Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
Biol Reprod. 2012 Jan 30;86(1):1-9. doi: 10.1095/biolreprod.111.094698. Print 2012 Jan.
Insulin-like growth factor binding protein 1 (IGFBP1), the main secretory product of the decidualized endometrium of a pregnant woman, has previously been shown to interact with the alpha(5)beta(1) integrin of extravillous trophoblast (EVT) cell surface to stimulate its migration in an IGF-independent manner. This migration stimulation has also been shown to require activation of extracellular signal regulated kinases 1 and 2 (ERK1/2; mitogen-activated protein kinase [MAPK] 3/1]) and focal adhesion kinase. The present study examined the roles of Rho GTPases RHOA, RHOC, RAC1, and CDC42 as well as RHO kinases ROCK1 and ROCK2 in IGFBP1-mediated migration of an immortalized EVT cell line HTR-8/SVneo. A nonselective RHO kinase inhibitor, Y27632, as well as siRNAs selective for ROCK1 and ROCK2 decreased the migration of these cells in a Transwell migration assay, and this inhibition could not be restored by IGFBP1. Clostridium difficile toxin B, which inhibits all the Rho GTPases, RAC inhibitor NSC23766, RAC1 siRNA, and CDC42 siRNA, decreased their basal migration, but none of these inhibitions except CDC42 siRNA-induced inhibition could be restored by IGFBP1. Clostridium botulinum C3 exoenzyme that inhibits RHOA, RHOB, and RHOC inhibited basal migration but not IGFBP1-induced migration. IGFBP1-induced activation of ERK1/2 (MAPK3/1), which did not require RHO proteins, might function as an alternate pathway for RHO action. However, selective siRNA-mediated downregulation of RHOA inhibited basal, but not IGFBP1-mediated, migration, whereas that of RHOC inhibited both basal and IGFBP1-mediated migration of these EVT cells. Therefore, RHO kinase, RHOC, and RAC1 are essential, but RHOA and CDC42 are not essential, for IGFBP1-induced EVT migration.
胰岛素样生长因子结合蛋白 1（IGFBP1）是孕妇蜕膜化子宫内膜的主要分泌产物，先前已显示其与滋养层细胞（EVT）细胞表面的 alpha(5)beta(1)整联蛋白相互作用，以 IGF 非依赖性方式刺激其迁移。这种迁移刺激也已显示需要激活细胞外信号调节激酶 1 和 2（ERK1/2；丝裂原活化蛋白激酶 [MAPK] 3/1）和粘着斑激酶。本研究检查了 Rho GTPases RHOA、RHOC、RAC1 和 CDC42 以及 Rho 激酶 ROCK1 和 ROCK2 在 IGFBP1 介导的永生化 EVT 细胞系 HTR-8/SVneo 迁移中的作用。非选择性 Rho 激酶抑制剂 Y27632 以及针对 ROCK1 和 ROCK2 的 siRNA 降低了这些细胞在 Transwell 迁移测定中的迁移，并且这种抑制不能被 IGFBP1 恢复。抑制所有 Rho GTPases 的艰难梭菌毒素 B、RAC 抑制剂 NSC23766、RAC1 siRNA 和 CDC42 siRNA 降低了它们的基础迁移，但除了 CDC42 siRNA 诱导的抑制外，这些抑制都不能被 IGFBP1 恢复。抑制 RHOA、RHOB 和 RHOC 的肉毒梭菌 C3 外毒素抑制基础迁移，但不抑制 IGFBP1 诱导的迁移。IGFBP1 诱导的 ERK1/2（MAPK3/1）激活不需要 Rho 蛋白，可能作为 Rho 作用的替代途径。然而，选择性 siRNA 介导的 RHOA 下调抑制了基础迁移，但不抑制 IGFBP1 介导的迁移，而 RHOC 下调抑制了这些 EVT 细胞的基础和 IGFBP1 介导的迁移。因此，RHO 激酶、RHOC 和 RAC1 是 IGFBP1 诱导的 EVT 迁移所必需的，但 RHOA 和 CDC42 不是必需的。
