Role of p27(Kip1) in cAMP- and TGF-beta2-mediated antiproliferation in rabbit corneal endothelial cells.
Role of p27(Kip1) in cAMP- and TGF-beta2-mediated antiproliferation in rabbit corneal endothelial cells.
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发表时间：
2001
影响因子：
4.4
通讯作者：
T. Y. Kim;W. Kim;R. Smith;E. D. Kay
中科院分区：
文献类型：
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作者：
T. Y. Kim;W. Kim;R. Smith;E. D. Kay
目的探讨p27（Kip1）在抗itogens （cAMP和tgf - β 2）介导的兔角膜内皮细胞（CECs）抗增殖中的作用。方法采用比色法测定细胞增殖，测定活细胞数。免疫荧光染色法检测细胞周期调控蛋白的亚细胞定位，免疫印迹法检测蛋白的表达。结果当细胞被cAMP或tgf - β 2处理时，血清介导的细胞增殖呈剂量依赖性抑制。同时使用这两种抗菌素对抑制细胞生长没有协同作用。免疫荧光染色法检测细胞周期调节蛋白cyclin-D1、cyclin-E、cdk2、cdk4、p21（Cip1）、p27（Kip1）的表达。p27（Kip1）呈强核染色。其他蛋白质没有染色，或者只有非常微弱的染色。用cAMP或tgf - β 2处理细胞时，除p27（Kip1）外，其他任何蛋白的染色电位都没有改变，但用tgf - β 2或cAMP处理细胞时，所有细胞的核p27（Kip1）均呈阳性。相反，含有丝裂原（FGF-2）的培养基减少了p27（Kip1）阳性细胞的数量。在FGF-2单独处理或FGF-2与cAMP联合处理24小时的细胞中，用免疫印迹法检测p27（Kip1）的表达水平，FGF-2显著降低p27（Kip1）水平，cAMP阻止FGF-2诱导的p27（Kip1）水平下降。在细胞短期暴露于FGF-2或cAMP或两者的组合时，没有发生这种现象。当细胞被磷酸化p27（Kip1）染色时，FGF-2显著增强了细胞核中磷酸化p27（Kip1）的染色，而cAMP和tgf - β 2都阻止了p27（Kip1）的磷酸化。结论：这两种抗丝裂原均可通过抑制有丝裂原介导的p27（Kip1）水平下降而上调p27（Kip1）的表达。此外，cAMP和tgf - β 2可能通过阻断p27（Kip1）的磷酸化来抑制G(1)到s的转变，而p27（Kip1）的磷酸化是抑制剂分子核输出降解的先决条件。
PURPOSE To determine whether p27(Kip1) plays a role in antiproliferation mediated by antimitogens (cAMP and TGF-beta2) in rabbit corneal endothelial cells (CECs). METHODS Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of cell cycle-regulatory proteins was determined by immunofluorescent staining, and expression of the proteins was analyzed by immunoblot analysis. RESULTS When cells were treated with cAMP or TGF-beta2, serum-mediated cell proliferation was inhibited in a dose-dependent manner. Simultaneous treatment of the two antimitogens did not show a synergistic effect on inhibition of cell growth. Expression of cell cycle-regulatory proteins, such as cyclin-D1, cyclin-E, cdk2, cdk4, p21(Cip1), and p27(Kip1) was determined using immunofluorescent staining. A strong nuclear staining was observed for p27(Kip1). The other proteins were not stained or were only very faintly stained. Treatment of cells with either cAMP or TGF-beta2 did not change the staining potential of any proteins other than p27(Kip1), but all cells were positive for nuclear p27(Kip1) when treated with either TGF-beta2 or cAMP. In contrast, mitogen (FGF-2)-containing medium decreased the number of p27(Kip1)-positive cells. When the expression level of p27(Kip1) was determined using immunoblot analysis in the cells treated either with FGF-2 alone or with a concomitant treatment with FGF-2 and cAMP for 24 hours, FGF-2 markedly decreased the p27(Kip1) level, and cAMP prevented the decrease in p27(Kip1) level induced by FGF-2. No such phenomenon occurred during a short-term exposure of cells to either FGF-2 or cAMP or to a combination of the two. When cells were stained for phosphorylated p27(Kip1), FGF-2 markedly enhanced the staining of phosphorylated p27(Kip1) in nuclei, whereas both cAMP and TGF-beta2 prevented the phosphorylation of p27(Kip1). CONCLUSIONS These findings suggest that both antimitogens upregulate the expression of p27(Kip1) as they prevent the decrease of the p27(Kip1) level mediated by mitogen. Furthermore, cAMP and TGF-beta2 may inhibit the G(1)-to-S transition by blocking phosphorylation of p27(Kip1), which is a prerequisite for nuclear export of the inhibitor molecule for degradation.