Arikawa Kayo, Takuwa Noriko, Yamaguchi Hironori, Sugimoto Naotoshi, Kitayama Joji, Nagawa Hirokazu, Takehara Kazuhiko, Takuwa Yoh
Department of Physiology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8640, Japan.
J Biol Chem. 2003 Aug 29;278(35):32841-51. doi: 10.1074/jbc.M305024200. Epub 2003 Jun 16.
We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.
我们研究了1-磷酸鞘氨醇（S1P）抑制B16黑色素瘤细胞迁移和侵袭的机制，S1P是Edg家族G蛋白偶联受体的配体，也被认为是一种细胞内第二信使。S1P、二氢-S1P和鞘氨醇磷酸胆碱抑制B16细胞迁移和侵袭，其相对效力符合作为S1P2受体激动剂的预期。S1P2选择性拮抗剂JTE013完全消除了对这些激动剂的反应。此外，JTE013消除了鞘氨醇（S1P的前体，但不是S1P受体的激动剂）的抑制作用，表明鞘氨醇的作用是通过S1P2刺激介导的，很可能是由鞘氨醇转化而来的S1P介导的。S1P分别诱导B16细胞中Rac和RhoA的抑制和激活，这被JTE013消除。腺病毒介导的N17Rac表达模拟了S1P对迁移的抑制作用，而C3毒素预处理（而非Rho激酶抑制剂）逆转了S1P的抑制作用。S1P2的过表达使B16细胞对S1P抑制Rac和迁移敏感，而S1P1或S1P3的过表达则使其脱敏。在JTE013预处理的、S1P3过表达的B16细胞中，S1P刺激细胞RhoA，但未能抑制Rac或迁移，表明RhoA刺激本身不足以抑制迁移。这些结果提供了令人信服的证据，即内源性表达的S1P2通过对细胞Rac和RhoA活性的配体依赖性相互调节来负向调节细胞运动性和侵袭。在JTE013存在的情况下，S1P反而刺激过表达S1P1或S1P3的B16细胞中的Rac和迁移，揭示了S1P2与S1P1或S1P3趋化受体之间的拮抗作用。
