June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Rho-kinase inhibitor enhances corneal endothelial cell proliferation via p27 degradation
Author Affiliations & Notes
  • Ryohei Numata
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Naoki Okumura
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • EunDuck Kay
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Makiko Nakahara
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Shinichiro Nakano
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Morio Ueno
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Footnotes
    Commercial Relationships Ryohei Numata, None; Naoki Okumura, None; EunDuck Kay, None; Makiko Nakahara, None; Shinichiro Nakano, None; Morio Ueno, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P); Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R); Noriko Koizumi, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1690. doi:
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      Ryohei Numata, Naoki Okumura, EunDuck Kay, Makiko Nakahara, Shinichiro Nakano, Morio Ueno, Shigeru Kinoshita, Noriko Koizumi; Rho-kinase inhibitor enhances corneal endothelial cell proliferation via p27 degradation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1690.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: We previously reported that Rho kinase (ROCK) inhibitor promotes wound healing of corneal endothelial cells in an animal model and showed the possibility of the clinical application for corneal endothelial deficient condition. The purpose of this study is to determine the mechanism by which ROCK inhibitor promotes cell proliferation in corneal endothelial cells.

Methods: Cultivate monkey corneal endothelial cells (MCECs) were used for this study. Cell proliferation was analyzed by two methods in the absence or presence of 10 μM the selective ROCK inhibitor (Y-27632): BrdU ELISA assay to determine the incorporation of BrdU into the newly synthesized DNA and CellTiter Glo assay to measure the numbers of viable cells. Activation of Akt and expression of Cdc25A and p27 were analyzed by western blotting.

Results: Incorporation of BrdU into the newly synthesized DNA was doubled in the cells treated with ROCK inhibitor when compared to that of the control cells. Likewise, the viable cell numbers of MCECs treated with the inhibitor for 24 h or 48 h were 50% greater than the cell numbers in the absence of ROCK inhibitor. In addition to the increased cell numbers, the ROCK inhibitor promoted cell adhesion. To elucidate the action of ROCK inhibitor in MCECs with or without ROCK inhibitor, phosphorylation of Akt was analyzed for 1 h up to 24 h in serum-containing medium: in the absence of ROCK inhibitor, phosphorylation of Akt was gradually increased to 12 h. Unlike the control pattern, phosphorylation of Akt reached maximum at 1 h following treatment of cells with ROCK inhibitor, after which the level of phosphorylated Akt was gradually decreased. The expression of Cdc25A, which activates Cdk2 that phosphorylates p27, was observed during the early time periods in the presence of ROCK inhibitor, while the control cells showed a late expression pattern of Cdc25A. Similarly, the amount of p27 was greatly reduced from 1 h following treatment of cells with ROCK inhibitor, whereas the control cells maintained high levels of p27 up to 12 h.

Conclusions: The findings of the study demonstrate that ROCK inhibitor activates PI 3-kinase signaling which subsequently promotes degradation of p27 via Cdc25A pathway, thus leading to cell proliferation of CECs. These data suggest that the ROCK might be a useful pharmaceutical agent for corneal endothelial disease.

Keywords: 481 cornea: endothelium • 480 cornea: basic science • 654 proliferation  
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