Purchase this article with an account.
J. Yin, K.–P. Xu, F.–S.X. Yu; Role of Rho–ROCK in Regulating Human Corneal Epithelial Wound Healing . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5023.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Small GTPase Rho and its downstream effector Rho–associated kinase (ROCK) are known to be involved in actin cytoskeleton rearrangement, cell proliferation and migration. In this study, we sought to elucidate the role of Rho–ROCK signaling pathway in human corneal epithelial wound healing.
The activity of Rho was assessed by pull–down assay using GST–fusion protein consisting of Rho downstream effector Rhotekin. Rho–specific inhibitor Clostridium botulinum exoenzyme C3 and ROCK inhibitor Y–27632 were used to block the biological functions of Rho and ROCK. The effects of C3 and Y–27632 on wound healing were assessed by measuring the rate of scratch wound closure. Cell proliferation and cell migration were determined by BrdU incorporation and Boyden chamber assay, respectively. Cell adhesion was assessed by counting the numbers of HCECs adhered to FNC (3:1 fibronectin and type I collagen) coated substratum within a given time. Trans–epithelial resistance (TER) was measured as an indicator of the barrier function.
Wounding induced a rapid increase of Rho activity, observed as early as 10 minutes post wounding and sustained up to 2 hours. C3 attenuated both spontaneous and heparin–binding EGF–like growth factor (HB–EGF) enhanced healing of a scratch wound. Y–27632, on the other hand, significantly accelerated HB–EGF–enhanced wound closure. The effect of Y–27632 on wound closure was probably due to an increase in cell migration, since it enhanced migratory responses of HCECs toward FNC and HB–EGF and decreased the number of BrdU–positive cells in wounded, HB–EGF treated cells. Furthermore, Y–27632 treatment resulted in an increase in cell adhesion to FNC. HCECs gradually formed barriers, indicated by a gradual increase in TER. Y–27632 blocked the barrier formation and disrupted pre–formed tight junction barriers.
Rho activity was increased during corneal epithelial wound healing. Rho–ROCK, in collaboration with HB–EGF, enhances cell proliferation, promotes epithelial differentiation, but negatively modulates cell migration and cell adhesion, therefore plays a role in regulating corneal epithelial wound healing.
This PDF is available to Subscribers Only