The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.

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.

BEST1 is highly and preferentially expressed in the retinal pigment epithelium (RPE) and causes Best macular dystrophy when mutated. We previously demonstrated that the human BEST1 upstream region −154 to +38 bp is sufficient to direct expression in the RPE of transgenic mice, and microphthalmia-associated transcription factor (MITF) and OTX2 regulate this BEST1 promoter. However, a number of questions remained. Here, we show that yeast one-hybrid screen with bait corresponding to BEST1 −120 to −88 bp identified the SOX-E factors, SOX8, SOX9, and SOX10. A paired SOX site was found in this bait, and mutation of either of the paired sites significantly decreased BEST1 promoter activity in RPE primary cultures. Among the SOX-E genes, SOX9 is highly and preferentially expressed in the RPE, and chromatin immunoprecipitation with fresh RPE cells revealed binding of SOX9, but not SOX10, to the BEST1 region where the paired SOX site is located. BEST1 promoter activity was increased by SOX9 overexpression and decreased by siRNA-mediated SOX9 knockdown. Importantly, SOX9 physically interacted with MITF and OTX2 and orchestrated synergistic activation of the BEST1 promoter with the paired SOX site playing essential roles. A combination of the expression patterns of SOX9, MITF, and OTX2 yielded tissue distribution remarkably similar to that of BEST1. Lastly, the BEST1 promoter was also active in Sertoli cells of the testis in transgenic mice where SOX9 is highly expressed. These results define SOX9 as a key regulator of BEST1 expression and demonstrate for the first time its functional role in the RPE.

Oxidative stress of the retinal pigment epithelium (RPE) is a major risk factor for age-related macular degeneration (AMD). As a dry AMD model via oxidative stress, sodium iodate (NaIO₃), which is primarily toxic to the RPE, has often been used at a high dose to cause RPE death for studying photoreceptor degeneration. Thus, characterization of RPE damage by a low dose of NaIO₃ is still limited. To quantify RPE damage caused by NaIO₃ in mice, we recently developed a morphometric method using RPE flat-mounts. Here, we report that NaIO₃ has a narrow range of dose–effect correlation at 11–18 mg/kg body weight in male C57BL/6J mice. We evaluated the usefulness of our quantification method in two experimental settings. First, we tested the effect of NF-κB inhibition on NaIO₃-induced RPE damage in male C57BL/6J mice. IKKβ inhibitor BAY 651942 suppressed upregulation of NF-κB targets and protected the RPE from oxidative stress. Second, we tested sex-specific differences in NaIO₃-induced RPE damage in C57BL/6J mice using a low dose near the threshold. NaIO₃ caused more severe RPE damage in female mice than in male mice. These results demonstrate the usefulness of the quantification method and the importance of fine-tuning of the NaIO₃ dose. The results also show the therapeutic potential of IKKβ inhibition for oxidative stress-related RPE diseases, and reveal previously-unrecognized sex-specific differences in RPE susceptibility to oxidative stress.