Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G1/S phase arrest in rat basilar arterial smooth muscle cells
Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G1/S phase arrest in rat basilar arterial smooth muscle cells
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DOI：
10.1111/j.1365-2184.2008.00551.x
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发表时间：
2008-10-01
影响因子：
8.5
通讯作者：
Guan, Y. -Y.
中科院分区：
文献类型：
--
作者：
Tang, Y. -B.;Liu, Y. -J.;Guan, Y. -Y.
目的：此前，我们发现ClC-3氯离子通道参与内皮素-1（ET-1）诱导的大鼠主动脉平滑肌细胞增殖。本研究旨在探讨 ClC-3 在细胞周期进展/分布中的作用以及增殖的潜在机制。材料和方法：小干扰 RNA (siRNA) 用于沉默 ClC-3 表达。分别通过细胞计数、溴脱氧尿苷（BrdU）掺入、蛋白质印迹和流式细胞术测定或检测细胞增殖、细胞周期分布和蛋白质表达。结果：ET-1 诱导的大鼠基底血管平滑肌细胞 (BASMC) 增殖与 ClC-3 蛋白内源性表达的显着增加同时发生。通过 siRNA 沉默 ClC-3 可抑制 ClC-3 蛋白的表达，阻止 ET-1 诱导的 BrdU 掺入和细胞数量的增加。 ClC-3的沉默还导致细胞周期停滞在G(0)/G(1)期并阻止细胞从G(1)期进展到S期。 ClC-3 的敲低可有效抑制细胞周期蛋白 D1 和细胞周期蛋白 E 的表达，并增加细胞周期蛋白依赖性激酶抑制剂 (CDKI) p27(KIP) 和 p21(CIP) 的表达。此外，ClC-3 敲低显着减弱了 ET-1 诱导的 Akt 和糖原合酶激酶 3 beta (GSK-3 beta) 的磷酸化。结论：沉默ClC-3蛋白可有效抑制Akt/GSK-3β信号通路的磷酸化，导致cyclin D1和cyclin E下调，p27(KIP)和p21(CIP)上调。在这些 BASMC 中，综合效应导致细胞周期 G(1)/S 停滞并抑制细胞增殖。
Objectives: Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3 beta (GSK-3 beta) induced by ET-1. Conclusion: Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3 beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.