ROCK Inhibitor Y-27632 Promotes Human Retinal Pigment Epithelium survival by Altering Cellular Biochemical Properties
Abstract: Purpose: Dysfunction or death of retinal pigment epithelial (RPE) cells is a common pathogenesis of various types of retinal degenerative diseases. Recent reports indicated that ROCK pathway inhibitors regulate cell proliferation or apoptosis in a cell- type-dependent manner. Here, we aim to investigate the effect of ROCK inhibitor Y- 27632 on the human retinal pigment epithelium (RPE) in vitro.Methods: Cell proliferation and apoptosis were analyzed by CCK-8 and flow cytometry respectively. Cell proliferation markers were detected by immunofluorescence and western blot. Cell morphology was evaluated using scanning electron microscopy. Thetopography and biomechanical properties of living cells were assessed using atomic force microscope (AFM). In addition, cytoskeleton and epithelial-mesenchymal transition (EMT) markers were detected by western blot and immunofluorescence. Results: 30μM Y-27632 significantly promoted cell proliferation and decreased apoptosis. Compared with control group, human retinal pigment epithelial cell line ARPE-19 cells treated with 30μM Y-27632 exhibited significantly decreased cytomembrane roughness (Ra: 41.04±1.63nm vs. 24.41±0.75nm, P<0.01; Rq: 51.56±2.03nm vs. 30.81±0.95nm, P<0.01) and increased elasticity modulus (16.66±0.83KPa vs. 32.55±1.48KPa, P<0.01). In addition, the inhibition of ROCK activity by Y-27632 caused cell elongation and reorganization of microfilaments and microtubules of cytoskeletons. Conclusion: Taken together, our data demonstrated that Y-27632 could alter biomechanical properties and reorganized cytoskeletons to promote RPE cell survival. These results are an important step toward the future application of Y-27632 in retinal degenerative diseases. 1.INTRODUCTION The retinal pigment epithelium (RPE) is located between the neurosensory retina and the choriocapillaris in the healthy eye. These monolayer cells are of vital importance to the maintenance and function of overlying rod and cone photoreceptors and, hence, for normal visual function [1, 2]. Dysfunction or death of RPE cells is a common pathogenesis of various types of degenerative eye diseases, including Stargardt's disease [3], retinitis pigmentosa (RP) [4], and age-related macular degeneration (AMD) [5], which is the major cause of irreversible blindness in the elderly worldwide. Although there were continuousattempts, many patients have no chance to restore vision. Recently, delaying/blocking the apoptosis of RPE cells has been suggested as an alternative approach to halt the progression of degenerative eye diseases [6-8].The Rho-associated coiled-coil containing kinases (ROCK), believed as effectors of the RhoA small GTPase, participate in actomyosin cytoskeleton contraction and RhoA and RhoC activation [9-11]. Since then, the roles of ROCK have been widely recognized in some other aspects of cell behavior, such as cell proliferation, apoptosis and transformation [12]. Although the underlying mechanism has yet to be thoroughly revealed, recent reports indicated that the been demonstrated as an effective inhibitor of apoptosis in embryonic stem cells [16, 17], cardiomyocyte [18, 19], and corneal endothelial cells in the eye [20]. Given the principal function of ROCK that mediated its downstream effects on actin organization, Y-27632 has been explored in some ocular diseases associated with the contractile activity of cells [21-26]. Nevertheless, its precise role in apoptosis-related degenerative eye diseases has yet to be established.In this study, we aimed to determine whether ROCK inhibitor Y-27632 would promote proliferation or block apoptosis in human retinal pigment epithelium, and be helpful for the survival of RPE cells in vitro. Since cellular mechanical properties are sensitive indicators for indicating cell states [27-29], we also analyzed the influence of Y-27632 on biophysical properties in alive RPE cells by Atomic force microscopy (AFM). ROCK inhibitor Y-27632 has already been used clinically in cardiovascular therapies, suggesting that it is safe for use with the human body [30]. We expect that our study will be valuable in establishing a clinically applicable way to use Y-27632 in apoptosis-related degenerative eye diseases. 2.MATERIALS AND METHODS Human retinal pigment epithelial cell line ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% HyClone™ Fetal Bovine Serum (FBS; GE Healthcare Life Sciences Company, USA). After 24h incubation at 37°C and 5% CO, in a humidified incubator for cell adherent growth, ARPE-19 cells were treated with or without Y-27632 (Selleckchem Company, USA) for further experiments. Y-27632 was diluted in DMSO at a final concentration of 0.03% in the DMEM, which showed no detectable effect on cell proliferation and apoptosis.Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8; Dojindo, GMBH, EU). The cells were seeded in 96-well plates at the density of 5.0×103 cells/well. After cell adherent growth, plates were washed twice with DMEM. Y-27632 was added to the medium to the final concentrations (0, 30μM). The cells were treated with or without Y-27632 for 24h, 36h or 48h. Subsequently, the cells were incubated with 10μL of the CCK-8 solution at 37°C for 2h in dark. The optical density (OD) values were determined using a 96-well microplate reader (Tecan Sunrise Reader, Switzerland) at 450nm.Flow cytometry reagents (Becton, Dickinson and Company, USA) were used according to the manufacturer’s protocol. Briefly, ARPE-19 cells were trypsinized and collected in 100μL 1× binding buffer. Then the cells were incubated with the labeling solution(5μL Annexin V-APC and 5μL propidium iodide (PI)) for 20min in the dark. After that, each sample was diluted with 1×binding buffer to 500μL at the cell density of 1×106/mL. The samples were analyzed by a flow cytometry (Beckman CytoFLEX S, Germany).To assess morphology, ARPE-19 cells were seeded on chamber slides and treated with or without Y-27632. Each group was fixed in 2.5% glutaraldehyde-4% paraformaldehyde in PBS for 0.5h at room temperature. After dehydrated through a graded ethanol series (50%, 70%, 85%, 100%), specimens were dried in a CO2 critical point dryer. Then the samples were sputter coated with gold and examined by an FEI QUANTA200 SEM.A Dimension Fastscan with ScanAsyst™ AFM (Bruker, Santa Barbara, CA) was used to analyzed biophysical changes in a single living cell [31-35]. The pyramidal tip of silicon nitride cantilevers (nominal spring constant k=0.01N/m, tip radius 20.0nm, tip half angle 18.0°, MLCT-C, Bruker, USA) was used for the contact mode of AFM. This experiment was performed as described previously [35]. The cells were imaged over an area of 20×20μm2 with a resolution of 128×128 pixels. NanoScope Analysis (Bruker, USA) was used to calculate cell elasticity values and roughness values. The Young's modulus was calculated using Hertz model, which was selected from 10-12loci (2μm×2μm) on each cell [33].In short, the cells were washed with PBS buffer and fixed using 4% paraformaldehyde for 30min at RT. After permeabilizing in PBS containing 0.1% Triton X- 100, the cells were blocked with 1% bovine serum albumin (BSA) for 1h. Then, the cells were incubated with primary antibodies and FITC-conjugated antibody (1:100; Cell Signaling Technology, Danvers, MA, USA). The CytoPainter Phalloidin-iFluor 488 reagent (1:100; Abcam, Cambridge, MA, USA) was used to label microfilaments (MFs) for 30min. Then, Hoechst33342 (1:1000; Abcam, Cambridge) was used to stain the cell nuclei for 30min. The images were taken using Zeiss LSM 800 microscope (LSM800, Zeiss, Germany). The sources of the primary antibodies are as follows: rabbit anti-β-tubulin antibody (1:50; Abcam), rabbit anti-Ki67 antibody (1:50; Abcam) and mouse anti-Vimentin (1:50; Abcam).ARPE-19 cells were washed with PBS and then lysed in RIPA buffer supplied with protease. The BCA-100 Protein Quantitative Analysis Kit (Biocolor Bioscience & Technology Co. Ltd., Shanghai, China) was used to detect protein concentration. Samples were separated in 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) andthen transferred to polyvinylidene fluoride membranes (PVDF membranes; Millipore, MA, USA). 5% BSA was used to block nonspecific binding sites for 1h at RT. Then the PVDF membranes were incubated with primary antibodies and horseradish peroxidase (HRP)- conjugated secondary antibodies. The signals of each band were detected using an eECL Western Blot Kit (ComWin Biotech Co., Ltd, Beijing, China). The sources of the antibodies used are as follows: mouse anti-cyclin E1 (1:1000; Cell Signaling Technology), mouse anti-cyclin D1 (1:2000; Cell Signaling Technology), rabbit anti-P21 (1:2000; Cell Signaling Technology), mouse anti-α-SMA (1:1000; Abcam) , rabbit anti-E-cadherin (1:1000; Cell Signaling Technology) and mouse anti-β-actin (1:2000; Cell Signaling Technology).Statistical tests were conducted with SPSS 13.0. Student’s t test was used to compare means between the control and the treatment group. Error bars represent standard deviation (SD). The level of statistical significance (P value) is indicated in the corresponding figure legends, *P<0.05, **P<0.01,***P<0.001. 3.RESULTS To investigate the effects of Y-27632 on cell growth. Cell proliferation of ARPE-19 cells was assessed with or without Y-27632 treatment. We evaluated cell proliferation marker Ki67 after 10μM Y-27632 and 30μM Y-27632 treatment for 48h. ARPE-19 cellstreated with 30μM Y-27632 showed more Ki67-positive cells (Fig. 1a), indicating promoted cell proliferation. The CCK-8 assay was also performed. Consistently, 30μM Y-27632 treatment for 24h, 36h and 48h markedly increased cell proliferation, as the group treated with Y-27623 had significantly higher OD values than the untreated group (0.559±0.021 vs. 0.768±0.050 at 48h, P<0.001) (Fig. 1b).We next tested the effect of Y-27632 on cell apoptosis. After 48h of treatment with 30μM Y-27632, apoptosis of cells in both early and late phase was inhibited, and the data are shown in Fig. 2.To further understand the mechanism of cell cycle, we assessed the expression level of key cell cycle regulators. During a normal cell cycle, Rb was phosphorylated by cyclin D1/CDK6/CDK4 complex and cyclin E1/CDK2 complex to entering S phase. P21 could inhibit CDK2 activation [36]. We found that treated with 30μM Y-27632 for 48h significantly increased the expression levels of cyclin E1, cyclin D1 and downregulated the expression level of P21 (Fig. 3). These data suggested that 30μM Y-27632 promotes cell cycle progression and inhibits apoptosis in ARPE- 19 cells.Knowing that Y-27632 strongly promoted cell proliferation, we next characterized the phenotype of ARPE-19 cells. The untreated ARPE-19 cells were round, or slightly elongated and remained tightly attached to each other and had a typical polygonal appearance (Fig. 4a). However, the ROCK inhibitor Y- 27632 caused morphological changes in the cells. The treated cells became elongated in shape (Fig. 4b).Different phenotypes of ARPE-19 cells might associate with different biomechanical properties. To quantitatively analyze the biomechanical change in the living cells, the morphological data (including the 2D images, peak force error images, elasticity maps and 3D images) was obtained using AFM (Fig. 5a). The surface roughness (Ra and Rq) was used to statistically characterize changes in cell topography. Compared with control group, the roughness of Y- 27632-treated ARPE-19 cells (Ra: 41.04±1.63nm vs. 24.41±0.75nm, P<0.01; Rq: 51.56±2.03nm vs.30.81±0.95nm, P<0.01) decreased showing a significant difference and these results are shown in Fig. 5b and 5c. Consistent with 3D images, the corrugations of cell surface were more evident in the control group in the peak force error images. After 48h of treatment with 30μM Y-27632, cell elasticity was also changed. Compared with control group, Young's modulus of Y-27632-treated cells (16.66±0.83KPa vs.32.55±1.48KPa, P<0.01) increased, showing a significant difference, and the result is shown in Fig. 5d.Cell morphology and biomechanical property were determined by the cytoskeleton [37]. ROCK pathway inhabitation had been shown to reduce actin stress fibers formation and myosin phosphorylation [38]. Indeed, we found that Y-27632 converted ARPE-19 cells from rounded to an elongated shape and decreased roughness of ARPE-19 cells membrane, suggesting that the inhibition of ROCK pathway might reorganize the cytoskeleton.Cytoskeleton morphology was illustrated after being exposed to Y-27632 exhibiting characteristic alterations including aggregation and distribution of microfilaments (MFs; Phalloidin) and microtubules (MTs; β -tubulin)(Fig. 6). Normally, the cells exhibited a clear distribution of MFs and MTs, which possessed a dense cortical layer and an isotropic network in the cytoplasm. However, after 30μM Y-27632 treatment for 48h, the obvious reorganization of MFs and MTs with elongated shape was observed. In order to determine whether Y- 27632 could induce epithelial–mesenchymal transition (EMT), we detected EMT markers. As compared to the expression level of the epithelial marker E-cadherin, fibrotic markers α -SMA and vimentin in control group, we found that the expression level of E-cadherin, α - SMA and vimentin remained unchanged in treated group (Fig. 7). 4.DISCUSSION In this study, we characterized the biomechanical properties in RPE cells after Y-27632 treatment using AFM for the first time. After being treated with Y-27632, the proliferation rate was significantly increased while apoptosis rate was significantly decreased. In addition, the roughness of RPE cells was significantly decreased whereas Young's modulus of RPE cells significantly increased. Moreover, the cytoskeleton underwent reorganization after treated with Y-27632. These data indicated that Y-27632 may promote proliferation and inhibit apoptosis of RPE cells through changing the cell membrane properties as well as reorganizing cytoskeleton. It is worth mentioning that ROCK activity inhibition leads to pro-apoptotic or pro-survival in a cell-type dependent manner. Most of the cancer studies supported the idea that inhibition of ROCK pathway suppressed cell cycle progression such as non-small cell lung cancer, bladder cancer and melanoma [39- 41]. The underlying mechanism might be Rho/ROCK signaling which regulated the G1/S progression and DNA synthesis in fibroblast [42]. In addition, ROCK inhibitor Y-27632 was shown to be involved in p53- mediated apoptosis in hemangioma cells [43]. However, ROCK inhibitor, Y-27632, was also found to promote limbal epithelial cell proliferation through increasing S-phase cells [44]. Y-27632 was also shown to deregulate P27 and upregulate the expression of cyclin D via PI3-kinase signaling to promote proliferation in corneal endothelial cells [45]. Moreover, ROCK inhibition was shown to enhance the survival of dissociated human embryonic stem cells [16]. Consistently, our result showed that Y-27632 promoted cell cycle progression through upregulating cyclin E1, cyclin D1 and downregulating P21. Different cell types in different physiological conditions possess a unique cell membrane structure with specific biomechanical properties. In our study, by using AFM to analyze cell membrane biomechanical properties, we found that cell elasticity was significantly altered with the treatment of 30μM Y-27632 as the Young's modulus of treatment group showing significant upregulation. Consistently, Abidine et al., also found that Y-27632 increased the plateau modulus of bladder cancer cell [46]. Recently, growing evidence suggests that the mechanical properties of the cell are closely related to cell proliferation, apoptosis, migration and adhesion. For example, Artesunate could inhibit the proliferation and migration of human glioma SHG44 cells by decreasing elasticity force or increasing adhesive force [47]. Consistently, Sodium nitroprusside (SNP)-induced apoptosis by decreasing cell elasticity in chondrocytes. Moreover, the biomechanical changes induced by SNP occurred before morphological changes, indicating that the cellular mechanical properties may be a more sensitive indicator of cell function [48]. Changes in the cell membrane may affect the signal transportation and subsequently, influence the cell cytoskeleton and survival [49]. Our study showed that Y-27632 treatment leads to the reorganization of MFs and MTs as well as the elongated shape of the cell. Known as an actin cytoskeletal organization regulator, ROCK participated in actin stress fiber formation and focal adhesion [50]. Changes in actomyosin structure might subsequently influence cell contractility, cell adhesion, cell migration and cell morphogenesis [12, 50]. In addition, the reorganization of cytoskeleton might influence chromosome stability and cell proliferation [51]. Further study is needed to reveal the underlying molecular mechanisms as well as the specific effects on the RPE cells due to changes in biomechanical properties. It has been previously established that specific inhibition of the ROCK pathway might have a therapeutic benefit for the prevention of various vitreoretinal interface diseases especially PVR [21]. In this study, we investigated the role of ROCK signaling by utilizing its inhibitor Y-27632 which caused multiple changes in ARPE-19 cells. Further in vivo experiments are needed to evaluate its therapeutic effect and safety properties as well as the cytoskeletal reorganization of ARPE-19 cells. The reorganization of the cytoskeletons combined with cell elongation is likely to bring changes in cellular elasticity and morphology. These results may provide insightful understanding and the reference value to the application of Y-27632 in RPE degeneration diseases.