Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing

• Published in: Investigative ophthalmology & visual science 2014-01, Vol.55 (1), p.318-329
• Main Authors: Okumura, Naoki, Nakano, Shinichiro, Kay, EunDuck P, Numata, Ryohei, Ota, Aya, Sowa, Yoshihiro, Sakai, Toshiyuki, Ueno, Morio, Kinoshita, Shigeru, Koizumi, Noriko
• Format: Article
• Language: English
• Subjects: Amides - pharmacology; Animals; Cell Division - drug effects; Cell Proliferation - drug effects; Cells, Cultured; Cyclin D - metabolism; Cyclin-Dependent Kinase Inhibitor p27 - metabolism; Endothelium, Corneal - drug effects; Endothelium, Corneal - injuries; Endothelium, Corneal - metabolism; Enzyme Inhibitors - pharmacology; Enzyme-Linked Immunosorbent Assay; Eye Injuries - drug therapy; Eye Injuries - metabolism; Eye Injuries - pathology; Humans; Immunoblotting; Macaca fascicularis; Pyridines - pharmacology; Rabbits; Signal Transduction; Wound Healing - drug effects
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.13-12225

To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect. The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated. Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM. These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.