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Naoki Okumura, Ai Odajima, EunDuck Kay, Wen Chen, Morio Ueno, Junji Hamuro, Shigeru Kinoshita, Noriko Koizumi; Activation of the Rho/ROCK Signaling Pathway in the Apoptosis of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2202. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In the pathological condition of corneal endothelium associated with Fuchs’ dystrophy or post keratoplasty, apoptosis is known to be involved. We previously reported that Rho kinase (ROCK)-inhibitor Y-27632 suppresses the apoptosis of cultured corneal endothelial cells (CECs). The purpose of this present study was to evaluate the involvement of the Rho/ROCK signaling pathway in the apoptosis of CECs and the effect of ROCK inhibitor on modulating apoptosis.
Monkey corneal endothelial cells (MCECs) were cultured and then exposed to ultra-violet (UV) radiation (100J/m2) to induce apoptosis. To elucidate the involvement of the Rho/ROCK signaling pathway in apoptosis, RhoA-GTPase, myosin light chain (MLC) phosphorylation, and the cleavage of caspase 3 and ROCK 1 was evaluated by western blotting. Anti-blebbing effect of Y-27632 was evaluated after exposure to UV radiation by time-lapse phase contrast microscopy, and actin and MLC phosphorylation were evaluated by immunostaining. Annexin V staining was employed to confirm the anti-apoptotic effect of Y-27632.
UV radiation caused cell death in MCECs in a dose-dependent manner; a significant cell death was observed at 100J/m2and higher dosages. Active GTPase pull-down assay showed that UV radiation activated RhoA of the MCECs. MLC phosphorylation and the cleavage of caspase 3 and ROCK 1 were increased by UV radiation. Compared to the control, Y-27632-treated MCECs exposed to UV radiation showed significantly decreased membrane blebbing (p<0.01). Moreover, actin contraction and MLC phosphorylation were both downregulated by Y-27632. In the human corneal tissue exposed to UV, a significantly decreased number of UV-induced Annexin V-positive apoptotic CECs was found in the Y-27632-treated tissue compared to the control (3.1±0.5% and 20.3±2.0%, respectively) (p<0.01).
The findings of this study indicate that Rho/ROCK signaling activation is involved in the UV-induced apoptosis of CECs. Rho/ROCK signaling might be a potent therapeutic target for the suppression of cell apoptosis in various corneal endothelial diseases. However, further investigation is needed to elucidate the anti-apoptotic mechanisms of ROCK inhibitor.
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