Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27 upregulation.
Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27 upregulation.
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DOI：
10.1371/journal.pone.0011668
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发表时间：
2010-07-22
期刊：
影响因子：
3.7
通讯作者：
Rao JS
中科院分区：
文献类型：
--
作者：
Gopinath S;Malla RR;Gondi CS;Alapati K;Fassett D;Klopfenstein JD;Dinh DH;Gujrati M;Rao JS
组织蛋白酶B和尿激酶纤溶酶原激活物受体（uPAR）在胶质瘤中都是过表达的。我们之前的工作和其他人的工作强烈表明胶质瘤的浸润表型与组织蛋白酶B和uPAR的表达之间存在关系。虽然它们在迁移和粘附中的作用已经得到了很好的研究，但这些分子对细胞周期进程的影响尚未得到彻底的研究。在SNB19和U251胶质瘤细胞中，使用Cathespin B和uPAR单、双电子siRNA质粒下调这些分子。FACS分析和BrdU掺入试验分别显示G0/G1阻滞和增殖降低。免疫印迹和免疫细胞分析显示p27Kip1的表达和核定位随着组织蛋白酶B和uPAR的下调而增加。αVβ3/PI3K/AKT/FOXO通路可能介导了这些作用，αVβ3表达降低，PI3K和AKT磷酸化，FOXO3a水平升高。用Ly294002（10µM）处理后p27Kip1和FOXO3a的表达增加，用siRNA和Ly294002处理后p27Kip1的FOXO结合启动子区荧光素酶的表达增加，进一步证实了上述结果。我们的处理还降低了cyclin D1、cyclin D2、p-Rb和cyclin E的表达，而Cdk2的表达不受影响。值得注意的是，Cdk2-cyclin E复合物的形成明显减少。我们的研究表明，组织蛋白酶B和uPAR敲低通过调节PI3K/AKT信号通路诱导G0/G1阻滞，并进一步增加p27Kip1的表达，同时FOXO3a与其启动子结合。综上所述，我们的研究结果为SNB19和U251胶质瘤细胞中组织蛋白酶B和uPAR下调诱导的G0/G1阻滞提供了分子机制。
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined. Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly. Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.
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