Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.
Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 μM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor). Likewise, diquafosol-treated cells showed acceleration of gap closure in cell migration assay, which was inhibited by suramin, BAPTA/AM, AG1478, and U0126 (MEK inhibitor). These studies demonstrate that diquafosol is effective in promoting corneal epithelial wound healing and that this effect may result from ERK-stimulated cell proliferation and migration via P2Y2R-mediated [Ca(2+)]i elevation.
Department of Ophthalmology and Visual Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea.
Yong-Soo
YS
Byun
Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea.
Young-Sik
YS
Yoo
Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea.
Ji-Young
JY
Kwon
Department of Ophthalmology and Visual Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea.
Jong-Soo
JS
Joo
Department of Ophthalmology and Visual Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea.
Sung-A
SA
Lim
Department of Ophthalmology and Visual Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea.
Woong-Joo
WJ
Whang
Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea.
Jee-Won
JW
Mok
Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea.
Jun-Sub
JS
Choi
Department of Ophthalmology and Visual Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Institute of Visual Science, Catholic University of Korea, Seoul, Republic of Korea. Electronic address: ckjoo@catholic.ac.kr.
Choun-Ki
CK
Joo
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D019573:Epithelium, Corneal / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D008297:Male; D009883:Ophthalmic Solutions; D010766:Phosphorylation; D011122:Polyphosphates / Q000494:pharmacology*; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D058473:Receptors, Purinergic P2Y2 / Q000378:metabolism; D014500:Uracil Nucleotides / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects*
D009883:Ophthalmic Solutions; D011122:Polyphosphates; D058912:Purinergic P2Y Receptor Agonists; D058473:Receptors, Purinergic P2Y2; D014500:Uracil Nucleotides; D004815:Epidermal Growth Factor; C403315:diquafosol; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium
Corneal epithelium; Diquafosol; P2Y2 receptor agonist; Purinergic P2Y2 receptor; Rat; Uridine triphosphate; Wound healing