May 2012
Volume 53, Issue 6
Free
Glaucoma  |   May 2012
The Effect of Rho-Associated Protein Kinase Inhibitor on Monkey Schlemm's Canal Endothelial Cells
Author Affiliations & Notes
  • Takanori Kameda
    Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the
  • Toshihiro Inoue
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Masaru Inatani
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Tomokazu Fujimoto
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Megumi Honjo
    Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the
  • Nanako Kasaoka
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Miyuki Inoue-Mochita
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Nagahisa Yoshimura
    Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the
  • Hidenobu Tanihara
    Department of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.
  • Corresponding author: Toshihiro Inoue, Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, Japan; noel@da2.so-net.ne.jp
Investigative Ophthalmology & Visual Science May 2012, Vol.53, 3092-3103. doi:10.1167/iovs.11-8018
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      Takanori Kameda, Toshihiro Inoue, Masaru Inatani, Tomokazu Fujimoto, Megumi Honjo, Nanako Kasaoka, Miyuki Inoue-Mochita, Nagahisa Yoshimura, Hidenobu Tanihara; The Effect of Rho-Associated Protein Kinase Inhibitor on Monkey Schlemm's Canal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(6):3092-3103. doi: 10.1167/iovs.11-8018.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells.

Methods.: SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632–induced changes in the intracellular localization of ZO-1, claudin-5, β-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system.

Results.: Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions.

Conclusions.: Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.

Introduction
The major route of drainage of the aqueous humor is the conventional pathway through the trabecular meshwork (TM) and Schlemm's canal in primates. 13 Although discrepancies exist among previous studies between clinical observations and basic examinations, 4,5 some experimental studies suggest that the aqueous outflow resistance is generated in the locus of the inner wall of Schlemm's canal and the juxtacanalicular tissue region in both normal and glaucomatous eyes. 68 The properties of the cytoskeletal structures, adhesive interactions, permeability of Schlemm's canal endothelial (SCE) cells, and secretion from TM cells are suggested to play important roles in the regulation of aqueous outflow. 1,9,10  
Previous studies, including ours, revealed that a selective Rho-associated coiled-coil–forming protein kinase (ROCK) inhibitor, Y-27632, caused retraction and rounding of human TM-cell bodies, disruption of filamentous actin (F-actin), impairment of focal adhesion formation, and decreased myosin light chain phosphorylation in TM cells and SCE cells, resulting in a significant intraocular pressure (IOP) decrease and an aqueous outflow facility increase. 1116 Another selective ROCK inhibitor, Y-39983, was reported to have similar effects on outflow facility, 17,18 and reduced IOP in healthy volunteers in our previous clinical trial. 19 Thus, ROCK inhibitors are recognized as potential glaucoma drugs to lower IOP. Previous works, however, mainly focused on the molecular mechanisms underlying the effects of ROCK inhibitors on TM cells, and effects on SCE cells have not been clarified thoroughly.  
The aim of the present study was to investigate the effect of Y-27632 on monkey SCE cells. Permeability of the SCE-cell monolayer and properties of the intercellular junctions were observed. Furthermore, we used DNA microarrays and gene ontology analysis to investigate whether Y-27632 modulates broad functions in SCE cells. 
Materials and Methods
Perfusion and Histologic Examination
Fresh cynomolgus monkey eyes were obtained from a commercial company (Keari Co., Ltd., Osaka, Japan). The eyes were stored on ice in eye storage medium (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) and perfused within 48 hours. Radial iridotomy was performed to prevent artificial deepening of the anterior chamber during perfusion. A 23-gauge perfusion needle was inserted through the cornea, placing the tip into the anterior chamber of the eye. The perfusion medium was Dulbecco's phosphate-buffered saline (DPBS), pH 7.4, with 5.5 mM glucose. Eyes were perfused at a constant pressure of 10 mm Hg at 25°C. After the initial baseline was recorded for 1 hour, the test eyes were perfused for 5 hours with DPBS containing 50 μM Y-27632 (Wako Pure Chemical Industries, Osaka, Japan). As controls, the contralateral fellow eyes were perfused with DPBS alone. To calculate the inflow rate, the weight of the perfusion medium reservoir was measured using a digital balance (Shimadzu, Tokyo, Japan) and the data were recorded on a computer every 30 seconds. The aqueous humor outflow facility (μL/min/mm Hg) was calculated as the ratio between the inflow rate (μL/min) and the perfusion pressure (mm Hg). Outflow facility values were calculated as the average over 10 minutes and expressed as percentage change from baseline. The baseline values of outflow facility were determined at least 10 minutes before changing the perfusion medium. Following drug perfusion, the eyes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M phosphate buffer at 10 mm Hg for 2 hours. After fixation, the eyes were dissected at the equator and the vitreous and lens were removed. The anterior segment of the eyes was immersed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C. For scanning electron microscopy (SEM), specimens were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer. After critical point drying, the anterior segment was mounted on an SEM stub, gold-coated, and examined with an SE microscope (JSM-6360LV; JEOL, Tokyo, Japan). All three pairs of eyes were examined under SEM, and 4 to 10 different sites in each eye were observed.  
Culture of Monkey Schlemm's Canal Endothelial Cells
The SCE explants were dissected from eyes of 6- to 12-month-old cynomolgus monkeys obtained from a commercial laboratory (Shin Nippon Biomedical Laboratories, Kagoshima, Japan) according to the method described previously by Alvarado et al. 20,21 Briefly, Schlemm's canal was identified by cannulating the lumen with a 9-0 nylon suture under microscopic observation. The corneoscleral and uveoscleral TM tissues were removed completely with fine forceps, and explants of the inner wall of Schlemm's canal were cut out and placed on plates coated with collagen gel. Primary SCE cells were expanded in Dulbecco's modified Eagle's medium (DMEM; WAKO Pure Chemical Industries, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) and antibiotics at 37°C in 5% CO2, and SCE cells between passages 6 and 8 were used in all experiments in the present study. 
Measurement of Monolayer Transendothelial Electron Resistance
SCE cells were seeded at 1 × 105 cells/well and were grown until confluent on polycarbonate membrane inserts (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich, St. Louis, MO) on 12-well culture plates (BD Falcon, Franklin Lakes, NJ) in DMEM supplemented with 10% FBS at 37°C in 5% CO2. The volume of the apical side (inside of the membrane inserts) was 0.5 mL and that of the basal side (outside of the membrane inserts) was 1.5 mL. Two weeks after seeding, the medium was changed to DMEM supplemented with Y-27632 (0, 1, 5, or 25 μM) or A23187 (a calcium ionophore that increases intracellular calcium ion levels; 10 μM; Calbiochem, Darmstadt, Germany) for TEER experiments. The TEER was measured using an electrical resistance system (Millicell ERS; Millipore, Billerica, MA) according to the manufacturer's instructions 30, 60, and 90 min after treatment with Y-27632, and recorded as net values (Ωcm2). Time-dependent changes after treatment were monitored and compared as percentage change from baseline values. Each experiment was repeated at least three times. The effect of EGTA (2 mM; Dojindo Laboratories, Kumamoto, Japan) was confirmed as a positive control. 
Measurement of Monolayer Cell Permeability
SCE cells were prepared by the same method for TEER measurement as described above. SCE-cell monolayers were then stimulated with Y-27632 at concentrations of 0, 1, 5, or 25 μM. A tracer, 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich), was simultaneously applied at 50 μM to the basal compartment of the wells. The medium was collected from the apical side for fluorescence measurements at 30, 60, and 90 minutes after adding the tracer and the same volume of the culturing medium was added to replace the medium removed. The concentration of FITC-dextran in the collected medium was measured using a multimode plate reader (Multi Microplate Reader, MTP-800AFC; Corona Electric, Ibaragi, Japan), with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Fluorescence intensity of the normal medium was measured as the background concentration in each experiment, and each experiment was repeated at least three times. 
Immunocytochemistry
SCE cells were cultured on gelatin-coated glass coverslips until confluent and then treated with Y-27632 (25 μM) or latrunculin B (an F-actin depolymerizing drug, 400 nM; Merck KGaA, Darmstadt, Germany). When needed, SCE cells were pretreated with jasplakinolide (an F-actin stabilizing drug, 15 nM; Enzo Life Sciences, Farmingdale, NY) for 1 hour before treatment with Y-27632. After a 30-minute treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 minutes, washed with cytoskeletal buffer (10 mM MES [2- morpholinoethansulfonic acid potassium salt], 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose, pH 6.1), and serum buffer (10% FBS and 0.2 mg/mL sodium azide in PBS). Cells were permeabilized with 0.5% Triton X-100 in PBS for 12 minutes at room temperature and blocked with serum buffer for at least 2 hours at 4°C. After blocking, the cells were incubated overnight at 4°C with the following primary antibodies: rabbit anti-ZO-1 (1.25 μg/mL; Invitrogen, Carlsbad, CA), anti-claudin-5 (2.5 μg/mL; Invitrogen), anti-β-catenin (1:1000 dilution; Sigma-Aldrich), and anti-pan-cadherin (1:100 dilution; Sigma-Aldrich). The cells were rinsed with serum buffer and then incubated with secondary antibody (Alexa Fluoro 488; Invitrogen) and Phalloidin-TRITC (Sigma-Aldrich) for F-actin stained for 30 minutes at room temperature. After washing the cells with PBS, the cells were mounted with commercial mounting medium (VECTASHIELD; Vector Laboratories, Burlingame, CA), with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and observed via a fluorescence microscope (BX51; Olympus, Tokyo, Japan). 
DNA Microarray Analysis
Custom cDNA microarray analysis was performed using DNA microarray technology (CombiMatrix, Mukilteo, WA). The cynomolgus monkey array was designed to detect directly labeled mRNA from 12,613 probes. Confluent SCE cells in 100-mm dishes were treated with Y-27632 (25 μM) or vehicle for 30 minutes. Total RNA was then extracted from the cells, and the integrity and concentration of total RNA were measured using a bioanalysis unit (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP or Cy3-UTP, using each RNA sample and an RNA transcript kit (RNA Transcript SureLABEL Core Kit; TaKaRa BIO, Shiga, Japan). The labeled antisense RNAs were hybridized simultaneously with the microarray chips. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed according to the manufacturer's instructions (Filgen, Nagoya, Japan). Fluorescence images of hybridized microarrays were obtained with a commercial scanner (GenePix 4000B Scanner; Molecular Devices, Sunnyvale, CA). The analyzer (Array-Pro Analyzer Ver4.5; Media Cybernetics, Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scan data images were analyzed using microarray data software (Microarray Data Analysis Tool Ver 3.2 software; Filgen). Signals from Y-27632–treated cells were compared with those from vehicle-treated cells, and genes that showed a >2-fold change in expression in at least one of the pairwise probe comparisons were considered upregulated, whereas those that showed a change of expression <0.5-fold were considered downregulated. These analyses were performed three times using SCE cells from three different monkeys independently, and genes with common differences in expression among the three experiments were identified as affected genes. The affected genes were further analyzed by gene ontology, in which putative functions of gene products were categorized as “biological process,” “cellular component,” or “molecular function” by a BLAST (Basic Local Alignment Search Tool) homology search of the expressed sequence tags available at the National Center for Biotechnology Information. 
Calcium Imaging
Confluent SCE cells were incubated with Fluo-4/AM (5 μM; Invitrogen) in serum-free DMEM for 60 minutes in the dark, followed by a wash with Tyrode's solution with 0.5 mM of calcium according to the manufacturer's instructions. Fluo-4 was then excited at 480 nm, and emission was recorded continuously with a band-pass filter between 505 and 550 nm using a fluorescence microscope system (IX71; Olympus). The archived photographs were converted to gray-scale images, and the signals were quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The percent change from baseline (before treatment) was compared among conditions. 
Statistical Analysis
Data are presented as means ± SEs. Statistical comparisons of multiple groups were performed using one-way or two-way repeated-measure ANOVA followed by Bonferroni/Dunn's post hoc test. Comparisons of two groups used Student's t-test with Bonferroni's correction. Differences were considered statistically significant at P < 0.05. 
Results
Effect of Y-27632 on Outflow Facility
Baseline values of outflow facility in the control eyes and drug-perfused eyes were 0.50 ± 0.15 and 0.48 ± 0.08, respectively (n = 3). After the drug perfusion, outflow facility increased progressively, and was significantly higher within 170 minutes after perfusion compared with control eyes. There was a 38% increase in outflow facility over control eyes at 3 hours (P = 0.026), and the corresponding value progressed to 61% (P = 0.008) at 4 hours after drug perfusion (Fig. 1). 
Figure 1. 
 
Effect of perfusion with Y-27632 on outflow facility in enucleated monkey eyes. After measuring the baseline value, eyes were perfused with 50 μM Y-27632 at a constant pressure (10 mm Hg) at 25°C. After drug perfusion, the percentage change of outflow facility from the baseline value increased significantly and time-dependently over control eyes. Values are mean ± SE, n = 3, *P < 0.05, # P < 0.01.
Figure 1. 
 
Effect of perfusion with Y-27632 on outflow facility in enucleated monkey eyes. After measuring the baseline value, eyes were perfused with 50 μM Y-27632 at a constant pressure (10 mm Hg) at 25°C. After drug perfusion, the percentage change of outflow facility from the baseline value increased significantly and time-dependently over control eyes. Values are mean ± SE, n = 3, *P < 0.05, # P < 0.01.
Effect of Y-27632 on Morphology of SCE
After drug perfusion, baseline values of outflow facility in the control eyes and drug-perfused eyes were subjected to SEM observations. As shown in a past study, 12 Y-27632–perfused eyes tended to have more giant vacuoles (Fig. 2B) on the SCE compared with control eyes (Fig. 2A). Although intercellular detachments, possibly paracellular drainage routes, were observed in both control eyes and drug-perfused eyes, they were more frequently seen in the latter. 
Figure 2. 
 
Scanning electron micrographs of SCE. Compared with control eyes (left), SCE of Y-27632 perfused eyes tended to have more giant vacuoles (right). Although intercellular detachments were also observed in control eyes, they were more frequently observed in the drug-perfused eyes.
Figure 2. 
 
Scanning electron micrographs of SCE. Compared with control eyes (left), SCE of Y-27632 perfused eyes tended to have more giant vacuoles (right). Although intercellular detachments were also observed in control eyes, they were more frequently observed in the drug-perfused eyes.
Effect of Y-27632 on SCE-Cell Monolayer Permeability
TEER of the SCE cell monolayer after adding 25 μM of Y-27632 was significantly lower compared with that of controls at 30 minutes. At 60 minutes after treatment, 1, 5, and 25 μM Y-27632 significantly decreased TEER (Fig. 3). Of interest, the TEER values were not significantly different among the three samples treated with Y-27632 at 1, 5, and 25 μM each 60 minutes after treatment. The permeability assays using a flux of FITC-dextran showed that treatment with Y-27632 increased the concentration of FITC-dextran in the apical side time-dependently and dose-dependently. One millimolar Y-27632 did not significantly change the concentration of the FITC-dextran, whereas 25 μM of Y-27632 increased the concentration nearly 2-fold (Fig. 4). 
Figure 3. 
 
Changes in TEER in the SCE-cell monolayer. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 3. 
 
Changes in TEER in the SCE-cell monolayer. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 4. 
 
Changes in the SCE-cell monolayer permeability of 4 kDa fluorescein isothiocyanate-dextran. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 4. 
 
Changes in the SCE-cell monolayer permeability of 4 kDa fluorescein isothiocyanate-dextran. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Effects of Y-27632 on F-Actin and Molecules Associated with Cell–Cell Contact in SCE Cells
Immunocytochemical staining revealed a modest disruption of tight-junction–related proteins, such as ZO-1 and claudin-5 in Y-27632–treated cells in conjunction with the disappearance of longitudinal F-actin (Figs. 5A–D). Meanwhile, the staining of molecules related to adherence junction, such as β-catenin and pan-cadherin, tended to be broad and sparse after treatment with Y-27632 (Figs. 5E–H). Those changes in adherence junctions were less prominent, if any, compared with tight junctions. We also investigated effects of latrunculin B, a Rho-independent cytoskeletal drug. As shown in Fig. 6, ZO-1 proteins were disrupted by treatment with latrunculin B. Moreover, pretreatment with jasplakinolide, an actin filament–stabilizing drug, rescued in part the effect of subsequent treatment with Y-27632 on both F-actin structure and intracellular localization of ZO-1 (Figs. 6C–F). 
Figure 5. 
 
Effects of the ROCK inhibitor Y-27632 on cell–cell contact and actin stress fibers in SCE cells. SCE cells in culture treated with 25 μM Y-27632 for 30 minutes were immunostained for molecules relating to cell–cell contact: ZO-1, claudin-5, β-catenin, and pan-cadherin (green). The left image of each figure is a merged image with F-actin staining (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
Figure 5. 
 
Effects of the ROCK inhibitor Y-27632 on cell–cell contact and actin stress fibers in SCE cells. SCE cells in culture treated with 25 μM Y-27632 for 30 minutes were immunostained for molecules relating to cell–cell contact: ZO-1, claudin-5, β-catenin, and pan-cadherin (green). The left image of each figure is a merged image with F-actin staining (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
Figure 6. 
 
Effects of latrunculin, jasplakinolide, and Y-27632 on the polymerization of F-actin and intracellular localization of ZO-1. The top images show ZO-1 expression (green) in untreated SCE cells and latrunculin-treated SCE cells. The effects of Y-27632 with or without pretreatment with jasplakinolide are shown in the middle (F-actin, red) and bottom images (ZO-1, green). Scale bar: 50 μm.
Figure 6. 
 
Effects of latrunculin, jasplakinolide, and Y-27632 on the polymerization of F-actin and intracellular localization of ZO-1. The top images show ZO-1 expression (green) in untreated SCE cells and latrunculin-treated SCE cells. The effects of Y-27632 with or without pretreatment with jasplakinolide are shown in the middle (F-actin, red) and bottom images (ZO-1, green). Scale bar: 50 μm.
Microarray Expression Profile in Y-27632–Treated SCE Cells
Affected genes analyzed by microarray are listed in Tables 1 and 2, in which 57 genes were upregulated and 15 genes were downregulated. Among those genes, 10 genes showed >5-fold expression changes, and one gene showed <0.2-fold expression change (Tables 1 and 2). Significantly upregulated and downregulated gene categories based on gene ontology analysis in Y-27632–treated SCE cells are listed in Tables 3 and 4. Gene ontology analysis revealed that affected genes were related to various cellular functions, including upregulation of a biological process, “regulation of extracellular matrix disassembly” (P = 0.023), downregulation of a cellular component, “integrin complex” (P = 0.039), and downregulation of a biological process, “calcium ion transport into cytosol” (P = 0.008). 
Table 1.  
 
Representative Genes That Are Upregulated in SCE Cells
Table 1.  
 
Representative Genes That Are Upregulated in SCE Cells
Accession Number Gene Name Fold Change
CJ435051 Myelin basic protein isoform 2 16.42917
CJ440786 Pleckstrin homology domain interacting protein 15.84593
BB889989 Chondroadherin precursor 7.27175
AB170326 Human syndecan 4 (amphiglycan, ryudocan) (SDC4) 4.79868
AB171586 Human adenylate cyclase 2 (brain) (ADCY2) 4.44696
DW528016 Thyroid hormone receptor interactor 3 4.38051
CJ492124 Splicing factor, arginine/serine-rich 6 [Rattus norvegicus] 4.285
AB168674 Human RIKEN cDNA 1700016G05 (LOC402604) 4.25099
DC621007 L-Threonine dehydrogenase 4.07265
BB898986 Complement factor I preproprotein 3.73984
AB172153 Human CDW92 antigen (CDW92) 2.9576
AB170860 Human WD repeat domain 8 (WDR8) 2.66228
AB070180 Human hypothetical protein FLJ46088, mRNA, AK127973 2.59433
DK578068 Hypothetical protein LOC348262 [Homo sapiens] 2.58971
CJ470450 Splicing factor 3b, subunit 1 isoform 1 [Danio rerio] 2.58217
EF208807 MHC class II antigen DP alpha chain (Mafa-DPA) mRNA, Mafa-DPA1*0202 allele 2.56233
AB174415 Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) (SPTAN1) 2.55997
AB169059 CD4 precursor 2.40463
DC645348 Sulfotransferase family, cytosolic, 1C, member 1 isoform a 2.40361
CJ446069 Protein phosphatase 3, catalytic subunit, alpha isoform 2.40133
CJ440484 Brain cDNA 2.38472
DK579312 Spliceosome RNA helicase BAT1 2.36141
AB168995 Human ring finger protein 36 (RNF36) 2.28256
DC850662 Fc gamma RIIIa 2.26894
AB179134 Human syntaxin binding protein 4 (STXBP4) 2.25772
AB071116 Human ankyrin repeat and SOCS box-containing 15 (ASB15) 2.22498
DC850740 Cytosolic phospholipase A2 gamma precursor (cPLA2-gamma) (Phospholipase A2 group IVC) 2.2098
AB174030 Human galactose mutarotase (aldose 1-epimerase) (GALM) 2.20885
AB168319 Human chromosome 6 open reading frame 10 (C6orf10) 2.1913
AB169280 Human hypothetical protein FLJ13305 (FLJ13305) 2.16347
CJ492687 USP6 N-terminal like 2.16298
AB169348 Human hypothetical protein MGC33600 (MGC33600) 2.15724
AB172168 Human protein tyrosine phosphatase, receptor type, fpolypeptide (PTPRF), interacting protein (liprin), alpha4 (PPFIA4) 2.12463
AB173609 Human cholinergic receptor, nicotinic, epsilon polypeptide (CHRNE) 2.12166
AB173849 Human zinc finger protein 24 (KOX 17) (ZNF24) 2.07481
AB168153 Human DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 (DDX43) 2.07426
Table 2.  
 
Representative Genes That Are Downregulated in SCE Cells
Table 2.  
 
Representative Genes That Are Downregulated in SCE Cells
Accession Number Gene Name Fold Change
DK576163 Human H3 histone, family 3B (H3.3B) (H3F3B) 0.28442
AB174375 Human dual specificity phosphatase 14 (DUSP14) 0.35283
DC631004 Nucleophosmin (nucleolar phosphoprotein B23, numatrin) 0.42112
AB072781 Chromodomain Y-like protein 2 (CDYL2) 0.44636
CJ440707 Brefeldin A–inhibited guanine nucleotide-exchange protein 1 0.46621
AB070198 Human DNA-damage inducible protein 1 (DDI1) 0.4856
DK581802 Cytokine receptor-like factor 3 [Homo sapiens] 0.49076
AB173178 Human hepatic leukemia factor (HLF) 0.49856
Table 3.  
 
Gene Ontology of Upregulated Genes in Y-27632–Treated SCE Cells
Table 3.  
 
Gene Ontology of Upregulated Genes in Y-27632–Treated SCE Cells
Ontology Term Total Genes P-Value
Molecular function MHC class II protein binding 3(3) 0.00131
Biological process Transmembrane receptor protein tyrosine kinase signaling pathway 104(27) 0.00169
Biological process Negative regulation of Ras protein signal transduction 6(3) 0.0036
Biological process Negative regulation of small GTPase-mediated signal transduction 6(0) 0.0036
Biological process Regulation of cytoskeleton organization 60(2) 0.0064
Biological process RNA processing 339(35) 0.00688
Biological process tRNA processing 35(28) 0.00961
Biological process Enzyme-linked receptor protein signaling pathway 152(1) 0.00999
Biological process Cellular response to insulin stimulus 39(18) 0.01265
Molecular function MHC protein binding 13(0) 0.01275
Biological process Cellular response to endogenous stimulus 78(0) 0.01507
Biological process Cellular response to hormone stimulus 78(12) 0.01507
Biological process Cellular response to peptide hormone stimulus 42(0) 0.01525
Molecular function Molecular transducer activity 621(0) 0.01768
Molecular function Signal transducer activity 621(134) 0.01768
Biological process Positive regulation of cytoskeleton organization 16(2) 0.01822
Biological process Regulation of cellular component organization 229(0) 0.01957
Molecular function 1-Acylglycerophosphocholine O-acyltransferase activity 1(1) 0.02316
Molecular function 1-Alkylglycerophosphocholine O-acetyltransferase activity 1(1) 0.02316
Molecular function Aldose 1-epimerase activity 1(1) 0.02316
Molecular function Cholesterol 24-hydroxylase activity 1(1) 0.02316
Molecular function Choline transmembrane transporter activity 1(1) 0.02316
Molecular function tRNA isopentenyltransferase activity 1(1) 0.02316
Biological process Elastin catabolic process 1(0) 0.02321
Biological process Extracellular matrix disassembly 1(0) 0.02321
Biological process Histolysis 1(0) 0.02321
Biological process Induction by virus of host cell–cell fusion 1(1) 0.02321
Biological process Negative regulation of Rac protein signal transduction 1(1) 0.02321
Biological process Negative regulation of blood vessel remodeling 1(1) 0.02321
Biological process Negative regulation of collagen catabolic process 1(1) 0.02321
Biological process Negative regulation of elastin catabolic process 1(1) 0.02321
Biological process Negative regulation of extracellular matrix disassembly 1(1) 0.02321
Biological process Negative regulation of histolysis 1(1) 0.02321
Biological process Positive regulation of centriole replication 1(1) 0.02321
Biological process Positive regulation of centrosome cycle 1(0) 0.02321
Biological process Regulation of blood vessel remodeling 1(0) 0.02321
Biological process Regulation of collagen catabolic process 1(0) 0.02321
Biological process Regulation of elastin catabolic process 1(0) 0.02321
Biological process Regulation of extracellular matrix disassembly 1(0) 0.02321
Biological process Regulation of histolysis 1(0) 0.02321
Biological process Tissue death 1(0) 0.02321
Biological process Trophoblast giant cell differentiation 1(1) 0.02321
Biological process Cellular response to organic substance 96(0) 0.02891
Biological process Insulin receptor signaling pathway 21(12) 0.02903
Molecular function Receptor activity 425(266) 0.0338
Cellular component Exon–exon junction complex 2(2) 0.03402
Molecular function 3′,5′-Cyclic-GMP phosphodiesterase activity 2(1) 0.03454
Molecular function O-Acetyltransferase activity 2(0) 0.03454
Molecular function Actinin binding 2(0) 0.03454
Molecular function alpha-Actinin binding 2(1) 0.03454
Molecular function Amine oxidase activity 2(2) 0.03454
Molecular function Arginase activity 2(2) 0.03454
Molecular function Isocitrate dehydrogenase (NADP+) activity 2(2) 0.03454
Biological process Factor XII activation 2(2) 0.03462
Biological process Cell differentiation involved in embryonic placenta development 2(1) 0.03462
Biological process Choline transport 2(2) 0.03462
Biological process Elastin metabolic process 2(1) 0.03462
Biological process Glyoxylate cycle 2(2) 0.03462
Biological process Kinin cascade 2(0) 0.03462
Biological process ncRNA catabolic process 2(0) 0.03462
Biological process Negative regulation of Rho protein signal transduction 2(2) 0.03462
Biological process Negative regulation of collagen metabolic process 2(0) 0.03462
Biological process Negative regulation of multicellular organismal metabolic process 2(0) 0.03462
Biological process Plasma kallikrein-kinin cascade 2(0) 0.03462
Biological process Positive regulation of focal adhesion assembly 2(2) 0.03462
Biological process rRNA catabolic process 2(2) 0.03462
Biological process Regulation of Rac GTPase activity 2(2) 0.03462
Biological process Regulation of centriole replication 2(1) 0.03462
Biological process Response to insulin stimulus 59(21) 0.03533
Biological process Cellular component disassembly 60(0) 0.0368
Biological process Regulation of organelle organization 104(0) 0.03688
Cellular component Nuclear speck 62(62) 0.03815
Biological process Regulation of small GTPase- mediated signal transduction 106(20) 0.03906
Biological process Nitrogen compound metabolic process 1974(14) 0.04265
Cellular component Compact myelin 3(2) 0.04511
Cellular component Spectrin 3(3) 0.04511
Molecular function Calcium- and calmodulin-responsive adenylate cyclase activity 3(3) 0.04579
Molecular function Histamine receptor activity 3(3) 0.04579
Molecular function Structural constituent of myelin sheath 3(3) 0.04579
Molecular function Thrombospondin receptor activity 3(3) 0.04579
Biological process Regulation of catalytic activity 382(4) 0.0458
Biological process Arginine catabolic process 3(3) 0.04589
Biological process Cytoplasmic microtubule organization 3(2) 0.04589
Biological process Positive regulation of fibrinolysis 3(3) 0.04589
Biological process Positive regulation of protein maturation by peptide bond cleavage 3(0) 0.04589
Biological process Regulation of cell-substrate junction assembly 3(0) 0.04589
Biological process Regulation of centrosome cycle 3(0) 0.04589
Biological process Regulation of centrosome duplication 3(1) 0.04589
Biological process Regulation of focal adhesion assembly 3(0) 0.04589
Biological process RNA splicing, via transesterification reactions with bulged adenosine as nucleophile 66(0) 0.04622
Biological process Nuclear mRNA splicing, via spliceosome 66(30) 0.04622
Biological process Regulation of cellular component biogenesis 67(0) 0.0479
Table 4.  
 
Gene Ontology of Downregulated Genes in Y-27632–Treated SCE Cells
Table 4.  
 
Gene Ontology of Downregulated Genes in Y-27632–Treated SCE Cells
Ontology Term Total Genes P-Value
Molecular function Hydroxymethylglutaryl-CoA synthase activity 2(2) 0.00033
Molecular function Transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer 6(3) 0.00152
Molecular function Exonuclease activity, active with either ribo- or deoxyribonucleic acids and producing  5′-phosphomonoesters 9(0) 0.00294
Biological process Regulation of release of sequestered calcium ion into cytosol 11(0) 0.00395
Biological process Negative regulation of sequestering of calcium ion 13(0) 0.00527
Biological process Regulation of sequestering of calcium ion 13(0) 0.00527
Biological process Release of sequestered calcium ion into cytosol 13(2) 0.00527
Molecular function 3′-5′ Exonuclease activity 13(7) 0.00551
Biological process Isoprenoid biosynthetic process 14(10) 0.006
Biological process Sequestering of calcium ion 15(2) 0.00677
Molecular function Glutathione transferase activity 15(15) 0.00707
Biological process Regulation of calcium ion transport into cytosol 16(0) 0.00758
Biological process Calcium ion transport into cytosol 17(0) 0.00843
Biological process Cytosolic calcium ion transport 17(0) 0.00843
Biological process Sequestering of metal ion 17(0) 0.00843
Cellular component Microtubule 122(101) 0.00956
Biological process Cholesterol biosynthetic process 20(15) 0.01123
Biological process DNA replication checkpoint 1(1) 0.01472
Biological process Cysteine biosynthetic process from serine 1(1) 0.01472
Biological process Cysteine biosynthetic process via cystathionine 1(1) 0.01472
Biological process Homocysteine catabolic process 1(1) 0.01472
Biological process Negative regulation of DNA-dependent DNA replication initiation 1(0) 0.01472
Molecular function L-Gulonate 3-dehydrogenase activity 1(1) 0.01504
Molecular function Bradykinin receptor activity 1(1) 0.01504
Molecular function Cystathionine beta-synthase activity 1(1) 0.01504
Molecular function Exodeoxyribonuclease III activity 1(1) 0.01504
Molecular function Monodehydroascorbate reductase (NADH) activity 1(1) 0.01504
Molecular function Polyribonucleotide nucleotidyltransferase activity 1(1) 0.01504
Molecular function Ribonuclease III activity 1(1) 0.01504
Biological process Sterol biosynthetic process 25(5) 0.01666
Biological process Regulation of calcium ion transport 26(1) 0.01786
Molecular function Exonuclease activity 26(19) 0.01864
Biological process Regulation of metal ion transport 28(0) 0.02037
Molecular function Nuclease activity 76(10) 0.02145
Biological process L-Ascorbic acid biosynthetic process 2(2) 0.022
Biological process L-Cysteine catabolic process 2(2) 0.022
Biological process L-Cysteine metabolic process 2(1) 0.022
Biological process Cysteine biosynthetic process 2(1) 0.022
Biological process Cysteine catabolic process 2(0) 0.022
Biological process Homocysteine metabolic process 2(0) 0.022
Biological process Hydrogen sulfide biosynthetic process 2(2) 0.022
Biological process Hydrogen sulfide metabolic process 2(0) 0.022
Biological process Regulation of DNA-dependent DNA replication 2(0) 0.022
Biological process Regulation of DNA-dependent DNA replication initiation 2(1) 0.022
Molecular function Transferase activity, transferring alkyl or aryl (other than methyl) groups 31(2) 0.02542
Biological process Isoprenoid metabolic process 32(0) 0.02578
Biological process L-Serine catabolic process 3(3) 0.02923
Biological process Positive regulation of ryanodine-sensitive calcium-release channel activity 3(3) 0.02923
Biological process Positive regulation of survival gene product expression 3(3) 0.02923
Biological process Regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum 3(3) 0.02923
Biological process Release of sequestered calcium ion into cytosol by sarcoplasmic reticulum 3(0) 0.02923
Biological process Sulfur amino acid catabolic process 3(0) 0.02923
Biological process Sulfur compound catabolic process 3(0) 0.02923
Molecular function N-Terminal myristoylation domain binding 3(3) 0.02987
Molecular function Exodeoxyribonuclease activity 3(0) 0.02987
Molecular function Exodeoxyribonuclease activity, producing 5′-phosphomonoesters 3(0) 0.02987
Molecular function Interleukin-2 binding 3(1) 0.02987
Molecular function Interleukin-2 receptor activity 3(3) 0.02987
Biological process Regulation of ion transport 36(1) 0.03171
Biological process Cell surface receptor linked signaling pathway 552(70) 0.03242
Molecular function Ribonuclease activity 36(16) 0.03305
Biological process L-Ascorbic acid metabolic process 4(2) 0.03641
Biological process Negative regulation of ryanodine-sensitive calcium-release channel activity 4(4) 0.03641
Biological process Sarcoplasmic reticulum calcium ion transport 4(1) 0.03641
Biological process Cytokine-mediated signaling pathway 39(20) 0.03648
Biological process Initiation of signal transduction 39(0) 0.03648
Biological process Leukocyte migration 39(5) 0.03648
Biological process Signal initiation by diffusible mediator 39(0) 0.03648
Biological process Signal initiation by protein/peptide mediator 39(0) 0.03648
Cellular component Barr body 5(5) 0.03939
Cellular component Integrin complex 5(5) 0.03939
Cellular component Receptor complex 47(4) 0.04198
Biological process Elevation of cytosolic calcium ion concentration 43(26) 0.04323
Biological process Ephrin receptor signaling pathway 5(5) 0.04353
Biological process Positive regulation of ion transmembrane transporter activity 5(0) 0.04353
Biological process Regulation of defense response to virus by virus 5(5) 0.04353
Biological process Regulation of ryanodine-sensitive calcium-release channel activity 5(1) 0.04353
Biological process Regulation of survival gene product expression 5(0) 0.04353
Molecular function MAP kinase phosphatase activity 5(0) 0.04447
Molecular function MAP kinase tyrosine/serine/threonine phosphatase activity 5(5) 0.04447
Biological process Signaling pathway 1024(0) 0.04488
Cellular component X chromosome 6(1) 0.0458
Biological process Cytosolic calcium ion homeostasis 46(4) 0.04858
Effects of Y-27632 on the Calcium Concentration in SCE Cells
TEER of the SCE cell monolayer after adding 2 mM EGTA was significantly lower compared with that of control at 30 minutes or more (Fig. 7A). Meanwhile, treatment with 10 μM A23187, a calcium ionophore, increased the TEER significantly (Fig. 7B). A23187 increased the intracellular concentration of calcium ions in SCE cells, and Y-27632, interestingly, partially diminished the A23187-induced increase of intracellular calcium ions (Fig. 7C). 
Figure 7. 
 
(A, B) Changes in TEER in the SCE cell monolayer. SCE cells were treated with EGTA (A) or A23187 (B), and TEER was measured at 30, 60, and 90 minutes after administration. White bars indicate the values in control condition, and black bars indicate the values with drug treatment. Mean values ± SEs from three separate filters are presented. *P < 0.05, Student's t-test comparing control and drug treatment at each time point. (C) Effects of A23187 with or without pretreatment by Y-27632 on the intracellular concentration of calcium ions estimated by Fluo-4 AM The mean ± SEs are presented as the ratio of F (value after treatment) to F0 (value before treatment). *P < 0.05, Student's t-test with Bonferroni correction comparing control and drug treatment at each time point.
Figure 7. 
 
(A, B) Changes in TEER in the SCE cell monolayer. SCE cells were treated with EGTA (A) or A23187 (B), and TEER was measured at 30, 60, and 90 minutes after administration. White bars indicate the values in control condition, and black bars indicate the values with drug treatment. Mean values ± SEs from three separate filters are presented. *P < 0.05, Student's t-test comparing control and drug treatment at each time point. (C) Effects of A23187 with or without pretreatment by Y-27632 on the intracellular concentration of calcium ions estimated by Fluo-4 AM The mean ± SEs are presented as the ratio of F (value after treatment) to F0 (value before treatment). *P < 0.05, Student's t-test with Bonferroni correction comparing control and drug treatment at each time point.
Discussion
In the present study, a specific inhibitor of Rho kinase, Y-27632, increased outflow facility (Fig. 1), decreased TEER (Fig. 3), increased permeability of FITC-dextran (Fig. 4), and decreased ZO-1 and claudin-5 expression in conjunction with depolymerization of F-actin (Fig. 5) in the SCE-cell monolayer compared with controls, suggesting that these changes are related to a decrease in IOP and an increase in aqueous outflow facility by Y-27632. 11,12 Moreover, microarray and subsequent gene ontology analyses first revealed that Y-27632 changed expression patterns of various genes, and affected broad range of cellular functions in SCE cells (Tables 14). Above all, the inhibitory effect on the increase in intracellular calcium ions was confirmed functionally (Fig. 7). 
It is widely believed that RhoA activation leads to macrovascular endothelial barrier breakdown. 22 Using intestinal epithelial cells, however, revealed that the dominant active RhoA increases TEER and decreases paracellular permeability. Of interest, Schlegel and colleagues 22 reported that both activation and inactivation of RhoA signaling increased paracellular permeability. Thus, the responses of cell monolayer against modulation of Rho signaling are assumed to be context dependent. In the present study, Y-27632 increased SCE-cell monolayer permeability and decreased TEER (Figs. 3 and 4), consistent with the study using human SCE cells, revealing that Y-27632 increased permeability of cell monolayer with cell–cell detachment in SCE cells. 12 Although SCE cells have properties similar to those of vascular endothelial cells, 2325 their responses to Y-27632 were different from those of other vascular endothelial cells. Breaks were found in the endothelial lining of the Schlemm's canal and aqueous plexus after perfusion with certain cytoskeletal drugs, including latrunculin-B, ethacrynic acid, and wiskostatin. 2628 Moreover, our examination with SEM in the present study revealed that perfusion with Y-27632 increased giant vacuoles accompanied by opening of the intercellular routes of SCE (Fig. 2). In a previous study, however, the endothelial lining was intact after perfusion with Y-27632, as assessed by transmission electron microscopy. 12 Taken together, although the results were reasonable to explain how Y-27632 increased aqueous outflow facility, 11,12 further exploration will be required to determine the full effect of ROCK inhibitors against paracellular permeability in SCE cells in vivo. Additionally, SCE cells have transcellular pores accompanied by giant vacuoles, 29 a different property from vascular endothelial cells, which were not evaluated in the present study. 
Tight junctions play an important role as a barrier to the movement of water, solutes, and immune cells, and their dysfunction leads to increased paracellular permeability. ZO-1 is a universal component of tight junctions and interacts with multiple other tight junction proteins. 30 In the present study, ROCK inhibition resulted in modest disruption of the cell–cell contact along with decreased staining of ZO-1, claudin-5 (Fig. 5), suggesting that Y-27632 induced paracellular permeability through changes in the tight junctions. In support, Walsh et al. 31 reported that an inhibitor inhibited redistribution of ZO-1 to the intercellular junction upon calcium repletion. Because microarray analysis did not detect changes in RNA expression of the genes coding these molecules, ROCK did not regulate transcription of the molecules associated with tight junction; rather, it regulated protein degradation or intracellular localization in SCE cells. These changes in the intracellular localization of tight junction–associated proteins were linked to the depolymerization of F-actin, because latrunculin B caused similar changes to those of Y-27632. Moreover, treatment with Y-27632 in the presence of jasplakinolide, which potentially inhibited ROCK with polymerized F-actin, showed less effect on ZO-1 localization compared with Y-27632 treatment only. Thus, the changes in the tight junction are thought to be secondary to the disassembly of F-actin. 
Gene ontology grouping of the affected genes based on microarray analysis revealed that ROCK inhibition changed expressions of genes that were related to various cellular functions in SCE cells (Tables 3 and 4). Identified ROCK-related function included regulation of the extracellular matrix disassembly and expression of the integrin complex, which may be involved in the regulation of aqueous outflow because the interaction between SCE and extracellular matrix is important for aqueous outflow. 9 These changes might explain the mechanism underlying the inner wall/juxtacanalicular tissue separation between the basal lamina of the inner wall and the extracellular matrix of the juxtacanalicular tissue after perfusion with ROCK inhibitors in eyes of various species. 1214 Moreover, these data indicate that ROCK inhibitors potentially affect not only aqueous outflow but also other unknown cell properties, providing insights into possible pharmacologic reactions after clinical application of ROCK inhibitors. 
Microarray analysis using TM cells revealed that dominant active Rho, which activates Rho-ROCK signaling, led to the changes in gene expression. 32 This study revealed greater expression of potential glaucoma-related genes, such as myocilin, fibronectin, and collagen, encoding the extracellular matrix and increased expression of genes, including gremlin-2, IL-1, TGF-β, BMP, Wnt, and CTGF, encoding secreted bioactive factors. Although ROCK inhibitors have opposite effects to dominant active Rho generally, affected genes were different from those in the present study. This may be explained by the difference of cell properties between TM cells and SCE cells, the difference of species, or the difference of methods between pharmacologic treatment and gene induction. Another possibility is that the gene upregulated by activation of an intracellular signal is not always downregulated by inhibition of the same signal. 
It is well known that changes in the intracellular calcium ions can affect the monolayer permeability of the endothelial cells, including SCE cells. 33 The present study, however, is the first to report the involvement of Rho-ROCK signaling in the regulation of intracellular calcium ions. This finding was unexpectedly derived from our data in microarray and gene ontology analyses. In rat arteries, a previous study reported that Rho-kinase is involved in noradrenaline activation of calcium ion entry. 34 Although those authors found that the involvement was distinct from voltage- or store-operated channels in arteries, the data in the present study are limited, and further studies are required to clarify the underlying mechanisms in SCE cells. In addition, it remains unknown whether the effect on the calcium ion transport of Y-27632 is specific to ROCK inhibition or is a nonspecific side effect. 
In the present study, the effect of Y-27632 on outflow facility was significant at 2.5 hours or later, which was relatively slow compared with results of previous studies. 12,35 The contradiction is likely due to the difference in study techniques. In the previous studies, the anterior chambers were washed with Y-27632–containing buffer just before switching from baseline buffer to Y-27632–containing buffer, whereas no wash was conducted in the present study. The absence of the wash may delay the time to reach the threshold amount of the drug for the increase of outflow facility. 
There is a limitation in SEM studies to compare the morphologic changes between control and Y-27632–treated eyes, because a previous study in which monkey eyes were perfused with Y-27632 showed that aqueous outflow differed by location and that some inactive areas exist. 
In summary, exposure to the ROCK inhibitor Y-27632 results in Rho/ROCK-dependent F-actin reorganization and disruption of proteins associated with tight junction, which may lead to increased aqueous humor outflow facility. Additionally, the current study revealed that Y-27632 induced changes in the expression of various genes in SCE cells, providing insight into possible pharmacologic reactions after clinical application of ROCK inhibitors. 
References
Tan JC Peters DM Kaufman PL . Recent developments in understanding the pathophysiology of elevated intraocular pressure. Curr Opin Ophthalmol . 2006;17:168–174. [PubMed]
Conventional Bill A . and uveo-scleral drainage of aqueous humour in the cynomolgus monkey (Macaca irus) at normal and high intraocular pressures. Exp Eye Res . 1966; 5:45–54. [CrossRef] [PubMed]
Pederson JE Gaasterland DE MacLellan HM . Uveoscleral aqueous outflow in the rhesus monkey: importance of uveal reabsorption. Invest Ophthalmol Vis Sci . 1977;16:1008–1007. [PubMed]
Rosenquist R Epstein D Melamed S Johnson M Grant WM . Outflow resistance of enucleated human eyes at two different perfusion pressures and different extents of trabeculotomy. Curr Eye Res . 1989;8:1233–1240. [CrossRef] [PubMed]
Grant WM . Experimental aqueous perfusion in enucleated human eyes. Arch Ophthalmol . 1963;69:783–801. [CrossRef] [PubMed]
Johnson M . What controls aqueous humour outflow resistance?. Exp Eye Res . 2006; 82:545–557 . [CrossRef] [PubMed]
Quigley HA . Open-angle glaucoma. N Engl J Med . 1993;328:1097–1106. [CrossRef] [PubMed]
Weinreb RN Khaw PT . Primary open-angle glaucoma. Lancet . 2004;363:1711–1720. [CrossRef] [PubMed]
Acott TS Kelley MJ . Extracellular matrix in the trabecular meshwork. Exp Eye Res . 2008;86:543–561. [CrossRef] [PubMed]
Rao VP Epstein DL . Rho GTPase/Rho kinase inhibition as a novel target for the treatment of glaucoma. BioDrugs . 2007;21:167–177. [CrossRef] [PubMed]
Honjo M Tanihara H Inatani M . Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci . 2001;42:137–144. [PubMed]
Rao PV Deng PF Kumar J Epstein DL . Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632. Invest Ophthalmol Vis Sci . 2001;42:1029–1037. [PubMed]
Lu Z Overby DR Scott PA Freddo TF Gong H . The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes. Exp Eye Res . 2008;86:271–281. [CrossRef] [PubMed]
Tian B Kaufman PL . Effects of the Rho kinase inhibitor Y-27632 and the phosphatase inhibitor calyculin A on outflow facility in monkeys. Exp Eye Res . 2005;80:215–225. [CrossRef] [PubMed]
Koga T Koga T Awai M Tsutsui J Yue BY Tanihara H . Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Exp Eye Res . 2006;82:362–370. [CrossRef] [PubMed]
Rosenthal R Choritz L Schlott S . Effects of ML-7 and Y-27632 on carbachol- and endothelin-1-induced contraction of bovine trabecular meshwork. Exp Eye Res . 2005;80:837–845. [CrossRef] [PubMed]
Tokushige H Inatani M Nemoto S . Effects of topical administration of Y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys. Invest Ophthalmol Vis Sci . 2007; 48:3216–3222. [CrossRef] [PubMed]
Nakajima E Nakajima T Minagawa Y Shearer TR Azuma M . Contribution of ROCK in contraction of trabecular meshwork: proposed mechanism for regulating aqueous outflow in monkey and human eyes. J Pharm Sci . 2005;94:701–708. [CrossRef] [PubMed]
Tanihara H Inatani M Honjo M Tokushige H Azuma J Araie M . Intraocular pressure-lowering effects and safety of topical administration of a selective ROCK inhibitor, SNJ-1656, in healthy volunteers. Arch Ophthalmol . 2008;126:309–315. [CrossRef] [PubMed]
Underwood JL Murphy CG Chen J . Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions. Am J Physiol Cell Physiol . 1999; 277:C330–C342.
Alvarado JA Betanzos A Franse-Carman L Chen J Gonzalez-Mariscal L . Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features. Am J Physiol Cell Physiol . 2004; 286:C621–C634. [CrossRef] [PubMed]
Spindler V Schlegel N Waschke J . Role of GTPases in control of microvascular permeability. Cardiovasc Res . 2010;87:243–253. [CrossRef] [PubMed]
Ramos RF Hoying JB Witte MH Stamer WD . Schlemm's canal endothelia, lymphatic, or blood vasculature?. J Glaucoma . 2007; 16:391–405. [CrossRef] [PubMed]
Heimark RL Kaochar S Stamer WD . Human Schlemm's canal cells express the endothelial adherens proteins, VE-cadherin and PECAM-1. Curr Eye Res . 2002;25:299–308. [CrossRef] [PubMed]
Hamanaka T Bill A Ichinohasama R Ishida T . Aspects of the development of Schlemm's canal. Exp Eye Res . 1992;55:479–488. [CrossRef] [PubMed]
Epstein DL Freddo TF Bassett-Chu S Chung M Karageuzian L . Influence of ethacrynic acid on outflow facility in the monkey and calf eye. Invest Ophthalmol Vis Sci . 1987;28:2067–2075. [PubMed]
Ethier CR Read AT Chan DW . Effects of latrunculin-B on outflow facility and trabecular meshwork structure in human eyes. Invest Ophthalmol Vis Sci . 2006;47:1991–1998. [CrossRef] [PubMed]
Inoue T Pattabiraman PP Epstein DL . Vasantha Rao P. Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway. Exp Eye Res . 2010; 90:360–367. [CrossRef] [PubMed]
Bill A Svedbergh B . Scanning electron microscopic studies of the trabecular meshwork and the canal of Schlemm—an attempt to localize the main resistance to outflow of aqueous humor in man. Acta Ophthalmol (Copenh) . 1972; 50:295–320. [CrossRef] [PubMed]
Hartsock A Nelson WJ . Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta . 2008;1778:660–669. [CrossRef] [PubMed]
Walsh SV Hopkins AM Chen J Narumiya S Parkos CA Nusrat A . Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology . 2001;121:566–579. [CrossRef] [PubMed]
Zhang M Maddala R Rao PV . Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork. Am J Physiol Cell Physiol . 2008; 295:C1057–C1070. [CrossRef] [PubMed]
Stamer WD Roberts BC Epstein DL . Hydraulic pressure stimulates adenosine 3′, 5′-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal. Invest Ophthalmol Vis Sci . 1999; 40:1983–1988. [PubMed]
Ghisdal P Vandenberg G Morel N . Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries. J Physiol . 2003;551:855–867. [CrossRef] [PubMed]
Lu Z Zhang Y Freddo TF Gong H . Similar hydrodynamic and morphological changes in the aqueous humor outflow pathway after washout and Y27632 treatment in monkey eyes. Exp Eye Res . 2011;93:397–404. [CrossRef] [PubMed]
Footnotes
 Disclosure: T. Kameda, None; T. Inoue, None; M. Inatani, None; T. Fujimoto, None; M. Honjo, None; N. Kasaoka, None; M. Inoue-Mochita, None; N. Yoshimura, None; H. Tanihara, None
Figure 1. 
 
Effect of perfusion with Y-27632 on outflow facility in enucleated monkey eyes. After measuring the baseline value, eyes were perfused with 50 μM Y-27632 at a constant pressure (10 mm Hg) at 25°C. After drug perfusion, the percentage change of outflow facility from the baseline value increased significantly and time-dependently over control eyes. Values are mean ± SE, n = 3, *P < 0.05, # P < 0.01.
Figure 1. 
 
Effect of perfusion with Y-27632 on outflow facility in enucleated monkey eyes. After measuring the baseline value, eyes were perfused with 50 μM Y-27632 at a constant pressure (10 mm Hg) at 25°C. After drug perfusion, the percentage change of outflow facility from the baseline value increased significantly and time-dependently over control eyes. Values are mean ± SE, n = 3, *P < 0.05, # P < 0.01.
Figure 2. 
 
Scanning electron micrographs of SCE. Compared with control eyes (left), SCE of Y-27632 perfused eyes tended to have more giant vacuoles (right). Although intercellular detachments were also observed in control eyes, they were more frequently observed in the drug-perfused eyes.
Figure 2. 
 
Scanning electron micrographs of SCE. Compared with control eyes (left), SCE of Y-27632 perfused eyes tended to have more giant vacuoles (right). Although intercellular detachments were also observed in control eyes, they were more frequently observed in the drug-perfused eyes.
Figure 3. 
 
Changes in TEER in the SCE-cell monolayer. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 3. 
 
Changes in TEER in the SCE-cell monolayer. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 4. 
 
Changes in the SCE-cell monolayer permeability of 4 kDa fluorescein isothiocyanate-dextran. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 4. 
 
Changes in the SCE-cell monolayer permeability of 4 kDa fluorescein isothiocyanate-dextran. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.
Figure 5. 
 
Effects of the ROCK inhibitor Y-27632 on cell–cell contact and actin stress fibers in SCE cells. SCE cells in culture treated with 25 μM Y-27632 for 30 minutes were immunostained for molecules relating to cell–cell contact: ZO-1, claudin-5, β-catenin, and pan-cadherin (green). The left image of each figure is a merged image with F-actin staining (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
Figure 5. 
 
Effects of the ROCK inhibitor Y-27632 on cell–cell contact and actin stress fibers in SCE cells. SCE cells in culture treated with 25 μM Y-27632 for 30 minutes were immunostained for molecules relating to cell–cell contact: ZO-1, claudin-5, β-catenin, and pan-cadherin (green). The left image of each figure is a merged image with F-actin staining (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
Figure 6. 
 
Effects of latrunculin, jasplakinolide, and Y-27632 on the polymerization of F-actin and intracellular localization of ZO-1. The top images show ZO-1 expression (green) in untreated SCE cells and latrunculin-treated SCE cells. The effects of Y-27632 with or without pretreatment with jasplakinolide are shown in the middle (F-actin, red) and bottom images (ZO-1, green). Scale bar: 50 μm.
Figure 6. 
 
Effects of latrunculin, jasplakinolide, and Y-27632 on the polymerization of F-actin and intracellular localization of ZO-1. The top images show ZO-1 expression (green) in untreated SCE cells and latrunculin-treated SCE cells. The effects of Y-27632 with or without pretreatment with jasplakinolide are shown in the middle (F-actin, red) and bottom images (ZO-1, green). Scale bar: 50 μm.
Figure 7. 
 
(A, B) Changes in TEER in the SCE cell monolayer. SCE cells were treated with EGTA (A) or A23187 (B), and TEER was measured at 30, 60, and 90 minutes after administration. White bars indicate the values in control condition, and black bars indicate the values with drug treatment. Mean values ± SEs from three separate filters are presented. *P < 0.05, Student's t-test comparing control and drug treatment at each time point. (C) Effects of A23187 with or without pretreatment by Y-27632 on the intracellular concentration of calcium ions estimated by Fluo-4 AM The mean ± SEs are presented as the ratio of F (value after treatment) to F0 (value before treatment). *P < 0.05, Student's t-test with Bonferroni correction comparing control and drug treatment at each time point.
Figure 7. 
 
(A, B) Changes in TEER in the SCE cell monolayer. SCE cells were treated with EGTA (A) or A23187 (B), and TEER was measured at 30, 60, and 90 minutes after administration. White bars indicate the values in control condition, and black bars indicate the values with drug treatment. Mean values ± SEs from three separate filters are presented. *P < 0.05, Student's t-test comparing control and drug treatment at each time point. (C) Effects of A23187 with or without pretreatment by Y-27632 on the intracellular concentration of calcium ions estimated by Fluo-4 AM The mean ± SEs are presented as the ratio of F (value after treatment) to F0 (value before treatment). *P < 0.05, Student's t-test with Bonferroni correction comparing control and drug treatment at each time point.
Table 1.  
 
Representative Genes That Are Upregulated in SCE Cells
Table 1.  
 
Representative Genes That Are Upregulated in SCE Cells
Accession Number Gene Name Fold Change
CJ435051 Myelin basic protein isoform 2 16.42917
CJ440786 Pleckstrin homology domain interacting protein 15.84593
BB889989 Chondroadherin precursor 7.27175
AB170326 Human syndecan 4 (amphiglycan, ryudocan) (SDC4) 4.79868
AB171586 Human adenylate cyclase 2 (brain) (ADCY2) 4.44696
DW528016 Thyroid hormone receptor interactor 3 4.38051
CJ492124 Splicing factor, arginine/serine-rich 6 [Rattus norvegicus] 4.285
AB168674 Human RIKEN cDNA 1700016G05 (LOC402604) 4.25099
DC621007 L-Threonine dehydrogenase 4.07265
BB898986 Complement factor I preproprotein 3.73984
AB172153 Human CDW92 antigen (CDW92) 2.9576
AB170860 Human WD repeat domain 8 (WDR8) 2.66228
AB070180 Human hypothetical protein FLJ46088, mRNA, AK127973 2.59433
DK578068 Hypothetical protein LOC348262 [Homo sapiens] 2.58971
CJ470450 Splicing factor 3b, subunit 1 isoform 1 [Danio rerio] 2.58217
EF208807 MHC class II antigen DP alpha chain (Mafa-DPA) mRNA, Mafa-DPA1*0202 allele 2.56233
AB174415 Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) (SPTAN1) 2.55997
AB169059 CD4 precursor 2.40463
DC645348 Sulfotransferase family, cytosolic, 1C, member 1 isoform a 2.40361
CJ446069 Protein phosphatase 3, catalytic subunit, alpha isoform 2.40133
CJ440484 Brain cDNA 2.38472
DK579312 Spliceosome RNA helicase BAT1 2.36141
AB168995 Human ring finger protein 36 (RNF36) 2.28256
DC850662 Fc gamma RIIIa 2.26894
AB179134 Human syntaxin binding protein 4 (STXBP4) 2.25772
AB071116 Human ankyrin repeat and SOCS box-containing 15 (ASB15) 2.22498
DC850740 Cytosolic phospholipase A2 gamma precursor (cPLA2-gamma) (Phospholipase A2 group IVC) 2.2098
AB174030 Human galactose mutarotase (aldose 1-epimerase) (GALM) 2.20885
AB168319 Human chromosome 6 open reading frame 10 (C6orf10) 2.1913
AB169280 Human hypothetical protein FLJ13305 (FLJ13305) 2.16347
CJ492687 USP6 N-terminal like 2.16298
AB169348 Human hypothetical protein MGC33600 (MGC33600) 2.15724
AB172168 Human protein tyrosine phosphatase, receptor type, fpolypeptide (PTPRF), interacting protein (liprin), alpha4 (PPFIA4) 2.12463
AB173609 Human cholinergic receptor, nicotinic, epsilon polypeptide (CHRNE) 2.12166
AB173849 Human zinc finger protein 24 (KOX 17) (ZNF24) 2.07481
AB168153 Human DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 (DDX43) 2.07426
Table 2.  
 
Representative Genes That Are Downregulated in SCE Cells
Table 2.  
 
Representative Genes That Are Downregulated in SCE Cells
Accession Number Gene Name Fold Change
DK576163 Human H3 histone, family 3B (H3.3B) (H3F3B) 0.28442
AB174375 Human dual specificity phosphatase 14 (DUSP14) 0.35283
DC631004 Nucleophosmin (nucleolar phosphoprotein B23, numatrin) 0.42112
AB072781 Chromodomain Y-like protein 2 (CDYL2) 0.44636
CJ440707 Brefeldin A–inhibited guanine nucleotide-exchange protein 1 0.46621
AB070198 Human DNA-damage inducible protein 1 (DDI1) 0.4856
DK581802 Cytokine receptor-like factor 3 [Homo sapiens] 0.49076
AB173178 Human hepatic leukemia factor (HLF) 0.49856
Table 3.  
 
Gene Ontology of Upregulated Genes in Y-27632–Treated SCE Cells
Table 3.  
 
Gene Ontology of Upregulated Genes in Y-27632–Treated SCE Cells
Ontology Term Total Genes P-Value
Molecular function MHC class II protein binding 3(3) 0.00131
Biological process Transmembrane receptor protein tyrosine kinase signaling pathway 104(27) 0.00169
Biological process Negative regulation of Ras protein signal transduction 6(3) 0.0036
Biological process Negative regulation of small GTPase-mediated signal transduction 6(0) 0.0036
Biological process Regulation of cytoskeleton organization 60(2) 0.0064
Biological process RNA processing 339(35) 0.00688
Biological process tRNA processing 35(28) 0.00961
Biological process Enzyme-linked receptor protein signaling pathway 152(1) 0.00999
Biological process Cellular response to insulin stimulus 39(18) 0.01265
Molecular function MHC protein binding 13(0) 0.01275
Biological process Cellular response to endogenous stimulus 78(0) 0.01507
Biological process Cellular response to hormone stimulus 78(12) 0.01507
Biological process Cellular response to peptide hormone stimulus 42(0) 0.01525
Molecular function Molecular transducer activity 621(0) 0.01768
Molecular function Signal transducer activity 621(134) 0.01768
Biological process Positive regulation of cytoskeleton organization 16(2) 0.01822
Biological process Regulation of cellular component organization 229(0) 0.01957
Molecular function 1-Acylglycerophosphocholine O-acyltransferase activity 1(1) 0.02316
Molecular function 1-Alkylglycerophosphocholine O-acetyltransferase activity 1(1) 0.02316
Molecular function Aldose 1-epimerase activity 1(1) 0.02316
Molecular function Cholesterol 24-hydroxylase activity 1(1) 0.02316
Molecular function Choline transmembrane transporter activity 1(1) 0.02316
Molecular function tRNA isopentenyltransferase activity 1(1) 0.02316
Biological process Elastin catabolic process 1(0) 0.02321
Biological process Extracellular matrix disassembly 1(0) 0.02321
Biological process Histolysis 1(0) 0.02321
Biological process Induction by virus of host cell–cell fusion 1(1) 0.02321
Biological process Negative regulation of Rac protein signal transduction 1(1) 0.02321
Biological process Negative regulation of blood vessel remodeling 1(1) 0.02321
Biological process Negative regulation of collagen catabolic process 1(1) 0.02321
Biological process Negative regulation of elastin catabolic process 1(1) 0.02321
Biological process Negative regulation of extracellular matrix disassembly 1(1) 0.02321
Biological process Negative regulation of histolysis 1(1) 0.02321
Biological process Positive regulation of centriole replication 1(1) 0.02321
Biological process Positive regulation of centrosome cycle 1(0) 0.02321
Biological process Regulation of blood vessel remodeling 1(0) 0.02321
Biological process Regulation of collagen catabolic process 1(0) 0.02321
Biological process Regulation of elastin catabolic process 1(0) 0.02321
Biological process Regulation of extracellular matrix disassembly 1(0) 0.02321
Biological process Regulation of histolysis 1(0) 0.02321
Biological process Tissue death 1(0) 0.02321
Biological process Trophoblast giant cell differentiation 1(1) 0.02321
Biological process Cellular response to organic substance 96(0) 0.02891
Biological process Insulin receptor signaling pathway 21(12) 0.02903
Molecular function Receptor activity 425(266) 0.0338
Cellular component Exon–exon junction complex 2(2) 0.03402
Molecular function 3′,5′-Cyclic-GMP phosphodiesterase activity 2(1) 0.03454
Molecular function O-Acetyltransferase activity 2(0) 0.03454
Molecular function Actinin binding 2(0) 0.03454
Molecular function alpha-Actinin binding 2(1) 0.03454
Molecular function Amine oxidase activity 2(2) 0.03454
Molecular function Arginase activity 2(2) 0.03454
Molecular function Isocitrate dehydrogenase (NADP+) activity 2(2) 0.03454
Biological process Factor XII activation 2(2) 0.03462
Biological process Cell differentiation involved in embryonic placenta development 2(1) 0.03462
Biological process Choline transport 2(2) 0.03462
Biological process Elastin metabolic process 2(1) 0.03462
Biological process Glyoxylate cycle 2(2) 0.03462
Biological process Kinin cascade 2(0) 0.03462
Biological process ncRNA catabolic process 2(0) 0.03462
Biological process Negative regulation of Rho protein signal transduction 2(2) 0.03462
Biological process Negative regulation of collagen metabolic process 2(0) 0.03462
Biological process Negative regulation of multicellular organismal metabolic process 2(0) 0.03462
Biological process Plasma kallikrein-kinin cascade 2(0) 0.03462
Biological process Positive regulation of focal adhesion assembly 2(2) 0.03462
Biological process rRNA catabolic process 2(2) 0.03462
Biological process Regulation of Rac GTPase activity 2(2) 0.03462
Biological process Regulation of centriole replication 2(1) 0.03462
Biological process Response to insulin stimulus 59(21) 0.03533
Biological process Cellular component disassembly 60(0) 0.0368
Biological process Regulation of organelle organization 104(0) 0.03688
Cellular component Nuclear speck 62(62) 0.03815
Biological process Regulation of small GTPase- mediated signal transduction 106(20) 0.03906
Biological process Nitrogen compound metabolic process 1974(14) 0.04265
Cellular component Compact myelin 3(2) 0.04511
Cellular component Spectrin 3(3) 0.04511
Molecular function Calcium- and calmodulin-responsive adenylate cyclase activity 3(3) 0.04579
Molecular function Histamine receptor activity 3(3) 0.04579
Molecular function Structural constituent of myelin sheath 3(3) 0.04579
Molecular function Thrombospondin receptor activity 3(3) 0.04579
Biological process Regulation of catalytic activity 382(4) 0.0458
Biological process Arginine catabolic process 3(3) 0.04589
Biological process Cytoplasmic microtubule organization 3(2) 0.04589
Biological process Positive regulation of fibrinolysis 3(3) 0.04589
Biological process Positive regulation of protein maturation by peptide bond cleavage 3(0) 0.04589
Biological process Regulation of cell-substrate junction assembly 3(0) 0.04589
Biological process Regulation of centrosome cycle 3(0) 0.04589
Biological process Regulation of centrosome duplication 3(1) 0.04589
Biological process Regulation of focal adhesion assembly 3(0) 0.04589
Biological process RNA splicing, via transesterification reactions with bulged adenosine as nucleophile 66(0) 0.04622
Biological process Nuclear mRNA splicing, via spliceosome 66(30) 0.04622
Biological process Regulation of cellular component biogenesis 67(0) 0.0479
Table 4.  
 
Gene Ontology of Downregulated Genes in Y-27632–Treated SCE Cells
Table 4.  
 
Gene Ontology of Downregulated Genes in Y-27632–Treated SCE Cells
Ontology Term Total Genes P-Value
Molecular function Hydroxymethylglutaryl-CoA synthase activity 2(2) 0.00033
Molecular function Transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer 6(3) 0.00152
Molecular function Exonuclease activity, active with either ribo- or deoxyribonucleic acids and producing  5′-phosphomonoesters 9(0) 0.00294
Biological process Regulation of release of sequestered calcium ion into cytosol 11(0) 0.00395
Biological process Negative regulation of sequestering of calcium ion 13(0) 0.00527
Biological process Regulation of sequestering of calcium ion 13(0) 0.00527
Biological process Release of sequestered calcium ion into cytosol 13(2) 0.00527
Molecular function 3′-5′ Exonuclease activity 13(7) 0.00551
Biological process Isoprenoid biosynthetic process 14(10) 0.006
Biological process Sequestering of calcium ion 15(2) 0.00677
Molecular function Glutathione transferase activity 15(15) 0.00707
Biological process Regulation of calcium ion transport into cytosol 16(0) 0.00758
Biological process Calcium ion transport into cytosol 17(0) 0.00843
Biological process Cytosolic calcium ion transport 17(0) 0.00843
Biological process Sequestering of metal ion 17(0) 0.00843
Cellular component Microtubule 122(101) 0.00956
Biological process Cholesterol biosynthetic process 20(15) 0.01123
Biological process DNA replication checkpoint 1(1) 0.01472
Biological process Cysteine biosynthetic process from serine 1(1) 0.01472
Biological process Cysteine biosynthetic process via cystathionine 1(1) 0.01472
Biological process Homocysteine catabolic process 1(1) 0.01472
Biological process Negative regulation of DNA-dependent DNA replication initiation 1(0) 0.01472
Molecular function L-Gulonate 3-dehydrogenase activity 1(1) 0.01504
Molecular function Bradykinin receptor activity 1(1) 0.01504
Molecular function Cystathionine beta-synthase activity 1(1) 0.01504
Molecular function Exodeoxyribonuclease III activity 1(1) 0.01504
Molecular function Monodehydroascorbate reductase (NADH) activity 1(1) 0.01504
Molecular function Polyribonucleotide nucleotidyltransferase activity 1(1) 0.01504
Molecular function Ribonuclease III activity 1(1) 0.01504
Biological process Sterol biosynthetic process 25(5) 0.01666
Biological process Regulation of calcium ion transport 26(1) 0.01786
Molecular function Exonuclease activity 26(19) 0.01864
Biological process Regulation of metal ion transport 28(0) 0.02037
Molecular function Nuclease activity 76(10) 0.02145
Biological process L-Ascorbic acid biosynthetic process 2(2) 0.022
Biological process L-Cysteine catabolic process 2(2) 0.022
Biological process L-Cysteine metabolic process 2(1) 0.022
Biological process Cysteine biosynthetic process 2(1) 0.022
Biological process Cysteine catabolic process 2(0) 0.022
Biological process Homocysteine metabolic process 2(0) 0.022
Biological process Hydrogen sulfide biosynthetic process 2(2) 0.022
Biological process Hydrogen sulfide metabolic process 2(0) 0.022
Biological process Regulation of DNA-dependent DNA replication 2(0) 0.022
Biological process Regulation of DNA-dependent DNA replication initiation 2(1) 0.022
Molecular function Transferase activity, transferring alkyl or aryl (other than methyl) groups 31(2) 0.02542
Biological process Isoprenoid metabolic process 32(0) 0.02578
Biological process L-Serine catabolic process 3(3) 0.02923
Biological process Positive regulation of ryanodine-sensitive calcium-release channel activity 3(3) 0.02923
Biological process Positive regulation of survival gene product expression 3(3) 0.02923
Biological process Regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum 3(3) 0.02923
Biological process Release of sequestered calcium ion into cytosol by sarcoplasmic reticulum 3(0) 0.02923
Biological process Sulfur amino acid catabolic process 3(0) 0.02923
Biological process Sulfur compound catabolic process 3(0) 0.02923
Molecular function N-Terminal myristoylation domain binding 3(3) 0.02987
Molecular function Exodeoxyribonuclease activity 3(0) 0.02987
Molecular function Exodeoxyribonuclease activity, producing 5′-phosphomonoesters 3(0) 0.02987
Molecular function Interleukin-2 binding 3(1) 0.02987
Molecular function Interleukin-2 receptor activity 3(3) 0.02987
Biological process Regulation of ion transport 36(1) 0.03171
Biological process Cell surface receptor linked signaling pathway 552(70) 0.03242
Molecular function Ribonuclease activity 36(16) 0.03305
Biological process L-Ascorbic acid metabolic process 4(2) 0.03641
Biological process Negative regulation of ryanodine-sensitive calcium-release channel activity 4(4) 0.03641
Biological process Sarcoplasmic reticulum calcium ion transport 4(1) 0.03641
Biological process Cytokine-mediated signaling pathway 39(20) 0.03648
Biological process Initiation of signal transduction 39(0) 0.03648
Biological process Leukocyte migration 39(5) 0.03648
Biological process Signal initiation by diffusible mediator 39(0) 0.03648
Biological process Signal initiation by protein/peptide mediator 39(0) 0.03648
Cellular component Barr body 5(5) 0.03939
Cellular component Integrin complex 5(5) 0.03939
Cellular component Receptor complex 47(4) 0.04198
Biological process Elevation of cytosolic calcium ion concentration 43(26) 0.04323
Biological process Ephrin receptor signaling pathway 5(5) 0.04353
Biological process Positive regulation of ion transmembrane transporter activity 5(0) 0.04353
Biological process Regulation of defense response to virus by virus 5(5) 0.04353
Biological process Regulation of ryanodine-sensitive calcium-release channel activity 5(1) 0.04353
Biological process Regulation of survival gene product expression 5(0) 0.04353
Molecular function MAP kinase phosphatase activity 5(0) 0.04447
Molecular function MAP kinase tyrosine/serine/threonine phosphatase activity 5(5) 0.04447
Biological process Signaling pathway 1024(0) 0.04488
Cellular component X chromosome 6(1) 0.0458
Biological process Cytosolic calcium ion homeostasis 46(4) 0.04858
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