Unraveling the role of CRX as a potent intrinsic suppressor of epithelial-mesenchymal transition in retinal pigment epithelial cells

Unraveling the role of CRX as a potent intrinsic suppressor of epithelial-mesenchymal transition in retinal pigment epithelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Unraveling the role of CRX as a potent intrinsic suppressor of epithelial-mesenchymal transition in retinal pigment epithelial cells Haibin Tian, Dongli Li, Qingjian Ou, Furong Gao, Xi Wang, Lilin Zhu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5969324/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is one of the significant pathogenic mechanisms for the formation of subretinal fibrosis in age-related macular degeneration (AMD). Multiple signaling pathways that promote EMT have been well described, yet the endogenous signaling pathways that inhibit EMT within RPE cells remain largely elusive. In this study, we confirmed the expression of CRX in human RPE cells and human embryonic stem cell-derived RPE (ESC-RPE) cells. By employing sub-culture to disrupt intercellular connections and thereby inhibit the Hippo signaling pathway, combined with TGF-β1 treatment in vitro to mimic the microenvironment for the formation of subretinal fibrosis, it was revealed that Hippo/YAP1 and TGF-β1 synergistically promoted the nuclear translocation of β-catenin, and the latter bound to TCF7 to inhibit the expression of CRX. Overexpression of CRX was capable of suppressing the occurrence of EMT in ESC-RPE cells. CRX exerted its inhibitory effect on EMT partly by upregulating the expression of PPP2R2B. In the laser-induced choroidal neovascularization mouse model, the nuclear translocation of CRX took place in RPE cells, and overexpression of CRX played an inhibitory role in the formation of subretinal fibrosis. This study has identified CRX as an endogenous signaling molecule that inhibits EMT in RPE cells and has provided a new research target and treatment strategy for the treatment of wet AMD and the inhibition of subretinal fibrosis formation. Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases Biological sciences/Cell biology/Cell signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Macular neovascularization (MNV) is an important cause of blindness in wet age-related macular degeneration (AMD) patients. There are mainly three types of MNV: Type 1 MNV refers to choroidal blood vessels expanding below the RPE, type 2 MNV is characterized by proliferating choroidal blood vessels breaking through Bruch's membrane and the RPE monolayer to spread in the subretinal space, and type 3 MNV originates from the retinal vasculature and progresses posteriorly into the subretinal space [ 1 ]. The risk of eyes with wet AMD developing subretinal fibrosis (SF) despite ongoing anti-VEGF therapy is reported to be 45% by two years [ 2 ] and 41% by ten years [ 3 ]. Among the three types, type 2 MNV is associated with more SF than other MNV types [ 4 ], which is a major risk factor for poor visual outcomes after treatment [ 5 ]. RPE cells undergoing epithelial-mesenchymal transition (EMT) to transform into myofibroblasts is the most important contribution to the formation of SF [ 6 ]. It has been reported that the proportion of RPE cells undergoing EMT in this fibrotic tissue even reaches 40% [ 7 ]. Therefore, inhibiting EMT in RPE cells has always been an attractive research topic in the treatment of SF [ 8 , 9 ]. Currently, multiple EMT-related signaling pathways have been reported, such as the TGF-β signaling pathway, BMP signaling pathway, Wnt/β-catenin signaling pathway, and Hippo/YAP signaling pathway, and the use of inhibitors to inhibit these signaling pathways can inhibit EMT in RPE cells [ 10 , 11 ]. However, whether there are endogenous signaling pathways that inhibit EMT in RPE cells, especially when EMT occurs, whether endogenous signaling pathways can be activated to inhibit EMT remains unclear. When we cultured embryonic stem cell-derived RPE (ESC-RPE) cells and induced pluripotent stem cell-derived RPE (iPSC-RPE) cells, the cells can be passaged multiple times in vitro at a 50% ratio while still maintaining an epithelial state [ 12 , 13 ]. The process of cell passage is actually a process of EMT followed by mesenchymal-epithelial transition (MET). This indicates the existence of endogenous signaling pathways in the cells that inhibit EMT or promote MET, ensuring that the cells can return to the epithelial state. We previously successfully obtained iRPE cells using key transcription factors, which have the function of resisting EMT, and determined that the key transcription factor cone-rod homeobox containing gene (CRX) plays the most important role in resisting EMT [ 12 , 14 ]. CRX is mainly expressed in cone cells and rod cells and is an important transcription factor that regulates the differentiation of photoreceptor cells [ 15 ], and the manifestation of its expression within RPE cells is controversial [ 16 – 19 ], and its function in RPE cells is not clear. In this article, we aim to ascertain the expression of endogenous CRX in RPE cells, and explore whether CRX exerts an inhibitory effect on the EMT in RPE cells. Additionally, we will elucidate the upstream and downstream regulatory signaling pathways of CRX. Moreover, a laser-induced choroidal neovascularization (CNV) mouse model will be used to verify that CRX inhibits EMT in RPE cells, consequently impeding the formation of SF. RESULTS CRX is expressed in RPE cells CRX is a crucial transcription factor that regulates the development of photoreceptor cells [ 15 ]. Nevertheless, the manifestation of its expression within RPE cells is controversial [ 16 – 19 ]. To determine whether CRX is expressed in RPE cells, we analyzed the single-cell sequencing data [ 19 ]. Various types of cells were observed from the retina and RPE/choroid, including amacrine, astrocyte, bipolar, cone, horizontal, muller, myeloid, RGC, rod, RPE, and vascular cells (Fig. 1 A). Our observations revealed that RPE65 was predominantly expressed in RPE cells (Fig. 1 B). Moreover, CRX was found to be expressed not only in rod and cone cells but also in RPE cells (Fig. 1 C). Notably, a total of 4359 RPE cells (expressing RPE65 ) were counted. Among them, 2334 RPE cells were found to express CRX (Fig. 1 D). Consequently, the proportion of RPE cells expressing CRX reached 53.54%. Western blotting results showed that CRX was expressed in both ESC-RPE cells and iPSC-RPE cells (Supplementary Fig. 1A). Furthermore, an analysis of previously published gene microarray data [ 20 ] indicated that the expression of CRX could be detected in RPE-choroid tissues (Supplementary Fig. 1B). Notably, the expression level of CRX was found to be up-regulated in RPE-choroid tissues of patients with dry AMD, whereas in those with wet AMD, it was down-regulated (Supplementary Fig. 1B). Collectively, these results demonstrate that there is a certain amount of CRX expression in RPE cells, and the alterations in CRX expression levels in RPE cells of AMD patients may be correlated with the etiology of AMD. The expression level and the nucleus translocation of CRX demonstrate dynamic alterations during the sub-culturing process of RPE cells. In the context of Type 2 MNV, the choroidal vasculature penetrates the Bruch's membrane and the RPE cell layer [ 1 ], that invariably results in the disruption of tight junctions among RPE cells. Cell sub-culturing, which also disrupts intercellular connections, can mimic this process. Our previous investigations have demonstrated that RPE cells are able to be sub-cultured at a 50% ratio for at least five passages [ 12 , 13 ]. When cells are sub-cultured, their tight junctions became discontinuously and subsequently re-established (Fig. 2 A). The expression level of α-SMA exhibits a gradual increase of sub-culture, followed by a gradual decline to the initial level when the cells demonstrated polygonal epithelial morphology again (Fig. 2 A, B). This phenomenon corroborates that the cells first experience the EMT process and then the MET process. We further analyzed the expression of CRX and observed that the expression level of CRX decreased on the first day of sub-culture. Subsequently, concomitant with the formation of tight junctions (as indicated by ZO-1 expression in Fig. 2 A), the expression of CRX gradually augmented, peaking on the fourth day, and then decreased subsequent to the initial level (Fig. 2 C-E). Additionally, we discovered that when the cells formed tight junctions and assumed a polygonal morphology, the proportion of cells with positive CRX nuclear staining was less than 15% (Fig. 2 F, G). On the first day of cell sub-culturing, CRX translocated into the nucleus, the nuclear retention ratio was over 75% (Fig. 2 F, G). Thereafter, the nuclear retention ratio of CRX was gradually reduced (Fig. 2 F, G). Collectively, these findings suggest that the expression and nuclear shuttling of CRX are intricately associated with the EMT and MET processes of RPE cells. β-Catenin/TCF7 mediates the inhibitory effect of YAP1 on CRX expression. Cell-cell junction disruption rapidly inhibits the Hippo signaling pathway, reducing phosphorylated transcriptional co-activator YAP1 [ 21 ]. The latter translocates into the nucleus and induces EMT in RPE cells [ 22 , 23 ]. We found that after cell sub-culturing, YAP1 also exhibits kinetic changes in nuclear entry and exit. One day after sub-culturing, a large amount of YAP1 enters the nucleus, and then the amount of YAP1 in the nucleus gradually decreases (Fig. 3 A, B). We further conducted a scratch assay and found that the cells at the edge of the scratch lost contact inhibition and underwent migration (Supplementary Fig. 2A). The cells migrating in the front were completely dispersed and named disseminated cells, while the cells migrating slowly behind were named slow-migrating cells (Supplementary Fig. 2A). Two days after the scratch, a large number of disseminated cells expressed CRX in their cell nuclei, along with a small number of α-SMA-positive cells (Supplementary Fig. 2B). Seven days later, the disseminated cells completely lost their epithelial-like tight junctions, taking on a dedifferentiated fibroblast-like appearance (Supplementary Fig. 2C). In addition, both slow-migrating cells and disseminated cells expressed α-SMA, and almost no disseminated cells demonstrated nuclear retention of CRX (Supplementary Fig. 2B, D). Meanwhile, YAP1 was still present in the cell nuclei of the disseminated cells (Supplementary Fig. 2D). These data suggest that YAP1 may inhibit the expression of CRX, leading to the occurrence of EMT in RPE cells. In order to address that the Hippo signaling pathway regulates CRX expression, we used the Hippo signaling pathway inhibitor XMU-MP-1 to treat cells for 4 days after cells were sub-cultured, and found that 5 µM XMU-MP-1 markedly down-regulated the expression level of CRX (Fig. 3 C). 5 µM XMU-MP-1 did not significantly increase the cell death, but enlarged the cells (Supplementary Fig. 3). We further displayed that XMU-MP-1 significantly increased the nucleus retention ratio of YAP1 (Fig. 3 D), decreased the amount of phosphorylated YAP1, and reduced the expression level of CRX protein (Fig. 3 F, G), indicating that the Hippo/YAP1 pathway indeed inhibits CRX expression. Additionally, we observed that XMU-MP-1 treatment did not alter the nuclear retention ratio of CRX in ESC-RPE cells, indicating that Hippo signaling pathway does not regulate the shuttling of CRX into nucleus (Supplementary Fig. 4). Employing transcription factor binding prediction software (TF-Target Finder, https://jingle.shinyapps.io/TF_Target_Finder/ ), we identified that TEAD has the potential to bind to the CRX promoter. Nevertheless, chromatin immunoprecipitation (ChIP) experiments demonstrated that neither TEAD nor YAP1 could directly bind to the CRX promoter region (Supplementary Fig. 5). This finding strongly indicates that YAP1 does not directly exert an inhibitory effect on CRX expression. Previous studies reported that YAP1 can bind to β-catenin, promoting its nuclear retention, and can form a transcription complex with β-catenin and TCF to regulate gene expression [ 24 , 25 ]. We found that in RPE cells with tight junctions, β-catenin is mainly distributed at the cell junctions (Fig. 3 H). After sub-culturing, β-catenin is distributed in the cytoplasm and the nucleus (Fig. 3 H). Co-immunoprecipitation (CoIP) experiments confirmed that YAP1 was able to bind to β-catenin, and when the Hippo signaling pathway is inhibited by XMU-MP-1, the entry of β-catenin into the nucleus increased significantly (Fig. 3 I-K). When RPE cells were treated with the β-catenin inhibitor MSAB, the expression level of β-catenin decreased, and the expression level of CRX increased significantly (Fig. 3 L-N). 5 µM MSAB did not significantly increase the cell death (Supplementary Fig. 3). These results suggest that when the Hippo signaling pathway is inactivated, YAP1 enters the nucleus and promotes the nuclear retention of β-catenin, thereby inhibiting CRX expression. To further validate the regulation of CRX gene expression by β-catenin, we utilized software to predict that TCF7 was capable of binding to the CRX promoter, and verified that by ChIP-quantitative real-time PCR (qRT-PCR) analysis (Fig. 3 O-R). We further found that β-catenin binded to TCF7 (Fig. 3 S). Upon knockdown of TCF7, the expression level of CRX increased significantly (Fig. 3 T, U). These results demonstrated that β-catenin/TCF7 inhibits CRX expression. The above mentioned results confirm that β-catenin/TCF7 mediates the inhibitory effect of YAP1 on CRX expression. With the formation of tight junctions, YAP1 in the nucleus gradually decreases, and the expression level of CRX increases. CRX suppresses EMT in RPE cells. Previously, we discovered that ESC-RPE cells were more prone to undergo EMT when sub-cultured at a low density [ 13 ]. When we performed sub-culturing at a 25% ratio, we observed that the ESC-RPE cells were not able to maintain epithelial morphology after being cultured for 8 days compared with those with the sub-culturing at a 50% ratio (Fig. 4 A). The expression of α-SMA was significant increased, while the expression of CRX was decreased (Fig. 4 B-E). To validate the function of CRX in inhibiting EMT, we overexpressed CRX (OE-CRX) in ESC-RPE cells (Fig. 4 F-H). It was found that on the 4th day of sub-culturing, overexpression of CRX could inhibit the occurrence of EMT in cells, the expression of E-cadherin and occludin increased, while the expression of α-SMA and vimentin decreased (Fig. 4 I-K). During the formation of fibrotic scar, apart from the disruption of cell-cell junctions in RPE cells activating the YAP1 signaling pathway, another crucial inducer is TGF-β1 [ 26 – 28 ]. To further validate the function of CRX in resisting EMT, when sub-culturing cells, we simultaneously used TGF-β1 to induce EMT in cells to simulate the conditions for the fibrotic scar formation in vivo. The results showed that TGF-β1 treatment could reduce the expression levels of E-cadherin and occludin, while increasing the expression of α-SMA and vimentin (Fig. 4 L-N). However, overexpression of CRX could counteract the effects of TGF-β1 (Fig. 4 L-N). These results demonstrate that CRX can play a role in inhibiting EMT in RPE cells. Further experiments were conducted by overexpressing and knocking down CRX in iPSC-RPE cells to verify the role of CRX in inhibiting EMT in RPE cells (Supplementary Fig. 6). Previously, we found TGF-β1 could promote β-catenin's nuclear translocation in ARPE19 cells [ 9 ]. Similarly, in ESC-RPE cells, TGF-β1 facilitated the nuclear translocation of β-catenin (Fig. 4 O, P), MSAB, in contrast, reduced this translocation (Fig. 4 O, P). TGF-β1 induced EMT, increasing α-SMA and decreasing CRX expression (Fig. 4 Q-S). However, inhibiting β-catenin with MSAB increased CRX expression and inhibited EMT (Fig. 4 Q-S). These results demonstrated that TGF-β1 inhibits CRX expression, at least in part, through the β-catenin pathway. CRX functions as a pivotal transcription factor in the differentiation process of photoreceptor cells [ 15 ]. Aiming to determine whether the overexpression of CRX in RPE cells instigates their differentiation into photoreceptor cells, the RNA-seq data was analyzed, photoreceptor-associated genes such as RCVRN , RHO , NRL , and NR2E3 exhibited no substantial alterations. In contrast, RPE-associated genes including MITF , RPE65 , RALBP1 , TYRP1 , and BEST1 underwent up-regulation (Supplementary Fig. 7A). Subsequently, the gene expression levels were verified by qRT-PCR. The expression of RPE-related genes demonstrated an upsurge, whereas that of photoreceptor-related genes displayed a mixed pattern with overall low levels (Supplementary Fig. 7B). Based on our previously published chromatin immunoprecipitation sequencing (ChIP-seq) data ( https://www.ncbi.nlm.nih.gov/geo , GSE174063) [ 14 ], it was evident that CRX binds to the promoter of RPE65 , that was further validated by ChIP-qRT-PCR (Supplementary Fig. 7C, D). These data suggest overexpressing CRX in RPE cells does not induce their differentiation into photoreceptor cells. However, it holds the potential to enhance the expression of RPE associated genes, particularly that of RPE65, thereby enhancing the physiological functions of RPE cells. PPP2R2B mediates the inhibitory effect of CRX on TGF-β1-induced EMT To elucidate the molecular mechanism by which CRX inhibits EMT in ESC-RPE cells, we conducted RNA-seq analysis on the normal control (Control) group, the CRX overexpression group (OE-CRX), the TGF-β1-induced group (Control-TGF-β1), and the TGF-β1-induced OE-CRX group (OE-CRX-TGF-β1) (Fig. 5 A). Volcano plots displayed the differentially expressed genes (DEGs) among different comparison groups (Fig. 5 B-D). Through Venn analysis of the DEGs, we screened out 9 CRX target genes that related to EMT (Fig. 5 E). The qRT-PCR detection determined that the expression level of PPP2R2B exhibited the most significant difference (Fig. 5 F). PPP2R2B encodes a member of the protein phosphatase 2A (PP2A) B-subunit family [ 29 ]. The B-subunit serves as a structural component of the PP2A complex, enabling PP2A to interact with diverse substrates and execute various functions in different cellular processes [ 29 ]. We selected PPP2R2B as the target gene for our further study and found that overexpression of CRX in ESC-RPE cells promoted the expression of PPP2R2B (Fig. 5 G, H). Based on our previously published ChIP-seq data ( https://www.ncbi.nlm.nih.gov/geo , GSE174063) [ 14 ], we next identified that CRX can bind to the promoter region of PPP2R2B, which was verified by ChIP-qRT-PCR (Fig. 6 A, B). To explore the regulatory role of PPP2R2B in EMT of ESC-RPE cells, we first overexpressed PPP2R2B in ESC-RPE cells (Fig. 6 C, D). Overexpression of PPP2R2B (OE-PPP2R2B) could partially counteract the alterations in EMT-related proteins induced by TGF-β1 (Fig. 6 E, F). We then knocked down PPP2R2B in CRX-overexpressed ESC-RPE cells (OE-CRX-sh PPP2R2B ) (Fig. 6 G-I). We detected the knockdown efficiency of PPP2R2B by qRT-PCR and Western blotting, and selected OE-CRX-sh PPP2R2B -1 for subsequent experiments. Overexpression of CRX could inhibit the down-regulation of epithelial markers such as E-cadherin and occludin, as well as the increase in the expression of mesenchymal markers like α-SMA and vimentin induced by TGF-β1 (Fig. 6 J, K). However, knockdown of PPP2R2B could counteract the inhibitory effect of CRX on EMT (Fig. 6 J, K). Furthermore, through scratch assays, we found that TGF-β1 significantly enhanced the migratory capacity of ESC-RPE cells. Nevertheless, these effects were inhibited by CRX overexpression, and the inhibitory effect was counteracted by knockdown of PPP2R2B (Fig. 6 L, M). The above results indicate that PPP2R2B exerts an inhibitory effect on EMT, and the anti-EMT effect of CRX is mediated, at least in part, by PPP2R2B. CRX reduced laser-induced CNV and subretinal fibrosis in mice To evaluate the role of CRX in inhibiting EMT of RPE cells in vivo, we first established a laser-induced CNV mouse model. On the 3rd, 7th, and 21st days after laser induction, the expression of CRX in RPE cells at the laser-induced lesion site, adjacent to the lesion site, and distal to the lesion site was detected. The results showed that on the 3rd and 7th days, the number of RPE cells with positive CRX expression in the nucleus at the site adjacent to the lesion site and distal to the lesion site was significantly increased compared with the control group, and the RPE cells adjacent to the lesion site showed the most significant change (Supplementary Fig. 8A, B). We further examined the expression of YAP1 and found that on the 3rd day, a large number of RPE cells at the lesion site were positive for YAP1 nuclear expression. Thereafter, the number of RPE cells with positive nuclear YAP1 expression gradually decreased (Supplementary Fig. 8A, B). In contrast, at 7 and 21 days, some RPE cells in the distal area still had positive YAP1 nuclear expression (Supplementary Fig. 8A, B). This result indicates that RPE cells at the lesion site were damaged, and some RPE cells rapidly underwent EMT. The RPE cells adjacent to the lesion site, similar to the slow-migrating cells in the scratch assay (Supplementary Fig. 2), expressed both CRX and YAP1 in the nucleus, with CRX playing a role in inhibiting EMT. The RPE cells in the distal area were still affected by the microenvironment, with some RPE cells showing positive nuclear expression of both CRX and YAP1 (Supplementary Fig. 8A, B). To determine the process of CRX in inhibiting EMT, we prepared lenti-CRX virus and administered it before laser photocoagulation via subretinal injection in the right eye, with empty vector serving as the control in the left eye (Fig. 7 A). We observed that the lentivirus mainly infected RPE cells (Fig. 7 B). We performed Masson staining and immunostaining on the choroidal flatmounts. Masson staining revealed that, compared to the empty vector group, significant disruptions were evident in the choroid and outer nuclear layer at the center of the laser burn 7 days after laser photocoagulation. Subsequently, 21 days after laser photocoagulation, retinal edema and newly-formed blood vessels extended into the subretinal space (Fig. 7 C). Compared with the empty vector group at days 7 and 21, the collagen area in the OE-CRX group was significantly reduced (Fig. 7 D). These results indicate that overexpression of CRX can reduce laser-induced SF in mouse retinas. DISCUSSION This study demonstrates that CRX functions as an endogenous signaling entity within RPE cells, effectively counteracting EMT. During the EMT process, the TGF-β1 and Hippo/YAP1 signaling cascades promote the nuclear translocation of β-catenin that combines with TCF7 to bind to promoter of CRX, thereby inhibiting CRX expression. Notably, overexpression of CRX leads to the up-regulation of PPP2R2B, which in turn suppresses EMT in RPE cells. The EMT-inhibitory efficacy of CRX has been experimentally validated in a laser-induced CNV mouse model. During retinal development, CRX is considered a pivotal transcription factor implicated in photoreceptor cell fate determination [ 15 ]. Mutations in the human CRX gene are associated with several retinal disorders, including cone-rod dystrophy and Leber’s congenital amaurosis [ 30 ]. Previous studies posited that CRX is not expressed in RPE cells [ 18 ]. However, other research has detected the expression of CRX in human RPE cells [ 16 , 17 , 19 ]. Noriko Esumi not only confirmed the expression of CRX in human and bovine RPE cells but also discovered that CRX binds to the BEST1 proximal promoter [ 17 ]. We further confirm that CRX is expressed in ESC-RPE and iPSC-RPE cells and can promote the expression of RPE65. Previously, we found that transcription factors such as CRX, MITF, NR2E1, and cMYC could convert dedifferentiated RPE cells into iRPE cells with anti-EMT properties [ 14 ]. CRX can regulate the expression of multiple genes related to EMT[ 14 ]. In this study, we further discovered that CRX can promote the expression of PPP2R2B, a member of the protein phosphatase 2A (PP2A) B-subunit family. PP2A belongs to the Ser/Thr protein phosphatase family, which consists of a structural scaffold A-subunit, a highly conserved catalytic C-subunit, and diverse regulatory B-subunit [ 29 ]. The B-subunit serves as a structural component of the PP2A complex, enabling PP2A to interact with different substrates and execute various functions in diverse cellular processes [ 29 ]. Research has shown that PPP2R2B can inhibit EMT in bladder cancer cells by suppressing the WNT signaling pathway and reducing the expression of β-catenin [ 31 ]. PPP2R2B inhibits the proliferation and migration of pancreatic cancer cells by suppressing the mTOR pathway [ 32 ]. Using okadaic acid to inhibit PP2A can induce EMT in pancreatic tumor cells, and okadaic acid can strongly inhibit the expression of PPP2R2B [ 33 ]. PP2A inhibits the EMT process of podocytes by suppressing the JIP4/p38-MAPK pathway [ 34 ]. A significant amount of β-catenin is sequestered by E-cadherin at the plasma membrane, and knockdown of E-cadherin leads to the redistribution of β-catenin in the cytoplasm, followed by nuclear translocation and subsequent activation of the Wnt signaling [ 35 ]. We found that overexpression of PPP2R2B can promote the expression of E-cadherin. It is highly likely that PPP2R2B inhibits the EMT process by increasing E-cadherin and reducing the nuclear translocation of β-catenin. Crx functions as a transcription activator and cooperates with the bZIP transcription factor Nrl to activate the expression of the rhodopsin [ 18 ]. This finding indicates that a high level of rhodopsin expression demands the function of at least two photoreceptor transcription factors [ 18 ]. Additionally, both Crx and NeuroD1 are necessary to induce monkey iris-derived cells to acquire the photoreceptor phenotype [ 36 ]. These results imply that the interaction with other photoreceptor transcription factors is crucial for Crx to facilitate photoreceptor differentiation. This also accounts for the fact that in our study, merely overexpressing CRX in RPE cells did not lead to their transformation into photoreceptor cells. Instead, it even promoted the expression levels of RPE cell-specific genes, as evidenced by the significant increase in RPE65 . Consequently, overexpressing CRX in RPE cells not only inhibits the occurrence of EMT in RPE cells but also holds the promise of enhancing the physiological functions of RPE cells. During the development of CNV, new blood vessels in the choroid rupture Bruch's membrane and grow beneath the RPE. They further extend into the subretinal space to form SF, the integrated RPE cells are damaged. The disruption of intercellular junctions inevitably inactivates the Hippo signaling pathway, leading to the activation and nuclear translocation of YAP1, thereby promoting the EMT of RPE cells [ 22 , 23 ]. The expression level of YAP1 is significantly elevated in CNV tissues. Employing RNA interference technology to reduce the expression of YAP can decrease the expression of α-SMA in CNV tissues [ 37 ]. In the early stage of the laser-induced CNV model, we also observed the nuclear translocation of YAP1 in RPE cells. Another crucial factor in inducing EMT in RPE cells is the TGF-β1. Research has shown that the expression of TGF-β1 is significantly increased in the vitreous humor of wet AMD [ 26 ], and the amount of TGF-β1 is also remarkably elevated in the CNV model [ 27 , 28 ]. TGF-β1 can rapidly promote EMT in RPE cells. We found that YAP1 and TGF-β1 can synergistically inhibit the expression of CRX by promoting the nuclear translocation of β-catenin. β-catenin is a key molecule in the WNT signaling pathway, and its nuclear translocation can promote the EMT process [ 10 ]. Previously, we demonstrated that β-catenin can translocate into the nucleus under the stimulation of TGF-β1, promoting the EMT process in ARPE19 cells [ 9 ]. The decrease in CRX expression induced by TGF-β1 is also achieved through β-catenin. The transcriptional co-activator proteins YAP/TAZ are the hubs of the Hippo pathway. Although YAP/TEAD is not able to directly bind to the promoter region of CRX, YAP1 can also bind to β-catenin and promote its nuclear translocation. It has been reported that YAP1 interacts with and stabilizes the β-catenin protein in the nucleus [ 24 ], and YAP1 could directly interact with β-catenin in the nucleus and form a transcriptional YAP/β-catenin/TCF4 complex [ 25 ]. Our results are consistent with these reports. Therefore, during the EMT process of RPE cells, signaling pathways such as TGF-β1, Wnt/β-catenin, and Hippo/YAP1 interact with each other and also have a synergistic regulatory effect on the expression of CRX. The two principal pathogenic manifestations of wet AMD are MNV and SF [ 38 – 40 ] Anti-VEGF therapy has achieved encouraging outcomes in the treatment of MNV. However, there remains no effective approach for managing SF, which typically leads to permanent visual impairment [ 41 , 42 ]. SF develops in the late stage of wet AMD. Multiple cell types, including RPE cells, macrophages, müller cells, pericytes, and endothelial cells, transdifferentiate into myofibroblasts, promoting the formation of SF and collagen deposition. Pathological analysis of surgically obtained MNV membranes has revealed that the majority of cells are co-stained with α-SMA and cytokeratin. Since only RPE cells express cytokeratin, RPE cells represent the predominant cell type within MNV membranes [ 6 ]. Thus, the EMT of RPE cells into myofibroblasts is the primary source of SF formation. Numerous inhibitors targeting signaling pathways associated with RPE cell EMT have been developed to suppress the formation of SF. For instance, knockdown of Galectin-1 can inhibit the TGF-β1/Smad2/Snail signaling axis, thereby attenuating subretinal fibrosis in mice [ 39 ]. The expression of YAP1 is significantly elevated in CNV tissues. Employing RNA interference technology to reduce YAP1 expression can decrease the expression of α-SMA in CNV tissues [ 37 ]. RO4929097, a selective γ-secretase inhibitor, directly inhibits the Notch signaling pathway and indirectly suppresses the ERK1/2 signaling pathway in ARPE-19 cells and a laser-induced mouse model [ 43 ]. We have demonstrated that treatment with H535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) effectively attenuates subretinal fibrosis and EMT in laser-induced CNV mice and ARPE19 cells [ 9 ]. This study reveals that CRX serves as an endogenous EMT-inhibitor within RPE cells that will provide novel research target for the treatment of wet AMD, especially in the prevention of the formation of SF. Although we have elucidated that the Hippo/YAP1/β-catenin and TGF-β1/β-catenin pathways synergistically inhibit the expression of CRX, and the nuclear translocation of CRX is not regulated by the Hippo signaling pathway, the signaling pathway that regulates the nuclear shuttling of CRX remains unclear. In addition, PPP2R2B can exert an inhibitory effect on EMT, but its downstream targets still require further investigation. In summary, this study utilized the published single-cell sequencing data to confirm the expression of CRX in human RPE cells and verified it in vitro-cultured ESC-RPE and iPSC-RPE. Sub-culturing was used to disrupt intercellular connections and thus inhibit the Hippo signaling pathway, combined with TGF-β1 treatment to simulate the microenvironment for the SF formation. It was found that Hippo/YAP1 and TGF-β1 synergistically promoted the nuclear translocation of β-Catenin, and the latter combined with TCF7 to bind to CRX promoter to inhibit the expression of CRX (Fig. 8 ). Overexpression of CRX could suppress the EMT of RPE cells by up-regulating the expression of PPP2R2B. In the laser-induced mouse CNV model, the nuclear translocation of CRX also occurred in RPE cells, and overexpression of CRX played an inhibitory role in the formation of subretinal fibrosis. This study identified CRX in RPE cells as an endogenous signal molecule that inhibits EMT, providing a new treatment strategy and research target for the treatment of wet AMD and the prevention of FS formation. It also offered a new treatment method for other ophthalmic diseases with EMT of RPE as the critical pathogenesis, such as proliferative vitreoretinopathy, diabetic retinopathy, and Inherited retinal degenerations [ 44 – 46 ]. METHODS Cell cultures According to our previously described protocol, retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (ESC-RPE) and induced pluripotent stem cells (iPSC-RPE) were cultured in medium (Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F-12, Gibco, Grand Island, NY, USA), 10% knockout serum replacement (KSR, Gibco), supplemented with 2 mM L-glutamine (Gibco, USA), 1% MEM nonessential amino acids (MEM NEAA, Gibco),1% penicillin-streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 10 mM nicotinamide (Sigma, St. Louis, MO, USA) in dishes precoated with 1% Matrigel (Corning Inc, Corning, NY, USA) at 37°C and 5% CO 2 . The RPE cells at passages 3–5 were used in this study. Analysis of the single-nuclei RNA-seq data of retinal tissues The single-nuclei RNA-seq data of 106 human retina and RPE/choroid with subtype resolution from over 100,000 cells (including the information of cell types) were obtained from GEO ( https://www.ncbi.nlm.nih.gov/geo/ ) database (GSE135133). The merged expression matrix was prepared for clustering using the Seurat 4.4.0, following the common pipeline. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was used to project cells in two dimensions. The function FeaturePlot and VlnPlot in Seurat were used to visualization the gene expressions. Analysis of microarray data The microarray data of human RPE/choroid, which includes 7 normal samples, 7 dry AMD samples, and 7 wet AMD samples, and the microarray data of human retina that includes 7 AMD samples (dry and wet AMD) (with the age of all the individuals being over 75) were obtained from the GEO ( https://www.ncbi.nlm.nih.gov/geo/ ) database (GSE29801). The relative level of CRX in each group was compared. Immunofluorescence staining For immunofluorescence analysis, fixed cells and cryosections from eyes were permeabilized with 0.25% Triton X-100 (Sigma) for 2 min, washed with PBS, and then blocked with 2% bovine serum albumin (BSA, Sigma) in PBS. The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech) overnight at 4℃. They were then washed three times with PBS, followed by incubation with the fluorescent secondary antibodies overnight. 4,6 diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) was used to indicate the nucleus. The samples were then examined by fluorescence microscope (Olympus IX73, Tokyo, Japan). Antibodies were listed in Table S1 . Quantitative real-time PCR ( qRT-PCR ) Total RNA was extracted and reverse transcription was performed using Primescript™ RT Master Mix kit (Takara, Shiga, Japan). qRT-PCR was performed in a Chromo4 instrument cycler (Bio-Rad, Hercules, USA) using Superreal Premix plus kit (Tiangen Biotech, Beijing, China). PCR amplification was carried out with the following cycling parameters: denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s. Primer sequences (Synthesized by Sangon Biotech, China) were listed in Table S2 . Western blotting The cells were lysed by RIPA buffer containing protease and phosphatase inhibitor (Sigma). The protein extracts (20µg per sample) were separated by 10% SDS-PAGE gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against α-SMA, E-cadherin (Cell Signaling Technology, Beverly, MA, USA), Vimentin (proteintech), YAP1, phospho-YAP1(Ser397) (proteintech), β-catenin, CRX, TCF7 (Cell Signaling Technology), PPP2R2B (abcam), and β-actin or GAPDH (proteintech) for 12h at 4℃, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. The blots were visualized with a chemiluminescence imaging system (Tanon 5200, Shanghai, China) and quantified with ImageJ software (Version 1.48v). Antibodies were listed in Table S1 . Co-immunoprecipitation (Co-IP) Protein A + G magnetic beads (20 µL) were incubated with 5 µg antibodies (against TCF7, β-Catenin) or normal 5 µg IgG for 1h. 5×10 6 cells were lysed by 200 µL IP lysis buffer (Beyotime, Shanghai, China) and incubated with prepared conjugated Protein A + G magnetic beads at 4℃ overnight. The beads were washed with TBS for three times and the binding proteins were eluted with protein loading buffer at 95℃ for 5min. After centrifugation, the samples were collected and used for Western blotting. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay The ChIP assay was performed using a SimpleChIP Plus Enzymatic Chromatin IP kit (Cell Signaling Technology). Briefly, Ten million cells were treated with 1% formaldehyde for 10min and unreacted formaldehyde was quenched with glycine for 5 min. The cells were collected by scraping and were lysed with lysate buffer, the nuclear pellet was digested by micrococcal nuclease. Immunoprecipitation was performed on the lysate with 2 µg antibody against YAP1, β-catenin, TCF7, CRX (proteintech), or control rabbit IgG at 4°C overnight. Then, the immune complexes were incubated with Protein G magnetic beads (Cell Signaling Technology) for 2 h. Chromatin was eluted from antibody/protein G magenetic beads and used for qRT-PCR. The ChIP-qPCR primer sequences for the promoters of CRX, PPP2R2B, and RPE65 are summarized in Table S3 . Scratch assay Cells were plated into 24-well culture plate and grow to confluency. Then, scratches on ESC-RPE cells monolayers were made with a sterilized 200-µL pipette tip and then gently washed twice with sterile PBS to remove floating debris. Images were recorded after 0, 24, and 48 h, and the wound recovery was analyzed using ImageJ software. Overexpression of CRX and PPP2R2B For generating lentivirus, human cDNAs of CRX and PPP2R2B were obtained by PCR amplification from ESC-RPE cells and cloned into lentiviral pLVX-mCMV-ZsGreen1-Puro vector (Takara). The packaging plasmids were psPAX2 and pMD2.G. HEK293FT cells were seeded at a density between 5.0–7.0×10 4 cells/cm 2 and transfected by Lipofectamine 2000 (Invitrogen) with each vector. Individual supernatants containing virus were harvested at 48 h post-transfection and filtered with a 0.45 µm PVDF membrane (Millipore, Boston, USA). ESC-RPE cells and iPSC-RPE cells were plated in 6-cm culture dishes, respectively. The next day, cells were infected with viruses. The positively transfected cells were sorted by FACS based on ZsGreen expression. The expression levels were determined by qRT-PCR and Western blotting. Generation of lentiviral vector to knockdown target genes HEK293T cells were seeded in 15 cm dishes at a density of 10 7 cells per dish 24 h before transfection. Lentiviral pLVX-shRNA2-ZsGreen1 (Takara) vector were used to prepare lentiviruses, the packaging plasmids are psPAX2 and pMD2.G. HEK293FT cells were transfected with vectors. Individual supernatants containing virus were harvested at 48h and used to infect cells. Reduced expression of target genes at the transcript level was determined by qRT-PCR. The targeting sequences of shRNAs for CRX , TCF7 , and PPP2R2B were included in Table S4 . The most effective shRNA sequence was selected. RNA sequencing Following the Illumina mRNA-seq protocol, pooled RNA libraries of the cells were established, with 50ng of RNA from RPE cells from different groups. Sequencing was performed by the MAJORBIO company (Shanghai, China). Filtering and quality control of the raw reads from RNA-seq was carried-out using FastQC. The clean reads were mapped to reference sequences using SOAP2 aligner. Gene expression levels were calculated using the TPM method. Log 2 fold change (FC) of TPM was used to identify differentially expressed genes (DEGs) between these two groups. Only those genes indicating log |FC| > 1 and adjusted p < 0.05 were regarded as DEGs. Calcein/PI staining Cells were cultured in 96-well culture plate. 1–5 µM XMU-MP-1 or MSAB (MedChemExpress, Shanghai, China) was used to treat ESC-RPE cells. For Calcein/PI staining, cells were cultured in 100µL of calcein AM/PI working solution (Beyotime) at 37°C for 1h. The samples were then examined by a fluorescence microscope (Olympus IX73) Animals In this study, 8-week-old C57BL/6J male mice (from the Laboratory Animal Center of Tongji University) were utilized. They were housed in groups and were allowed a period to acclimatize to the laboratory environment with a 12 / 12 h light / dark cycle and provided with food and water. Subretinal injection of virus and laser-induced CNV model A total of 40 C57BL/6J male mice were divided into laser-empty vector groups and laser-overexpression of CRX (OE-CRX) groups. mice were firstly anaesthetized by 1.25% tribromoethanol, their pupils were dilated with amethocaine (0.5%) and tropicamide (0.5%). A channel was created by inserting a 30-gauge needle, behind the limbus, into the vitreous chamber. A 33-gauge needle was inserted into the subretinal space of the central retina, and 2 µL lentivirus carrying the CRX gene was injected. The control eyes received an injection of lentivirus carrying an empty vector. Seven days after subretinal injection of virus, the laser-induced subretinal fibrosis model was established as previously described [ 9 , 47 ]. In brief, four to six laser spots (532 nm, 120 mW, 100 ms; Novus Spectra, Japan) were selected at each fundus around the optic disc and avoid tiny blood vessels. The disruption of Bruch’s membrane was confirmed by visually subretinal bubble formation immediately after laser application. After laser burns, the mice were randomly sacrificed on day 7 and day 21 for further quantification of CNV and subretinal fibrosis. Mice were randomly allocated to experimental groups and no blinding method was used for subretinal injection. Eyes with a damaged lens or detached retina due to subretinal injection were excluded from experiments. Masson’s trichrome staining The eyes were obtained at days 7 and 21 after laser induction. The samples were fixed with 4% PFA and embedded in paraffin to prepare the 3 µmthick sections. Masson’s trichrome staining was performed with a trichrome staining kit (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Collagen fibers were stained blue, and images were obtained by microscope (Olympus IX73). RPE/choroid Flatmount Staining The areas of CNV and collagen fibers were determined on RPE/choroidal flatmounts on day 7 and day 14. Mouse eyecups were fixed in 4% Paraformaldehyde (PFA), and anterior segments were removed before cutting six to eight radial incisions to be flattened. The RPE-choroid complexes were washed, blocked with 5% BSA and permeabilized with 0.3% Triton X-100, and then incubated with collagen type I antibody (abcam) (for evaluating subretinal fibrosis) at 4°C overnight. They were washed three times with PBS, followed by incubation with the fluorescent secondary antibodies overnight. Samples were finally observed under fluorescence microscope (Olympus IX73). Statistical analysis All values are expressed as the mean ± SD. Data were analyzed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). All statistical analyses were performed using unpaired Student’s t-test, or one-way ANOVA and post hoc Bonferroni’s test. Statistical significance was set at P < 0.05. Declarations DATA AVAILABILITY. All study data are included in the main text and/or SI Appendix. The raw data of RNA-seq have been deposited in the NCBI Sequence Read Archive (SRA) database under accession number: PRJNA1169190. ACKNOWLEDGMENTS We thank Zhenzhen Zhao, Xueying Wang, and Qian Wang for their expertise and technical assistance. Author contributions Research design and conceptualization: DL, QO, FG, and HT. Methodology: DL, QO, FG, XW, LZ, and YZ. Data analysis: J-YX, CJ, JW. Writing-original draft: DL, QO, F.G., and HT. Writing-review & editing: JZ, JL,YB, LL, and G-TX. Supervision and funding: QO, LL, G-TX, and HT. FUNDING. This work is supported by grants from the Natural Science Foundation of Shanghai (24ZR1470100), National Natural Science Foundation of China (82471073, 82271108), and Shanghai Municipal Health Commission (20234Y0113). COMPETING INTERESTS The authors declare no competing interest. 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Li","suffix":""},{"id":433735913,"identity":"9eb68827-c3ed-417b-b644-ef12292879c3","order_by":11,"name":"Yanlong Bi","email":"","orcid":"","institution":"Department of Ophthalmology of Tongji Hospital and Laboratory of Clinical and Visual Sciences of Tongji Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"Yanlong","middleName":"","lastName":"Bi","suffix":""},{"id":433735914,"identity":"140e31af-e293-4e16-a346-88948dd9c55e","order_by":12,"name":"Lixia Lu","email":"","orcid":"https://orcid.org/0000-0003-0268-4656","institution":"Tongji Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"Lixia","middleName":"","lastName":"Lu","suffix":""},{"id":433735915,"identity":"d1a816af-b419-4957-854e-0c73438d38bf","order_by":13,"name":"Guo-Tong Xu","email":"","orcid":"","institution":"Tongji Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"Guo-Tong","middleName":"","lastName":"Xu","suffix":""},{"id":433735916,"identity":"0cb2b798-69f7-44fe-9ed4-bda418d135c7","order_by":14,"name":"Ye Zhou","email":"","orcid":"","institution":"Department of Ophthalmology of Tongji Hospital and Laboratory of Clinical and Visual Sciences of Tongji Eye Institute","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-02-06 02:30:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5969324/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5969324/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79791141,"identity":"d62d870f-a099-4ac2-859e-c27f0b70d10b","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10762986,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of \u003cem\u003eCRX\u003c/em\u003e in human RPE cells is determined by the single-nuclei RNA-seq. \u003cstrong\u003eA\u003c/strong\u003e Uniform manifold approximation and projection (UMAP) dimensionality reductionplot showed the profiled cells including amacrine, astrocyte, bipolar, cone, horizontal, muller, myeloid, RGC, rod, RPE, vascular cells. \u003cstrong\u003eB-C\u003c/strong\u003e The violin plots of \u003cem\u003eRPE65\u003c/em\u003e and \u003cem\u003eCRX\u003c/em\u003e expression in all cell types. \u003cstrong\u003eD\u003c/strong\u003e The co-expressions of \u003cem\u003eRPE65 \u003c/em\u003eand \u003cem\u003eCRX\u003c/em\u003e. Red indicates the expression of \u003cem\u003eRPE65 \u003c/em\u003e(4359 RPE cells)\u003cem\u003e, \u003c/em\u003egreen indicates the expression of \u003cem\u003eCRX \u003c/em\u003e(2334 RPE cells);\u003cem\u003e \u003c/em\u003eyellow indicates the co-expressions of \u003cem\u003eRPE65 \u003c/em\u003eand \u003cem\u003eCRX, \u003c/em\u003ethe ratio of CRX+ RPE cells was 53.54%.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/ab403cd47b8374621777f2e2.png"},{"id":79791139,"identity":"80ea6040-57a5-44cf-b332-9f9af5966cd8","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14485650,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression level of CRX and its kinetic changes of nuclear retention during the sub-culturing of ESC-RPE cells. \u003cstrong\u003eA\u003c/strong\u003e Changes in cell morphology, and the expressions of ZO-1 and α-SMA at different time points during the cell sub-culturing process. \u003cstrong\u003eB\u003c/strong\u003e Quantitative calculation of the percentage of α-SMA\u003csup\u003e+\u003c/sup\u003e cells (n = 8). \u003cstrong\u003eC-E\u003c/strong\u003e Detection of the mRNA and protein expression levels of CRX at different time points by (\u003cstrong\u003eC\u003c/strong\u003e) qRT-PCR, (\u003cstrong\u003eD\u003c/strong\u003e) Western blotting, and (\u003cstrong\u003eE\u003c/strong\u003e) quantitative analysis (n = 3). \u003cstrong\u003eF-G\u003c/strong\u003e Nuclear retention ratio of CRX at different time points during the cell sub-culturing process (n = 8). Scale bar = 50 μm. Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 using one-way ANOVA and post hoc Bonferroni’s test. ns: no significance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/00042aaacb1f60d5054737ed.png"},{"id":79791724,"identity":"69e91a80-24ab-496b-ad96-767c7f47c055","added_by":"auto","created_at":"2025-04-02 19:08:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17682649,"visible":true,"origin":"","legend":"\u003cp\u003eβ-Catenin/TCF7 mediates the inhibitory effect of YAP1 on CRX expression in ESC-RPE cells. \u003cstrong\u003eA-B\u003c/strong\u003e The images of nuclear retention of YAP1 at different time points during sub-culturing of ESC-RPE cells and quantitative analysis (n = 8). \u003cstrong\u003eC\u003c/strong\u003e The CRX expression level in ESC-RPE cells after being treated with the Hippo pathway inhibitor XMU-MP-1 for four days was detected by qRT-PCR (n = 3). \u003cstrong\u003eD\u003c/strong\u003e After treatment with 5 μM XMU-MP-1 for four days, the nuclear retention of YAP1 was detected by immunostaining. \u003cstrong\u003eE \u003c/strong\u003eThe quantitative analysis of YAP1 nuclear retention ratio (n = 8). \u003cstrong\u003eF-G\u003c/strong\u003e The expression levels of p-YAP1, YAP1, and CRX in ESC-RPE cells after being treated with 5 μM XMU-MP-1 for four days were determined and quantified by Western blotting (n = 3). \u003cstrong\u003eH \u003c/strong\u003eThe cellular localization of β-catenin at different time points during sub-culturing was demonstrated by immunostaining. \u003cstrong\u003eI\u003c/strong\u003e Co-IP analysis of the interaction between β-catenin and YAP1. \u003cstrong\u003eJ-K\u003c/strong\u003e The protein level of β-catenin in the nucleus and cytoplasm of cells after being treated with 5 μM XMU-MP-1 for four days was determined and quantified by Western blotting (n = 3). \u003cstrong\u003eL \u003c/strong\u003eThe CRX expression level in ESC-RPE cells after being treated with β-catenin inhibitor MSAB for four days was detected by qRT-PCR (n = 3). \u003cstrong\u003eM-N\u003c/strong\u003e The expression levels of β-catenin and CRX in ESC-RPE cells after being treated with 3 μM MSAB for four days were determined and quantified by Western blotting (n = 3). \u003cstrong\u003eO \u003c/strong\u003eVenn analysis was conducted on Hippo signaling pathway-related proteins (\u003ca href=\"https://www.genecards.org/\" target=\"https://www.doubao.com/chat/_blank\"\u003ehttps://www.genecards.org/\u003c/a\u003e), transcription factors predicted to bind to the CRX promoter in the UCSC database (\u003ca href=\"https://genome.ucsc.edu/\" target=\"https://www.doubao.com/chat/_blank\"\u003ehttps://genome.ucsc.edu/\u003c/a\u003e) and the JAPSAR database (\u003ca href=\"https://jaspar.elixir.no/\" target=\"https://www.doubao.com/chat/_blank\"\u003ehttps://jaspar.elixir.no/\u003c/a\u003e), the candidate protein TCF7 was screened out. \u003cstrong\u003eP\u003c/strong\u003e The regions of the CRX gene paired with qRT-PCR primers (#1-5: promoter, #6: intron). \u003cstrong\u003eQ\u003c/strong\u003e The matrices of β-catenin and TCF7 proteins binding to the CRX gene promoter predicted by the JASPAR database. \u003cstrong\u003eR\u003c/strong\u003e The binding sites of β-catenin and TCF7 to the \u003cem\u003eCRX\u003c/em\u003e promoter were verified by ChIP-qRT-PCR (n = 3). \u003cstrong\u003eS\u003c/strong\u003e CoIP analysis of the interaction between TCF7 and β-catenin. \u003cstrong\u003eT-U\u003c/strong\u003e TCF7 was knocked down in ESC-RPE cells, and the expression levels of TCF7 and CRX were determined and quantified by Western blotting (n = 3). Scale bar = 50 μm. Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 using unpaired two-sided t-tests in Figs. E, K, and N and one-way ANOVA and post hoc Bonferroni’s test in the others.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/8a475c9efa69dd56ff34ab08.png"},{"id":79791132,"identity":"6c493d90-1e95-4624-860d-11082e3e7bf3","added_by":"auto","created_at":"2025-04-02 19:00:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16956762,"visible":true,"origin":"","legend":"\u003cp\u003eCRX inhibits EMT in ESC-RPE cells. \u003cstrong\u003eA\u003c/strong\u003e Representative images of ESC-RPE cells sub-cultured at a 50% ratio and a 25% ratio after 4 days and 8 days of culture. \u003cstrong\u003eB-C\u003c/strong\u003e The expression levels of (\u003cstrong\u003eB\u003c/strong\u003e) α-SMA and (\u003cstrong\u003eC\u003c/strong\u003e) CRX were determined by qRT-PCR (n = 3). \u003cstrong\u003eD-E\u003c/strong\u003e The expression levels of α-SMA and CRX were determined and quantified by Western blotting (n = 3). \u003cstrong\u003eF-H\u003c/strong\u003e CRX was overexpressed in ESC-RPE cells, and the expression level was detected by (\u003cstrong\u003eF\u003c/strong\u003e) qRT-PCR (n = 3), (\u003cstrong\u003eG\u003c/strong\u003e) Western blotting and (\u003cstrong\u003eH\u003c/strong\u003e) quantitative analysis (n = 3). \u003cstrong\u003eI-K\u003c/strong\u003e The expression levels of EMT-related genes in control- and OE-CRX-ESC-RPE cells after being sub-cultured for four days were determined by (\u003cstrong\u003eI\u003c/strong\u003e) qRT-PCR (n = 3), (\u003cstrong\u003eJ\u003c/strong\u003e) Western blotting and (\u003cstrong\u003eK\u003c/strong\u003e) quantitative analysis (n = 3). \u003cstrong\u003eL-N\u003c/strong\u003e The sub-cultured control- and OE-CRX-ESC-RPE cells were treated with 5 ng/mL TGF-β1 for four days, The expression levels of EMT-related genes were determined by (L) qRT-PCR (n = 3), (M) Western blotting and (N) quantitative analysis (n = 3). \u003cstrong\u003eO-P\u003c/strong\u003e The protein level of β-catenin in the nucleus and cytoplasm of cells after being treated with 5 ng/mL TGF-β1 and 3 μM MSAB for four days was determined and quantified by Western blotting (n = 3). \u003cstrong\u003eQ\u003c/strong\u003e Representative images of sub-cultured ESC-RPE cells treated with TGF-β1 and MSAB for 4 days and 8 days. \u003cstrong\u003eR-S\u003c/strong\u003eThe expression levels of α-SMA and CRX in sub-cultured ESC-RPE cells treated with TGF-β1 and MSAB for 8 days were determined and quantified by Western blotting (n = 3). Scale bar = 50 μm. Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 using unpaired two-sided t-tests in Fig. B, C, F, and H, and one-way ANOVA and post hoc Bonferroni’s test in the others.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/c70232410435bd071f413d49.png"},{"id":79791128,"identity":"f31a0886-6645-442e-81ec-12fe7578912d","added_by":"auto","created_at":"2025-04-02 19:00:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3845945,"visible":true,"origin":"","legend":"\u003cp\u003ePPP2R2B is a downstream target of CRX. \u003cstrong\u003eA\u003c/strong\u003e Heatmap of RNA sequencing of control- and OE-CRX-ESC-RPE cells after being treated with TGF-β1. \u003cstrong\u003eB-D\u003c/strong\u003e Volcano plots of DEGs. \u003cstrong\u003eE\u003c/strong\u003e Nine target genes of CRX were screened out by venn analysis. The EMT gene set was extracted from the EMT pathway analysis, and the CRX target gene set was obtained from the TF_Target_Finder website (\u003ca href=\"https://jingle.shinyapps.io/TF_Target_Finder/\" target=\"https://www.doubao.com/chat/_blank\"\u003ehttps://jingle.shinyapps.io/TF_Target_Finder/\u003c/a\u003e). \u003cstrong\u003eF\u003c/strong\u003e The expression levels of the 9 targeted genes were verified by qRT-PCR (n = 3). \u003cstrong\u003eG-H\u003c/strong\u003e The protein expression level of the target gene PPP2R2B in control- and OE-CRX-ESC-RPE cells was determined and quantified by Western blotting (n = 3). Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 using one-way ANOVA and post hoc Bonferroni’s test in the Fig. 5F and unpaired two-sided t-tests in Fig. 5H.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/0ace322fda7742772d7ceefa.png"},{"id":79791134,"identity":"596e97ae-9e6a-42e0-90a9-09511d9993ed","added_by":"auto","created_at":"2025-04-02 19:00:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13736389,"visible":true,"origin":"","legend":"\u003cp\u003ePPP2R2B mediates the effect of CRX in resisting EMT. \u003cstrong\u003eA\u003c/strong\u003e Enriched peaks of CRX binding to the promoter of \u003cem\u003ePPP2R2B\u003c/em\u003e. \u003cstrong\u003eB\u003c/strong\u003e The fold enrichment of CRX immunoprecipitation relative to the IgG control, as determined by qRT-PCR (n = 3). \u003cstrong\u003eC-D\u003c/strong\u003e The overexpression of PPP2R2B in ESC-RPE cells was determined and quantified by Western blotting (n = 3). \u003cstrong\u003eE-F\u003c/strong\u003e The control- and OE-PPP2R2B-ESC-RPE cells were treated with TGF-β1, the expression levels of EMT-related proteins were determined and quantified by Western blotting (n = 3). \u003cstrong\u003eG-I\u003c/strong\u003e PPP2R2B was knocked down in OE-CRX-ESC-RPE cells, and expression level of PPP2R2B was determined by (\u003cstrong\u003eG\u003c/strong\u003e) qRT- PCR (n = 3), (\u003cstrong\u003eH\u003c/strong\u003e) western blotting, and (\u003cstrong\u003eI\u003c/strong\u003e) quantitative analysis (n = 3). \u003cstrong\u003eJ-K\u003c/strong\u003e Cells in different groups were treated with TGF-β1, the expression levels of EMT-related proteins were determined and quantified by Western blotting (n = 3). \u003cstrong\u003eL-M\u003c/strong\u003e Scratch assays were performed on cells in different groups treated with TGF-β1. The black lines represent the edges of the scratch. The cell migration rate was quantified as (distance between the black lines at the start of the experiment-distance between the black lines at the end of the experiment) / distance between the black lines at the start of the experiment (n = 3). Scale bar = 200 μm. Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01 using unpaired two-sided t-tests in Fig. 6B and D, and one-way ANOVA and post hoc Bonferroni’s test in the others. ns: no significance.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/c479324403b757be66e9f464.png"},{"id":79791170,"identity":"8fc4f1ce-5b0b-4878-a192-20c148a82d72","added_by":"auto","created_at":"2025-04-02 19:00:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12872441,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of CRX in RPE cells reduces the area of laser-induced subretinal fibrosis in mice. \u003cstrong\u003eA\u003c/strong\u003eSchematic diagram of the laser-induced modeling and in vivo treatment in mice. \u003cstrong\u003eB\u003c/strong\u003eCo-localization staining of GFP and CRX in the RPE cell layer one week after lentiviral infection. \u003cstrong\u003eC\u003c/strong\u003e Masson staining was performed to analyze the changes in the fibrotic area at different time points after laser induction. \u003cstrong\u003eD\u003c/strong\u003eCollagen 1 immunostaining demonstrated the fibrotic area in the choroidal flat-mount at different time points after laser induction. \u003cstrong\u003eE\u003c/strong\u003e The quantitative analysis of fibrotic area in the choroidal flat-mount (n = 3). Scale bar = 50 μm. Data are mean ± SD, *P \u0026lt; 0.05, **P \u0026lt; 0.01 using unpaired two-sided t-tests.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/46dfc65aa382a8244c632eeb.png"},{"id":79791148,"identity":"99c0804f-0790-4a1a-8b7c-259e5c831713","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5093566,"visible":true,"origin":"","legend":"\u003cp\u003eThe Hippo/YAP1 and TGF-β signaling pathways synergistically inhibit the expression of CRX. In normal and healthy RPE cells, the Hippo signaling pathway is activated. Under this condition, YAP1 is phosphorylated and subsequently degraded, while the TGF-β signaling pathway remains inactivated. The translocation of β-catenin into the nucleus is blocked, leading to the activation of CRX expression. This activation of CRX then promotes the expression of PPP2R2B and, in turn, inhibits the EMT. Conversely, in the RPE cells of wet AMD, the Hippo signaling pathway is inactivated, and the phosphorylation of YAP1 is blocked. This inactivation of the Hippo pathway, in synergy with the activated TGF-β signaling pathway, promotes the translocation of β-catenin into the nucleus. β-catenin binds to TCF7, which results in the inhibition of CRX expression. Subsequently, the repression of PPP2R2B expression promotes EMT.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/3d7a1a8e0677bee88d68cbd1.png"},{"id":81832131,"identity":"d0057298-2d7b-4b84-9468-499c405b00b9","added_by":"auto","created_at":"2025-05-02 13:59:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":88653510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/cb4e80c5-72aa-4560-9a25-92c131915836.pdf"},{"id":79791150,"identity":"cdfb8977-9bfe-448b-a77f-094cb3a18f8e","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26984,"visible":true,"origin":"","legend":"Supplementary tables","description":"","filename":"Supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/7fdbb8f395834915e3bc839e.docx"},{"id":79791137,"identity":"3ef0437b-cca5-496c-bb39-64bae97f7126","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6374420,"visible":true,"origin":"","legend":"Supplementary figure 1","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/c444bca40404d8ee17fd3763.tif"},{"id":79791145,"identity":"cab314c8-ca99-408a-9903-f1942c300d02","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":52415836,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 2\u003c/p\u003e","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/bb03235d9be83542bc32b501.tif"},{"id":79791136,"identity":"51b99e76-dd14-4d0c-89e4-629c2f064835","added_by":"auto","created_at":"2025-04-02 19:00:51","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20618228,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 3\u003c/p\u003e","description":"","filename":"Supplementaryfigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/1be242d672a1262ca1b4c960.tif"},{"id":79791130,"identity":"c98ac2cd-cd24-4194-b484-091cd7537a84","added_by":"auto","created_at":"2025-04-02 19:00:51","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10961640,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 4\u003c/p\u003e","description":"","filename":"Supplementaryfigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/624ab92d2951bb58b36de258.tif"},{"id":79791726,"identity":"55cc8a63-729f-47e5-8d83-9baf23ccd73f","added_by":"auto","created_at":"2025-04-02 19:08:52","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7018400,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 5\u003c/p\u003e","description":"","filename":"Supplementaryfigure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/de0ca77a74cdaf43cd3f8a11.tif"},{"id":79791180,"identity":"ca845846-2dbc-4fc6-a81d-1257ebccbe63","added_by":"auto","created_at":"2025-04-02 19:00:54","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":42922464,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 6\u003c/p\u003e","description":"","filename":"Supplementaryfigure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/f5065f4af897dd8cc4a57b57.tif"},{"id":79791725,"identity":"3aa54085-cbb9-45e4-a017-14ec869327e0","added_by":"auto","created_at":"2025-04-02 19:08:52","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":22184748,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 7\u003c/p\u003e","description":"","filename":"Supplementaryfigure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/c18d412cad73b59748987b27.tif"},{"id":79791147,"identity":"0c83ee0a-ff8a-4c89-bc79-e51b9306ff70","added_by":"auto","created_at":"2025-04-02 19:00:52","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":72991716,"visible":true,"origin":"","legend":"Supplementary figure 8","description":"","filename":"Supplementaryfigure8.tif","url":"https://assets-eu.researchsquare.com/files/rs-5969324/v1/eab1c62663ba2dbe1c08c69f.tif"}],"financialInterests":"(Not answered)","formattedTitle":"Unraveling the role of CRX as a potent intrinsic suppressor of epithelial-mesenchymal transition in retinal pigment epithelial cells","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMacular neovascularization (MNV) is an important cause of blindness in wet age-related macular degeneration (AMD) patients. There are mainly three types of MNV: Type 1 MNV refers to choroidal blood vessels expanding below the RPE, type 2 MNV is characterized by proliferating choroidal blood vessels breaking through Bruch's membrane and the RPE monolayer to spread in the subretinal space, and type 3 MNV originates from the retinal vasculature and progresses posteriorly into the subretinal space [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The risk of eyes with wet AMD developing subretinal fibrosis (SF) despite ongoing anti-VEGF therapy is reported to be 45% by two years [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and 41% by ten years [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the three types, type 2 MNV is associated with more SF than other MNV types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which is a major risk factor for poor visual outcomes after treatment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. RPE cells undergoing epithelial-mesenchymal transition (EMT) to transform into myofibroblasts is the most important contribution to the formation of SF [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has been reported that the proportion of RPE cells undergoing EMT in this fibrotic tissue even reaches 40% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, inhibiting EMT in RPE cells has always been an attractive research topic in the treatment of SF [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, multiple EMT-related signaling pathways have been reported, such as the TGF-β signaling pathway, BMP signaling pathway, Wnt/β-catenin signaling pathway, and Hippo/YAP signaling pathway, and the use of inhibitors to inhibit these signaling pathways can inhibit EMT in RPE cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, whether there are endogenous signaling pathways that inhibit EMT in RPE cells, especially when EMT occurs, whether endogenous signaling pathways can be activated to inhibit EMT remains unclear.\u003c/p\u003e \u003cp\u003eWhen we cultured embryonic stem cell-derived RPE (ESC-RPE) cells and induced pluripotent stem cell-derived RPE (iPSC-RPE) cells, the cells can be passaged multiple times in vitro at a 50% ratio while still maintaining an epithelial state [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The process of cell passage is actually a process of EMT followed by mesenchymal-epithelial transition (MET). This indicates the existence of endogenous signaling pathways in the cells that inhibit EMT or promote MET, ensuring that the cells can return to the epithelial state. We previously successfully obtained iRPE cells using key transcription factors, which have the function of resisting EMT, and determined that the key transcription factor cone-rod homeobox containing gene (CRX) plays the most important role in resisting EMT [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CRX is mainly expressed in cone cells and rod cells and is an important transcription factor that regulates the differentiation of photoreceptor cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and the manifestation of its expression within RPE cells is controversial [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and its function in RPE cells is not clear. In this article, we aim to ascertain the expression of endogenous CRX in RPE cells, and explore whether CRX exerts an inhibitory effect on the EMT in RPE cells. Additionally, we will elucidate the upstream and downstream regulatory signaling pathways of CRX. Moreover, a laser-induced choroidal neovascularization (CNV) mouse model will be used to verify that CRX inhibits EMT in RPE cells, consequently impeding the formation of SF.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCRX is expressed in RPE cells\u003c/h2\u003e \u003cp\u003eCRX is a crucial transcription factor that regulates the development of photoreceptor cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nevertheless, the manifestation of its expression within RPE cells is controversial [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To determine whether CRX is expressed in RPE cells, we analyzed the single-cell sequencing data [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Various types of cells were observed from the retina and RPE/choroid, including amacrine, astrocyte, bipolar, cone, horizontal, muller, myeloid, RGC, rod, RPE, and vascular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Our observations revealed that \u003cem\u003eRPE65\u003c/em\u003e was predominantly expressed in RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, \u003cem\u003eCRX\u003c/em\u003e was found to be expressed not only in rod and cone cells but also in RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, a total of 4359 RPE cells (expressing \u003cem\u003eRPE65\u003c/em\u003e) were counted. Among them, 2334 RPE cells were found to express \u003cem\u003eCRX\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Consequently, the proportion of RPE cells expressing \u003cem\u003eCRX\u003c/em\u003e reached 53.54%. Western blotting results showed that CRX was expressed in both ESC-RPE cells and iPSC-RPE cells (Supplementary Fig.\u0026nbsp;1A). Furthermore, an analysis of previously published gene microarray data [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] indicated that the expression of \u003cem\u003eCRX\u003c/em\u003e could be detected in RPE-choroid tissues (Supplementary Fig.\u0026nbsp;1B). Notably, the expression level of CRX was found to be up-regulated in RPE-choroid tissues of patients with dry AMD, whereas in those with wet AMD, it was down-regulated (Supplementary Fig.\u0026nbsp;1B). Collectively, these results demonstrate that there is a certain amount of \u003cem\u003eCRX\u003c/em\u003e expression in RPE cells, and the alterations in \u003cem\u003eCRX\u003c/em\u003e expression levels in RPE cells of AMD patients may be correlated with the etiology of AMD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression level and the nucleus translocation of CRX demonstrate dynamic alterations during the sub-culturing process of RPE cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the context of Type 2 MNV, the choroidal vasculature penetrates the Bruch's membrane and the RPE cell layer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], that invariably results in the disruption of tight junctions among RPE cells. Cell sub-culturing, which also disrupts intercellular connections, can mimic this process. Our previous investigations have demonstrated that RPE cells are able to be sub-cultured at a 50% ratio for at least five passages [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. When cells are sub-cultured, their tight junctions became discontinuously and subsequently re-established (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The expression level of α-SMA exhibits a gradual increase of sub-culture, followed by a gradual decline to the initial level when the cells demonstrated polygonal epithelial morphology again (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). This phenomenon corroborates that the cells first experience the EMT process and then the MET process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further analyzed the expression of CRX and observed that the expression level of CRX decreased on the first day of sub-culture. Subsequently, concomitant with the formation of tight junctions (as indicated by ZO-1 expression in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the expression of CRX gradually augmented, peaking on the fourth day, and then decreased subsequent to the initial level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). Additionally, we discovered that when the cells formed tight junctions and assumed a polygonal morphology, the proportion of cells with positive CRX nuclear staining was less than 15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). On the first day of cell sub-culturing, CRX translocated into the nucleus, the nuclear retention ratio was over 75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). Thereafter, the nuclear retention ratio of CRX was gradually reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). Collectively, these findings suggest that the expression and nuclear shuttling of CRX are intricately associated with the EMT and MET processes of RPE cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eβ-Catenin/TCF7 mediates the inhibitory effect of YAP1 on CRX expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell-cell junction disruption rapidly inhibits the Hippo signaling pathway, reducing phosphorylated transcriptional co-activator YAP1 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The latter translocates into the nucleus and induces EMT in RPE cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We found that after cell sub-culturing, YAP1 also exhibits kinetic changes in nuclear entry and exit. One day after sub-culturing, a large amount of YAP1 enters the nucleus, and then the amount of YAP1 in the nucleus gradually decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). We further conducted a scratch assay and found that the cells at the edge of the scratch lost contact inhibition and underwent migration (Supplementary Fig.\u0026nbsp;2A). The cells migrating in the front were completely dispersed and named disseminated cells, while the cells migrating slowly behind were named slow-migrating cells (Supplementary Fig.\u0026nbsp;2A). Two days after the scratch, a large number of disseminated cells expressed CRX in their cell nuclei, along with a small number of α-SMA-positive cells (Supplementary Fig.\u0026nbsp;2B). Seven days later, the disseminated cells completely lost their epithelial-like tight junctions, taking on a dedifferentiated fibroblast-like appearance (Supplementary Fig.\u0026nbsp;2C). In addition, both slow-migrating cells and disseminated cells expressed α-SMA, and almost no disseminated cells demonstrated nuclear retention of CRX (Supplementary Fig.\u0026nbsp;2B, D). Meanwhile, YAP1 was still present in the cell nuclei of the disseminated cells (Supplementary Fig.\u0026nbsp;2D). These data suggest that YAP1 may inhibit the expression of CRX, leading to the occurrence of EMT in RPE cells. In order to address that the Hippo signaling pathway regulates CRX expression, we used the Hippo signaling pathway inhibitor XMU-MP-1 to treat cells for 4 days after cells were sub-cultured, and found that 5 \u0026micro;M XMU-MP-1 markedly down-regulated the expression level of CRX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 5 \u0026micro;M XMU-MP-1 did not significantly increase the cell death, but enlarged the cells (Supplementary Fig.\u0026nbsp;3). We further displayed that XMU-MP-1 significantly increased the nucleus retention ratio of YAP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), decreased the amount of phosphorylated YAP1, and reduced the expression level of CRX protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G), indicating that the Hippo/YAP1 pathway indeed inhibits CRX expression. Additionally, we observed that XMU-MP-1 treatment did not alter the nuclear retention ratio of CRX in ESC-RPE cells, indicating that Hippo signaling pathway does not regulate the shuttling of CRX into nucleus (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEmploying transcription factor binding prediction software (TF-Target Finder, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jingle.shinyapps.io/TF_Target_Finder/\u003c/span\u003e\u003cspan address=\"https://jingle.shinyapps.io/TF_Target_Finder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we identified that TEAD has the potential to bind to the \u003cem\u003eCRX\u003c/em\u003e promoter. Nevertheless, chromatin immunoprecipitation (ChIP) experiments demonstrated that neither TEAD nor YAP1 could directly bind to the \u003cem\u003eCRX\u003c/em\u003e promoter region (Supplementary Fig.\u0026nbsp;5). This finding strongly indicates that YAP1 does not directly exert an inhibitory effect on CRX expression.\u003c/p\u003e \u003cp\u003ePrevious studies reported that YAP1 can bind to β-catenin, promoting its nuclear retention, and can form a transcription complex with β-catenin and TCF to regulate gene expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We found that in RPE cells with tight junctions, β-catenin is mainly distributed at the cell junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). After sub-culturing, β-catenin is distributed in the cytoplasm and the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Co-immunoprecipitation (CoIP) experiments confirmed that YAP1 was able to bind to β-catenin, and when the Hippo signaling pathway is inhibited by XMU-MP-1, the entry of β-catenin into the nucleus increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K). When RPE cells were treated with the β-catenin inhibitor MSAB, the expression level of β-catenin decreased, and the expression level of CRX increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-N). 5 \u0026micro;M MSAB did not significantly increase the cell death (Supplementary Fig.\u0026nbsp;3). These results suggest that when the Hippo signaling pathway is inactivated, YAP1 enters the nucleus and promotes the nuclear retention of β-catenin, thereby inhibiting CRX expression.\u003c/p\u003e \u003cp\u003eTo further validate the regulation of CRX gene expression by β-catenin, we utilized software to predict that TCF7 was capable of binding to the CRX promoter, and verified that by ChIP-quantitative real-time PCR (qRT-PCR) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO-R). We further found that β-catenin binded to TCF7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS). Upon knockdown of TCF7, the expression level of CRX increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eT, U). These results demonstrated that β-catenin/TCF7 inhibits CRX expression.\u003c/p\u003e \u003cp\u003eThe above mentioned results confirm that β-catenin/TCF7 mediates the inhibitory effect of YAP1 on CRX expression. With the formation of tight junctions, YAP1 in the nucleus gradually decreases, and the expression level of CRX increases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRX suppresses EMT in RPE cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we discovered that ESC-RPE cells were more prone to undergo EMT when sub-cultured at a low density [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. When we performed sub-culturing at a 25% ratio, we observed that the ESC-RPE cells were not able to maintain epithelial morphology after being cultured for 8 days compared with those with the sub-culturing at a 50% ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The expression of α-SMA was significant increased, while the expression of CRX was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). To validate the function of CRX in inhibiting EMT, we overexpressed CRX (OE-CRX) in ESC-RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H). It was found that on the 4th day of sub-culturing, overexpression of CRX could inhibit the occurrence of EMT in cells, the expression of E-cadherin and occludin increased, while the expression of α-SMA and vimentin decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the formation of fibrotic scar, apart from the disruption of cell-cell junctions in RPE cells activating the YAP1 signaling pathway, another crucial inducer is TGF-β1 [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To further validate the function of CRX in resisting EMT, when sub-culturing cells, we simultaneously used TGF-β1 to induce EMT in cells to simulate the conditions for the fibrotic scar formation in vivo. The results showed that TGF-β1 treatment could reduce the expression levels of E-cadherin and occludin, while increasing the expression of α-SMA and vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL-N). However, overexpression of CRX could counteract the effects of TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL-N). These results demonstrate that CRX can play a role in inhibiting EMT in RPE cells. Further experiments were conducted by overexpressing and knocking down CRX in iPSC-RPE cells to verify the role of CRX in inhibiting EMT in RPE cells (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003ePreviously, we found TGF-β1 could promote β-catenin's nuclear translocation in ARPE19 cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, in ESC-RPE cells, TGF-β1 facilitated the nuclear translocation of β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO, P), MSAB, in contrast, reduced this translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO, P). TGF-β1 induced EMT, increasing α-SMA and decreasing CRX expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ-S). However, inhibiting β-catenin with MSAB increased CRX expression and inhibited EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ-S). These results demonstrated that TGF-β1 inhibits CRX expression, at least in part, through the β-catenin pathway.\u003c/p\u003e \u003cp\u003eCRX functions as a pivotal transcription factor in the differentiation process of photoreceptor cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Aiming to determine whether the overexpression of CRX in RPE cells instigates their differentiation into photoreceptor cells, the RNA-seq data was analyzed, photoreceptor-associated genes such as \u003cem\u003eRCVRN\u003c/em\u003e, \u003cem\u003eRHO\u003c/em\u003e, \u003cem\u003eNRL\u003c/em\u003e, and \u003cem\u003eNR2E3\u003c/em\u003e exhibited no substantial alterations. In contrast, RPE-associated genes including \u003cem\u003eMITF\u003c/em\u003e, \u003cem\u003eRPE65\u003c/em\u003e, \u003cem\u003eRALBP1\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, and \u003cem\u003eBEST1\u003c/em\u003e underwent up-regulation (Supplementary Fig.\u0026nbsp;7A). Subsequently, the gene expression levels were verified by qRT-PCR. The expression of RPE-related genes demonstrated an upsurge, whereas that of photoreceptor-related genes displayed a mixed pattern with overall low levels (Supplementary Fig.\u0026nbsp;7B). Based on our previously published chromatin immunoprecipitation sequencing (ChIP-seq) data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, GSE174063) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it was evident that CRX binds to the promoter of \u003cem\u003eRPE65\u003c/em\u003e, that was further validated by ChIP-qRT-PCR (Supplementary Fig.\u0026nbsp;7C, D). These data suggest overexpressing CRX in RPE cells does not induce their differentiation into photoreceptor cells. However, it holds the potential to enhance the expression of RPE associated genes, particularly that of RPE65, thereby enhancing the physiological functions of RPE cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePPP2R2B mediates the inhibitory effect of CRX on TGF-β1-induced EMT\u003c/h3\u003e\n\u003cp\u003eTo elucidate the molecular mechanism by which CRX inhibits EMT in ESC-RPE cells, we conducted RNA-seq analysis on the normal control (Control) group, the CRX overexpression group (OE-CRX), the TGF-β1-induced group (Control-TGF-β1), and the TGF-β1-induced OE-CRX group (OE-CRX-TGF-β1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Volcano plots displayed the differentially expressed genes (DEGs) among different comparison groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). Through Venn analysis of the DEGs, we screened out 9 CRX target genes that related to EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The qRT-PCR detection determined that the expression level of \u003cem\u003ePPP2R2B\u003c/em\u003e exhibited the most significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). \u003cem\u003ePPP2R2B\u003c/em\u003e encodes a member of the protein phosphatase 2A (PP2A) B-subunit family [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The B-subunit serves as a structural component of the PP2A complex, enabling PP2A to interact with diverse substrates and execute various functions in different cellular processes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We selected PPP2R2B as the target gene for our further study and found that overexpression of CRX in ESC-RPE cells promoted the expression of PPP2R2B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on our previously published ChIP-seq data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, GSE174063) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we next identified that CRX can bind to the promoter region of PPP2R2B, which was verified by ChIP-qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). To explore the regulatory role of PPP2R2B in EMT of ESC-RPE cells, we first overexpressed PPP2R2B in ESC-RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Overexpression of PPP2R2B (OE-PPP2R2B) could partially counteract the alterations in EMT-related proteins induced by TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). We then knocked down PPP2R2B in CRX-overexpressed ESC-RPE cells (OE-CRX-sh\u003cem\u003ePPP2R2B\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I). We detected the knockdown efficiency of PPP2R2B by qRT-PCR and Western blotting, and selected OE-CRX-sh\u003cem\u003ePPP2R2B\u003c/em\u003e-1 for subsequent experiments. Overexpression of CRX could inhibit the down-regulation of epithelial markers such as E-cadherin and occludin, as well as the increase in the expression of mesenchymal markers like α-SMA and vimentin induced by TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, K). However, knockdown of PPP2R2B could counteract the inhibitory effect of CRX on EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, K). Furthermore, through scratch assays, we found that TGF-β1 significantly enhanced the migratory capacity of ESC-RPE cells. Nevertheless, these effects were inhibited by CRX overexpression, and the inhibitory effect was counteracted by knockdown of PPP2R2B (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL, M). The above results indicate that PPP2R2B exerts an inhibitory effect on EMT, and the anti-EMT effect of CRX is mediated, at least in part, by PPP2R2B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCRX reduced laser-induced CNV and subretinal fibrosis in mice\u003c/h3\u003e\n\u003cp\u003eTo evaluate the role of CRX in inhibiting EMT of RPE cells in vivo, we first established a laser-induced CNV mouse model. On the 3rd, 7th, and 21st days after laser induction, the expression of CRX in RPE cells at the laser-induced lesion site, adjacent to the lesion site, and distal to the lesion site was detected. The results showed that on the 3rd and 7th days, the number of RPE cells with positive CRX expression in the nucleus at the site adjacent to the lesion site and distal to the lesion site was significantly increased compared with the control group, and the RPE cells adjacent to the lesion site showed the most significant change (Supplementary Fig.\u0026nbsp;8A, B). We further examined the expression of YAP1 and found that on the 3rd day, a large number of RPE cells at the lesion site were positive for YAP1 nuclear expression. Thereafter, the number of RPE cells with positive nuclear YAP1 expression gradually decreased (Supplementary Fig.\u0026nbsp;8A, B). In contrast, at 7 and 21 days, some RPE cells in the distal area still had positive YAP1 nuclear expression (Supplementary Fig.\u0026nbsp;8A, B). This result indicates that RPE cells at the lesion site were damaged, and some RPE cells rapidly underwent EMT. The RPE cells adjacent to the lesion site, similar to the slow-migrating cells in the scratch assay (Supplementary Fig.\u0026nbsp;2), expressed both CRX and YAP1 in the nucleus, with CRX playing a role in inhibiting EMT. The RPE cells in the distal area were still affected by the microenvironment, with some RPE cells showing positive nuclear expression of both CRX and YAP1 (Supplementary Fig.\u0026nbsp;8A, B).\u003c/p\u003e \u003cp\u003eTo determine the process of CRX in inhibiting EMT, we prepared lenti-CRX virus and administered it before laser photocoagulation via subretinal injection in the right eye, with empty vector serving as the control in the left eye (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We observed that the lentivirus mainly infected RPE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). We performed Masson staining and immunostaining on the choroidal flatmounts. Masson staining revealed that, compared to the empty vector group, significant disruptions were evident in the choroid and outer nuclear layer at the center of the laser burn 7 days after laser photocoagulation. Subsequently, 21 days after laser photocoagulation, retinal edema and newly-formed blood vessels extended into the subretinal space (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Compared with the empty vector group at days 7 and 21, the collagen area in the OE-CRX group was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results indicate that overexpression of CRX can reduce laser-induced SF in mouse retinas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrates that CRX functions as an endogenous signaling entity within RPE cells, effectively counteracting EMT. During the EMT process, the TGF-β1 and Hippo/YAP1 signaling cascades promote the nuclear translocation of β-catenin that combines with TCF7 to bind to promoter of CRX, thereby inhibiting CRX expression. Notably, overexpression of CRX leads to the up-regulation of PPP2R2B, which in turn suppresses EMT in RPE cells. The EMT-inhibitory efficacy of CRX has been experimentally validated in a laser-induced CNV mouse model.\u003c/p\u003e \u003cp\u003eDuring retinal development, CRX is considered a pivotal transcription factor implicated in photoreceptor cell fate determination [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Mutations in the human CRX gene are associated with several retinal disorders, including cone-rod dystrophy and Leber\u0026rsquo;s congenital amaurosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previous studies posited that CRX is not expressed in RPE cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, other research has detected the expression of CRX in human RPE cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Noriko Esumi not only confirmed the expression of CRX in human and bovine RPE cells but also discovered that CRX binds to the \u003cem\u003eBEST1\u003c/em\u003e proximal promoter [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We further confirm that CRX is expressed in ESC-RPE and iPSC-RPE cells and can promote the expression of RPE65.\u003c/p\u003e \u003cp\u003ePreviously, we found that transcription factors such as CRX, MITF, NR2E1, and cMYC could convert dedifferentiated RPE cells into iRPE cells with anti-EMT properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CRX can regulate the expression of multiple genes related to EMT[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this study, we further discovered that CRX can promote the expression of PPP2R2B, a member of the protein phosphatase 2A (PP2A) B-subunit family. PP2A belongs to the Ser/Thr protein phosphatase family, which consists of a structural scaffold A-subunit, a highly conserved catalytic C-subunit, and diverse regulatory B-subunit [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The B-subunit serves as a structural component of the PP2A complex, enabling PP2A to interact with different substrates and execute various functions in diverse cellular processes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Research has shown that PPP2R2B can inhibit EMT in bladder cancer cells by suppressing the WNT signaling pathway and reducing the expression of β-catenin [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. PPP2R2B inhibits the proliferation and migration of pancreatic cancer cells by suppressing the mTOR pathway [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Using okadaic acid to inhibit PP2A can induce EMT in pancreatic tumor cells, and okadaic acid can strongly inhibit the expression of PPP2R2B [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. PP2A inhibits the EMT process of podocytes by suppressing the JIP4/p38-MAPK pathway [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A significant amount of β-catenin is sequestered by E-cadherin at the plasma membrane, and knockdown of E-cadherin leads to the redistribution of β-catenin in the cytoplasm, followed by nuclear translocation and subsequent activation of the Wnt signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We found that overexpression of PPP2R2B can promote the expression of E-cadherin. It is highly likely that PPP2R2B inhibits the EMT process by increasing E-cadherin and reducing the nuclear translocation of β-catenin.\u003c/p\u003e \u003cp\u003eCrx functions as a transcription activator and cooperates with the bZIP transcription factor Nrl to activate the expression of the rhodopsin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This finding indicates that a high level of rhodopsin expression demands the function of at least two photoreceptor transcription factors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, both Crx and NeuroD1 are necessary to induce monkey iris-derived cells to acquire the photoreceptor phenotype [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results imply that the interaction with other photoreceptor transcription factors is crucial for Crx to facilitate photoreceptor differentiation. This also accounts for the fact that in our study, merely overexpressing CRX in RPE cells did not lead to their transformation into photoreceptor cells. Instead, it even promoted the expression levels of RPE cell-specific genes, as evidenced by the significant increase in \u003cem\u003eRPE65\u003c/em\u003e. Consequently, overexpressing CRX in RPE cells not only inhibits the occurrence of EMT in RPE cells but also holds the promise of enhancing the physiological functions of RPE cells.\u003c/p\u003e \u003cp\u003eDuring the development of CNV, new blood vessels in the choroid rupture Bruch's membrane and grow beneath the RPE. They further extend into the subretinal space to form SF, the integrated RPE cells are damaged. The disruption of intercellular junctions inevitably inactivates the Hippo signaling pathway, leading to the activation and nuclear translocation of YAP1, thereby promoting the EMT of RPE cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The expression level of YAP1 is significantly elevated in CNV tissues. Employing RNA interference technology to reduce the expression of YAP can decrease the expression of α-SMA in CNV tissues [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the early stage of the laser-induced CNV model, we also observed the nuclear translocation of YAP1 in RPE cells.\u003c/p\u003e \u003cp\u003eAnother crucial factor in inducing EMT in RPE cells is the TGF-β1. Research has shown that the expression of TGF-β1 is significantly increased in the vitreous humor of wet AMD [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and the amount of TGF-β1 is also remarkably elevated in the CNV model [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. TGF-β1 can rapidly promote EMT in RPE cells. We found that YAP1 and TGF-β1 can synergistically inhibit the expression of CRX by promoting the nuclear translocation of β-catenin. β-catenin is a key molecule in the WNT signaling pathway, and its nuclear translocation can promote the EMT process [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previously, we demonstrated that β-catenin can translocate into the nucleus under the stimulation of TGF-β1, promoting the EMT process in ARPE19 cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The decrease in CRX expression induced by TGF-β1 is also achieved through β-catenin. The transcriptional co-activator proteins YAP/TAZ are the hubs of the Hippo pathway. Although YAP/TEAD is not able to directly bind to the promoter region of CRX, YAP1 can also bind to β-catenin and promote its nuclear translocation. It has been reported that YAP1 interacts with and stabilizes the β-catenin protein in the nucleus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and YAP1 could directly interact with β-catenin in the nucleus and form a transcriptional YAP/β-catenin/TCF4 complex [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our results are consistent with these reports. Therefore, during the EMT process of RPE cells, signaling pathways such as TGF-β1, Wnt/β-catenin, and Hippo/YAP1 interact with each other and also have a synergistic regulatory effect on the expression of CRX.\u003c/p\u003e \u003cp\u003eThe two principal pathogenic manifestations of wet AMD are MNV and SF [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] Anti-VEGF therapy has achieved encouraging outcomes in the treatment of MNV. However, there remains no effective approach for managing SF, which typically leads to permanent visual impairment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. SF develops in the late stage of wet AMD. Multiple cell types, including RPE cells, macrophages, m\u0026uuml;ller cells, pericytes, and endothelial cells, transdifferentiate into myofibroblasts, promoting the formation of SF and collagen deposition. Pathological analysis of surgically obtained MNV membranes has revealed that the majority of cells are co-stained with α-SMA and cytokeratin. Since only RPE cells express cytokeratin, RPE cells represent the predominant cell type within MNV membranes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, the EMT of RPE cells into myofibroblasts is the primary source of SF formation.\u003c/p\u003e \u003cp\u003eNumerous inhibitors targeting signaling pathways associated with RPE cell EMT have been developed to suppress the formation of SF. For instance, knockdown of Galectin-1 can inhibit the TGF-β1/Smad2/Snail signaling axis, thereby attenuating subretinal fibrosis in mice [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The expression of YAP1 is significantly elevated in CNV tissues. Employing RNA interference technology to reduce YAP1 expression can decrease the expression of α-SMA in CNV tissues [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. RO4929097, a selective γ-secretase inhibitor, directly inhibits the Notch signaling pathway and indirectly suppresses the ERK1/2 signaling pathway in ARPE-19 cells and a laser-induced mouse model [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We have demonstrated that treatment with H535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) effectively attenuates subretinal fibrosis and EMT in laser-induced CNV mice and ARPE19 cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This study reveals that CRX serves as an endogenous EMT-inhibitor within RPE cells that will provide novel research target for the treatment of wet AMD, especially in the prevention of the formation of SF.\u003c/p\u003e \u003cp\u003eAlthough we have elucidated that the Hippo/YAP1/β-catenin and TGF-β1/β-catenin pathways synergistically inhibit the expression of CRX, and the nuclear translocation of CRX is not regulated by the Hippo signaling pathway, the signaling pathway that regulates the nuclear shuttling of CRX remains unclear. In addition, PPP2R2B can exert an inhibitory effect on EMT, but its downstream targets still require further investigation.\u003c/p\u003e \u003cp\u003eIn summary, this study utilized the published single-cell sequencing data to confirm the expression of CRX in human RPE cells and verified it in vitro-cultured ESC-RPE and iPSC-RPE. Sub-culturing was used to disrupt intercellular connections and thus inhibit the Hippo signaling pathway, combined with TGF-β1 treatment to simulate the microenvironment for the SF formation. It was found that Hippo/YAP1 and TGF-β1 synergistically promoted the nuclear translocation of β-Catenin, and the latter combined with TCF7 to bind to \u003cem\u003eCRX\u003c/em\u003e promoter to inhibit the expression of CRX (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Overexpression of CRX could suppress the EMT of RPE cells by up-regulating the expression of PPP2R2B. In the laser-induced mouse CNV model, the nuclear translocation of CRX also occurred in RPE cells, and overexpression of CRX played an inhibitory role in the formation of subretinal fibrosis. This study identified CRX in RPE cells as an endogenous signal molecule that inhibits EMT, providing a new treatment strategy and research target for the treatment of wet AMD and the prevention of FS formation. It also offered a new treatment method for other ophthalmic diseases with EMT of RPE as the critical pathogenesis, such as proliferative vitreoretinopathy, diabetic retinopathy, and Inherited retinal degenerations [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures\u003c/h2\u003e \u003cp\u003eAccording to our previously described protocol, retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (ESC-RPE) and induced pluripotent stem cells (iPSC-RPE) were cultured in medium (Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium: Nutrient Mixture F-12 (DMEM/F-12, Gibco, Grand Island, NY, USA), 10% knockout serum replacement (KSR, Gibco), supplemented with 2 mM L-glutamine (Gibco, USA), 1% MEM nonessential amino acids (MEM NEAA, Gibco),1% penicillin-streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 10 mM nicotinamide (Sigma, St. Louis, MO, USA) in dishes precoated with 1% Matrigel (Corning Inc, Corning, NY, USA) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The RPE cells at passages 3\u0026ndash;5 were used in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of the single-nuclei RNA-seq data of retinal tissues\u003c/h3\u003e\n\u003cp\u003eThe single-nuclei RNA-seq data of 106 human retina and RPE/choroid with subtype resolution from over 100,000 cells (including the information of cell types) were obtained from GEO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database (GSE135133). The merged expression matrix was prepared for clustering using the Seurat 4.4.0, following the common pipeline. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was used to project cells in two dimensions. The function FeaturePlot and VlnPlot in Seurat were used to visualization the gene expressions.\u003c/p\u003e\n\u003ch3\u003eAnalysis of microarray data\u003c/h3\u003e\n\u003cp\u003eThe microarray data of human RPE/choroid, which includes 7 normal samples, 7 dry AMD samples, and 7 wet AMD samples, and the microarray data of human retina that includes 7 AMD samples (dry and wet AMD) (with the age of all the individuals being over 75) were obtained from the GEO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database (GSE29801). The relative level of \u003cem\u003eCRX\u003c/em\u003e in each group was compared.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eFor immunofluorescence analysis, fixed cells and cryosections from eyes were permeabilized with 0.25% Triton X-100 (Sigma) for 2 min, washed with PBS, and then blocked with 2% bovine serum albumin (BSA, Sigma) in PBS. The samples were incubated with the primary antibodies against α-SMA (Abcam, Cambridge, UK), ZO-1 (Proteintech, Chicago, USA), CRX (Proteintech), YAP1 (Proteintech), β-catenin (Proteintech) overnight at 4℃. They were then washed three times with PBS, followed by incubation with the fluorescent secondary antibodies overnight. 4,6 diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) was used to indicate the nucleus. The samples were then examined by fluorescence microscope (Olympus IX73, Tokyo, Japan). Antibodies were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative real-time PCR (\u003c/b\u003eqRT-PCR\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal RNA was extracted and reverse transcription was performed using Primescript\u0026trade; RT Master Mix kit (Takara, Shiga, Japan). qRT-PCR was performed in a Chromo4 instrument cycler (Bio-Rad, Hercules, USA) using Superreal Premix plus kit (Tiangen Biotech, Beijing, China). PCR amplification was carried out with the following cycling parameters: denaturation at 95\u0026deg;C for 5 min, followed by 40 cycles of 95\u0026deg;C for 10 s, 60\u0026deg;C for 30 s. Primer sequences (Synthesized by Sangon Biotech, China) were listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe cells were lysed by RIPA buffer containing protease and phosphatase inhibitor (Sigma). The protein extracts (20\u0026micro;g per sample) were separated by 10% SDS-PAGE gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against α-SMA, E-cadherin (Cell Signaling Technology, Beverly, MA, USA), Vimentin (proteintech), YAP1, phospho-YAP1(Ser397) (proteintech), β-catenin, CRX, TCF7 (Cell Signaling Technology), PPP2R2B (abcam), and β-actin or GAPDH (proteintech) for 12h at 4℃, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. The blots were visualized with a chemiluminescence imaging system (Tanon 5200, Shanghai, China) and quantified with ImageJ software (Version 1.48v). Antibodies were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eProtein A\u0026thinsp;+\u0026thinsp;G magnetic beads (20 \u0026micro;L) were incubated with 5 \u0026micro;g antibodies (against TCF7, β-Catenin) or normal 5 \u0026micro;g IgG for 1h. 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were lysed by 200 \u0026micro;L IP lysis buffer (Beyotime, Shanghai, China) and incubated with prepared conjugated Protein A\u0026thinsp;+\u0026thinsp;G magnetic beads at 4℃ overnight. The beads were washed with TBS for three times and the binding proteins were eluted with protein loading buffer at 95℃ for 5min. After centrifugation, the samples were collected and used for Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay\u003c/h2\u003e \u003cp\u003eThe ChIP assay was performed using a SimpleChIP Plus Enzymatic Chromatin IP kit (Cell Signaling Technology). Briefly, Ten million cells were treated with 1% formaldehyde for 10min and unreacted formaldehyde was quenched with glycine for 5 min. The cells were collected by scraping and were lysed with lysate buffer, the nuclear pellet was digested by micrococcal nuclease. Immunoprecipitation was performed on the lysate with 2 \u0026micro;g antibody against YAP1, β-catenin, TCF7, CRX (proteintech), or control rabbit IgG at 4\u0026deg;C overnight. Then, the immune complexes were incubated with Protein G magnetic beads (Cell Signaling Technology) for 2 h. Chromatin was eluted from antibody/protein G magenetic beads and used for qRT-PCR. The ChIP-qPCR primer sequences for the promoters of CRX, PPP2R2B, and RPE65 are summarized in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eScratch assay\u003c/h2\u003e \u003cp\u003eCells were plated into 24-well culture plate and grow to confluency. Then, scratches on ESC-RPE cells monolayers were made with a sterilized 200-\u0026micro;L pipette tip and then gently washed twice with sterile PBS to remove floating debris. Images were recorded after 0, 24, and 48 h, and the wound recovery was analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of CRX and PPP2R2B\u003c/h2\u003e \u003cp\u003eFor generating lentivirus, human cDNAs of CRX and PPP2R2B were obtained by PCR amplification from ESC-RPE cells and cloned into lentiviral pLVX-mCMV-ZsGreen1-Puro vector (Takara). The packaging plasmids were psPAX2 and pMD2.G. HEK293FT cells were seeded at a density between 5.0\u0026ndash;7.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and transfected by Lipofectamine 2000 (Invitrogen) with each vector. Individual supernatants containing virus were harvested at 48 h post-transfection and filtered with a 0.45 \u0026micro;m PVDF membrane (Millipore, Boston, USA). ESC-RPE cells and iPSC-RPE cells were plated in 6-cm culture dishes, respectively. The next day, cells were infected with viruses. The positively transfected cells were sorted by FACS based on ZsGreen expression. The expression levels were determined by qRT-PCR and Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of lentiviral vector to knockdown target genes\u003c/h2\u003e \u003cp\u003eHEK293T cells were seeded in 15 cm dishes at a density of 10\u003csup\u003e7\u003c/sup\u003e cells per dish 24 h before transfection. Lentiviral pLVX-shRNA2-ZsGreen1 (Takara) vector were used to prepare lentiviruses, the packaging plasmids are psPAX2 and pMD2.G. HEK293FT cells were transfected with vectors. Individual supernatants containing virus were harvested at 48h and used to infect cells. Reduced expression of target genes at the transcript level was determined by qRT-PCR. The targeting sequences of shRNAs for \u003cem\u003eCRX\u003c/em\u003e, \u003cem\u003eTCF7\u003c/em\u003e, and \u003cem\u003ePPP2R2B\u003c/em\u003e were included in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e. The most effective shRNA sequence was selected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003eFollowing the Illumina mRNA-seq protocol, pooled RNA libraries of the cells were established, with 50ng of RNA from RPE cells from different groups. Sequencing was performed by the MAJORBIO company (Shanghai, China). Filtering and quality control of the raw reads from RNA-seq was carried-out using FastQC. The clean reads were mapped to reference sequences using SOAP2 aligner. Gene expression levels were calculated using the TPM method. Log\u003csub\u003e2\u003c/sub\u003e fold change (FC) of TPM was used to identify differentially expressed genes (DEGs) between these two groups. Only those genes indicating log |FC| \u0026gt; 1 and adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as DEGs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCalcein/PI staining\u003c/h2\u003e \u003cp\u003eCells were cultured in 96-well culture plate. 1\u0026ndash;5 \u0026micro;M XMU-MP-1 or MSAB (MedChemExpress, Shanghai, China) was used to treat ESC-RPE cells. For Calcein/PI staining, cells were cultured in 100\u0026micro;L of calcein AM/PI working solution (Beyotime) at 37\u0026deg;C for 1h. The samples were then examined by a fluorescence microscope (Olympus IX73)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eIn this study, 8-week-old C57BL/6J male mice (from the Laboratory Animal Center of Tongji University) were utilized. They were housed in groups and were allowed a period to acclimatize to the laboratory environment with a 12 / 12 h light / dark cycle and provided with food and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSubretinal injection of virus and laser-induced CNV model\u003c/h2\u003e \u003cp\u003eA total of 40 C57BL/6J male mice were divided into laser-empty vector groups and laser-overexpression of CRX (OE-CRX) groups. mice were firstly anaesthetized by 1.25% tribromoethanol, their pupils were dilated with amethocaine (0.5%) and tropicamide (0.5%). A channel was created by inserting a 30-gauge needle, behind the limbus, into the vitreous chamber. A 33-gauge needle was inserted into the subretinal space of the central retina, and 2 \u0026micro;L lentivirus carrying the \u003cem\u003eCRX\u003c/em\u003e gene was injected. The control eyes received an injection of lentivirus carrying an empty vector. Seven days after subretinal injection of virus, the laser-induced subretinal fibrosis model was established as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In brief, four to six laser spots (532 nm, 120 mW, 100 ms; Novus Spectra, Japan) were selected at each fundus around the optic disc and avoid tiny blood vessels. The disruption of Bruch\u0026rsquo;s membrane was confirmed by visually subretinal bubble formation immediately after laser application. After laser burns, the mice were randomly sacrificed on day 7 and day 21 for further quantification of CNV and subretinal fibrosis. Mice were randomly allocated to experimental groups and no blinding method was used for subretinal injection. Eyes with a damaged lens or detached retina due to subretinal injection were excluded from experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMasson\u0026rsquo;s trichrome staining\u003c/h2\u003e \u003cp\u003eThe eyes were obtained at days 7 and 21 after laser induction. The samples were fixed with 4% PFA and embedded in paraffin to prepare the 3 \u0026micro;mthick sections. Masson\u0026rsquo;s trichrome staining was performed with a trichrome staining kit (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Collagen fibers were stained blue, and images were obtained by microscope (Olympus IX73).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eRPE/choroid Flatmount Staining\u003c/h2\u003e \u003cp\u003eThe areas of CNV and collagen fibers were determined on RPE/choroidal flatmounts on day 7 and day 14. Mouse eyecups were fixed in 4% Paraformaldehyde (PFA), and anterior segments were removed before cutting six to eight radial incisions to be flattened. The RPE-choroid complexes were washed, blocked with 5% BSA and permeabilized with 0.3% Triton X-100, and then incubated with collagen type I antibody (abcam) (for evaluating subretinal fibrosis) at 4\u0026deg;C overnight. They were washed three times with PBS, followed by incubation with the fluorescent secondary antibodies overnight. Samples were finally observed under fluorescence microscope (Olympus IX73).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll values are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Data were analyzed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). All statistical analyses were performed using unpaired Student\u0026rsquo;s t-test, or one-way ANOVA and post hoc Bonferroni\u0026rsquo;s test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY.\u0026nbsp;\u003c/strong\u003eAll study data are included in the main text and/or SI Appendix. The raw data of RNA-seq have been deposited in the NCBI Sequence Read Archive (SRA) database under accession number: PRJNA1169190.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Zhenzhen Zhao, Xueying Wang, and Qian Wang for their expertise and technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch design and conceptualization: DL, QO, FG, and HT. Methodology: DL, QO, FG, XW, LZ, and YZ. Data analysis: J-YX, CJ, JW. Writing-original draft: DL, QO, F.G., and HT. Writing-review \u0026amp; editing: JZ, JL,YB, LL, and G-TX. Supervision and funding: QO, LL, G-TX, and HT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by grants from the Natural Science Foundation of Shanghai (24ZR1470100), National Natural Science Foundation of China (82471073, 82271108), and Shanghai Municipal Health Commission (20234Y0113).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were carried out in accordance with the institutional guidelines and the Guide for the Care and Use of Laboratory Animals issued by the NIH, and the guidelines of the animal experimentation ethics committee of Tongji University (Approved NO. TJAA09624101), and in line with the Association for Research in Vision and Ophthalmology Statement for the use of Animals in Ophthalmic and Vision Research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMathis T, Holz FG, Sivaprasad S, Yoon YH, Eter N, Chen LJ, et al. Characterisation of macular neovascularisation subtypes in age-related macular degeneration to optimise treatment outcomes. Eye (London, England). 2023;37(9):1758\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaniel E, Toth CA, Grunwald JE, Jaffe GJ, Martin DF, Fine SL, et al. Risk of scar in the comparison of age-related macular degeneration treatments trials. Ophthalmology. 2014;121(3):656\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeo KYC, Joe AW, Nguyen V, Invernizzi A, Arnold JJ, Barthelmes D, et al. 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Cell Death Discov. 2023;9(1):243.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, Zhang J, Liu Z, Gong Y, Zheng Z. Human umbilical cord mesenchymal stem cell-derived exosomal miR-27b attenuates subretinal fibrosis via suppressing epithelial\u0026ndash;mesenchymal transition by targeting HOXC6. Stem Cell Research \u0026amp; Therapy. 2021;12:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5969324/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5969324/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is one of the significant pathogenic mechanisms for the formation of subretinal fibrosis in age-related macular degeneration (AMD). Multiple signaling pathways that promote EMT have been well described, yet the endogenous signaling pathways that inhibit EMT within RPE cells remain largely elusive. In this study, we confirmed the expression of CRX in human RPE cells and human embryonic stem cell-derived RPE (ESC-RPE) cells. By employing sub-culture to disrupt intercellular connections and thereby inhibit the Hippo signaling pathway, combined with TGF-β1 treatment in vitro to mimic the microenvironment for the formation of subretinal fibrosis, it was revealed that Hippo/YAP1 and TGF-β1 synergistically promoted the nuclear translocation of β-catenin, and the latter bound to TCF7 to inhibit the expression of CRX. Overexpression of CRX was capable of suppressing the occurrence of EMT in ESC-RPE cells. CRX exerted its inhibitory effect on EMT partly by upregulating the expression of PPP2R2B. In the laser-induced choroidal neovascularization mouse model, the nuclear translocation of CRX took place in RPE cells, and overexpression of CRX played an inhibitory role in the formation of subretinal fibrosis. This study has identified CRX as an endogenous signaling molecule that inhibits EMT in RPE cells and has provided a new research target and treatment strategy for the treatment of wet AMD and the inhibition of subretinal fibrosis formation.\u003c/p\u003e","manuscriptTitle":"Unraveling the role of CRX as a potent intrinsic suppressor of epithelial-mesenchymal transition in retinal pigment epithelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 19:00:43","doi":"10.21203/rs.3.rs-5969324/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d544715-905b-4b36-a652-612c8097108d","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46184084,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases"},{"id":46184085,"name":"Biological sciences/Cell biology/Cell signalling"}],"tags":[],"updatedAt":"2025-05-02T13:50:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-02 19:00:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5969324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5969324","identity":"rs-5969324","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}