Nomura Naoko, Nomura Motohiro, Sugiyama Kazuhisa, Hamada Jun-Ichiro
Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, Kanazawa 920-0935, Japan.
Biochem Pharmacol. 2007 Sep 1;74(5):690-701. doi: 10.1016/j.bcp.2007.06.018. Epub 2007 Jun 19.
We investigated the mechanism of phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells focusing on the p38 mitogen-activated protein kinase (MAPK)/heat shock protein 27 (Hsp27) pathway. PMA-induced cell migration and activation of p38MAPK in A172 glioblastoma cells. PMA-induced formation of lamellipodia and focal complexes was blocked by inhibiting p38MAPK with SB203580 or small interfering RNA (siRNA). Furthermore, activation of p38MAPK resulted in phosphorylation of an F-actin polymerization regulator, Hsp27. Immunohistochemical analysis showed that upon PMA stimulation, both unphosphorylated and phosphorylated Hsp27 were translocated to the lamellipodia. SB203580 or p38MAPK siRNA blocked these phenomena, indicating that Hsp27 phosphorylation and translocation from cytosol to membrane were mediated by p38MAPK. To address the question of whether endogenous Hsp27 participates in PMA-induced migration, we inhibited the expression of Hsp27 using Hsp27 siRNA. Although knockdown of Hsp27 by siRNA had little effect on p38MAPK activation, lamellipodia and focal complex formation was markedly inhibited. Migration was also abolished in Hsp27 siRNA-transfected cells. In conclusion, p38MAPK activation followed by Hsp27 phosphorylation was required for PMA-induced migration. Furthermore, Hsp27 itself played critical roles in PMA-induced migration. Our data provide substantial evidence for a model elucidating the molecular mechanisms of regulation of actin dynamics and migration by PMA-activated protein kinase C in glioblastoma cells.
我们研究了佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）诱导胶质母细胞瘤细胞迁移的机制，重点关注p38丝裂原活化蛋白激酶（MAPK）/热休克蛋白27（Hsp27）信号通路。PMA可诱导A172胶质母细胞瘤细胞迁移并激活p38MAPK。用SB203580或小干扰RNA（siRNA）抑制p38MAPK可阻断PMA诱导的片状伪足和粘着斑复合物的形成。此外，p38MAPK的激活导致F -肌动蛋白聚合调节因子Hsp27的磷酸化。免疫组织化学分析表明，PMA刺激后，未磷酸化和磷酸化的Hsp27均转位至片状伪足。SB203580或p38MAPK siRNA可阻断这些现象，表明Hsp27的磷酸化以及从胞质溶胶向细胞膜的转位是由p38MAPK介导的。为了探讨内源性Hsp27是否参与PMA诱导的迁移，我们使用Hsp27 siRNA抑制Hsp27的表达。尽管siRNA敲低Hsp27对p38MAPK的激活影响不大，但片状伪足和粘着斑复合物的形成受到明显抑制。Hsp27 siRNA转染的细胞中迁移也被消除。总之，PMA诱导的迁移需要p38MAPK激活后Hsp27磷酸化。此外，Hsp27自身在PMA诱导的迁移中起关键作用。我们的数据为阐明PMA激活的蛋白激酶C在胶质母细胞瘤细胞中调节肌动蛋白动力学和迁移的分子机制的模型提供了大量证据。
