Comparative Proteome Analysis of Native Differentiated and Cultured Dedifferentiated Human RPE Cells

Alge, Claudia S.; Suppmann, Sabine; Priglinger, Siegfried; Neubauer, Aljoscha S.; May, Christian Albrecht; Hauck, Stefanie M.; Welge–Lüßen, Ulrich; Ueffing, Marius

doi:10.1167/iovs.02-1225

Cited by 92 publications

(93 citation statements)

References 54 publications

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“…RPE cells are among the most phagocytically active of all cells in the body and several members of the annexin family have been identified in mature human pigment epithelium (Alge et al, 2003;West et al, 2003). However, in a subtractive PCR analysis only annexins A2 and A4 were up-regulated when ARPE19 cells were allowed to differentiate on porcine lens capsule, used here as a surrogate for Bruch's membrane (Turowski et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

Law

Hajjar

et al. 2009

MBoC

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The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane-cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2 ؊/؊ mice the phagocytosis of outer segments was impaired, and in ANX A2 ؊/؊ mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2 ؊/؊ mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells. INTRODUCTIONThe retinal pigment epithelium (RPE) performs a range of functions that directly support the retina (Strauss, 2005), including the daily phagocytosis and digestion of shed photoreceptor outer segments (POS). The importance of this activity is exemplified in the dystrophic Royal College of Surgeons rat, which due to a failure in phagocytosis undergoes a progressive and rapid retinal degeneration characterized by accumulation of cellular debris and photoreceptor cell death. The gene responsible for this pathology was identified by positional cloning as the Mer tyrosine kinase (MerTK), a key signaling intermediate that is expressed in RPE cells and plays an essential role in the internalization of bound POS (Chaitin and Hall, 1983;D'Cruz et al., 2000). Recent studies have provided insight into other RPE cell proteins that have distinct roles either in the binding or the internalization of shed POS. For example, the apically expressed ␣v␤5 integrin is required for efficient binding of POS, and RPE cells from ␤5 Ϫ/Ϫ mice show markedly reduced binding of POS in vitro (Nandrot et al., 2004). Intriguingly, engagement of the ␣v␤5 integrin by its cognate ligand, milk fat globule-EGF8 (MFG-E8), is essential for the circadian synchronization of phagocytosis (Nandrot and Finnemann, 2006;Nandrot et al., 2007), and although phagocytosis of POS occurs in MFG-E8 Ϫ/Ϫ and ␤5 Ϫ/Ϫ mice, in both mutants the daily rhythm is absent. In contrast, focal adhesion kinase (FAK) and MerTK are not required for the initial binding of POS to the apical surface of the RPE, but they are sequentially activated by ␣v␤5 receptors and are essential for phagosome internalization (Feng et al., 2002;Finnemann, 2003).Between the pigment epithelium and neuroretina RPE cells extend apical processes into the interphotorecept...

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Section: Discussionmentioning

confidence: 99%

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

Law

Hajjar

et al. 2009

MBoC

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“…A challenge in these studies was that the available RPE cell lines generally do not maintain their original differentiated state as indicated by cell morphology, gene expression patterns, and physiological properties (66,67). To find host cells suitable for transfection analysis, we screened nine human cell cultures for expression of endogenous VMD2 and chose three RPE and a neuroepithelioma cell lines.…”

Section: Fig 4 Effect Of Mutation In E-box Elements On Vmd2 Promotementioning

confidence: 99%

Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation

Esumi

Oshima

et al. 2004

Journal of Biological Chemistry

124

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The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5-upstream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacZ constructs demonstrated that a ؊253 to ؉38 bp fragment is sufficient to direct RPEspecific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, ؊585 to ؊541 bp for high level expression and ؊56 to ؊42 bp for low level expression. Mutation of a canonical E-box located in the ؊56 to ؊42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression. The retinal pigment epithelium (RPE)1 is a monolayer of cuboidal cells located between the photoreceptors and choroid of the eye. It has many specialized functions that support and nourish photoreceptors, including important roles in retinoid metabolism (visual cycle), phagocytosis of shed photoreceptor outer segments, maintenance of the blood-retina barrier, movement of ions and water, and synthesis and transport of substances that constitute the interphotoreceptor matrix (1). The importance of the RPE in maintaining retinal photoreceptors is highlighted by the Royal College of Surgeons rat that exhibits a markedly reduced capacity for phagocytosis of outer segments by the RPE that results in the degeneration and loss of photoreceptor cells (2, 3). In addition, in humans, RPE dysfunction has been implicated in the pathogenesis of age-related macular degeneration, which is the leading cause of irreversible blindness in elderly people in western countries (4, 5).Mutations in several genes that are specifically or preferentially expressed in the RPE, such as RPE65, RLBP1, RGR, TIMP3, and VMD2, are associated with inherited human retinal dystrophies (6 -15). Mutations in VMD2 result in Best disease (vitelliform macular dystrophy (VMD)), an autosomal dominant, juvenile onset macular dystrophy characterized by a striking accumulation of lipofuscin-like material within and beneath the RPE (16 -18). VMD2 encodes a multispan transmembrane protein, bestrophin, tha...

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“…However, after repeated passage it has been established that these adult human cell lines can dedifferentiate and lose their morphology and RPE gene expression, and express neuronal cell markers instead. 59,60 Figure 2 (a-c): Schematic diagrams of (a) human eye (b) mouse eye and (c) rabbit eye (shown to scale). Retinal surgery in the human eye is facilitated by the large vitreous cavity.…”

Section: Eyementioning

confidence: 99%

Retinal pigment epithelium transplantation: concepts, challenges, and future prospects

Alexander

Thomson

Luff

et al. 2015

Eye

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The retinal pigment epithelium (RPE) is a single layer of cells that supports the light-sensitive photoreceptor cells that are essential for retinal function. Age-related macular degeneration (AMD) is a leading cause of visual impairment, and the primary pathogenic mechanism is thought to arise in the RPE layer. RPE cell structure and function are well understood, the cells are readily sustainable in laboratory culture and, unlike other cell types within the retina, RPE cells do not require synaptic connections to perform their role. These factors, together with the relative ease of outer retinal imaging, make RPE cells an attractive target for cell transplantation compared with other cell types in the retina or central nervous system. Seminal experiments in rats with an inherited RPE dystrophy have demonstrated that RPE transplantation can prevent photoreceptor loss and maintain visual function. This review provides an update on the progress made so far on RPE transplantation in human eyes, outlines potential sources of donor cells, and describes the technical and surgical challenges faced by the transplanting surgeon. Recent advances in the understanding of pluripotent stem cells, combined with novel surgical instrumentation, hold considerable promise, and support the concept of RPE transplantation as a regenerative strategy in AMD.

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Comparative Proteome Analysis of Native Differentiated and Cultured Dedifferentiated Human RPE Cells

Cited by 92 publications

References 54 publications

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

Analysis of the VMD2 Promoter and Implication of E-box Binding Factors in Its Regulation

Retinal pigment epithelium transplantation: concepts, challenges, and future prospects

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