Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1583, USA.
J Cell Physiol. 2011 Nov;226(11):2881-93. doi: 10.1002/jcp.22631.
The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20 kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA-induced migration is insensitive to pertussis toxin (PTX) but does require the LPA(1) receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP-bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA-induced migration and invasion are attenuated by specific inhibitors including C3 cell-permeable transferase and Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II-A or II-B. Inhibition or loss of either NM II-A or NM II-B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II-A or NM II-B also rescued LPA-induced migration. Taken together, these results suggest defined pathways for signaling through the LPA(1) receptor to promote LPA-mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells.
在肿瘤细胞中发现的增强迁移通常是由外部刺激和细胞骨架相关信号分子的顺序参与引起的。然而，直到现在，对于 LPA 诱导的迁移，还没有详细分析溶血磷脂酸（LPA）受体和非肌肉肌球蛋白 II（NM II）之间的分子连接。在这里，我们证明 LPA 通过激活 LPA(1)受体诱导迁移，该受体通过激活 Rho 激酶（ROCK）促进 20kDa NM II 轻链的磷酸化。我们表明，LPA 诱导的迁移对百日咳毒素（PTX）不敏感，但确实需要 LPA(1)受体，这可以通过 siRNA 和受体拮抗剂来确定。LPA 激活 ROCK 并增加 GTP 结合的 RhoA 活性，同时增强 RhoA 的膜募集。特异性抑制剂包括 C3 细胞通透性转移酶和 Y-27632 可减弱 LPA 诱导的迁移和侵袭。我们通过用 blebbistatin 和针对 NM II-A 或 II-B 的 shRNA 慢病毒抑制其细胞功能，证明 NM II 在 LPA 诱导的迁移和侵袭中起重要作用。在 4T1 细胞中抑制或丧失 NM II-A 或 NM II-B 会导致迁移和侵袭减少。NM II-A 或 NM II-B 的表达恢复也挽救了 LPA 诱导的迁移。总之，这些结果表明，LPA(1)受体通过明确的信号通路促进 LPA 介导的 NM II 激活，并随后在 4T1 乳腺癌细胞中促进细胞迁移。
