Kaur Sukhbir, Samant Ganesh V, Pramanik Kallal, Loscombe Philip W, Pendrak Michael L, Roberts David D, Ramchandran Ramani
Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
BMC Cell Biol. 2008 Nov 3;9:61. doi: 10.1186/1471-2121-9-61.
Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro.
Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.
This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.
环行蛋白是最近被发现也在血管导向中发挥作用的轴突导向分子。在本研究中，我们使用小干扰RNA技术在体外研究了内皮细胞中Robo蛋白的基因敲低分析，特别是内皮细胞中主要的Robo蛋白——环行蛋白4（robo4）。
Robo1和Robo4敲低的细胞在内皮细胞迁移试验中表现出不同的活性。robo4的敲低消除了内皮细胞对血清的趋化反应，但增强了对Slit2的化学动力学反应，而Robo1敲低的细胞对血清或VEGF不表现出趋化反应。Robo4敲低的内皮细胞意外地显示出Rho GTP酶的上调。斑马鱼Robo4可挽救robo4敲低细胞中的Rho GTP酶稳态和血清降低的趋化性。Robo1和Robo4相互作用并共享诸如Slit2、Mena和一种Cdc42-GAP即Vilse等分子。此外，本研究从机制上表明IRSp53参与了活化的Cdc42和Mena之间的信号连接，这两者先前已被证明在内皮细胞中与Robo4信号传导有关。
本研究确定了共同指导内皮细胞定向迁移的Robo信号传导装置的特定组成部分。
