Adult Retinal Pigment Epithelial Cells Research Articles

Secretion of αB-Crystallin via exosomes: New clues to the function of human retinal pigment epithelium

αB-crystallin (αB) is an archetypical small heat shock protein whose physiological function is not clearly defined. The interest in this protein arises from its well-established but poorly understood association with a myriad of neurodegenerative diseases, cancer and cardiomyopathies. The discovery of the secretion of αB from human adult retinal pigment epithelial cells (ARPE19) via exosomes not only points to the involvement of this protein in lateral transfer of information between cells in the visual system but also to the status of this protein as a potential ligand that may activate or modulate immune and stress responses, normal growth and oncogenic pathways. Retinal pigment epithelium (RPE) is a single layer of polarized cells that supports photoreceptor physiology and function. We have initiated investigations on understanding the origin of the elevated levels of αB in extracellular sub-retinal proteolipid deposits (known as "drusen") associated with the death of photoreceptor neurons in age-related macular degeneration (AMD). Here we discuss the potential implications of the presence and transport of αB in exosomes across cell membranes in RPE.

Human adult retinal pigment epithelial cells as potential cell source for retina recovery

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.

Hypoxia Specific SDF-1 Expression by Retinal Pigment Epithelium Initiates Bone Marrow-derived Cells to Participate in Choroidal Neovascularization in a Laser-induced Mouse Model

Purpose: Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age-related macular degeneration (AMD). Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) plays a critical role in homing of bone marrow-derived cells (BMCs) to choroidal neovascularization (CNV). In this study, we investigated the contribution of hypoxia specific HIF-1α-induced SDF-1 expression in retinal pigment epithelium (RPE) cells and the potential role of SDF-1 in CNV formation.Materials and Methods: Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells of gfp+/+ transgenic mice to sublethally irradiated C57BL/6J mice. CNV was induced by laser photocoagulation. Ocular tissue was processed for immunofluorescence to detect HIF-1α and SDF-1 expression, and cell surface markers such as CXCR4, CD34 and CD31 and so on during CNV formation. In vitro, adult human RPE (hRPE) cells were cultured under conditions of chemical hypoxia using CoCl2 administration. And RNAi technique was used to knock down HIF-1α gene to observe the expression of HIF-1α and SDF-1 in hRPE cells.Results: BMCs trafficked around laser lesion adjacent to RPE layer 4 h after laser photocoagulation, where SDF-1 expression was relatively higher. With increasing expression of SDF-1, more BMCs were infiltrated into laser lesion to participate in CNV, and both reached peak at 3 d (p < 0.05). About 81% BMCs involved in CNV were CXCR4+. Many of them acquired the surface marker of endothelial precursor cells (CD34+) and endothelial cells (CD31+). The constituent ratio of CD34+ and CD31+ BMCs increased with SDF-1 expression. In vitro, we proved that hypoxia specific-HIF-1α influenced SDF-1 expression in hRPE cells.Conclusions: These findings suggested that hypoxia-induced SDF-1 expression in RPE might be a critical initiator for recruitment of BMCs in CNV. SDF-1 might be another important factor in BMCs’ differentiation into endothelial cells to participate in the CNV.

Characterization of Conditioned Media Collected from Cultured Adult versus Fetal Retinal Pigment Epithelial Cells

To characterize trophic factor secretion of cultured adult and fetal retinal pigment epithelial (RPE) cells and to assess the impact on porcine retinal survival in vitro. Conditioned media (CM) were collected from cultured adult and fetal RPE cells and analyzed for trophic factor composition and concentration by multiplex ELISA. Trophic factor receptor occupancy was calculated to evaluate the potential biological effectiveness of the differences in trophic factor concentrations. Retina-preserving activity of the collected CM was evaluated using an in vitro model of degenerating porcine retina. Isobaric tag for relative and absolute quantification (iTRAQ) was used to identify additional proteins with a potential effect on porcine retinal survival. The overall trophic factor secretion profile of cultured fetal RPE cells remained stable over multiple passages and extended culture duration. Compared with CM from adult RPE cells, fetal RPE-CM had significantly higher concentrations of vascular endothelial growth factor-A (VEGF-A), brain-derived neurotrophic factor (BDNF), and pigment epithelium-derived factor (PEDF) and significantly lower concentrations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Fetal RPE-CM was significantly better than adult RPE-CM at improving degenerating porcine retina survival. iTRAQ analysis identified insulin-like growth factor binding protein-3 (IGFBP-3), semaphorin-3B, transforming growth factor (TGF)-β, hepatoma-derived growth factor (HDGF), and gelsolin as factors potentially contributing to this activity. Co-culture of fetal RPE and porcine retina was significantly better than fetal RPE-CM at preserving porcine retinal survival. Adult and fetal RPE cells have significantly different trophic factor secretion profiles, which correlate with differences in their ability to support porcine retina survival. Combined with trophic factor receptor occupancy calculations, these data implicate VEGF-A and PEDF as key RPE-derived factors promoting preservation of retinal structure and function.

Light Filtering in a Retinal Pigment Epithelial Cell Culture Model

We tested for protection of blue light-exposed A2E-containing retinal pigment epithelial (RPE) from damage through the implementation of polycarbonate filters containing varying levels of a pigment that absorbs short wavelength light. Human adult RPE cells (ARPE-19) that had accumulated synthesized A2E were exposed to either a light line delivered from a tungsten halogen source (430 ± 20 nm; 8 mW/cm) or to the entire area of a 35 mm dish (1 mW/cm). Blue-light absorbing polycarbonate filters (2.5 × 4 cm) containing varying levels of short-wavelength light absorbing pigment (1.0, 1.9, 3.8, 7.5, 15, and 35 ppm) or no dye (PC) were placed in the light path. Cytotoxicity was measured by the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric microtiter assay (Roche Diagnostics Corporation, Indianapolis, IN) or by fluorescent staining of non-viable cells. When filters containing blue-light absorbing dye were placed in the light path, protection of 430 nm irradiated A2E-laden RPE was observed. The extent of protection was dependent on the concentration of the dye. By MTT assay and fluorescence labeling, statistically significant differences (p < 0.05) between irradiation in the absence of a filter and irradiation in the presence of a filter were observed. The series of filters tested in this work provided protection against blue light damage in a culture model.

Supercritical carbon dioxide anti-solvent precipitation of anti-oxidative zeaxanthin highly recovered by elution chromatography from Nannochloropsis oculata

Zeaxanthin, a kind of xanthophylls, rich in Marigold flowers and a few micro algae, acts as a strong antioxidant capable of protecting human eye-sight from age-related macular degeneration. This study examined elution chromatography coupled with supercritical anti-solvent precipitation applied for generation of zeaxanthin-rich particulates from micro-algal Nannochloropsis oculata species. Crude of the ultrasonic acetone extract subjected to the column fractionation successfully yields a fraction containing 303.02 mg/g (30%) of zeaxanthin with recovery of 90%. Supercritical anti-solvent precipitation of the algal solution at 200 bar and 313 K was used in producing submicron-sized particulates of 50% zeaxanthin. These extracts were subjected to tests of antioxidant capacities in 2,2-diphenyl-2-picrylhydrazyl hydrate free-radical assay and human adult retinal pigment epithelium-19 cells. This work provided a fast and environmental benign process to generate anti-oxidative capable algal micronized powders from the N. oculata.

Application of continuous supercritical anti-solvents for rapid recrystallization and purification of zeaxanthin dipalmitates from de-glycosides of Lycium barbarum fruits

This study employed column elution fractionation coupled with supercritical anti-solvent recrystallization to isolate the purest zeaxanthin dipalmitates (ZP), one of the eye-protecting compounds in the Lycium barbarum fruits. The amount of ZP increased to 983 mg/g in liquid anti-solvent precipitates with 29.1% recovery and increased to 929 mg/g in supercritical anti-solvent precipitates with 59.6% recovery, respectively from the ultrasonic acetone extract. Experimental data showed that supercritical anti-solvent recrystallization is feasible in producing platelet type precipitates within a few minutes. These ZP compounds exhibit anti-oxidative abilities by proliferating human adult retinal pigment epithelial cells (from 22% to 31%); though it was a meager increase when compared to those of algal 95% zeaxanthin (from 43% to 94%).

αB-Crystallin Is Found in Detergent-resistant Membrane Microdomains and Is Secreted via Exosomes from Human Retinal Pigment Epithelial Cells

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.

523. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to Adult Murine Retinal Pigment Epithelial Cells

523. Non-Viral Phi C31 Integrase Mediated In Vivo Gene Delivery to Adult Murine Retinal Pigment Epithelial Cells

Low Power Laser Irradiation Stimulates the Proliferation of Adult Human Retinal Pigment Epithelial Cells in Culture

We investigated the effects of low power laser irradiation on the proliferation of retinal pigment epithelial (RPE) cells. Adult human RPE cells were artificially pigmented by preincubation with sepia melanin, and exposed to a single sublethal laser pulse (590 nm, 1 µs, <200 mJ/cm2). DNA synthesis, cell number, and growth factor activity in irradiated RPE cells were subsequently monitored. The effect of sublethal laser irradiation on the "wound" healing response of an RPE monolayer in an in vitro scratch assay was also investigated. Single pulsed laser irradiation increased DNA synthesis in pigmented RPE cells measured 6 h post-treatment. In the scratch assay, laser irradiation increased the rates of cell proliferation and wound closure. Conditioned medium, collected 48 h following laser treatment, increased cell proliferation of unirradiated cells. Irradiation increased RPE cell secretion of platelet-derived growth factor (PDGF)-B chain, and increased mRNA levels of several growth factors and their receptors, including PDGF, transforming growth factor-β1, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, as well as heat shock proteins. This demonstrates, for the first time, that low power single pulsed laser irradiation stimulates the proliferation of RPE cells, and upregulates growth factors that are mitogenic for RPE cells.

Celecoxib Inhibits Proliferation of Retinal Pigment Epithelial and Choroid-Retinal Endothelial Cells by a Cyclooxygenase-2-Independent Mechanism

Age-related macular degeneration (ARMD) is a leading cause of blindness. The major reason for severe vision loss in ARMD is choroidal neovascularization due to an elevation in the expression of angiogenic factors such as vascular endothelial growth factor (VEGF). Drugs with anti-VEGF and antiproliferative activities can be beneficial for the treatment of this disorder. We have previously demonstrated that celecoxib [a selective cyclooxygenase (Cox)-2 inhibitor] inhibits VEGF expression in retinal pigment epithelial cells. In this study, we investigated the antiproliferative effects of celecoxib in adult retinal pigment epithelial (ARPE-19) and choroidal endothelial (RF/6A) cells. The results indicate that celecoxib 1) causes a dose-dependent antiproliferative effect in ARPE-19 and RF/6A cells (IC(50) of 23 and 13 microM, respectively); 2) leads to a G(2)-M phase cell cycle arrest in these cell types; and 3) inhibits VEGF-induced proliferation of RF/6A cells (IC(50) of 20 microM). In addition, 4) the concentrations of celecoxib required for antiproliferative effects are lower than those required for the cytotoxicity. These effects of celecoxib are by mechanisms independent of its Cox-2 inhibitory activity because rofecoxib (another Cox-2 inhibitor) had no effects on the proliferation or cell cycle distribution of the two cell types, and flurbiprofen (an inhibitor of Cox-1 and Cox-2) had weak antiproliferative effects on ARPE-19 cells, with IC(50) of 90 microM. In summary, celecoxib has potent antiproliferative effects in RF/6A and ARPE-19 cells; thus, it can be a potential new treatment in proliferative disorders of the choroid-retina such as choroidal neovascularization in age-related macular degeneration.

Culture-induced increase in alpha integrin subunit expression in retinal pigment epithelium is important for improved resurfacing of aged human Bruch's membrane

The purpose of this study was to examine the change in integrin expression in adult human retinal pigment epithelium (RPE) after culturing and to characterize the role of integrins in RPE adhesion to aged submacular human Bruch's membrane. Expression of α integrin subunits 1 through 6 in adult RPE cells, cultured or uncultured, was examined by reverse transcription/real-time polymerase chain reaction (PCR) and Western blotting. RPE was cultured on bovine corneal endothelial cell-secreted extracellular matrix (BCE-ECM). The role of α integrin subunits in RPE attachment was examined by immunofluorescent localization of these subunits at sites of focal adhesions in cultured adult RPE attached to laminin or collagen-I-coated culture dishes. Additionally, the effect of function-blocking antibodies to α integrin subunits on RPE attachment to laminin, collagen I, and aged submacular human Bruch's membrane was determined. Cultured adult RPE had increased expression of α1–5 integrin subunits by PCR compared to uncultured RPE. Western blots showed that α2, 3, and 5 subunit levels were low or absent in uncultured adult RPE. Cultured adult RPE had a substantially higher expression of these integrins. Alpha 1–3 subunits co-localized with phosphorylated focal adhesion kinase (FAK) at focal adhesions in RPE cells spread on laminin. Only α2 and α3 co-localized with phosphorylated FAK in focal adhesions of RPE on collagen I. Using function blocking antibodies, blocking α1 subunit singly or in combination with α2 and/or α3 significantly decreased RPE adhesion to laminin. Blocking α1 and α2 or blocking α1, α2, and α3 subunits significantly decreased RPE adhesion to collagen I. Compared to controls, significantly fewer RPE cells were able to spread on aged submacular human Bruch's membrane when α1–6 integrin subunits were blocked. These results indicate that alpha 1–5 subunits that are upregulated by culturing on BCE-ECM are necessary for RPE attachment to aged submacular human Bruch's membrane. Relative lack of these integrin subunits in uncultured adult RPE may be responsible for poor resurfacing of aged submacular human Bruch's membrane by these cells.

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor-2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan-retinal-neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1-3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK-mediated misexpression of PRN markers in the initial process of transdifferentiation.

Melanosome Maturation Defect in Rab38-deficient Retinal Pigment Epithelium Results in Instability of Immature Melanosomes during Transient Melanogenesis

Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.

Evidence for Notch signaling involvement in retinal regeneration of adult newt

Involvement of Notch signaling in retinal regeneration by transdifferentiation of pigment epithelium cells was investigated using the adult newt Cynops pyrrhogaster. During retinal regeneration, cells expressing Notch-1 first appeared in the regenerating retina one to two cells thick (stage E-3) originated from the retinal pigment epithelium (RPE) cells, and increased in number as the regenerating retina increased in thickness. Notch-1 expression was decreased in the central retina in association with cell differentiation and became restricted to the peripheral retina. Administration of a Notch signaling blocker DAPT resulted in the appearance of a cluster of neurons, earlier than in normal regeneration, along the regenerating retina 1–3 cells thick (stage E-3 to I-1). Immunoblot analysis suggested that DAPT could perturb the processing of Notch-1. Similar results were obtained in the newt embryonic retinal development. These results suggest that the Notch-1 signaling system may be reset to regulate neurogenesis during retinal regeneration. However, PCR analysis revealed that the adult newt RPE cells express Hes-1, neurogenin1 and sometimes Delta-1 Hes-1, neurogenin1 and sometimes Delta-1 all of which are differently regulated in association with retinal regeneration, implying that Notch signaling might also be involved early in the process of transdifferentiation.

Bone morphogenetic protein‐4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

Proteomic Analysis for the Assessment of Different Lots of Fetal Bovine Serum as a Raw Material for Cell Culture. Part IV. Application of Proteomics to the Manufacture of Biological Drugs

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE-19) using three different lots of fetal bovine serum (FBS-Ia, FBS-Ib, and FBS-II). We found that the growth rate of the culture was significantly and consistently higher in the FBS-II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional-related and cell-spreading-related proteins, decreased over time.

Migration and proliferation of retinal pigment epithelium on extracellular matrix ligands

We compared aged adult and fetal retinal pigment epithelium (RPE) migration and proliferation in cell culture. Aged adult RPE resurfaced on dishes coated with human or mouse laminin, human or rat collagen I, human or mouse collagen IV, or human fibronectin. Resurfacing was best on bovine corneal endothelial cell extracellular matrix. Resurfacing was worst on mouse laminin. Fetal RPE resurfacing was robust on all surfaces studied except human and mouse laminin. In general, fetal RPE resurfacing was much greater than that seen with aged RPE on comparable surfaces. The degree of cell proliferation (labeled by Ki-67) was much greater in fetal than adult RPE cells.

Adult retinal pigment epithelium cells express neural progenitor properties and the neuronal precursor protein doublecortin

The adult mammalian retina is devoid of any detectable neurogenesis. However, different cell types have been suggested to potentially act as neural progenitors in the adult mammalian retina in vitro, such as ciliary body (CB), Müller glia, and retinal pigment epithelium (RPE) cells. In rodents and humans, strong evidence for neural stem or progenitor properties exists only for CB-derived cells, but not for other retinal cell types. Here, we provide a comparative analysis of adult rat CB- and RPE-derived cells suggesting that the two cell types share certain neural progenitor properties in vitro. CB and RPE cells expressed neural progenitor markers such as Nestin, Flk-1, Hes1, and Musashi. They proliferated under adherent and neurosphere conditions and showed limited self-renewal. Moreover, they differentiated into neuronal and glial cells based on the expression of differentiation markers such as the young neuronal marker β-III tubulin and the glial and progenitor markers GFAP and NG2. Expression of β-III tubulin was found in cells with neuronal and non-neuronal morphology. A subpopulation of RPE- and CB-derived progenitor cells expressed the neurogenesis-specific protein doublecortin (DCX). Interestingly, DCX expression defined a β-III tubulin-positive CB and RPE fraction with a distinct neuronal morphology. In summary, the data suggest that RPE cells share with CB cells the potential to de-differentiate into a cell type with neural progenitor-like identity. In addition, DCX expression might define the neuronal-differentiating RPE- and CB-derived progenitor population.

Plasma membrane localization and function of TRPC1 is dependent on its interaction with β-tubulin in retinal epithelium cells

Mammalian homologues of the Drosophila canonical Transient Receptor Potential (TRPC) protein have been proposed to encode the store-operated Ca2+ influx (SOC) channel(s). This study examines the role of TRPC1 in the SOC mechanism of retinal cells. htrpc1 transcript was detected in bovine retinal and in human adult retinal pigment epithelial (ARPE) cells. Western blot analysis also confirmed the expression of TRPC1 protein in neuronal cells including retina and ARPE cells. To determine the role of TRPC1 protein in retinal cells, TRPC1 was recombinantly expressed in ARPE cells and changes in intracellular Ca2+ were analyzed. ARPE cells stably transfected with htrp1 cDNA displayed 2-fold higher Ca2+ influx with no significant increase in the basal influx. Consistent with this the overexpressed TRPC1 protein was localized in the plasma membrane region of ARPE cells. Interestingly, both bovine retinal tissues and ARPE cells showed that TRPC1 protein co-localizes and could be co-immunoprecipitated with beta-tubulin. Disruption of tubulin by colchicine significantly decreased both plasma membrane staining of the TRPC1 protein and Ca2+ influx in ARPE cells. These results suggest that TRPC1 channel protein is expressed in retinal cells, further, targeting/retention of the TRPC1 protein to the plasma membrane in retinal cells is mediated via its interaction with beta-tubulin.