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Regulation of a Rho-Associated Kinase Expression during the Corneal Epithelial Cell Cycle

From the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

PURPOSE. It has been recognized that an increased expression of the Rho-associated kinase (ROCK-I), a downstream target of Rho (a Ras-related small guanosine triphosphatase [GTPase]), is associated with limbal-to-corneal epithelial transition. The purpose of the present study was to determine whether the expression of ROCK-I is regulated during the cell cycle of corneal epithelial cells.

METHODS. Rabbit corneal epithelial cells in culture were subjected to different culture conditions to enrich them in the G₀, G₁, and S phases of the cell cycle. Indirect immunofluorescence staining and western blot techniques were used for analyzing the changes in the relative intracellular concentrations of ROCK-I. Northern blot analysis of the isolated cellular RNA was performed to estimate the relative concentrations of ROCK-I mRNA.

RESULTS. Serum deprivation did not cause all the corneal epithelial cells in culture to be arrested in the G₀ phase of the cell cycle. However, the cells could be arrested in G₀ by treating them with culture medium supplemented with transforming growth factor (TGF)-ß1. The relative concentration of ROCK-I in the G₀-arrested cells was higher than in the corresponding control untreated cultures. G₀-arrested cells were induced to enter G₁, followed by the S phase of the cell cycle, by refeeding them with the medium devoid of TGF-ß1. The total intracellular concentration of ROCK-I significantly decreased during the G₁ phase of the cell cycle and increased again during the S phase. The decrease in intracellular ROCK-I during the G₁ phase was confirmed by arresting the cells in G₁ with isoleucine deprivation and thymidine-mimosine treatments. ROCK-I mRNA levels were also found to be decreased during the G₁ phase of the cell cycle.

CONCLUSIONS. The levels of ROCK-I in the corneal epithelial cells were significantly lower in the G₁ phase than those in the S and G₀ phases of the cell cycle. Therefore, a Rho signaling pathway(s) involving ROCK-I may be regulated during the corneal epithelial cell cycle. The downregulation of ROCK-I during the G₁ phase, at least in part, is due to the decreased levels of its mRNA. Based on these findings, ROCK-I may have a role in the progression of the cell cycle in the corneal epithelial cells as they migrate centripetally from the limbal to the corneal surface.

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