Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells.

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and ß-catenin, which was reduced in the presence of activated TGF-ß1. TGF-ß1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced ß-catenin translocation and GSK-3ß phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active ß-catenin and GSK-3ß phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8.

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

Complement Stimulates Retinal Pigment Epithelial Cells to Undergo Pro-Inflammatory Changes.

BACKGROUND/AIMS: We examined the effect of human complement sera (HCS) on retinal pigment epithelial (RPE) cells with respect to pro-inflammatory mediators relevant in early age-related macular degeneration (AMD). METHODS: RPE cells were treated with complement-containing HCS or with heat-inactivated (HI) HCS or C7-deficient HCS as controls. Cells were analysed for C5b-9 using immunocytochemistry and flow cytometry. Interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were quantified by ELISA and RT-PCR. Tumour necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were analysed by Western blotting. The intracellular distribution of nuclear factor (NF)-x03BA;B was investigated by immunofluorescence. RESULTS: A concentration-dependent increased staining for C5b-9 but no influence on cell viability was observed after HCS treatment. ELISA and RT-PCR analysis revealed elevated secretion and expression of IL-6, IL-8, and MCP-1. Western blot analysis showed a concentration-dependent increase in ICAM-1, VCAM-1, and TNF-α in response to HCS, and immunofluorescence staining revealed nuclear translocation of NF-x03BA;B. CONCLUSION: This study suggests that complement stimulates NF-x03BA;B activation in RPE cells that might further create a pro-inflammatory environment. All these factors together may support early AMD development.

Retinal Pigment Epithelial Cells Mitigate the Effects of Complement Attack by Endocytosis of C5b-9.

Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration. The alternative pathway of complement activation is strongly implicated in RPE cell dysfunction and loss in age-related macular degeneration; therefore, it is critical that RPE cells use molecular strategies to mitigate the potentially harmful effects of complement attack. We show that the terminal complement complex C5b-9 assembles rapidly on the basal surface of cultured primary porcine RPE cells but disappears over 48 h without any discernable adverse effects on the cells. However, in the presence of the dynamin inhibitor dynasore, C5b-9 was almost completely retained at the cell surface, suggesting that, under normal circumstances, it is eliminated via the endocytic pathway. In support of this idea, we observed that C5b-9 colocalizes with the early endosome marker EEA1 and that, in the presence of protease inhibitors, it can be detected in lysosomes. Preventing the endocytosis of C5b-9 by RPE cells led to structural defects in mitochondrial morphology consistent with cell stress. We conclude that RPE cells use the endocytic pathway to prevent the accumulation of C5b-9 on the cell surface and that processing and destruction of C5b-9 by this route are essential for RPE cell survival.

Complement and UV-irradiated photoreceptor outer segments increase the cytokine secretion by retinal pigment epithelial cells.

PURPOSE: Age-related macular degeneration (AMD) is accompanied by increased complement activation, and by lipofuscin accumulation in retinal pigment epithelial (RPE) cells due to incomplete degradation of photoreceptor outer segments (POS). The influence of POS, ultraviolet (UV)-irradiated POS and human complement sera (HCS) on cytokine secretion from RPE cells was therefore examined. METHODS: RPE cells were incubated with POS or UV-POS every other day for 1 week. The autofluorescence (AF) was measured photometrically and by flow cytometry. Senescence-associated genes were analyzed by RT-PCR. Internalization and degradation of POS were determined using phagocytosis and degradation assays, and lysosomal function by neutral red uptake. RPE cells in polycarbonate cell culture inserts were incubated apically with POS or UV-POS and afterward basally with HCS. C7-deficient HCS was used as control. The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TER) and the permeability. Interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor were quantified by ELISA. RESULTS: POS treatment led to an increased AF and senescence marker expression, which were further elevated in response to UV-POS. UV-POS were preferentially accumulated over POS and the lysosomal function was impaired due to UV-POS. HCS intensified the cytokine production compared with controls. POS had no effect, though UV-POS combined with HCS induced a significant increase in all cytokines. CONCLUSIONS: RPE cultivation with UV-POS might serve as a model to investigate the accumulation of lipofuscin-like structures. The enhanced cytokine secretion due to UV-POS with HCS may account for an increased susceptibility for lipofuscin-loaded cells to complement, inducing a proinflammatory environment as observed in AMD.

Increased vitronectin production by complement-stimulated human retinal pigment epithelial cells.

PURPOSE: A variation in the complement factor H gene was associated with an enhanced risk to develop especially early age-related macular degeneration. Drusen and basal laminar deposits are hallmarks of this AMD manifestation that contain vitronectin as a major component. In this study, the correlation between complement stimulation and vitronectin production of retinal pigment epithelial (RPE) cells was investigated. METHODS: ARPE-19 cells, a permanent cell line of human RPE cells, were supplemented with and without human complement competent serum in medium with and without heat inactivated fetal calf serum. The cells were examined in situ for their vitronectin production as an effective inhibitor of alternatively activated complement by immunohistochemistry. Semi-quantitative RT-PCR and Western blots were performed to analyze vitronectin mRNA and protein. RESULTS: A strong immunohistochemical staining for vitronectin was observed after complement supplementation. The enhanced production of this complement inactivator by ARPE-19 cells was confirmed by Western blot, whereas the expression analysis revealed unaltered mRNA amounts. CONCLUSIONS: A stimulation of RPE cells with complement resulted in an upregulated production of vitronectin. This may support the concept of a protective mechanism, since vitronectin is the major inhibitor of complement activated by the alternative pathway. On the other hand, this increased vitronectin production after complement stimulation may contribute to focal or diffuse deposits in Bruch's membrane, as observed in early AMD.