Regulatory role of PI 3-kinase on expression of cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells
Regulatory role of PI 3-kinase on expression of cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells
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DOI：
10.1167/iovs.02-0637
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发表时间：
2003-04-01
影响因子：
4.4
通讯作者：
Kay, EP
中科院分区：
文献类型：
--
作者：
Lee, HT;Kay, EP
目的。FGF-2是兔角膜内皮细胞（CECs）的一种有效的丝裂原。本研究旨在探讨PI 3-激酶是否参与cec细胞周期调控以响应FGF-2的刺激。通过计数检测细胞增殖情况。免疫荧光染色检测蛋白的亚细胞定位，免疫印迹检测细胞周期蛋白依赖性激酶4 （Cdk4）、p27(Kip1) （p27）、磷脂酰肌醇3 （PI 3）激酶、蛋白激酶B/Akt （Akt）和β -肌动蛋白的表达。通过测定磷脂酰肌醇-3-磷酸的产量来测定PI 3-激酶活性。LY294002对PI 3-激酶有抑制作用。CEC需要长时间持续暴露于FGF-2。FGF-2在10 ng/mL时显著刺激PI 3-激酶活性，同时FGF-2的刺激也引起Akt的活化。LY294002抑制细胞增殖和PI 3-激酶活性呈浓度依赖性。PI 3-激酶在细胞周期刺激中的作用被确定：FGF-2显著上调Cdk4的表达并刺激易位。而LY294002明显阻断了Cdk4表达上调，促进了Cdk4的核输出。相反，FGF-2显著下调p27的表达并促进p27的磷酸化。LY294002完全阻断FGF-2对p27的表达和磷酸化的作用。这些数据表明，pi3 -激酶最终导致FGF-2对细胞周期机制的激活。它通过上调Cdk4的表达，促进Cdk4的核输入，并将Cdk4隔离在细胞核中，同时下调p27的表达，并通过磷酸化促进该分子的蛋白水解。
PURPOSE. FGF-2 is a potent mitogen of rabbit corneal endothelial cells (CECs). This study was undertaken to investigate whether PI 3-kinase participates in cell cycle regulation in response to stimulation with FGF-2 in CECs.METHODS. Cell proliferation was assayed by counting the cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of cyclin-dependent kinase 4 (Cdk4), p27(Kip1) (p27), phosphatidylinositol 3 (PI 3)kinase, protein kinase B/Akt (Akt), and beta-actin was analyzed by immunoblot. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. LY294002 was used to inhibit PI 3-kinase.RESULTS. CEC required prolonged and continuous exposure to FGF-2. FGF-2 at 10 ng/mL markedly stimulated PI 3-kinase enzyme activity, and stimulation, with FGF-2 also caused activation of Akt. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase in cell cycle stimulation was determined: FGF-2 markedly upregulated expression of Cdk4 and stimulated translocation. of Cdk4 into nuclei, whereas LY294002 markedly blocked upregulation of Cdk4 expression, and the inhibitor facilitated nuclear export of Cdk4. In contrast, FGF-2 significantly downregulated expression of p27 and facilitated phosphorylation of p27. LY294002 completely blocked the action of FGF-2 on the expression and phosphorylation of p27.CONCLUSIONS. These data indicate that PI 3-kinase ultimately leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating expression of Cdk4, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei as it simultaneously downregulates expression of p27 and facilitates the proteolysis of the molecule by phosphorylation.