Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid.

<p>(A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers (<i>Sox9</i>, <i>Otx2</i>, and <i>Rpe65</i>) in three biological replicates was analyzed by RT-qPCR in triplicate using <i>Gapdh</i>, <i>Hprt</i>, and <i>Actb</i> as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers (<i>Vwf</i> and <i>Col6a1</i>) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for <i>Cdh1</i> (gene for E-cadherin), <i>Cdh2</i> (N-cadherin), and <i>Cdh3</i> (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p < 0.05) and ** (p < 0.01).</p

P-cadherin is the dominant cadherin in mouse and human RPE <i>in situ</i>.

<p>(A) Absolute quantification of cDNA to assess the mRNA quantity of <i>Cdh1</i>, <i>Cdh2</i>, and <i>Cdh3</i> in mouse RPE <i>in situ</i>. Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of <i>CDH1</i>, <i>CDH2</i>, and <i>CDH3</i> in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).</p

P-cadherin is co-localized with other junctional proteins at the RPE cell border in mice.

<p>Immunofluorescence of mouse RPE flat-mounts. Double staining: P-cadherin (red; A, E, I) and either ZO-1 (green; B), β-catenin (green; F), or F-actin (green; J), with nuclear stain by DAPI (blue; C, G, K). Merged images (D, H, L) show the co-localization of P-cadherin with ZO-1 (tight junction), β-catenin (adherens junction), and F-actin (adherens junction) at the cell-cell border.</p

Oxidative stress-induced dissociation of adherens junctions results in nuclear translocation of β-catenin and an increase of EMT-related factors in mouse RPE.

<p>(A) Immunofluorescence of mouse RPE flat-mounts. Mice were injected with NaIO₃ (15 mg/kg body weight) on Day 0, and the localization of β-catenin (green) and P-cadherin (red) was analyzed along with nuclear stain by DAPI (blue) on Days 0 (a-c), 1 (d-f), 3 (g-i) and 7 (j-l). Double staining: β-catenin (a, d, g, j), P-cadherin (b, e, h, k), and merged images with DAPI (c, f, i, l). The localization of β-catenin and P-cadherin at the cell-cell border was significantly disrupted, and instead prominently detected on/in the nucleus on Day 3. (B) Immunofluorescence of mouse retinal sections with a focus on the RPE nuclei. Mice were injected with NaIO₃ (15 mg/kg body weight) on Day 0, and the localization of β-catenin (green) and P-cadherin (red) was analyzed along with nuclear stain by DAPI (blue) on Days 0 (m-o) and 3 (two representative nuclei; p-r and s-u). Double staining: β-catenin (m, p, s), P-cadherin (n, q, t), and merged images with DAPI (o, r, u). On Day 3, β-catenin was detected in the nuclei of mouse RPE. (C) Western blot analyses of mouse RPE proteins. Mice were injected with NaIO₃ (15 mg/kg body weight) on Day 0, and RPE protein lysates were prepared on Days 0, 1, 3, and 7. The protein levels were analyzed using Western blotting with antibodies against P-cadherin, β-catenin, SNAI1 (Snail), vimentin, and control β-actin. The protein levels of β-catenin and SNAI1 increased similarly on Day 1 following oxidative stress.</p

Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid.

<p>(A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers (<i>Sox9</i>, <i>Otx2</i>, and <i>Rpe65</i>) in three biological replicates was analyzed by RT-qPCR in triplicate using <i>Gapdh</i>, <i>Hprt</i>, and <i>Actb</i> as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers (<i>Vwf</i> and <i>Col6a1</i>) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for <i>Cdh1</i> (gene for E-cadherin), <i>Cdh2</i> (N-cadherin), and <i>Cdh3</i> (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p < 0.05) and ** (p < 0.01).</p

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

<div><p>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 <i>in vivo</i>. We found that P-cadherin (CDH3) is highly dominant in both mouse and human RPE <i>in situ</i>. The degree of dominance of P-cadherin is surprisingly large, with mouse <i>Cdh3</i> and human <i>CDH3</i> 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 <i>in situ</i> 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.</p></div