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

Invest Ophthalmol Vis Sci. 2014 Jan 15;55(1):318-29. doi: 10.1167/iovs.13-12225.


Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: ROCK inhibitor; bullous keratopathy; cell proliferation; corneal endothelial cells.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*


  • Amides
  • Cyclin D
  • Enzyme Inhibitors
  • Pyridines
  • Y-39983
  • Y 27632
  • Cyclin-Dependent Kinase Inhibitor p27