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

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 downregulated 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.In vivo, retinal pigment epithelial (RPE) cell phenotype is sustained by the retinal microenvironment. However, once removed from the retina and placed in culture, RPE cells dedifferentiate within a few rounds of division, losing signature characteristics such as pigment granules and expression of genes such as MerTk and RPE65. The widely used human RPE cell line, ARPE19, is typical in this respect, though several studies have shown that under appropriate culture conditions ARPE19 cells will re-adopt a more mature phenotype that includes restoration of pigment granules and expression of key RPE-associated genes 1-3 . Interest in RPE de-differentiation has also been driven by the need to understand the process in proliferative vitreoretinopathy (PVR) where epithelial mesenchymal transition (EMT) plays a key role in the pathogenesis of this condition. More recently, interest in RPE cell differentiation and maturation has intensified with advances in regenerative medicine that utilize RPE cells derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells 4-6 . 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 7-10 . Key to these clinical advances is a better understanding of the signaling pathways that regulate and maintain RPE cell phenotype.The plasticity of RPE cells in culture is evident from studies showing that not only can they d...

Fine-tuning of Epithelial EGFR signals Supports Coordinated Mammary Gland Development

“…Modulators of Wnt signals, Anxa8 (Anexin A8), Trp63 (p63), and Fgfr2 are also in this cluster. Anxa8 is expressed in c-kit-positive luminal progenitor cells in the mammary epithelium (Iglesias et al, 2015) and can enhance Wnt target gene expression (Lueck, Carr, Yu, Greenwood, & Moss, 2020). p63 is essential for proliferation of epithelial stem cells (Senoo, Pinto, Crum, & McKeon, 2007) and promotes beta-catenin signaling (Lee et al, 2014), and Fgfr2 can promote beta-catenin signaling as well (Krejci et al, 2012).…”

Identification of Diagnostic Biomarkers and Their Correlation with Immune Infiltration in Age-Related Macular Degeneration

“…The above results suggested that immune dysregulation plays an important role in AMD. According to previous experimental studies, the Wnt signaling was found aberrantly increased in wet AMD, contributing to pathological angiogenesis [21,22]. The complement system was also demonstrated to have a causative role in AMD development and had been introduced into emerging clinical trials as a potential therapeutic target [23,24].…”

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells

“…TGF-β2 promotes the expression of MMP-2, a mesenchymal marker, the transformation of RPE cells into myofibroblasts and the production of the components of extracellular matrix, that is, controls the development of EMT [20] . In addition, a synthetic retinoid derivative, fenretinide, has been shown to promote trans-differentiation of ARPE-19 cells towards a neuronal-like phenotype [21][22] . Whether the EMT mechanism exists in RPE cells during myopia deserves further investigation.…”