Lueck, K; Carr, A; Greenwood, J; Moss, S; (2014) Regulation of epithelial cell phenotype by annexin A8 and Wnt signalling. Presented at: 13th International Meeting of the European Calcium Society Centre De Congrès, Aix aux Provence, France.

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Abstract

The retinoic acid derivative Fenretinide (FR) is capable of trans-differentiating retinal pigment epithelial (RPE) cells towards a neuron-like phenotype in culture. Microarray analysis of FR-treated ARPE-19 cells revealed down-regulation of Annexin (Anx)A8 and specific Wnt signalling proteins in transdifferentiated cells. AnxA8, a calcium-dependent phospholipid-binding protein, is expressed in the RPE cell cytosol, where it may be involved in membrane and cytoskeletal organisation and cell proliferation. The aim here was to analyse the role of AnxA8 and its interaction with Wnt signalling in RPE cell transdifferentiation. Human RPE cells were seeded at a concentration of 2,200/ml and treated with 3% charcoal dextran-treated foetal bovine serum (FBS) for 24h. 3µM FR or vehicle (0.1% dimethylsulfoxide) was added every day for 7 days. As a second approach, AnxA8 was suppressed in RPE cells using short interfering RNA (siRNA). FR and AnxA8 siRNA treatment both induced a decrease in AnxA8 expression and inhibited cell proliferation. It further led to transdifferentiation of ARPE-19 cells into neuron-like cells and a concomitant up-regulation of the neuronal markers Calbindin and Calretinin analysed by qPCR and immunofluorescence. Additionally, expression of Wnt signalling proteins such as β-Catenin, Frizzled-1, Frizzled-4, Wnt2b and Wnt3a was decreased. The reduction in AnxA8 and cell morphology changes induced by FR, were not reversible by inhibiting Wnt signalling using Dickkopf-1 and DAPT. Wnt signalling activators such as recombinant Wnt3a or SB216763 (glycogen synthase kinase-3β inhibitor) were able to partially reverse the FR effect. These data imply an important role for AnxA8 in maintaining RPE phenotype. Down-regulation of AnxA8 appears to be sufficient for neuronal transdifferentiation of RPE cells and the expression of neuronal markers. Further, the interdependence of AnxA8 and Wnt proteins suggests that AnxA8 might be an important regulator of Wnt signalling.

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