Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.     

CLCA protein and chloride transport in canine retinal pigment epithelium

Problems in ion and fluid transfer across the retinal pigment epithelium (RPE) are a probable cause of inappropriate accumulations of fluid between the photoreceptors of the retina and the RPE. The activities of Cl- transporters involved in basal fluid transfer across the RPE have been compared to determine whether Ca2+- or cAMP-dependent channels may be responsible for basal housekeeping levels of secretory activity in this tissue. The role of a candidate Ca2+-dependent CLCA protein in the basal RPE transport of Cl- has been investigated. Low concentrations of the Cl- conductance inhibitors glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoate reduced the short-circuit current in dog RPE preparations mounted in Ussing chambers and decreased the Ca2+-dependent Cl- efflux from fibroblasts expressing the pCLCA1 Cl- conductance regulator. However, these same agents did not inhibit the rate of Cl- release from cultured fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive Cl- channel. Addition of ionomycin to primary cultures of canine RPE cells or to fibroblasts expressing the pCLCA1 channel regulator increased the rate of release of Cl- from both types of cultured cells. However, the presence of pCLCA1 also increased cAMP-dependent Cl- release from fibroblasts expressing CFTR. We conclude that Ca2+-dependent Cl- transport may be more important than cAMP-dependent Cl- transport for normal fluid secretion across the RPE. Furthermore, CLCA proteins expressed in the RPE appear to regulate the activity of other Cl- transporters, rather than functioning as primary ion transport proteins.     

Protein database,human retinal pigment epithelium

The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome.     

Endocytosis in the rat retinal pigment epithelium

Endocytosis in the retinal pigment epithelium (RPE) of rats was studied using horseradish peroxidase, microperoxidase and ferritin tracers. Tracer uptake was mediated by coated pits and coated vesicles. Coated pits formed at two discrete regions at the RPE plasma membrane: that portion of basal membrane directly opposing Bruch's membrane, and at the bases of the apical lamellae and villi. Two populations of coated vesicles were identified and distinguished by size, location and function. Large coated vesicles (91.8 +/- 14.7 nm in diameter) were located near the cell surface and incorporated tracer. Small coated vesicles (64.5 +/- 15.7 nm diameter) located more deeply within the cell were not tracer-labeled, and were often fused with the endoplasmic reticulum or the Golgi apparatus. Observations of the endocytic pathway in rat RPE cells are presented. Tracer was also found in organelles of the lysosomal system, e.g. the multivesicular body, but was not identified in the smooth endoplasmic reticulum or Golgi apparatus.     

Characterization of a plasma retinol-binding protein membrane receptor expressed in the retinal pigment epithelium.

A specific membrane receptor for plasma retinol-binding protein (RBP) is expressed in the retinal pigment epithelium (RPE). When chemically cross-linking RBP to RPE membranes, an 86-kDa RBP.RBP receptor complex is formed, and a 63-kDa protein was identified as the RBP-binding membrane protein (B?vik, C.-O., Eriksson, U., Allen, R., and Peterson, P. (1991) J. Biol. Chem. 266, 14978-14985). To explore in more detail the characteristics of this membrane receptor, we have generated a monoclonal antibody, A52, to the 63-kDa protein (p63). A52 binds the 86-kDa RBP.RBP receptor complex and p63. Several lines of evidence suggest that p63 is not a regular integral membrane protein, and it occurs in different forms. One form is firmly attached to membranes, is part of a high molecular weight complex, and is able to bind RBP. The other form of p63 can be removed from membranes by treatment with an alkaline buffer and is unable to bind RBP. Both forms of p63 contain extensive hydrophobic domains and are found in the detergent phase upon extraction with Triton X-114. The expression of p63 is restricted to RPE, and immunohistochemical localization of tissue sections from bovine retina showed highest expression in the basolateral portion of RPE cells. Immunofluorescence localization, using isolated RPE cells, showed that p63 is exposed on the cell surface of newly isolated RPE cells.     

The retinal pigment epithelium as a gateway for monocyte trafficking into the eye

The choroid plexus epithelium within the brain ventricles orchestrates blood‐derived monocyte entry to the central nervous system under injurious conditions, including when the primary injury site is remote from the brain. Here, we hypothesized that the retinal pigment epithelium (RPE) serves a parallel role, as a gateway for monocyte trafficking to the retina following direct or remote injury. We found elevated expression of genes encoding leukocyte trafficking determinants in mouse RPE as a consequence of retinal glutamate intoxication or optic nerve crush (ONC). Blocking VCAM‐1 after ONC interfered with monocyte infiltration into the retina and resulted in a local pro‐inflammatory cytokine bias. Live imaging of the injured eye showed monocyte accumulation first in the RPE, and subsequently in the retina, and peripheral leukocytes formed close contact with the RPE. Our findings further implied that the ocular milieu can confer monocytes a phenotype advantageous for neuroprotection. These results suggest that the eye utilizes a mechanism of crosstalk with the immune system similar to that of the brain, whereby epithelial barriers serve as gateways for leukocyte entry.     

Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.     

Neurotransmitter-related features of the retinal pigment epithelium

Various neurotransmitter-related biochemical features of the separated pigment epithelium and neural retina of the cow have been examined. The pigment epithelium contains high affinity binding sites for several pharmacological agents thought to attach to neurotransmitter receptor sites with a high degree of specificity. Thus, serotonergic, adrenergic and opiate receptors appear to be present in the pigment epithelium. Serotonin has also been detected in this region.Several neuropeptides were found in the pigment epithelium. Relatively large amounts of neurotensin and met-enkephalin were present, but substance P was not detected.     

Retinyl ester hydrolases in retinal pigment epithelium

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.     

Light-induced phosphorylation of crystallins in the retinal pigment epithelium

Protein phosphorylations have essential regulatory roles in visual signaling. Previously, we found that phosphorylation of several proteins in the retina and retinal pigment epithelium (RPE) is involved in anti-apoptotic signaling under oxidative stress conditions, including light exposure. In this study, we used a phosphoprotein enrichment strategy to evaluate the light-induced phosphoproteome of primary bovine RPE cells. Phosphoprotein-enriched extracts from bovine RPE cells exposed to light or dark conditions for 1h were separated by 2D SDS-PAGE. Serine and tyrosine phosphorylations were visualized by 2D phospho Western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. Light induced a marked increase in tyrosine phosphorylation of beta crystallin A3 and A4. The most abundant light-induced up-regulated phosphoproteins were crystallins of 15-25 kDa, including beta crystallin S and zeta crystallin. Phosphorylation of beta crystallin suggests an anti-apoptotic chaperone function of crystallins in the RPE. Other chaperones, cytoskeletal proteins, and proteins involved in energy balance were expressed at higher levels in the dark. A detailed analysis of RPE phosphoproteins provides a molecular basis for understanding of light-induced signal transduction and anti-apoptosis mechanisms. Our data indicates that phosphorylation of crystallins likely represents an important mechanism for RPE shielding from physiological and pathophysiological light-induced oxidative injury.     

Characterization of the retinal pigment epithelium in Friedreich ataxia

We assessed structural elements of the retina in individuals with Friedreich ataxia (FRDA) and in mouse models of FRDA, as well as functions of the retinal pigment epithelium (RPE) in FRDA using induced pluripotent stem cells (iPSCs). We analyzed the retina of the FRDA mouse models YG22R and YG8R containing a human FRATAXIN (FXN) transgene by histology. We complemented this work with post-mortem evaluation of eyes from FRDA patients. Finally, we derived RPE cells from patient FRDA-iPSCs to assess oxidative phosphorylation (OXPHOS) and phagocytosis. We showed that whilst the YG22R and YG8R mouse models display elements of retinal degeneration, they do not recapitulate the loss of retinal ganglion cells (RGCs) found in the human disease. Further, RPE cells differentiated from human FRDA-iPSCs showed normal OXPHOS and we did not observe functional impairment of the RPE in Humans.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

Culture of rat retinal pigment epithelium.

A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been culured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be usefull in studying the defect(s) associated with this mutation.     

The retinal pigment epithelium of Cr-deficient rats

The purpose of this study is to investigate the effect of Cr deficiency on the rat retina. Three-week-old Wistar Kyoto rats were divided into 2 groups. Cr-deficient rats were fed AIN-93G diet without Cr and deionized distilled water. Control rats were fed AIN-93G diet and deionized distilled water. The Cr and sugar concentrations in the whole blood and cholesterol concentration in the serum were measured. We observed the retina with an electron microscope, and counted phagocytized lamellar structures in the retinal pigment epithelium (RPE) before and after the start of light exposure on negative electron microscopic films. The whole blood Cr level of Cr-deficient rats was less than 0.2 microg/l. The blood sugar level of Cr-deficient rats was significantly higher than that of normal rats (p < 0.05). There were significantly more phagocytized lamellar structures in the RPE of Cr-deficient rats 1, 2, 7, 11 and 12 h after the start of light exposure than in that of normal rats (p < 0.05). However, no morphological abnormalities were found in the photoreceptor cells of Cr-deficient rats. Phagocytosis in the photoreceptor outer segment discs in the RPE was accelerated, but the pattern of the retinal circadian rhythm with maximum phagocytosis 2 h after exposure to light was unchanged. The Cr-deficient state may cause the membrane to degenerate, and phagocytosis of the photoreceptor outer segment discs in the RPE may be accelerated. This study provided an evidence of the nutritional importance of Cr in rat retina.     

Potassium transport across the frog retinal pigment epithelium

Summary Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic NaK pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K] _o ([Rb] _o ). The net rate of retina-to-choroid⁴²K(⁸⁶Rb) transport increased monotonically as [K] _o ([Rb] _o ), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K] _o ([Rb] _o ) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K] _o . The net K transport was completely inhibited by 10^–4 m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable⁴²K(⁸⁶Rb) flux increased with [K] _o ([Rb] _o ). These results show that the apical membrane NaK pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K] _o changes that modulate potassium transport coincide with the light-induced [K] _o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

Potassium transport across the frog retinal pigment epithelium

Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic Na:K pumps. In the present experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K]0 ( [Rb]0). The net rate of retina-to-choroid 42K(86Rb) transport increased monotonically as [K]0 ( [Rb]0) increased from approximately 0.2 to 5 mM on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K]0 ( [Rb]0) was elevated to 10 mM. Net sodium transport was also stimulated by elevating [K]0. The net K transport was completely inhibited by 10-4 M ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had no effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable 42K(86Rb) flux increased with [K]0 ( [Rb]0). These results show that the apical membrane Na:K pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K]0 changes that modulate potassium transport coincide with the light-induced [K]0 changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

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.