antibodies specific for RFT-1 demonstrated an apical distribution of this protein in cultured human and intact mouse RPE, which contrasts with the basolateral distribution of FRalpha in these cells. The expression of RFT-1 in the RPE cell apical membrane was confirmed by functional studies with purified apical membrane vesicles from bovine RPE. These studies, done with N(5)-methyltetrahydrofolate (the predominant folate derivative in blood) and folate as substrates, have shown that RFT-1 functions in a Na(+)- and C1(-)-independent manner. The transporter is specific for folate and its analogs. A transmembrane H(+) gradient influences the transport function of this protein markedly; the transport mechanism is likely to be either folate/H(+) co-transport or folate/OH(-) exchange. Based on the differential polarization of FRalpha and RFT-1 in RPE, we suggest that these two proteins work in a concerted manner to bring about the vectorial transfer of folate across the RPE cell layer from the choroidal blood to the neural retina. This constitutes the first report of the differential polarization of the two folate transport proteins in any polarized epithelium.     

The glucose transport in retinal pigment epithelium is via passive facilitated diffusion

The glucose transport across the bovine retinal pigment epithelium (RPE) was studied in a modified Ussing chamber. Unidirectional fluxes were recorded with radioactive tracers L-[14C]-glucose (LG) and 3-O-methyl-D-[3H]-glucose (MDG). There was no significant difference between the unidirectional MDG fluxes (retina to choroid, and choroid to retina directions) with or without ouabain. The effects of two glucose transporter inhibitors, phloretin and cytochalasin B, on the glucose fluxes from choroid to retina cells were also investigated. The MDG flux was found to be inhibited by 45.5% by phloretin (10(-4) M) and 87.4% by cytochalasin B (10(-4) M). These inhibitory characteristics resembled the facilitated diffusion mode of glucose transport. The glucose transporter protein in the plasma membrane of RPE was located by means of photolabeling [3H]-cytochalasin B. The labeled plasma membrane enriched fraction was analysed by SDS-PAGE. The glucose transporter of bovine RPE was found to have a molecular weight range of 46-53 kDa. The molecular weight range of this transporter protein agreed with those of facilitated glucose transporters in other tissues indicating a molecular similarity between them. The results indicated that the glucose transport across the RPE is via passive facilitated diffusion.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

Retinyl palmitate hydrolase activity in the bovine retina

Retinyl palmitate hydrolase (RPH) activity of bovine tissues was estimated from retinol formation following incubation of tissue homogenates with all-trans retinyl palmitate. The quantity of retinol produced in the incubation mixture was analyzed by high-performance liquid chromatography. RPH activities of retinal pigment epithelium (RPE), liver, retina, muscle and brain were 194.2, 138.0, 72.5, 25.0 and 5.1 units/gm protein respectively. The RPH activity in the retina was far above that attributable solely to RPE contaminations. The presence of RPH in the retina suggests that retina can utilize retinyl esters for the formation of visual pigments and/or cellular metabolism.     

Localization of the gene for interphotoreceptor retinoid-binding protein to mouse chromosome 14 near Np-1

Interphotoreceptor retinoid-binding protein (IRBP) is a large glycoprotein known to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the retinal pigment epithelium and the photoreceptor cells. It is thought to transport retinoids between the retinal pigment epithelium and the photoreceptors, a critical role in the visual process. We have used a 900-bp bovine IRBP cDNA fragment to map the corresponding gene, Rbp-3, to mouse chromosome 14 with somatic cell hybrids and have positioned the gene near Np-1 (nucleoside phosphorylase-1) by analysis of the progeny of an intersubspecific backcross. In the human genome, NP maps to human chromosome 14 and RBP3 to human chromosome 10. Thus, these two genes span the putative site of a chromosomal translocation which contributed to divergent karyotype evolution of man and mouse.     

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3β,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3β-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.     

Localization of the gene for interphotoreceptor retinoid-binding protein to mouse chromosome 14 near Np-1

Interphotoreceptor retinoid-binding protein (IRBP) is a large glycoprotein known to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the retinal pigment epithelium and the photoreceptor cells. It is thought to transport retinoids between the retinal pigment epithelium and the photoreceptors, a critical role in the visual process. We have used a 900-bp bovine IRBP cDNA fragment to map the corresponding gene, Rbp-3, to mouse chromosome 14 with somatic cell hybrids and have positioned the gene near Np-1 (nucleoside phosphorylase-1) by analysis of the progeny of an intersubspecific backcross. In the human genome, NP maps to human chromosome 14 and RBP3 to human chromosome 10. Thus, these two genes span the putative site of a chromosomal translocation which contributed to divergent karyotype evolution of man and mouse.     

Heme carrier protein 1 transports heme and is involved in heme-Fe metabolism

Heme-Fe is an important source of dietary iron in humans; however, the mechanism for heme-Fe uptake by enterocytes is poorly understood. Heme carrier protein 1 (HCP1) was originally identified as mediating heme-Fe transport although it later emerged that it was a folate transporter. We asked what happened to heme-Fe and folate uptake and the relative abundance of hcp1 and ho1 mRNA in Caco-2 cells after knockdown by transfection with HCP1-directed short hairpin (sh)RNA. Control Caco-2 cells were cultured in bicameral chambers with 0-80 μM heme-Fe for selected times. Intracellular Fe and heme concentration increased in Caco-2 cells reflecting higher external heme-Fe concentrations. Maximum Fe, heme, and heme oxygenase 1 (HO1) expression and activity were observed between 12 and 24 h of incubation. Quantitative RT-PCR for hcp1 revealed that its mRNA decreased at 20 μM heme-Fe while ho1 mRNA and activity increased. When shRNA knocked down hcp1 mRNA, heme-(55)Fe uptake and [(3)H]folate transport mirrored the mRNA decrease, ho1 mRNA increased, and flvcr mRNA was unchanged. These data argue that HCP1 is involved in low-affinity heme-Fe uptake not just in folate transport.     

Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

Background

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods

We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results

We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier.

Conclusion

These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

Haem carrier protein 1 (HCP1): Expression and functional studies in cultured cells

Haem released from digestion and breakdown of meat products provides an important source of dietary iron, which is readily absorbed in the proximal intestine. The recent cloning and characterization of a haem carrier protein 1 (HCP 1) has provided a candidate intestinal haem transporter. The current studies describe the expression and functional analysis of HCP1 in cultured Caco-2 cells, a commonly used model of human intestinal cells. HCP1 mRNA expression in other cell types was also studied. The uptake of ⁵⁵Fe labeled haem was determined in cells under different experimental conditions and HCP1 expression was measured by RT-PCR and immunohistochemistry. mRNA and protein expressions increased in Caco-2 cells transduced with HCP1 adenoviral plasmid, and consequently ⁵⁵Fe haem uptake was higher in these cells. Haem uptake was also increased in fully differentiated Caco-2 cells compared to undifferentiated cells. Preincubation of cells with desferrioxamine (DFO, to deplete cells of iron) had no effect on HCP1 expression or haem uptake. Treatment with CdCl₂ (to induce haem oxygenase, HO-1) enhanced HCP1 expression and increased haem uptake into the cells. HCP1 expression and function were found to be adaptive to the rate of haem degradation by HO-1. Furthermore, HCP1 expression in different cells implies a functional role in tissues other than the duodenum.     

Study on RNA synthesis in the retina and retinal pigment epithelium of mice by light microscopic radioautography.

With the aim of determining the distribution of the incorporation of 3H-uridine in both retina and retinal pigment epithelium (RPE), the mouse eyes at embryonic day 9.5 (E 9.5), E 12.5, E 14.5, E 16.5, E 18.5 of gestational ages, and postnatal day 1 (P 1), P 3, P 7, P 14 were analyzed by light microscopic radioautography. Small pieces of the ocular tissues were labelled with 3H-uridine in vitro and light microscopic radioautographs were prepared. The average grain numbers per cell of the respective regions of tissues were calculated. In the retina, the grain numbers increased gradually from E 9.5 to P 1 and reached the maximal value at P 1, and then decreased until P 14. However, the grain numbers were more in the vitreal portion than those in the scleral portion at E 16.5 and then became more in the scleral portion from E 18.5 to P 14. It is considered that the ganglion and bipolar cells finish the RNA synthesis earlier, while the photoreceptor cells do it later during the fetal and postnatal development. In the RPE, the grain numbers gradually increased from E 12.5 to P 7 and then decreased until P 14. Considering the same ages, the grain numbers increased in the following order, anterior, equatorial and posterior regions during embryonic stages, but decreased in the same order after birth. Therefore, it is suggested that the activity of RNA synthesis in PE cells is higher in the posterior region than in the anterior region during embryonic stages. But the activity ascends generally and becomes relatively higher in the anterior region, after birth. Comparing the retina and RPE, it was noted that the grain numbers in the RPE were more important than in the retina and that the maximal value was at P 1 in the retina, while it was at P 7 in the RPE. From these results, it can be concluded that the RNA synthesis ceases earlier in the retina than in the RPE.     

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells.In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises.The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.     

Microsomal glutathione S-transferase 1 in the retinal pigment epithelium: protection against oxidative stress and a potential role in aging

High oxygen tension, exposure to light, and the biochemical events of vision generate significant oxidative stress in the retina and the retinal pigment epithelium (RPE). Understanding the mechanisms and basis of susceptibility to progressive retinal diseases involving oxidative damage such as age-related macular degeneration (AMD) remains a major challenge. Here microsomal glutathione S-transferase (MGST1) is shown to be a dominant, highly expressed enzyme in bovine and mouse RPE microsomes that displays significant reduction activity toward synthetic peroxides, oxidized RPE lipids, and oxidized retinoids. This enzymatic reduction activity (GPx) can be partially neutralized with a monoclonal anti-MGST1 antibody developed in this study. MGST1-transfected HEK293 cells exhibited greater viability (70 +/- 4% survival) compared with untransfected control cells (46 +/- 4% survival) when challenged with 20 microM H(2)O(2), and greater viability of MGST1-transfected cells following challenge with oxidized docosahexaenoic acid was also observed. Cultured ARPE19 cells transfected with silencing MGST1 siRNAs exhibited lower expression of MGST1 (12% and 26% of the controls) and significantly lower GPx activity (44 +/- 13%) and, thus, were more susceptible to oxidative damage. Immunoblotting revealed that the in vivo expression of MGST1 in mouse RPE decreases 3-4-fold with age, to trace levels in 18-month-old mice. GPx activity in the RPE was also found to be reduced in 12-month-old mice to approximately 67%. These results support an important protective function for MGST1 against oxidative insult in the RPE that decreases with age and suggest that this enzyme may play a role in the development of age-related diseases such as AMD.