Regulation of ADAM10 and ADAM17 by Sorafenib Inhibits Epithelial-to-Mesenchymal Transition in Epstein-Barr Virus–Infected Retinal Pigment Epithelial Cells

Abstract

Purpose: The a-disintegrin-and-metalloprotease (ADAM) family proteins are widely expressed in the different layers of the retina throughout development. The effect of ADAM proteins on the epithelial-to-mesenchymal transition (EMT) in proliferative vitreoretinopathy (PVR) or AMD is yet to be elucidated. In this study we used Epstein-Barr virus (EBV)-transformed adult retinal pigment epithelial (ARPE) cells to investigate how sorafenib, a multikinase inhibitor, modulates ADAM proteins to control EMT.

Methods: Epithelial to mesenchymal transition and related mechanisms in EBV-infected ARPE cells were determined by RT-PCR, Western blot, invasion assay, ELISA assay, and gene silencing with siRNA.

Results: Mesenchymal-like ARPE/EBV cells exhibited considerably increased cellular migration and invasion compared with ARPE cells and produced EMT-related cytokines. Sorafenib significantly inhibited production of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α and blocked the activation of migration-related signaling molecules, such as HIF-1α, p-STAT3, MMP2, and Ang-1. The expression of mature ADAM10, ADAM17, and cleaved Notch 1 proteins in ARPE/EBV cells was downregulated after treatment with sorafenib through the regulatory activity of nardilysin (NRD-1). Gene silencing of NRD-1 in ARPE/EBV cells attenuated secretion of EMT-related cytokines and expression of ADAM10 and 17 and upregulated epithelial markers.

Conclusions: Sorafenib controls the mesenchymal characteristics of EBV-infected ARPE cells. Nardilysin and ADAM family proteins might be new targets for the prevention or control of EMT in retinal diseases.

Epithelial to mesenchymal transition (EMT) is an essential morphologic conversion that occurs in adults during wound healing, tumor progression, and organ fibrosis.¹ Epithelial to mesenchymal transition is characterized by the disassembly of cell–cell contacts, remodeling of the actin cytoskeleton, and separation of cells, and generates fibroblast-like cells that express mesenchymal markers and have migratory properties.^2–4 This transition is characterized by loss of epithelial proteins such as E-cadherin and the acquisition of new mesenchymal markers, including vimentin and α-smooth muscle actin (α-SMA).⁵ 

Epithelial to mesenchymal transition has been linked to the optical conditions of proliferative vitreoretinopathy (PVR) and wet AMD. Proliferative vitreoretinopathy is a dynamic scarring process that develops with some cases of retinal detachment (RD) and is characterized by the formation of fibrotic tissue.^6,7 After detachment from their basement membrane, RPE cells can attach to the vitreous or the retinal surface and undergo EMT. During this process, the RPE cells lose their epithelial morphology and transform into fibroblast-like cells.^8,9 Wet AMD is characterized by the formation of choroidal neovascularization (CNV).¹⁰ Upon CNV, RPE cells have been shown to lose their junctional integrity.¹¹ Although inflammatory or angiogenesis-related cytokines, such as VEGF and connective tissue growth factor (CTGF), are known to trigger EMT changes, little is known about the molecular mechanisms of EMT in PVR and CNV. 

Sorafenib (SRF) (Nexavar; Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ, USA), the first oral multikinase inhibitor that blocks multiple signaling pathways, was initially approved as an oral agent for the treatment of advanced renal cell carcinoma.¹² Recently, SRF has also been approved for the treatment of hepatocellular carcinoma (HCC).¹³ Recent studies show that SRF has antifibrotic activity in vitro. Sorafenib inhibits the activation, growth, and collagen accumulation of hepatic stellate cells (HSCs),^14,15 and also suppresses TGF-β1-induced EMT of HCC through blocking the upregulation of Snail and recovering the expression of E-cadherin.^16,17 Signal transducer and activator of transcription 3 (STAT3) cooperates with hypoxia-inducible factor 1-alpha (HIF-1α), and the subsequent accumulation of HIF-1α in the nucleus upregulates Twist-related protein 1 (TWIST1) and TGF-β1 expression.^18,19 Interestingly, HSCs show increased expression of the sheddases a-disintegrin-and-metalloprotease 10 (ADAM10) and ADAM17 that are associated with the severity of liver fibrosis in patients with chronic liver diseases.^20,21 The ADAM family is the major protein family that mediates ectodomain shedding through activity similar to that of α-secretase.²² Both ADAM10 and ADAM17 are widely expressed in the different layers of the retina throughout the whole embryonic period, whereas ADAM12 is mainly expressed in the ganglion cell layer at a later stage.²³ The ADAM10 activated by TGF-β1 specifically cleaves E-cadherin in its ectodomain.²⁴ Furthermore, the shedding of E-cadherin by ADAM10 modulates the subcellular localization of β-catenin and its downstream signaling.²⁵ Both ADAM10 and ADAM17 have an important role in maintaining the epidermal integrity through regulation of Notch-mediated signaling.^26,27 Nardilysin (N-arginine dibasic convertase [NRD-1]), a metalloendopeptidase of the M16 family, promotes releasing of the precursor forms of various growth factors and cytokines by enhancing the protease activities of ADAM proteins.²⁸ However, the role of ADAM family proteins or NRD-1 in PVR or CNV and the relationship between EMT and changes in EMT-related proteins in RPE cells are not yet fully understood. 

Ocular manifestations of Epstein-Barr virus (EBV) infection as a cause of intraocular inflammation are rare and typically mild²⁹; however, they can involve various parts of the eye, including the conjunctiva, cornea, retina, uvea, and optic nerve.^30,31 We previously developed EBV-infected human corneal epithelial cells (EBV-HCECs) as a model of viral keratitis.³² The EBV-HCECs exhibit morphologic change into a spindle-like shape and express several mesenchymal markers.³² In this study, we established adult retinal pigment epithelial (ARPE) cells that secrete TGF-β and VEGF after transformation by EBV infection. These cells lose expression of E-cadherin and N-cadherin, which is the most common adherens junction (AJ) proteins in RPE cells.^33,34 They also gain expression of mesenchymal markers, including vimentin and α-SMA. We investigated the molecular mechanisms of EMT in PVR or CNV conditions using these EBV-transformed ARPE cells as model of RD or AMD. We also focused on the development of new possible treatments or therapeutic targets by investigating the effects of the multikinase inhibitor SRF on the regulation of ADAM proteins and EMT-triggering cytokines. 

Materials and Methods

Cell Culture and Reagents

Primary human retina pigment epithelial (HRPEpi) cells were purchased from ScienCell (Carlsbad, CA, USA). A human retinal pigment epithelial cell line, ARPE-19, was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DME/F12 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone) and antibiotics under a humidified atmosphere with 5% CO₂. Sorafenib (BAY43-9006, Nexavar) was purchased from LC Laboratories (Woburn, MA, USA). GI254023X, Marimastat, and ONO4817 were purchased from TOCRIS Biosciences (Bristol, UK). 

Preparation of EBV Virions and Generation of EBV-Infected HRPEpi and ARPE-19 Cells

Cell-free EBV virions were prepared from the B95-8 cell line (EBV type I; ATCC), which has been described previously.³² After HRPEpi and ARPE-19 cells (2 × 10⁵ cells/T25 flask/4 mL media) were completely attached, an equal volume of EBV supernatant (4 mL, 73 ± 11 colony-forming units per milliliter) was added. The cultures were incubated for durations of 1 day to 4 weeks. 

Reverse-Transcription PCR

After infection for 2 weeks, total RNA was isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany). The RNA was transcribed into cDNA using oligo(dT) primers (Bioneer, Daejeon, Korea) and reverse transcriptase. To investigate the expression of EBV genes in EBV-infected ARPE-19 and parental ARPE-19 cells, PCR amplification was performed using specific primer sets (Table; Bioneer) and Prime Taq Premix (GeNet Bio, Chungnam, Korea). The PCR products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide under UV light using the multiple Gel DOC system (Fujifilm, Tokyo, Japan). Data were analyzed using ImageJ 1.38 software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Experiments were performed in triplicate. 

Table

 

Specific Primer Sequences Used for RT-PCR

 

Immunoblotting

After infection for 4 weeks, cells were harvested, lysed, and subjected to SDS-PAGE and immunoblotting using standard techniques. Primary antibodies against the following proteins were used: phospho-Stat3 (Tyr⁷⁰⁵), Stat3, HIF-1α_, metalloproteinase (MMP)-2, MMP-9, E-cadherin, N-cadherin, β-catenin, Vimentin, Snail, TCF8/Zeb1, PARP, ZO-1, Claudin-1, cleaved Notch, and β-actin (Cell Signaling Technology, Beverly, MA, USA); phospho-Fyn (Thr¹²), Epstein-Barr nuclear antigen (EBNA)-2, EBNA-3A, latent membrane protein (LMP)-1, LMP-2A, Ang-1, VEGF, nardilysin, ADAM10, ADAM12, and ADAM17 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); EBNA-1 (Thermo Scientific, Rockford, IL, USA); RPE65 and PEDF (Abcam, Cambridge, UK); and β-tubulin (BD Biosciences, San Diego, CA, USA). 

Enzyme-Linked Immunosorbent Assay

At 24 hours, the conditioned media were collected and the concentration of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 secreted by ARPE-19 or EBV-infected ARPE-19 cells was measured using the Single Analyte ELISArray Kit (Qiagen) according to the manufacturer's instructions. The VEGF, TNF-α, and active TGF-β1 were quantified using the Single Cytokine ELISA Assay Kit (R&D Systems, Minneapolis, MN, USA). Data are expressed as the mean value for biological replicates ± SD. 

Wound Healing and E-Cadherin Repressors Assay

Wound-healing assays were performed to measure the migration ability of ARPE-19 cells. Epstein-Barr virus–infected or uninfected ARPE-19 cells were plated in six-well plates. After the cell layers had reached confluence, we inflicted a uniform wound, such as a straight line in each well using a 200-μL micropipette tip and washed the wounded layers with PBS to remove all cell debris. The cells were cultured in 5% CO₂ at 37°C, and images were taken at 0 and 24 hours after scratching using an inverted phase-contrast microscope (Olympus, Tokyo, Japan) at ×100 magnification. After infection for 4 weeks, cytosol and nuclear cellular fractions were prepared to detect E-cadherin repressor proteins using a Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA, USA) according to the manufacturer's instructions. All fractions were stored at −80°C until use. Cytosolic extracts or nuclear extracts were analyzed by Western blotting using antibodies against Snail and TCF8/Zeb1. 

Detection of Migration and Invasion

After infection for 4 weeks, transendothelial migration of EBV-infected ARPE-19 cells was detected using a CytoSelect Tumor Transendothelial Migration Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's instructions. The fluorescence (in relative fluorescence units [RFU]) of migrated cells was measured using a microplate reader. The invasion assay was performed using the CultureCoat 96-well Medium BME Cell Invasion Assay Kit (R&D Systems) according to the manufacturer's instructions. Invaded cells were stained with calcein AM and quantified using a microplate reader. 

Statistical Analysis

Data were expressed as the mean ± SD. Statistical analysis was conducted using one-way ANOVA. A P value less than 0.05 was considered to be statistically significant. 

Results

ARPE Cells Show Mesenchymal Characteristics and Enhanced Migration and Invasion After EBV Infection

We developed EBV-infected HRPEpi (HRPEpi/EBV) and ARPE-19 (ARPE/EBV) cells to determine whether ARPE cells similarly show EMT after transformation with EBV. As expected, EBV-infected RPE cells (HRPEpi/EBV and ARPE/EBV) acquired a fibroblast-like, mesenchymal appearance (Fig. 1A; Supplementary Fig. S1A) and stably expressed EBV-related viral mRNA or proteins, including EBNA1, EBNA2, EBNA3A, LMP1, and LMP2A (Figs. 1B, 1C; Supplementary Fig. S1B). The expression of mRNA and proteins encoding epithelial markers (such as E-cadherin, ZO-1, claudin-1, and β-catenin) was downregulated in HRPEpi/EBV and ARPE/EBV cells, whereas mesenchymal markers (such as Vimentin and Snail) were upregulated (Figs. 1D, 1E). The expression of N-cadherin, the most common AJ proteins in ARPE cells, was reduced at mRNA and protein levels after infection with EBV (Figs. 1D, 1E). Next, we examined whether the mesenchymal characteristics of ARPE/EBV cells were associated with enhanced cell migration and invasion. A scratch assay and migration chamber assay revealed that cellular migration and invasion activities were enhanced approximately 2-fold in ARPE/EBV cells compared with those of ARPE cells (Figs. 2A, 2B). We also observed that the E-cadherin repressors Snail and TCF8/ZEB1 were present in the nuclear fraction of ARPE/EBV, but not ARPE cells (Fig. 2C). Our data suggest that the mesenchymal-like ARPE cells generated by EBV infection acquire EMT phenotypes and could be an in vitro model of pathologic PVR and CNV conditions. 

Figure 1

 

Epstein-Barr virus induces EMT-like transformation in the human RPE cell line ARPE-19. (A) Mesenchymal morphology elicited by EBV infection. Morphology was observed under an inverted phase-contrast microscope. Magnification bar is 100 μm. Photographs were taken at ×100 magnification using a digital camera (Olympus, Tokyo, Japan). (B, C) Messenger RNA (B) and protein (C) levels of EBV-related gene expression in EBV-infected and uninfected ARPE-19 cells measured using RT-PCR and Western blotting, respectively. Epstein-Barr virus–transformed B cells were used as a positive control. (D, E) Effect of EBV infection on expression of epithelial and mesenchymal markers, as analyzed by RT-PCR (D) and Western blotting (E). The HRPEpi is a human primary retina epithelial cell; RPE65 is an RPE-specific marker; β-actin served as the loading control. Data are representative of three independent experiments.

Figure 2

 

Epstein-Barr virus increases cell migration and invasion and induces nuclear translocation of Snail and TCF8/Zeb1. (A) Cell motility of ARPE-19 cells was increased by EBV infection as measured with a wound-healing assay. Epstein-Barr virus–infected and uninfected ARPE-19 cells were wounded (0 h) and maintained for 12 hours in complete medium. Arrows point to the edges of the wounds. Wound closure (measured after 12 hours) was faster in EBV-infected ARPE-19 cells than in uninfected ARPE-19 cells. (B) The migratory capacity of ARPE-19 cells was increased by EBV infection, as determined by a transwell migration assay kit. Epstein-Barr virus also enhanced invasiveness, as detected by a BME cell invasion assay kit. Each value is the mean ± SD of three determinations. *P < 0.05. (C) Cytosolic extracts (left) or nuclear extracts (right) were analyzed by Western blotting using antibodies against Snail and TCF8/Zeb1. The nuclear marker PARP and the cytosol marker β-tubulin were used to check the purity of each fraction. Data are representative of three independent experiments.

ARPE/EBV Cells Produce EMT-Related Cytokines Through Upregulation of Various Signaling Molecules

To determine the effect of inflammatory or angiogenesis-related cytokines on EMT in ARPE/EBV cells, we compared the expression level of these cytokines in ARPE/EBV and ARPE cells. First, to investigate whether EBV infection affects the cellular expression of signaling proteins involved in the induction of EMT-related cytokines, we examined the mRNA and protein levels of HIF-1α, phosphorylated STAT3 (p-STAT3), MMP-2, and MMP-9. The mRNA and protein levels of HIF-1α, p-STAT3, Angiopoietin-1 (Ang-1), MMP2, and MMP9 were upregulated concomitant with VEGF production in ARPE/EBV cells compared with the expression level of these molecules in ARPE cells (Figs. 3A, 3B). Next, culture media of each cell line were collected and the concentration of secreted TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α was measured using a sandwich ELISA method. The TGF-β1 and TNF-α, critical cytokines for EMT, were barely detectable in the culture media of ARPE cells. However, the production of these cytokines by ARPE/EBV cells was approximately 2- to 5-fold higher than that in ARPE cells (Figs. 3A, 3C). Our data suggest that ARPE/EBV cells unconventionally produce EMT-related cytokines and signaling molecules to regulate cell migration and invasion. 

Figure 3

 

Epstein-Barr virus promotes the production of proinflammatory cytokines through upregulation of various signaling molecules. (A, B) Total RNA and protein were extracted from EBV-infected and uninfected ARPE-19 cells. Reverse-transcriptase PCR (A) and Western blot (B) analysis of HIF-1α, STAT3, VEGF, PEDF, IL-6, IL-8, TNF-α, TGF-β, MCP-1, Ang-1, MMP2, and MMP9 were performed; β-actin served as the loading control. (C) Concentrations of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α in the culture supernatants of ARPE-19 and ARPE/EBV cells were quantified by ELISA assay. Cells were seeded into six-well plates (1 × 10⁵/well) and incubated for 24 hours. *P < 0.05. Data are presented as the mean of three independent experiments and error bars represent SDs of the means.

Sorafenib Inhibits the EMT Through Regulation of Cytokine Secretion and Related Signaling Pathways

To investigate whether SRF affects the EMT characteristics of ARPE/EBV cells and related signaling pathways, we first conducted dose-response experiments to investigate the effect of SRF on retinal vascularization using a mouse model of laser-induced neovascularization. Treatment with intravitreal SRF at daily interval for 5 days considerably inhibited the neovascularization (Supplementary Fig. S2). Next, we examined the changes in epithelial or mesenchymal markers in the presence of SRF. In ARPE/EBV cells, SRF markedly recovered expression of epithelial markers such as E-cadherin, N-cadherin, ZO-1, claudin-1, and β-catenin, whereas the expression of mesenchymal markers, including vimentin, Snail, and TCF/ZEB1, decreased in a dose-dependent manner (Fig. 4A). Sorafenib also reversed the epithelial characteristics of EBV-infected primary human retina pigment cells (HRPEpi/EBV) (Fig. 4B). Migration-related signaling pathways involving HIF-1α, p-STAT3, MMP-2, MMP-9, and Ang-1 were also blocked after treatment of ARPE/EBV cells with SRF (Fig. 4C). Productions of EMT-related cytokines, including TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α were significantly inhibited after treatment with SRF (Fig. 4D). Migration activity was also blocked after treatment of ARPE/EBV cells with SRF (Fig. 4E). Our data suggest that SRF might control the effect of inflammatory or angiogenesis-related cytokines on EMT in ARPE/EBV cells. 

Figure 4

 

Sorafenib inhibits the EMT through regulation of cytokine secretion and related signaling pathways. (A) The ARPE/EBV cells were treated with 1, 2, 5, 10, and 20 μM SRF for 24 hours. Cells were harvested and Western blotting was performed using an antibody to EMT markers; β-actin served as the loading control. (B) The HRPEpi/EBV cells were treated with 5 μM SRF for 24 hours. The cells were collected and immunoblotting was performed using an antibody to EMT markers; β-actin served as the loading control. (C) Expression of EBV-induced proinflammatory and proangiogenic signaling molecules was inhibited by SRF treatment. Cells of ARPE-19, DMSO-treated ARPE/EBV, and SRF-treated (5 μM, 24 hours) ARPE/EBV were harvested and subjected to Western blot analysis with the indicated antibodies. (D) After treatment with 5 μM SRF, concentrations of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α in the culture supernatants of ARPE19/EBV cells were quantified by ELISA assay. Cells were seeded into six-well plates (1 × 10⁵/well) and incubated for 24 hours. *P < 0.05. Data are presented as the mean of three independent experiments and error bars represent SDs of the means. (E) The migratory capacity and invasiveness of ARPE/EBV cells was inhibited by SRF as detected by transwell migration assay and BME cell invasion assay. *P < 0.05. Each value is the mean ± SD of three determinations.

ADAM10 and 17 Control the Mesenchymal Phenotype and Cell Migration Activity of ARPE/EBV Cells

Although the expression of ADAM in the retina during development has been extensively investigated, the role of ADAM family proteins on AMD or RD is still poorly understood. To examine whether SRF affects the activity of ADAM family proteins, we analyzed the expression of ADAM proteins in ARPE/EBV cells by immunoblot analysis. Expression of the mature forms of ADAM10 and ADAM17, a close homologue of ADAM10, was significantly increased in ARPE/EBV cells compared with ARPE cells (Fig. 5A). The expression of mature ADAM10 and ADAM17 was blocked in SRF-treated HRPEpi cells (Fig. 5B). Treatment with SRF also downregulated the generation of cleaved form ADAM10 and ADAM17 in a dose-dependent manner at 48 hours in ARPE/EBV cells (Figs. 5C, 5D). Next, to investigate whether inhibition of ADAM protein activity influences secretion of EMT-related cytokines and EMT characteristics of ARPE/EBV cells, we cultured ARPE/EBV cells in the presence of various ADAM inhibitors, MMP inhibitors, and SRF. GI254023X (ADAM10 inhibitor, 10 μM) and Marimastat (ADAM17 inhibitor, 50 nM) recovered the expression of epithelial markers and downregulated expression of mesenchymal markers compared with nontreated ARPE/EBV cells. Treatment with SRF or ONO4817 (MMP inhibitor) also blocked the EMT characteristics of ARPE/EBV cells (Fig. 6A). Inhibitors of ADAM10 and 17 modulated cytokine secretion and migration activity in ARPE/EBV cells (Figs. 6B, 6C). Together, our data suggest that SRF controls the EMT activity of ARPE/EBV cells through regulation of ADAM family proteins. 

Figure 5

 

Epstein-Barr virus activates ADAM10 and ADAM17 in ARPE19 cells. (A) Epstein-Barr virus–infected and uninfected ARPE19 cells were harvested and subjected to Western blot analysis with the indicated antibodies. Epstein-Barr virus infection increased the activity of ADAM10 and ADAM17, but not ADAM12. (B) Effect of SRF on the activity of ADAM family proteins in HRPEpi/EBV cells. Sorafinib efficiently blocked the expression of mature ADAM10 and ADAM17. The ARPE/EBV cells were treated with 2 μM and 5 μM SRF for 24 hours (C), and with 5 μM SRF for 24, 48, and 72 hours (D). After SRF treatment, cells were harvested and subjected to Western blot analysis with the indicated antibodies; β-actin served as the loading control. Data are representative of three independent experiments.

Figure 6

 

Both ADAM10 and ADAM17 regulate mesenchymal phenotype and cell migration activity of ARPE/EBV cells. The ARPE/EBV cells were preincubated with the ADAM10 inhibitor GI254023X (10 μM), the ADAM17 inhibitor Marimastat (50 nM), the pan-MMP inhibitor ONO4817 (50 nM or 5 μM) for 24 hours. (A) Western blots were performed using the indicated antibodies; β-actin served as the loading control. (B) Concentrations of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α in the culture supernatants of ARPE/EBV cells were quantified by ELISA assay. Cells were seeded into six-well plates (1 × 10⁵/well) and incubated for 24 hours. *P < 0.05. Data are presented as the mean of three independent experiments and error bars represent SDs of the means. (C) The migratory capacity and invasiveness of ARPE/EBV cells were inhibited by GI254023X, Marimastat, ONO4817, or SRF as detected by transwell migration assay and BME cell invasion assay. *P < 0.05. Each value is the mean ± SD of three determinations.

Knockdown of NRD-1 Expression Attenuates Expression of ADAM10 and ADAM17 and the Mesenchymal Characteristics of ARPE/EBV Cells

To investigate whether SRF affects the activity of ADAM family proteins through regulation of NRD-1 in ARPE/EBV cells, we first showed that the expression of NRD-1 was increased by infection with EBV, but was downregulated after treatment with SRF (Figs. 7A, 7B). Next, we analyzed the expression of ADAM proteins after silencing NRD-1 with small-interfering RNA (siRNA) in ARPE/EBV cells and showed that the expression of mature ADAM10 and ADAM17 was downregulated (Fig. 7C), whereas expression of epithelial markers was recovered (Fig. 7D). Gene silencing of ADAM10 and ADAM17 also upregulated the epithelial characteristics of ARPE/EBV cells (Fig. 7E). Production of EMT-related cytokines (TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α) was significantly attenuated (Fig. 7F) and migration and invasion activities were decreased (Fig. 7G) after NRD-1 knockdown in ARPE/EBV cells. We also observed that overexpressing ADAM10 and ADAM17 in ARPE/EBV cells induced the expression of cleaved Notch (Fig. 7A); however, SRF or transfection with siRNA-ADAM10 and siRNA-ADMA17 efficiently blocked the expression of cleaved Notch (Figs. 7B, 7E). Our data suggest that SRF modulates ADAM family activity through regulation of NRD-1 expression in ARPE/EBV cells. 

Figure 7

 

Silencing of NRD-1 attenuates ADAM10 and ADAM17 expression and the mesenchymal characteristics of ARPE/EBV cells. (A) Epstein-Barr virus infection significantly increased NRD-1 and cleaved Notch1 expression in ARPE19 cells. (B) Nardilysin and cleaved Notch1 expression in ARPE/EBV cells was inhibited after treatment with SRF. (C, D) After transfection with either NRD-1-siRNA or control-siRNA for 48 hours, protein levels of ARPE/EBV cells were detected by Western blotting with antibody to NRD-1, ADAM proteins (C), and EMT markers (D); β-actin was used as a loading control. (E) Gene silencing with ADAM10-siRNA or ADAM17-siRNA not only decreased the expression of mesenchymal markers and cleaved Notch1 but also recovered the expression of epithelial markers in ARPE/EBV cells. (F) After transfection with either NRD-1-siRNA or control-siRNA, ARPE/EBV cells were seeded into six-well plates (1 × 10⁵/well) and incubated for 48 hours. Concentrations of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α in the culture supernatants of ARPE/EBV cells were quantified by ELISA assay. *P < 0.05. Data are presented as the mean of three independent experiments and error bars represent SDs of the means. (G) After transfection with either NRD-1-siRNA or control-siRNA for 48 hours, the migratory capacity and invasiveness of ARPE/EBV cells were inhibited by the knockdown of NRD-1 expression as detected by transwell migration assay and BME cell invasion assay. *P < 0.05. Each value is the mean ± SD of three determinations.

Discussion

Proliferative vitreoretinopathy is a common complication of posterior segmental ocular trauma and surgical procedures. Epithelial to mesenchymal transition of RPE cells is the key event leading to visual impairment after retinal damage. Additionally, CNV associated with wet AMD is a leading cause of irreversible blindness.¹⁰ In this study, we developed EBV-infected ARPE19 cells as a model of retinal disease to investigate the effect of several drugs on PVR or wet AMD in vitro. During EMT, epithelial cells lose intracellular junctions, dissociate from surrounding cells, acquire mesenchymal-like characteristics, and become able to migrate away from their original location.³⁵ Migration of RPE cells is the major cause of severe eye diseases such as PVR, an ocular fibrotic disease. Recently, several studies have revealed the exact molecular mechanisms of EMT in RPE cells after exposure to various cytokines.^36–39 There is also clinical and experimental evidence that RPE and glial cells contribute to the final outcome of PVR.^40,41 Proliferative vitreoretinopathy involves a process of fibrocellular proliferation in the vitreous cavity and on both surfaces of the retina that may lead to the formation of contractile epiretinal membranes.^7,42,43 Fibrotic changes in the foveal CNV lesion result in permanent visual impairment in patients with wet AMD.⁴⁴ Inflammatory or angiogenesis-related cytokines, such as VEGF, CTGF, TNF-α, and TGF-β, which are known to trigger EMT changes, are expressed in CNV tissues.^45–47 Generally, binding of such ligands to the appropriate receptor upregulates the expression of EMT-regulating transcription factors, including SNAI1, SNAI2, ZEB1, ZEB2, and TWIST.⁴⁸ These findings led us to propose that tyrosine kinase inhibitors could be used to control the EMT in RPE cells and might inhibit fibrotic scar formation in AMD. Tyrosine kinase inhibitors are particularly attractive agents because they inhibit fibrotic cellular changes and might therefore also prevent PVR.^49,50 Sorafenib is a small molecule that inhibits the kinase activities of Raf-1, B-Raf, VEGFRs, PDGFR-b (platelet-derived growth factor receptor b), Flt-3, and c-KIT.⁵¹ There are also data suggesting that SRF has antifibrotic activities. Sorafenib can inhibit the activation, growth, and collagen accumulation of hepatic HSCs in vitro.^14,15 Sorafenib inhibits the secretion of VEGF, which is associated with progression of AMD in ARPE cells.⁵² Astrocytes in optic nerve that are exposed to light also reduce the secretion of VEGF as well as platelet-derived growth factor (PDGF) after treatment with SRF.⁵³ These results suggest that SRF has effect on the retinal neovascularization in both RPE cell and glial cells of retina. Recently, dasatinib, a tyrosine kinase inhibitor approved by the Food and Drug Administration, was shown to significantly inhibit PVR-related changes in RPE in vitro and prevent traction RD in an experimental PVR model in swine. Dasatinib was also shown to prevent RPE cell migration and EMT.⁴⁹ However, these reports did not reveal the mechanisms by which these drugs inhibit PVR-related changes in RPE cells. In the current study, we showed that the multikinase inhibitor SRF controlled migration and invasion of ARPE19/EBV cells by regulating the secretion of EMT-related cytokines and transcription factors. Thus, our data on the inhibition of EMT and cell migration by SRF may provide a possible explanation for its activity in tumor control and reduced cancer metastasis. Transforming growth factor-β can promote EMT during carcinogenesis and enhance the migratory and invasive properties of tumor cells.⁵⁴ Sorafenib inhibits STAT3 phosphorylation in a variety of tumors, including medulloblastoma, cholangiocarcinoma, and HCC.^55–57 Moreover, SRF also inhibits TGF-β–induced STAT3 phosphorylation during TGF-β–mediated EMT in mouse hepatocytes.¹⁶ However, although our data showed that migration-related signaling molecules, such as HIF-1α, p-STAT3, and MMP2, were downregulated after treatment with SRF in ARPE/EBV cells, further studies are needed to investigate the precise signaling pathway that initiates the process of EMT in retinal diseases. 

Both ADAM10 and ADAM17 (also known as TNF-α–converting enzyme or TACE) show very prominent expression in all epithelial tissues and are required for proper epithelial tissue development in mice.^58,59 Both ADAM10 and ADAM17 are also essential for adult epidermis maintenance through activation of Notch pathway.^26,27 The ADAM10/E-cadherin interaction represents a common regulatory mechanism in inflammatory epidermal diseases, which are characterized by loss of E-cadherin expression and loss of epithelial integrity.²⁴ In contrast, downregulation of ADAM10 leads to a more scattered cell phenotype, which is accompanied by the induction of Slug and the loss of E-cadherin, as observed during EMT.⁶⁰ ADAM10 is coexpressed with classic cadherins in the developing retina.²³ Although ADAM10-mediated E-cadherin shedding generally affects epithelial cell–cell adhesion and cell migration, there are no specific data on the modulation of ADAM10-mediated EMT processes in ocular disease. Our results suggest that ADAM family proteins might be involved in the migration and invasion of RPE cells through the control of E-cadherin expression and secretion of EMT-related cytokines. 

Nardilysin is a zinc peptidase of the M16 family that is localized diffusely in the cytoplasm and is secreted to the cell surface by a currently undetermined mechanism.^61,62 Nardilysin binds to the extracellular domain of ADAM17 and directly enhances its catalytic activity.^63,64 In this study, knockdown of NRD-1 downregulated the EMT process in ARPE19/EBV cells through regulation of ADAM proteases and the secretion of EMT-related cytokines. Our results suggest that regulation of NRD-1 and ADAM family proteins by the multikinase inhibitor SRF might be a new therapeutic approach to control PVR or CNV and provide experimental evidence supporting the application of ADAM inhibitors to prevent blindness from other retina-related ocular diseases. 

Acknowledgments

Supported by a grant from the Korea Healthcare Technology R&D Project of the Ministry of Health and Welfare Affairs, Republic of Korea (Grant HI12C0005). 

Disclosure: G.B. Park, None; D. Kim, None; Y.S. Kim, None; J.W. Kim, None; H. Sun, None; K.-H. Roh, None; J.W. Yang, None; D.Y. Hur, None