June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Regulation of retinal pigment epithelial cell phenotype by Annexin A8 and Wnt signalling
Author Affiliations & Notes
  • Katharina Lueck
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • John Greenwood
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • Stephen E Moss
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • Footnotes
    Commercial Relationships Katharina Lueck, None; John Greenwood, None; Stephen Moss, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4245. doi:https://doi.org/
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    • Get Citation

      Katharina Lueck, John Greenwood, Stephen E Moss; Regulation of retinal pigment epithelial cell phenotype by Annexin A8 and Wnt signalling . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4245. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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 trans-differentiated cells. AnxA8, a calcium-dependent phospholipid-binding protein, is expressed in RPE cells, where it may be involved in membrane and cytoskeletal organisation and cell proliferation. The purpose of this study was to analyse the role of anxA8 and its interaction with Wnt signalling in RPE cell differentiation.

Methods: Human ARPE-19 and primary porcine RPE cells were seeded at 2,200 cells/cm2 and treated with 3% charcoal dextran-treated foetal bovine serum (FBS) for 24 h. 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). Cells were then analysed for expression of anxA8, neuronal markers (Calbindin, Calretinin) and Wnt signalling proteins (β-Catenin, Frizzled-1, Frizzled-4, Wnt2b, Wnt3a) using immunofluorescence staining and qPCR. Further, cells were transfected with an anxA8-GFP construct to overexpress anxA8.<br /> To analyse the relationship between anxA8 and Wnt signalling, Wnt3a and the GSK3β-inhibitor SB216763 were used to activate Wnt signalling, and ɤ-secretase inhibitor DAPT and Dickkopf (DKK)-1 to inhibit Wnt signalling.

Results: FR and anxA8 siRNA treatment both induced a decrease in AnxA8 expression and inhibited cell proliferation. They also led to RPE trans-differentiation into neuron-like cells and a concomitant up-regulation of neuronal markers. Overexpression of anxA8 in ARPE-19 cells led to cell death within a week. FR did not affect anxA8-GFP transfected cells. Additionally, expression of Wnt signalling proteins was decreased and the transdifferentiating effect of FR was partially reversible by Wnt activation.

Conclusions: These data reveal an important role for anxA8 in maintaining RPE phenotype. Down-regulation of anxA8 appears to be sufficient for neuronal trans-differentiation of RPE cells and the expression of neuronal markers, whereas high levels of anxA8 seem to be lethal for cultured cells. Further, the interdependence of anxA8 and Wnt proteins expression suggests that anxA8 might be an important regulator of Wnt signalling.

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