Diquafosol Sodium Inhibits Apoptosis and Inflammation of Corneal Epithelial Cells Via Activation of Erk1/2 and RSK: In Vitro and In Vivo Dry Eye Model

Abstract

Purpose: To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model.

Methods: Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1β, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1β, and used the TUNEL assay in corneal tissue.

Results: The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1β, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1β was downregulated by DIQUAS.

Conclusions: Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.

Diquafosol tetrasodium (Santen Pharmaceutical Co., Inc., Osaka, Japan), a P2Y2 receptor agonist, acts via a novel mechanism that activates P2Y2 receptors in the ocular surface, which improves fluid transport, mucin secretion from the conjunctival epithelium, and stimulation of lipid production leading to improved quantity and quality of tear film in patients with dry eye disease.^1,2 The role of P2Y2R in the eye has been previously reviewed.³ Dinucleotide INS365 (diquafosol, also known as DIQUAS) is a mixed P2Y2R and P2Y4R agonist that has been shown to improve corneal barrier function.⁴ In a rat dry eye model, this agonist increased both tear fluid secretion and corneal epithelial resistance and induced the release of glycoprotein-containing moieties from goblet cells. These successful preclinical results have led to the introduction of diquafosol as a treatment for dry eye disease in Japan and South Korea.⁵ As a topical ophthalmic solution (3%, six times daily), it stimulates the secretion of tears and mucin in patients with dry eye disease, maintaining its effectiveness for 12 months.⁶ A large randomized, double-blind trial showed that among those with dry eye disease, diquafosol had fluorescein-staining scores that were comparable to those of 0.1% sodium hyaluronate and rose bengal subjective symptom scores that were significantly better than those of hyaluronate.⁷ The encouraging clinical results of diquafosol in dry eye disease were recently reviewed.⁸ It was shown to be effective against various types of dry eye diseases, such as aqueous deficiency, short tear film breakup time, and obstructive meibomian gland dysfunction. Diquafosol was also useful in treating dry eye disease resulting from surgery (e.g., in situ keratomileusis and cataracts) and from the use of contact lenses and visual display terminals.¹ Therefore, purinergic receptor agonists (including diquafosol tetrasodium) are a promising new class of drugs that may help restore physiological tear flow in patients with dry eye disease. 

Recent reports have shown that diquafosol promotes corneal epithelial wound healing through P2Y2 receptor activation,¹ with such an effect resulting from extracellular signal–regulated kinase (ERK)-stimulated cell proliferation.¹ However, the exact mechanism through which diquafosol enhances goblet cell mucin production and stimulates lipid production by improving meibomian gland dysfunction has yet to be elucidated. 

P2 receptors, which consist of P2X (ionotropic) and P2Y (metabotropic, G-protein–coupled) subtypes, respond to a variety of nucleotide agonists. The role of P2 receptors, particularly P2X receptors, in apoptosis had been demonstrated in both nonneuronal and neuronal cells, including the prominent role of the P2X7 receptor in initiating spinal neuron apoptosis.⁹ A report had shown that ATP, UTP, and ATPγS protected PC12 (pheochromocytoma 12) cells and dorsal root ganglion neurons from serum starvation-induced apoptosis, an effect mediated via P2Y2 receptors.¹⁰ In contrast, the role of P2Y receptors in nonneuronal apoptosis remains mostly unexplored. There is little evidence to suggest that survival of corneal epithelial cells in dry eye disease, particularly the inhibition of apoptosis, is regulated by the P2Y2 receptor agonist diquafosol. Moreover, to our knowledge, no previous studies have been conducted on downstream signaling events that mediate this effect and no evidence has shown the anti-inflammatory effects of diquafosol on the ocular surface. 

In the present study, we hypothesized that diquafosol tetrasodium, a P2Y2 receptor agonist, may modulate sensitive reactive oxygen species (ROS)-induced apoptosis and inflammation in human corneal epithelial cells (hCECs) using Transwell culture systems to mimic dry eye conditions. As such, we evaluated an unappreciated molecular signaling mechanism through which diquafosol improves the survival of corneal epithelial cells in an in vitro dry eye model. Furthermore, to test this hypothesis in vivo, we topically applied the DIQUAS (ophthalmic solution 3%; Santen Pharmaceutical Co., Ltd.) to the corneas of a scopolamine-induced dry eye model, a rat model sharing the pathogenesis and pathologies with human aqueous-deficient dry eye disease.^11,12 

Methods

Cell Culture

Telomerase-immortalized hCECs were kindly donated to us by James V. Jester, MD, PhD (Gavin Herbert Eye Institute, University of California Irvine, CA, USA). The cells were cultured at 37°C under 5% CO₂ atmosphere in bronchial epithelium growth medium (Lonza, Inc., Walkersville, MD, USA) supplemented with 5 mg/mL insulin (Clonetics, San Diego, CA, USA), 0.5 mg/mL hydrocortisone (Clonetics), 6.5 ng/mL triiodothyronine (Clonetics), 10 ng/mL transferrin (Clonetics), 10 ng/mL retinoic acid (Clonetics), 0.13 mg/mL bovine pituitary extract (Clonetics), a mixture of 50 mg/mL gentamicin and 50 ng/mL amphotericin (Clonetics), 5 ng/mL human epidermal growth factor (Sigma-Aldrich Corp., St. Louis, MO, USA), and 0.15 mg/mL BSA (Sigma-Aldrich Corp.). They were then subcultured with 0.25% trypsin-EDTA every 3–4 days prior to use in this study. hCECs were seeded onto a ϕ2.4-cm Transwell membrane with a 0.4 μm pore size (Corning, Inc., Corning, NY, USA) and cultivated overnight to allow the cells to attach to the membrane. To desiccate hCECs, the upper layer of the medium was discarded and the cells were incubated for 24 hours with or without 3% diquafosol tetrasodium media diluted at 1:100 in the lower layer. 

Measuring Intracellular ROS Levels in hCECs

Intracellular ROS levels in hCECs were measured by incubating the cells with the redox-sensitive fluorescent dye 2′7′-dichlorofluorescein diacetate (DCFH-DA) (10 μM; Molecular Probes, Life Technologies, Grand Island, NY, USA) at 37°C for 30 minutes in the dark. The cells were then detached from the culture wells using 0.25% trypsin-EDTA and washed twice using ice-cold PBS. Flow cytometry measurements (BD Biosciences, Inc., San Diego, CA, USA) were performed three times for each treatment. Mean fluorescence intensity was quantified using software (CELLQuest; BD Biosciences, Inc.). Cells that had been incubated without DCFH-DA were used as negative control. The distribution of fluorescent dichlorofluorescein (DCF) on the cell monolayer was visualized and photographed using a fluorescence microscope (Olympus, Tokyo, Japan). 

Assessing Apoptosis Using Annexin V-PI Staining

To assess apoptosis, cells were washed twice with ice-cold PBS, seeded onto a membrane, and incubated with 1× FITC-labeled annexin V and PI-binding buffer (BD Biosciences, San Diego, CA, USA) at room temperature for 15 minutes in the dark. Cells were then photographed using a fluorescence microscope. 

Western Blot Analysis

To evaluate whether 3% diquafosol tetrasodium could reduce the activation of NF-κB in hCECs exposed to dry conditions, Western blotting was used to quantify the nuclear translocation of the NF-κB protein subunit p65. We subsequently measured pERK1/2, p90 ribosomal s6 kinase (p90RSK), and pAkt levels using Western blot analysis to examine whether dry conditions induced hCEC apoptosis via ERK1/2, ribosomal S6 kinase, and PI3K/Akt. hCECs were collected and lysed by adding a lysis solution (10 mM Tris, 10 mM NaCl, 2 mM EDTA, 25 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, proteinase and phosphatase inhibitor cocktail, 0.5% Triton X-100, pH 7). After addition of 10% NP-40 (3 μL for every 100 μL cell lysis buffer), the cell lysates were centrifuged at 10,000g for 1 minute and the supernatants were collected as cytosolic fractions. A nuclear pellet was suspended in ice-cold extraction buffer (20 mM HEPES, pH 7.9/0.4 M NaCl/1 mM EDTA/1 mM EGTA/1 mM dithiolthreitol) and incubated on ice for 30 minutes with intermittent vortex. The nuclear extract was then subjected to centrifugation at 10,000g for 5 minutes, and the supernatant was saved as nuclear fraction. Protein concentrations in the supernatants were determined using the Bradford method. Protein aliquots (30 μg) were boiled in equal volumes of Laemmli sample buffer, resolved using 12% SDS-PAGE, and electrophoretically transferred to nitrocellulose filters (Amersham, Little Chalfont, UK). The blots were treated with antibodies against phospho-Erk1/2 (1:1000, catalog no. 4370S; Cell Signaling Technology), nonphosphorylated Erk1/2 (1:1000, catalog no. 9102s; Cell Signaling Technology), phospho-p90RSK (1:1000, catalog no. 9341; Cell Signaling Technology), nonphosphorylated p90RSK (1:1000, catalog no. 9326; Cell Signaling Technology), phospho-Akt (1:1000, catalog no. 4060S; Cell Signaling Technology), nonphosphorylated Akt (1:1000, catalog no. 9272s; Cell Signaling Technology), IκBα (1:1000; catalog no. 9247; Cell Signaling Technology), NF-κB-p65 (1:1000; catalog no. sc-109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and an antibody for β-actin (1:10,000; Sigma-Aldrich Corp.) used as a loading control for overnight at 4°C. After three 10-minute washes using Tris-buffered saline (TBS) containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase–conjugated anti-IgGs (1:10,000; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The respective target proteins of the antibodies were developed using enhanced chemiluminescence reagents (Santa Cruz Biotechnology), exposed to film (Fujifilm, Tokyo, Japan), underwent colorimetric detection for visualization with developer and fix solution without the need for specialized equipment, and finally scanned for analysis with ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). All experiments were performed at least three times. 

Multiplex Cytokine Analysis

Concentrations of the proinflammatory cytokines IL-1β and TNF-α in the supernatants of hCECs were determined using inflammatory cytokine human magnetic five-plex panel that include IL-1β, TNF-α, IL-6, IL-8, and GM-CSF (catalog number LHC0003M; Thermo Fisher Scientific, Inc., Rockford, IL, USA) that uses multiplex bead technology. The human inflammatory magnetic five-plex panel contains all the reagents that are intended for use with the Luminex 200 dual laser detection system with software (xPONENT; Luminex, Inc., Austin, TX, USA). 

Animal Model

Scopolamine hydrobromide was purchased from Sigma-Aldrich Corp. and dissolved in 0.9% sterilized NaCl solution at 6 mg/mL prior to injections. Wistar female rats (6 weeks of age, body weight 160–180 g) were maintained at 25° ± 1°C with relative humidity of 40% ± 5% under 12-hour light-dark illumination cycles (8 AM to 8 PM). The animals were given food and water. All experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences at the Asan Medical Center. The committee abides by the Institute of Laboratory Animal Resources guide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol. The rats were subjected to subcutaneous (SC) injections with scopolamine hydrobromide four times a day (8 AM, 12 AM, 4 PM, and 8 PM; 0.5 mL at each time point) for 28 days. This approach has been demonstrated by others to successfully induce the dry eye condition in rats.^11,12 The scopolamine-induced dry eye rats were then randomly divided into two groups (eight rats per group), and they received topical administrations of DIQUAS eyedrop solution on the left eyes; the right eyes of these rats were treated with sterilized 0.9% NaCl and served as vehicle controls. Both the DIQUAS eyedrop solutions and normal saline were administered four times daily (8 AM, 12 AM, 4 PM, and 8 PM; the total volume 40 μL at each time point). Another group (eight rats) was included as saline control to receive SC injections and topical applications of sterilized normal saline with the identical volume and frequency as the dry eye groups. 

Histopathology and Immunohistochemistry in the Rat Cornea

In all three groups, 28 days after SC injections the animals were euthanized by CO₂ inhalation, and the left eyes were enucleated; all the corneas were buttoned and fixed in 10% phosphate-buffered formalin for 24 hours before being embedded in paraffin wax. The eyes were then cut into 4-μm-thick sections and stained using hematoxylin and eosin. Phosphorylated p90RSK, phosphorylated Erk1/2, and IL-1β expression were analyzed by immunohistochemistry (Envision-HRP Detection System; Dako, Carpinteria, CA, USA). After deparaffinization and antigen retrieval using 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker at full power for 10 minutes, tissue sections were treated with 3% H₂O₂ for 5 minutes. The anti-phospho p90RSK antibody (1:50; Thermo Fisher Scientific), anti-phospho Erk1/2 (1:450; Cell Signaling Technology), and anti-IL-1β antibody (1:50; Santa Cruz Biotechnology) were added to the slides, which were then incubated at 4°C overnight. The slides were then incubated with the Envision reagent for 30 minutes and with 3,3′-diaminobenzidine chromogen for 10 minutes; they were then counterstained with Meyer's hematoxylin and mounted. Careful rinses using several changes of TBS-0.1% Tween buffer were performed at each step. As a negative control, rat IgG1 isotype control was used to exclude the primary antibody. The slides were evaluated and photographed using a microscope (Microphoto FXA; Leica, Wetzlar, Germany). 

Apoptosis Assay in the Scopolamine-Induced Rat Cornea

Apoptosis was detected under different conditions using the TUNEL technique (Cell Death Detection Kit; Roche Diagnostics, Mannheim, Baden-Wuttemberg, Germany), which labels the cut ends of DNA fragments in the nuclei of apoptotic cells. The nuclei of all cells were stained by incubation with 4′-6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). 

Statistical Analysis

All quantitative experiments were performed at least three times in triplicate, and the data shown are means ± SDs of one representative experiment. Statistical significance was determined using the Student's t-test between highlighted groups, and a 95% confidence level was taken at a P < 0.05. 

Results

Diquafosol Significantly Decreases ROS Generation in Dry-Conditioned hCECs

As shown in Figure 1, cells cultured with diquafosol media diluted at 1:100 exhibited a greater decrease in the distribution of highly fluorescent DCF compared with dry condition exposed cells cultured without diquafosol, indicating the anti-ROS activity of diquafosol during oxidative stress in dry-conditioned hCECs. 

Figure 1

 

Diquafosol tetrasodium inhibits DCF-sensitive ROS. (A) Distribution of fluorescent DCF on the cell monolayer was photographed using a fluorescence microscope. (B) Intracellular ROS production was measured using flow cytometry after staining with 10 μM DCFH-DA dye. (C) Data are expressed as mean ± SD of three independent experiments: *P < 0.05 compared with cells without diquafosol.

Diquafosol Inhibits Apoptosis in Dry-Conditioned hCECs and Scopolamine-Induced Rat Cornea

Apoptosis analysis involved culturing hCECs in the presence of diquafosol media diluted at 1:100 for 24 hours under dry conditions, incubating with FITC-labeled annexin V and PI, and analyzing using flow cytometry (Fig. 2). Distributions of FITC- and PI-positive cells were photographed using a fluorescence microscope, with diquafosol effectively decreasing the number of positive cells under dry conditions (Fig. 2A). The percentage of FITC-positive cells was lower with diquafosol (8.7%) than without it (15.4%) (P < 0.05; Figs. 2B, 2C). This observation suggested that diquafosol inhibited dry condition–induced cell death. In the corneas of the control group, few TUNEL-positive nuclei were detected (data not shown). In the dry eye corneas, TUNEL-positive cells were seen in many corneal epithelial cells in particular (Fig. 3). Conversely, barely any TUNEL-positive nuclei were observed in the dry eye diquafosol group (Fig. 3). That is, the diquafosol treatment markedly reversed the apoptotic response in the dry eye cornea. 

Figure 2

 

Diquafosol tetrasodium inhibits dry-conditioned apoptosis in hCECs cultivated on ϕ2.4-cm Transwell membranes with a 0.4-μm pore size for 24 hours. (A) Cellular apoptosis was photographed using a fluorescence microscope. (B) Cellular apoptosis was determined using flow cytometry. (C) The number of FITC-positive cells was calculated as the sum of the values in quadrants Q2 and Q4. The number of PI-positive cells was calculated as the sum of the values in quadrants Q1 and Q2. *P < 0.05 compared with cells without diquafosol.

Figure 3

 

TdT-mediated nick-end labeling (TUNEL) assay of experimental groups: scopolamine-induced dry eye rat and diquafosol-treated dry eye rat. Many apoptotic cells (green, i.e., TUNEL-positive, nuclei) were detected in the corneal epithelium of the dry eye rat, whereas barely any TUNEL-positive apoptotic cells were seen in the corneal epithelial layer of the dry eye rat after diquafosol treatment. Magnification: ×100.

Diquafosol Activates ERK1/2, P90RSK, and AKT in Dry-Conditioned hCECs and Scopolamine-Induced Rat Cornea

We performed Western blot analysis to identify signal transduction cascades induced by diquafosol, focusing on PI3K/AKT and ERK1/2 mitogen-activated protein kinase (MAPK) pathways predominantly involved in cell survival. Moreover, p90RSK, which is the downstream substrate of ERK, regulates cell proliferation and survival.¹³ Thus, to investigate whether diquafosol induces p90rsk activation in dry-conditioned hCECs, we measured the amount of phosphorylated-p90rsk after diquafosol treatment for 24 hours. As shown in Figure 4, Western blot analysis revealed that the levels of phospho-Erk1/2, phospho-Akt, and phospho-p90RSK were significantly increased in dry-conditioned hCECs treated with diquafosol. These results indicated that diquafosol activated the ERK1/2- and P90RSK-mediated MAPK pathway and PI3K/AKT signaling dry-conditioned hCECs. To confirm the effects of diquafosol on dry eye in vivo, we used immunohistochemistry to detect the expression of phospho-p90RSK and phospho-Erk1/2 in scopolamine-induced dry eye rats and diquafosol-treated dry eye rats. Similar to the results of the corneal epithelial cell analysis, cornea of diquafosol-treated dry eye rats showed intense staining for phospho-Erk1/2 and phospho-p90RSK (see Fig. 7). 

Figure 4

 

Diquafosol tetrasodium increased survival regulators, phospho-Erk1/2, and phospho-Akt expression in hCECs. (A) Cells were cultivated on a ϕ2.4-cm Transwell membrane with a 0.4-μm pore size for 24 hours. After the cells were harvested, whole-cell extracts were prepared and probed for phosphorylated Erk1/2, 90RSK, and Akt. (B) Relative protein levels were normalized to those for each total protein. Data are expressed as mean ± SD of three independent experiments. *P < 0.05 compared with cells without diquafosol.

Diquafosol Regulates the Expression of Proinflammatory Mediators in Dry-Conditioned hCECs and Scopolamine-Induced Rat Cornea

NF-κB activation results in the phosphorylation and ubiquitination of the inhibitory subunit IκB, as well as translocation of p50 and p65 from the cytoplasm to the nucleus.¹⁴ In the present study, IκB and NF-κB protein levels were measured using Western blot analysis (Fig. 5). Compared with dry-conditioned hCECs without diquafosol, those with diquafosol had considerably lower NF-κB-p65 levels and markedly higher IκB-α levels, suggesting that diquafosol prevented the degradation of IκB-α. NF-κB promotes the expression of target genes that mediate the expression of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6.^5,13 The ROS production (Fig. 1), as well as the expression of proinflammatory mediators IL-1β and TNF-α (Fig. 6), in hCEC cultured under dry conditions were attenuated by incubation with diquafosol. However, the increase in IL-6, IL-8, and GM-CSF levels induced by the dry condition was not significantly reduced by simultaneous exposure to diquafosol (Fig. 6). Immunohistochemically, the epithelium, stroma, and endothelium of the dry cornea showed intense staining for IL-1β (Fig. 7). However, weak immunohistochemical staining for IL-1β was observed in the dry cornea after diquafosol treatment (Fig. 7). 

Figure 5

 

Diquafosol tetrasodium regulates the expression of inflammatory regulators. (A) Cells were cultivated on a ϕ2.4-cm Transwell membrane with a 0.4-μm pore size for 24 hours. The expression of phospho-NF-κB-p65 (nuclear fraction) and IκBα with or without diquafosol tetrasodium treatment for 24 hours was analyzed and quantified using Western blotting, with β-actin as the control. (B) Representative data of three experiments and quantitative mean densitometry results are shown. The data are expressed as mean ± SD of three independent experiments. *P < 0.05 compared with cells without diquafosol.

Figure 6

 

Diquafosol tetrasodium attenuated the effects of dry conditions on the secretion of inflammatory cytokines. Representative data of three experiments and quantitative mean densitometry results are shown. After an identical treatment, IL-1β, TNF-α, IL-6, IL-8, and GM-CSF expressions were quantified using multiplex cytokine analysis. The data are expressed as mean ± SD of three independent experiments.

Figure 7

 

Immunohistologic staining for phospho-p90RSK, phospho-Erk1/2, and IL-1β in the cornea of the experimental groups: scopolamine-induced dry eye rats and diquafosol-treated dry eye rats. Representative photomicrographs of the corneas of rats (magnification ×200). Diquafosol-treated corneal epithelial cells showed strong immunoreactivity for phospho-Erk1/2 and phospho-p90RSK. Only weak immunoreactivity for phospho-p90RSK and phospho-Erk1/2 were seen in the scopolamine-induced cornea. Diquafosol-treated corneal epithelial cells showed weak immunoreactivity for IL-1β. Strong immunoreactivity for IL-1β was seen in the scopolamine-induced cornea.

Discussion

Dry eye disease is a common ocular surface disorder described as a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film and accompanied by ocular symptoms in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiologic roles.¹⁴ It is well established that diquafosol improves fluid transport, soluble and membrane-associated mucin secretion, and stimulation of lipid production leading to improved quantity and quality of tear film. These effects indicated that diquafosol may act on all of the ocular surface, including cornea, conjunctiva, meibomian glands, and lacrimal glands. It has been demonstrated that diquafosol is a dinucleotide derivative of UTP, and nucleotides including ATP and UTP control the process of tearing, wound healing, and protection from superficial infections.¹⁵ Therefore, we suspected that diquafosol may promote cell survival and modulate ROS-induced apoptosis and inflammation in corneal epithelial cells of dry eye condition. In this study, we obtained the following results from experiments, and the results are consistent with our assumptions. 

First, although the pathogenesis of dry eye disease has not been entirely understood, inflammation has been recognized to play a key role in its development. Phosphorylation of stress-activated MAPKs p38 and c-Jun N-terminal kinase (JNK), followed by the activation of transcription factors AP-1 and NF-κB, results in increased levels of proinflammatory cytokines such as IL-1β, TNF-α, IL-8, and IL-6.^16–20 There was also a report that GM-CSF is significantly upregulated at the ocular surface in dry eye disease.²¹ On the basis of in vitro and in vivo experiments using hCECs, we suspect that the inhibition of the NF-κB signaling pathway by diquafosol led to the observed decrease in proinflammatory cytokine expression (Figs. 4, 5, 7). However, diquafosol does not inhibit IL-6, IL-8, and GM-CSF secretion (Fig. 5). Another contributor to ocular surface damage in dry eye disease is ROS overproduction and the resultant oxidative stress.²² A close association among lipid peroxidation-related membrane damage, protein oxidation, ROS production, and inflammation has been demonstrated in patients and animal models of dry eye disease.²³ Antioxidants such as oral sea buckthorn oil, green tea polyphenol epigallocatechin gallate, and topical selenoprotein P have been used in animal models and patients with dry eye disease. These antioxidants have been proven to alleviate symptoms of dry eye disease, indicating that ROS plays an important role in the pathogenesis of dry eye disease.^24–27 Given its potent antioxidative properties, this study found that diquafosol application for 24 hours decreased ROS generation in dry-conditioned hCECs (Fig. 1). 

Second, p90rsk is a family of serine/threonine kinases located downstream from the MAPK cascade.²⁸ In humans, three p90rsk isoforms have been identified, showing overall structures similar to those for the amino- and carboxy-terminal kinase domains. The amino-terminal domain is similar to the p70 ribosomal s6 kinase (approximately 60% sequence identity), whereas the carboxy-terminal domain is more closely related to calcium/calmodulin-dependent kinases (35% sequence identity).²⁹ p90rsk plays an essential role in cell survival and cell cycle regulation given its ability to phosphorylate and regulate the activity of several substrates, including many transcription factors and kinases, cyclin-dependent kinase inhibitors, tumor suppressors, and several cell survival factors.³⁰ p90rsk has antiapoptotic effects and is generally overexpressed in various cancer cells for apoptosis regulation.^28,30–32 Moreover, a recent study reported that p90rsk directly promotes cancer cell survival by interacting with heat shock protein 27.³³ However, detailed mechanisms related to p90rsk in dry eye disease remain unknown. P2Y2 receptor activation also leads to Src activation/phosphorylation, which in turn activates B-Raf and PI3 kinase. These events lead to ERK1/2 and Akt activation, respectively, which subsequently inhibits apoptosis by suppressing various molecules such as JNK, p38, and various caspases.^34–36 Nevertheless, detailed mechanisms downstream of ERK are not well understood. P90rsk, a well-known downstream substrate of ERK and an important regulator of apoptosis, is associated with cancer progression in various cell types.³³ Therefore, we aimed to determine whether p90rsk played an important role in diquafosol-induced survival of hCECs cultured under dry conditions. Diquafosol treatment enhanced the activities of ERK1/2, p90RSK, and Akt and inhibited apoptosis (Figs. 2, 3, 6, 7), suggesting that diquafosol, a P2Y2 receptor agonist, activates ERK1/2 and Akt, possibly subsequently inhibiting apoptosis in dry-conditioned hCECs through p90rsk, a downstream ERK substrate. 

In conclusion, the current findings shed light on a previously unappreciated function of nucleotides/P2Y2 receptors in dry eye disease, in addition to the enhancement of corneal wound healing by P2Y2 receptor agonists.¹ These studies demonstrate that diquafosol is effective in promoting cell survival, which may have resulted from ERK–p90RSK-mediated apoptosis inhibition, as well as anti-inflammation through NF-κB signaling pathway inhibition. 

Acknowledgments

The authors thank Kang Hyun Kim, MD, DVM (Laboratory Animal Research, Asan Institute for Life Science, Asan Medical Center) for technical support in the animal experiment. 

Supported by a Student Research Grant (2016) from the University of Ulsan College of Medicine, Seoul Korea, and by a grant (NRF-2016R1A2B1011341) from the National Research Foundation of Korea, Seoul, Republic of Korea. 

Disclosure: J.H. Park, None; S.-H. Moon, None; D.H. Kang, None; H.J. Um, None; S.-S. Kang, None; J.Y. Kim, None; H. Tchah, None