Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy

The polarized retinal pigment epithelial cell

The human RPE cell achieves terminal differentiation at four to six weeks of gestation and subsequently remains mitotically quiescent (Lutty & McLeod, 2018; Stern & Temple, 2015). The RPE, which is situated between the photoreceptors and the choroid, plays many complex roles indispensable to the health of the neural retina and the choroid. These roles include recycling of components of the visual cycle, absorption of light to protect from photo-oxidative stress, production of essential growth factors, immunological regulation of the eye, phagocytosis of photoreceptor outer segments generated during daily photoreceptor renewal, and transportation across the blood retina barrier (BRB) (Ferrington, Sinha & Kaarniranta, 2016; Fields et al., 2019; Mateos et al., 2014; Naylor et al., 2019; Strauss, 2005; Vigneswara et al., 2015). In order to maintain these multiple functions, RPE cells display a highly specialized structural and functional polarity.

Similar to other epithelia, the RPE displays three characteristics of the epithelial phenotype: apical plasma membrane, junctional complexes, and basolateral domain. RPE cells display structural polarity, with apical microvilli and melanosomes, and basal microinfolds. The abundant melanin granules in RPE cells absorb stray light, a process that is essential for visual function (Strauss, 2005). In a polarized cell, the distributions of surface proteins on the apical and basal plasma membranes are different, contributing to the performance of cellular functions (Khristov et al., 2018). However, a highly polarized distribution of ion channels, transporters and receptors in RPE is different from that observed in conventional extraocular epithelia (Lehmann et al., 2014). For example, Na, K-ATPase (Sonoda et al., 2009) and monocarboxylate transporters (MCT) 1 (Deora et al., 2005) are localized to the apical aspect of RPE cells, while chloride transporter CFTR (Maminishkis et al., 2006) is basally located. On the apical plasma membrane, RPE cells phagocytize the photoreceptor outer segments, which are regulated by polarized receptors. Bulloj et al. (2018) found that binding of Semaphorin 4D (sema4D) to RPE apical receptor Plexin-B1 suppresses outer segment internalization, contributing to the maintenance of photoreceptor function and longevity. The RPE also transports fluid out of the subretinal space, and regulates bidirectional nutrient transport between the outer retina and the choroid, in a manner dependent on the polarized distribution of membrane channels and transporters (Strauss, 2005). The RPE basolaterally secretes extracellular matrix components and factors, which participate in ECM remodeling and maintain the outer BRB (oBRB) function (Caceres & Rodriguez-Boulan, 2020). Therefore, the polarized phenotype of the RPE is vital to both the oBRB and is the basis of the homeostasis of the outer retina (Caceres & Rodriguez-Boulan, 2020; Lehmann et al., 2014). The disruption of RPE polarity contributes to the development of several retinal diseases, such as PVR and age-related macular degeneration (AMD). A comprehensive understanding of the way in which this polarity is achieved may provide insights into the pathogenesis of PVR.

However, most available data on RPE polarity is contributed by studies performed on RPE-immortalized cell lines that show partial preservation of the RPE phenotype, and were extrapolated from data obtained from the prototype Madin-Darby Canine Kidney (MDCK) cell line (Lehmann et al., 2014). The detailed mechanisms that determine RPE polarization remain unclear. Some scholars believe that junctional complexes, including adherens junctions (AJs) and tight junctions (TJs), are essential for building epithelial cell polarity and maintaining the integrity of epithelial layers such as RPE (Niessen, 2007; Pei et al., 2019; Tamiya & Kaplan, 2016).

Tight junctions are complex cell–cell junctions formed by transmembrane proteins interactions with peripheral cytoplasmic proteins (Fig. 1). Transmembrane proteins include occludin, members of the claudin family, and junctional adhesion molecules (JAMs). Peripheral cytoplasmic proteins, such as zonula occludens (ZOs), form bridges between transmembrane proteins and the actin filament cytoskeleton and play a key role in the assembly and organization of TJs (Bazzoni & Dejana, 2004; Bazzoni et al., 2000; Naylor et al., 2019).

The RPE tight junctions regulate the paracellular movement of solutes via size and charge selectivity (Benedicto et al., 2017; Caceres et al., 2017; Naylor et al., 2019).Occludin and claudins determine the permeability and semi-selectivity of the TJs, and as such play critical roles in the oBRB (Balda et al., 2000; Fields et al., 2019; Furuse et al., 1998; Günzel & Yu, 2013; Rosenthal et al., 2017). JAMs regulate TJ assembly and function by recruiting other proteins to the TJ and play an important role in the barrier property of TJs (Balda & Matter, 2016; Orlova et al., 2006; Shin, Fogg & Margolis, 2006). In patients with RRD, damage to TJs elicits the breakdown of oBRB and promotes the penetration of growth factors and cytokines, aggravating PVR. As well as having a barrier function, TJs define the physical separation between apical and basal domains of the plasma membrane, to maintain RPE cell polarity (Campbell, Maiers & DeMali, 2017; González-Mariscal et al., 2014; Sluysmans et al., 2017). The two extracellular loops of occludin mediate adhesion of adjacent cells and block the movement of plasma components. The C-terminal domain combines directly with ZOs, subsequently interacting with the actin cytoskeleton, which is essential to organizing and maintaining cell polarization (Balda & Matter, 2016; Furuse et al., 1994; Shin, Fogg & Margolis, 2006; Tarau et al., 2019). Feng et al. (2019) demonstrated that during EMT, the breakdown of TJs resulting from loss of claudin-1 causes ARPE-19 cells to lose their epithelial phenotype and transform into fibroblasts, promoting the development of PVR. TJs are involved in the regulation of signaling pathways that govern various cellular functions such as proliferation, migration, and differentiation (Bhat et al., 2018; Shi et al., 2018; Sluysmans et al., 2017). Vietor et al. (2001) found that decreased amounts of occludin can cause up-regulation and translocation of the adhesion junction protein β-catenin, which interacts with the transcription factor lymphoid enhancer-binding factor (LEF)/T cell factor (TCF) in the nucleus, leading to a loss of the polarized epithelial phenotype in EpH4 cells. ZOs, adaptor proteins within the TJ complex, exhibit dual localization at TJs and in the nucleus. Under injury or stress, the disruption of TJs increases ZO-2 nuclear accumulation, driving its interaction with transcription factors, and inducing MDCK epithelial cell proliferation (Islas et al., 2002; Shi et al., 2018; Traweger et al., 2003). In differentiated RPE cells, the interaction between ZO-1 with ZO-1-associated nucleic acid-binding protein (ZONAB) maintains cell–cell contact by sequestering ZONAB at the TJ or in the cytoplasm, maintaining cells dormancy. However, when damage to TJs decreases ZO-1 levels, ZONAB is translocated into the nucleus, leading to the up-regulation of cyclin D1 (CD1) and subsequent cell proliferation (Balda, Garrett & Matter, 2003; González-Mariscal et al., 2014). Therefore, TJs provide a structural foundation for the maintenance of cell–cell contact. Georgiadis et al. (2010) demonstrated that the overexpression of ZONAB or knockdown of ZO-1 could result in increased RPE proliferation and the development of EMT. Recent research has confirmed that during EMT, ZO-1 is decreased in ARPE-19 cells, and the knockdown of either ZO-1 or AJ protein E-cadherin leads to the downregulation of the other protein, indicating the existence of an interaction between the two junctional complexes (Bao et al., 2019). Due to the importance of TJs in the maintenance of integrity and functionality of epithelial cells, several researchers have focused on novel factors that stimulate the formation of TJs, such as nicotinamide (Hazim et al., 2019) and lysophosphatidic acid (Lidgerwood et al., 2018). Studies into these factors may produce well-differentiated RPE cell lines and a platform to enable the rapid expansion of our understanding of many RPE functions and retinal pathologies. This approach could be conducive to finding novel therapeutic interventions for PVR.

Besides the TJ complex described above, another type of junctional complex called AJs plays a key role in the maintenance of the integrity of epithelial cells and cell–cell contact (Fig. 1). Cadherins, the major proteins of AJs, belong to the glycoprotein superfamily, of which there are more than 20 members. The cytoplasmic domain of cadherins regulates interactions between cadherins and catenins, including β-catenin, α-catenin, and p120-catenin, and other scaffolding proteins such as ZO-1, to maintain cell shape and modulate cell proliferation (Aberle et al., 1994; Nelson & Nusse, 2004; Wheelock & Johnson, 2003). In quiescent adult RPE cells, epithelial cadherins (E- and/or P-cadherin) sequester β-catenin at the AJs to maintain cell–cell contact. Reduction of cadherin levels or dissociation of AJs allows β-catenin to translocate into the nucleus, where it interacts with the transcription factor LEF, and activates the transcription of various genes, including Snail and cyclin D1, which participate in RPE cell EMT via the canonical Wnt/β-catenin signaling pathway (Gonzalez & Medici, 2014; Lamouille, Xu & Derynck, 2014; Nelson & Nusse, 2004; Yang et al., 2018) . Tamiya, Liu & Kaplan (2010) suggested that the loss of P-cadherin causes the loss of cell–cell contact and initiates RPE cell migration and EMT. These events coincide with a switch in cadherin isoform expression from P- to N-cadherin. In addition, hepatocyte growth factor (HGF) and its receptor c-Met can destabilize cell–cell adhesion and elicit nuclear translocation of β-catenin, resulting in RPE cell migration (Lilien & Balsamo, 2005; Liou et al., 2002). Jin et al. found that HGF induces loss or redistribution of junctional proteins ZO-1, occludin, and β-catenin in RPE explants, potentially damaging barrier function and increasing the migration of RPE cells, resulting in retinal detachment(RD) and PVR (Jin et al., 2002; Jin et al., 2004). Given the importance of HGF in the interruption of RPE junction, HGF may be a potential target for the prevention and treatment of PVR. However, this possibility needs further study.

Under physiological conditions in the eye, TJs and AJs maintain the specialized structural and functional polarity of RPE cells and play a pivotal role in the maintenance of cell–cell contact; they sequester EMT signaling effectors ZONAB and β-catenin at the junction or cytoplasm to prevent cells from responding to mitotic factors, causing cells to leave the cell-cycle (Fig. 1). Thus, normally, RPE cells form a cobblestone-like monolayer of immotile, polarized, and mitotically quiescent cells. However, once junctional complexes break down, RPE cells undergo EMT, which is an important contributor to proliferative vitreoretinopathy. In this pathological process, RPE cells lose their structural and functional polarity and transdifferentiate into mesenchymal cells, which proliferate, resist apoptosis, possess migratory ability, and produce abundant ECM, leading to the formation of an aberrant scar-like fibrocellular membrane.

De-differentiated RPE and fibrocellular membrane

Proliferative vitreoretinopathy is characterized by the formation of fibrocellular membranes composed of proliferative and migratory cells and excessive, aberrant ECM. Histopathological analysis of PVR has demonstrated that PVR membranes have contractile activity and strain the retina, leading to tractional retinal detachment (TRD), which is responsible for blurring vision.

Several studies (Feist et al, 2014; Takahashi et al., 2010) have found that the cellular components of PVR membranes include RPE cells, myofibroblasts, fibroblasts, glial cells and macrophages, and that myofibroblasts are critical for the formation and contractile activity of fibrocellular membranes. Based on the indirect immunofluorescence evaluation of human PVR membranes, Feist et al (2014) showed that myofibroblasts originate principally from RPE cells through EMT. Myofibroblasts are characterized by increased expression of alpha-smooth muscle actin (α-SMA) and incorporation of α-SMA into newly formed actin stress fibers, which enhances their contractile properties. Myofibroblasts also secrete excessive matrix and pro-fibrogenic factors, promoting the contraction of PVR membranes that ultimately cause irreversible loss of vision (Gamulescu et al., 2006; Hinz et al., 2001; Shu & Lovicu, 2017; Tamiya & Kaplan, 2016; Tomasek et al., 2002).

In addition to myofibroblasts, abnormally increased ECM reinforces the continuous contractile tension of PVR membranes, and this mechanical tension, together with specialized ECM proteins, regulates myofibroblast differentiation and its function, contributing to PVR. In PVR membranes, the primary components of ECM are collagen and fibronectin. The majority of collagen fibrils are type I collagen, which is synthesized by RPE cells and Müller cells. Collagen fibrils provide tensile strength to the ECM, and activate Rho, resulting in the translocation of myocardin-related transcription factor (MRTF) into the nucleus and promoting RPE cell EMT (Guettler et al., 2008; Miralles et al., 2003). Fibronectin may also play a significant role in PVR. During pathological ECM remodeling, fibronectin is one of the earliest ECM components recruited, serving as a scaffold for other ECM proteins (Kadler, Hill & Canty-Laird, 2008; Miller et al., 2017; Miller et al., 2014). Extra domain (ED)-A fibronectin, a splice variant of fibronectin, is increased in transforming growth factor (TGF)-β2-induced RPE cells and induces myofibroblast differentiation, participating in PVR (Khankan et al., 2011).

Under normal conditions, ECM breakdown by proteases such as matrix-metalloproteases (MMPs) plays a crucial role in ECM remodeling and the release of growth factors, maintaining tissue homeostasis in cooperation with ECM synthesis, reassembly, and chemical modification (Bonnans, Chou & Werb, 2014; Craig et al., 2015; Lindsey et al., 2016). As mentioned above, the polarized RPE is able to basolaterally secrete the extracellular matrix components fibronectin and collagens, MMP and tissue inhibitors of MMPs (TIMPs), which participate in ECM remodeling. However, under pathological conditions such as inflammation and retinal injury, RPE cells lose their apical-basal polarity, undergo EMT and abnormally secrete MMPs, TIMPs and ECM proteins, leading to dysregulated ECM remodeling (Greene et al., 2017). Such ECM has aberrant composition and organization and mechanical properties, and enhances matrix stiffness and strain, which disrupts the normal structure and function of the retina, exacerbating the progression of PVR.

RPE and epithelial-mesenchymal transition

Signaling pathways of EMT

During RPE cell EMT, extracellular signals change the expression of genes encoding epithelial and mesenchymal proteins and mediate cellular behavior such as cell migration, proliferation, and apoptosis through a network of interacting signaling pathways that contribute to the development of PVR (Chen et al., 2014a; Chen et al., 2014b; Lee-Rivera et al., 2015). Among these, TGF-β and its intracellular cascades play a key role in the EMT of RPE cells.

TGF-β induces EMT of RPE cells via two pathways: the classical SMAD-dependent pathway and the SMAD-independent pathway (Fig. 2) (Cai et al., 2018; He et al., 2017; Heffer et al., 2019; Ishikawa et al., 2015; Takahashi, Haga & Tanihara, 2015; Yao et al., 2019; Zhang et al., 2017; Zhang et al., 2018b; Zhou et al., 2017). In the SMAD dependent pathway, TGF-β binds to cell surface receptor complexes, and activates type I TGF-β receptors, which phosphorylate SMAD2 and SMAD3. The activated SMADs combine with SMAD4 to form a SMAD complex, which then enters the nucleus and combines with regulatory elements to regulate the expression of key genes associated with EMT. In addition to SMAD-dependent signaling, TGF β induces EMT through SMAD independent signaling pathways including Rho GTPase-dependent pathways (Lee, Ko & Joo, 2008), PI3K/Akt pathway (Huang et al., 2017; Yokoyama et al., 2012), mitogen-activated kinase (MAPK) pathways (Chen et al., 2017; Lee et al., 2020; Matoba et al., 2017; Schiff et al., 2019) and Jagged/Notch signaling pathway (Zhang et al., 2017). The MAPK signaling pathways include extracellular signal-regulated kinase(ERK) MAPK pathway, p38 MAPK pathway, and JUN N-terminal kinase (JNK) pathway (Parrales et al., 2013; Schiff et al., 2019; Xiao et al., 2014; Zhang et al., 2018a).

The Rho pathway has been reported to regulate the assembly and organization of the actin cytoskeleton and associated gene expression, and may be essential for the fibrotic response of RPE cells in PVR. In TGF-β1-treated ARPE-19 cells, activated RhoA or its downstream effector Rho kinase (ROCK) increase the kinase activity of LIM kinase (LIMK) which then phosphorylates cofilin. This phosphorylation attenuates the activity of cofilin, which promotes actin polymerization and reorganizes the actin cytoskeleton, leading to stress fiber formation (Lee, Ko & Joo, 2008). TGF-β-induced RhoA activation also facilitates cell migration and increases α-SMA expression in primary RPE cells (Tsapara et al., 2010). Itoh et al. (2007) demonstrated that ROCK inhibitor Y27632 and RhoA inhibitor, simvastatin, suppress TGF-β2-induced type I collagen expression in ARPE-19 cells, and confirmed the existence of crosstalk between the SMAD pathway and the Rho pathway. Some studies have suggested that activated SMAD3 induces NET1 gene expression to regulate RhoA activation in RPE cells (Lee et al., 2010). Moreover, thrombin can activate Rho and ROCK, leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in EMT of RPE cells (Fig. 3) (Ruiz-Loredo, López & López-Colomé, 2011). Therefore, ROCK inhibitor and RhoA inhibitor may be new potential therapeutic target drugs for PVR.

The PI3K/Akt pathway mediates a broad range of cellular functions, such as cell transformation, migration, proliferation, apoptosis, and gene expression (Aguilar-Solis et al., 2017; Liu et al., 2019). During PVR, binding of TGF-β to its receptor activates PI3K, resulting in the phosphorylation of Akt; activated Akt inhibits glycogen synthase kinase 3β (GSK-3β), promoting EMT in RPE cells (Shukal et al., 2020; Zhang et al., 2018a). Researchers have found that inhibition or knockdown of GSK-3β promotes cell migration and collagen contraction in ARPE-19 cells, while GSK-3β overexpression and PI3K/Akt inhibitor reverse these cellular responses (Huang et al., 2017). Some studies have shown that thrombin can activate PI3K, resulting in increased cyclin D1 expression and RPE cell proliferation, processes that are involved in the development of PVR through PDK1/Akt and PKCζ/mTORC signaling (Fig. 3) (Lee-Rivera et al., 2015; Palma-Nicolás & López-Colomé, 2013; Parrales et al., 2013).

In addition to the PI3K-AKT pathway, other kinase pathways contribute to EMT in cooperation with the SMAD-dependent signaling pathways. In human RPE cells, TGF-β activates TGF-β-activated kinase 1 (TAK1), which subsequently transduces signals to several downstream effectors, including p38 (Heffer et al., 2019), JNK (Kimura et al., 2015) and nuclear factor-κB (NF-κB) (Chen et al., 2016b), which participate in EMT. Dvashi et al. (2015) found that TAK1 inhibitor caused a reduction in both p38 and SMAD2/3 activity, attenuating cell migration, cell contractility and α-SMA expression in TGF-β1-induced RPE cells. Moreover, the ERK MAPK pathway plays a role in TGF-β-induced EMT and cooperates with other signaling pathways in the regulation of EMT in RPE cells. Recent studies (Chen et al., 2014b; Tan et al., 2017; Xiao et al., 2014) have shown that blocking the ERK1/2 pathway inhibits the phosphorylation of SMAD2 and the Jagged/Notch pathway. Inhibition of the Jagged/Notch signaling pathway can alleviate TGF-β2-induced EMT by regulating the expression of Snail, Slug and Zeb1 (Fig. 3); this also suppresses the ERK1/2 signaling (Chen et al., 2014b).

The contribution of growth factors other than TGF-β, such as HGF, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) should also be factored in with regard to the induction of RPE EMT. These factors bind to and stimulate the autophosphorylation of transmembrane receptors on Tyr, subsequently participating in RPE cell EMT via PI3K/Akt pathway, ERK MAPK pathway, p38 MAPK pathway (Fig. 3) (Chen et al., 2016a; Ozal et al., 2020). Chen et al. (2012) explored the role of Wnt/β-catenin signaling in PVR, and found that when EGTA disrupted contact inhibition in RPE cells, EGF+FGF2 could activate Wnt signaling and increase nuclear levels of β-catenin, which interacts with TCF and/or LEF, leading to cell proliferation (Fig. 3); and EGF+FGF2 cooperated with TGF-β1 to induce EMT through SMAD/Zeb1/2 signaling. Acting together, various inductive signals received by RPE cells from their niche can trigger the activation of EMT programs by individual intracellular cascades or the crosstalk of multiple intracellular signaling pathways.