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Naoki Okumura, Noriko Koizumi, Kenta Yamasaki, Morio Ueno, Yuji Sakamoto, Yoshihiro Sowa, Toshiyuki Sakai, Junji Hamuro, Shigeru Kinoshita; ROCK Inhibitor Regulates the G1-S Progression of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6594.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that the Rho kinase (ROCK) inhibitor Y-27632 promotes wound healing of corneal endothelial cells (CECs) in a primate model and showed the possibility of the clinical application for humans who are in a corneal endothelial deficient condition (ARVO, 2009). This current study was conducted to demonstrate the effect of Y-27632 on the cell cycle of corneal endothelial cells in vitro.
CECs of cynomolgus monkeys were cultured and were seeded at a density of 2.5×104/cm2. The culture medium was then replaced with fresh medium containing 10 µM of Y-27632 at 24 hours after cell seeding. Culture medium without Y-27632 was used for the control. The phosphorylation of Rb protein, which regulates G1-S progression, was examined by Western blot assay at 6, 12, and 24 hours after treatment with Y-27632. In addition, labeling assay using BrdU, which is a marker of S phase, was performed at 48 hours after treatment.
Western blot assay demonstrated that phosphorylation of Rb protein at serine 807/811 and serine 780 was promoted in the Y-27632-treated CECs compared to the control cells at 12 and 24 hours. BrdU-labeling assay of 48 hours showed a statistically higher proportion of BrdU-positive CECs among cell populations cultured with Y-27632 compared to among control cells (32.4±4.2% and 18.8±0.7%, respectively; p<0.01).
The findings of this study demonstrate that ROCK inhibitor Y-27632 promotes cell proliferation of corneal endothelial cells through G1-S progression via the phosphorylation of Rb protein.
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