title: Activation of the Rho/Rho Kinase Signaling Pathway Is Involved in Cell Death of Corneal Endothelium.
abstract: Rho kinase (ROCK) pathways control fundamental cell functions, making ROCK an important therapeutic target in several pathophysiologic conditions. The purpose of this study was to investigate whether inhibition of ROCK can suppress apoptosis of the corneal endothelium and to determine the role of ROCK signaling in regulating apoptosis.
  
  
  
  The effects of inhibitors of ROCK or myosin light chain (MLC) were evaluated in cultured monkey corneal endothelial cells (MCECs) irradiated with ultraviolet (UV) (100 J/m2) to induce apoptosis. Annexin V and TUNEL staining and Western blot for apoptosis-related proteins and focal adhesion complexes were then performed. RhoA activation was further evaluated by pull-down assays. ROCK inhibitor and caspase inhibitor effects on apoptosis were also evaluated in MCECs treated with ethylene glycol tetraacetic acid (EGTA) to induce MLC phosphorylation.
  
  
  
  ROCK or MLC inhibition suppressed the caspase-3 cleavage and Annexin V and TUNEL expression typically seen during UV-mediated apoptosis of MCECs. The apoptotic stimulus activated RhoA and then induced phosphorylation of MLC via ROCK activation. EGTA-mediated phosphorylation of MLC was sufficient to induce the loss of cell contact with the substrate and subsequent apoptosis. Western blot showed that ROCK inhibition upregulated the expression of the focal adhesion complex in adhered cells, following UV stress.
  
  
  
  Apoptotic stimuli activated Rho/ROCK/MLC phosphorylation in the corneal endothelium, and subsequent actomyosin contraction induced apoptosis by loss of cell adhesion. ROCK inhibition suppressed MLC phosphorylation and subsequent cell death, and it counteracted the loss of cell adhesion by activating the focal adhesion complex.
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okumura
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Fujii
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Takato
authors.initials: T
authors.lastname: Kagami
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Nakahara
authors.initials: N
authors.lastname: Makiko
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Miu
authors.initials: M
authors.lastname: Kitahara
authors.affiliation: Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000502:physiology*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008252:Macaca fascicularis; D010766:Phosphorylation; D011817:Rabbits; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D054460:rho-Associated Kinases