Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.
OBJECTIVE
The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression.


METHODS
Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively.


RESULTS
ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition.


CONCLUSIONS
ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).
Chen
Jian
J
UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, 203 Lothrop Street, Pittsburgh, PA 15213, USA.
Guerriero
Emily
E
Lathrop
Kira
K
SundarRaj
Nirmala
N
D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D007150:Immunohistochemistry; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
D000577:Amides; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; C108830:Y 27632; D054460:rho-Associated Kinases