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Yuki Tsujimoto, Naoki Okumura, EunDuck Kay, Ryohei Numata, Shigeru Kinoshita, Noriko Koizumi; Rho kinase inhibitor promotes cell adhesion of corneal endothelial cells through inhibiting phosphorylation of MLC. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1694.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that Rho kinase (ROCK) inhibitor promoted cell adhesion of cultivated corneal endothelial cells, and enabled cell injection therapy combined with ROCK inhibitor in cornea endothelial dysfunction animal model. The purpose of this study is to determine the mechanism how ROCK inhibitor promotes corneal endothelial cell adhesion on the substrate.
Cultivate monkey corneal endothelial cells (MCECs) were used for this study. To examine the involvement of Rho/ROCK signaling in cell adhesion, RhoA activity and phosphorylation of MLC in MCECs cultured on non-adhesion or on adhesion culture plate was evaluated by G-LISA RhoA activation assay and western blotting. Next, to elucidate the effect of ROCK inhibitor on cell adhesion, phosphorylation of MLC, FAK, and paxillin of the MCECs seeded with or without ROCK inhibitor (Y-27632) was evaluated by western blotting. The expression of actin and vinculin was also determined by immunostaining. Then, adhere cell numbers of the MCECs seeded with either C3 (RhoA inhibitor) or blebbistatin (MLC inhibitor) were evaluated by CellTiter Glo assay.
The activity of RhoA and phosphorylation of MLC was higher in MCECs cultured on non-adhesion culture plate compared to on adhesion culture plate. In MCECs seeded with ROCK inhibitor, phosphorylation of MLC was suppressed, and phosphorylation of FAK and paxillin was promoted compared to the control. ROCK inhibitor also promoted actin spreading and the expression of vinculin. Adhered cell numbers were significantly enhanced both by C3 and blebbistatin compared to the control (125.4±4.7%, 172.3±1.7%, respectively) (p<0.05).
Our findings indicate that ROCK inhibitor activates focal adhesion proteins through phosphorylation of MLC by inhibiting Rho/ROCK signaling pathway. Modulation of cell adhesion property on the substrate by ROCK inhibitor might provide a new tool for cell based tissue engineering therapy.
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