Chen Jian, Guerriero Emily, Lathrop Kira, SundarRaj Nirmala
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.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):175-83. doi: 10.1167/iovs.07-0488.
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.
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.
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.
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).
作者之前的研究表明，Rho相关丝氨酸/苏氨酸激酶（ROCK）的表达在角膜上皮细胞的细胞周期进程中受到调控。本研究旨在确定Rho/ROCK信号是否以及如何调节细胞周期进程。
培养的兔角膜上皮细胞（RCECs）通过血清剥夺被阻滞在细胞周期的G(0)期，然后在补充血清的培养基中，在有或无ROCK抑制剂（Y27632）的情况下使其重新进入细胞周期。分别通过BrdU标记、蛋白质印迹和免疫细胞化学分析以及实时RT-PCR测定S期细胞数量、特定细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）的相对水平及其细胞内分布以及mRNA的相对水平。
ROCK抑制延迟了G(1)期到S期的进程，并导致进入S期的RCECs数量在12至24小时之间从31.5%±4.5%降至8.1%±2.6%。在细胞周期进程中，细胞周期蛋白D1和D3以及细胞周期蛋白依赖性激酶CDK4和CDK6的蛋白质和mRNA水平在ROCK抑制的细胞中显著降低，而CDK抑制剂p27(Kip1)的蛋白质水平较高。细胞周期蛋白E的细胞内mRNA或蛋白质水平以及CDK2的蛋白质水平未受到显著影响，但它们的核转位因ROCK抑制而延迟。
ROCK信号参与RCECs的细胞周期进程，可能是通过上调细胞周期蛋白D1和D3以及CDK4、-6和-2；CDK2和细胞周期蛋白E的核转位；以及下调p27(Kip1)。
