Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of slingshot or LIM kinase
Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of slingshot or LIM kinase
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DOI：
10.1074/jbc.m706538200
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发表时间：
2008-03-07
影响因子：
4.8
通讯作者：
Mizuno, Kensaku
中科院分区：
文献类型：
--
作者：
Horita, Yuji;Ohashi, Kazumasa;Mizuno, Kensaku
肌动蛋白细胞骨架重组对于肿瘤细胞迁移、粘附和侵袭至关重要。 Cofilin 和肌动蛋白解聚因子 (ADF) 通过刺激肌动蛋白丝的解聚和切断，充当肌动蛋白细胞骨架动力学的关键调节剂。 Cofilin/ADF 通过 LIM 激酶-1 (LIMK1) 磷酸化 Ser-3 失活，并通过 Slingshot-1 (SSH1) 和 -2 (SSH2) 蛋白磷酸酶去磷酸化重新激活。在本研究中，我们使用体外跨细胞迁移测定检查了 cofilin/ADF、LIMK1 和 SSH1/SSH2 在肿瘤细胞侵袭中的作用。在该测定中，将大鼠腹水肝癌 (MM1) 细胞覆盖在原代培养的大鼠间皮细胞单层上，并计数在存在溶血磷脂酸 (LPA) 的情况下迁移到单层下方的细胞灶的数量。通过小干扰 RNA (siRNA) 敲低 cofilin/ADF、LIMK1 或 SSH1/SSH2 表达，可显着降低 LPA 诱导的 MM1 细胞跨细胞迁移及其在二维培养中的运动性。 LIMK1 的敲低还抑制了纤连蛋白介导的细胞附着和粘着斑形成。我们的结果表明，LIMK1 介导的 cofilin/ADF 磷酸化和 SSH1/SSH2 介导的去磷酸化对于肿瘤细胞的迁移和侵袭至关重要，并且 LIMK1 通过增强细胞的粘附和运动来参与肿瘤细胞的跨细胞迁移。
Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.