Abstract
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 β-catenin, which was reduced in the presence of activated TGF-β1. TGF-β1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced β-catenin translocation and GSK-3β phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β 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.
Highlights
Wnt signalling mediates complex cell-cellinteractions during development and proliferation
FR-induced Annexin A8 (AnxA8) loss correlated with decreased expression of the Wnt-related genes Frizzled-1, Frizzled-4 and Wnt2b (Table 1), leading us to hypothesize that AnxA8 may regulate retinal pigment epithelial (RPE) phenotype via modulation of Wnt signaling
Consistent with this, immunofluorescence analysis revealed reduced staining at cell-cell contact sites for both β-catenin and active β-catenin in AnxA8-depleted or FR-treated RPE cells compared to controls
Summary
Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β 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. RPE cells derived from ES or iPS cells exhibit many characteristics of mature fully differentiated RPE cells, and first-in-man transplantation studies in dry and wet age-related macular degeneration (AMD) have yielded encouraging results[7,8,9,10] Key to these clinical advances is a better understanding of the signaling pathways that regulate and maintain RPE cell phenotype. We identify a novel signaling nexus that has implications for strategies aimed at preventing dedifferentiation and at yielding mature RPE cells from ES or iPS cells
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