Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo

RESEARCH ARTICLE Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo Xue Yang☯, Jin-Yong Chung☯, Usha Rai, Noriko Esumi* Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Yang X, Chung J-Y, Rai U, Esumi N (2018) Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo. PLoS ONE 13(1): e0191279. https://doi.org/ 10.1371/journal.pone.0191279 Editor: Alfred S Lewin, University of Florida, UNITED STATES Received: August 5, 2017 Accepted: January 2, 2018 Published: January 16, 2018 Copyright: © 2018 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. ☯ These authors contributed equally to this work. * Abstract The retinal pigment epithelium (RPE) supports the health and function of retinal photoreceptors and is essential for normal vision. RPE cells are post-mitotic, terminally differentiated, and polarized epithelial cells. In pathological conditions, however, they lose their epithelial integrity, become dysfunctional, even dedifferentiate, and ultimately die. The integrity of epithelial cells is maintained, in part, by adherens junctions, which are composed of cadherin homodimers and p120-, β-, and α-catenins linking to actin filaments. While E-cadherin is the major cadherin for forming the epithelial phenotype in most epithelial cell types, it has been reported that cadherin expression in RPE cells is different from other epithelial cells based on results with cultured RPE cells. In this study, we revisited the expression of cadherins in the RPE to clarify their relative contribution by measuring the absolute quantity of cDNAs produced from mRNAs of three classical cadherins (E-, N-, and P-cadherins) in the RPE in vivo. We found that P-cadherin (CDH3) is highly dominant in both mouse and human RPE in situ. The degree of dominance of P-cadherin is surprisingly large, with mouse Cdh3 and human CDH3 accounting for 82–85% and 92–93% of the total of the three cadherin mRNAs, respectively. We confirmed the expression of P-cadherin protein at the cell-cell border of mouse RPE in situ by immunofluorescence. Furthermore, we found that oxidative stress induces dissociation of P-cadherin and β-catenin from the cell membrane and subsequent translocation of β-catenin into the nucleus, resulting in activation of the canonical Wnt/ β-catenin pathway. This is the first report of absolute comparison of the expression of three cadherins in the RPE, and the results suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated. Funding: This work was supported by grants from the BrightFocus Foundation (M2015220 to NE), US National Institutes of Health (Core grant EY001765 to Wilmer Eye Institute), Wilmer Pooled Professor Research Fund (NE), and unrestricted funds from Research to Prevent Blindness, Inc. (Wilmer Eye Institute). The funders had no role in study design, PLOS ONE | https://doi.org/10.1371/journal.pone.0191279 January 16, 2018 1 / 20 P-cadherin is the dominant cadherin in the RPE data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction The retinal pigment epithelium (RPE), located between retinal photoreceptor cells and the choroid of the eye, is a single layer of pigmented epithelial cells with cobblestone-like morphology [1]. The RPE is essential for normal vision through multiple activities that support the health and function of retinal photoreceptors. The RPE constantly faces oxidative stress due to its large oxygen consumption and daily phagocytosis of photoreceptor outer segments, leading to accumulation of oxidative damage with age, which is thought to contribute to the loss of epithelial integrity and the development of diseases such as age-related macular degeneration (AMD) [1–3]. RPE cells are known to dedifferentiate and lose their fully matured state as a result of a variety of stresses, including oxidative stress and mechanical dissociation of cell-cell junctions [4– 10]. Dissociation of cultured RPE cells leads to dedifferentiation of the cells into fibroblast-like cells through epithelial to mesenchymal transition (EMT) [5, 9]. EMT is a process in which cells lose cell-cell junctions and epithelial morphology and become fibroblast-like with increased mesenchymal markers [11–13]. RPE cells undergoing EMT contribute to scarring and wound contractions in proliferative vitreoretinopathy (PVR) as well as subretinal fibrosis in advanced AMD [14–16]. To maintain the integrity of epithelial cells, adherens junctions are critical by forming cellcell contacts as protein complexes consisting of cadherin homodimers and p120-, β-, and αcatenins that link to actin filaments (F-actin) [17–19]. Cadherins are Ca2+-dependent cell adhesion molecules that connect neighboring cells through homophilic interaction of two homodimers on the cell surface [18, 20–22]. In most epithelial cell types, E-cadherin is the major cadherin responsible for forming and maintaining their epithelial phenotype [18, 20, 23]. However, it has been reported that RPE cells are different from other epithelial cells in terms of the major cadherin subtype that they express [24, 25]. Results of the expression of cadherin subtypes in the RPE have been conflicting. In cultured human RPE cells, N-cadherin rather than E-cadherin was dominantly expressed [25–27]. In the center of cultured porcine RPE sheets, where intact RPE cells were located, P-cadherin was abundantly detected, but it was lost at the edge of RPE sheets, where cells were migrating away and showed fibroblastic morphology with the expression of N-cadherin and vimentin [9]. In situ hybridization with mouse embryos showed that the outer layer (RPE) of the optic cup expressed N-cadherin until embryonic day 10.5 (E10.5) but switched to P-cadherin from E12 onward [28]. This study also showed that E-cadherin expression was not detectable in the RPE throughout embryonic and postnatal stages, indicating that each cadherin displays unique spatial and temporal expression patterns [28]. However, drawing general conclusions from these studies is challenging because they differ with regards to species (human, pig, and mouse), RPE source (cultured RPE cells, cultured RPE sheet, and in situ RPE), temporal stage (embryonic, postnatal, and adult), and methodology (Western blot, immunofluorescenc (...truncated)