Gonzalez Eva, Kou Ruqin, Michel Thomas
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2006 Feb 10;281(6):3210-6. doi: 10.1074/jbc.M510434200. Epub 2005 Dec 8.
Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.
1-磷酸鞘氨醇（S1P）是一种血小板衍生的鞘脂，可激活G蛋白偶联的S1P受体，并在血管内皮细胞中引发广泛的反应。小GTP酶Rac1参与内皮细胞中多种S1P调节的细胞反应，然而，S1P介导的Rac1激活所涉及的分子机制尚未完全清楚。我们研究了牛主动脉内皮细胞（BAEC）中S1P介导的Rac1激活途径，发现通过转染βARKct螯合G蛋白βγ亚基后，S1P诱导的Rac1激活受到损害。用Src酪氨酸激酶抑制剂PP2处理可完全减弱S1P介导的Rac1激活；然而，用渥曼青霉素（一种磷酸肌醇（PI）3激酶抑制剂）预处理BAEC对Rac1激活没有影响，同时完全阻断S1P诱导的Akt磷酸化。我们使用Rac1特异性小干扰RNA（siRNA）双链体来“敲低”内源性Rac1表达，发现siRNA介导的Rac1敲低显著损害了蛋白激酶Akt的基础磷酸化以及S1P诱导的磷酸化，以及Akt的几个下游靶点，包括内皮型一氧化氮合酶和糖原合酶激酶3β。相比之下，S1P诱导的丝裂原活化蛋白激酶ERK1/2的磷酸化不受siRNA介导的Rac1敲低的影响。我们发现Rac1鸟嘌呤核苷酸交换因子（GEF）Tiam1的过表达显著增强了Rac1活性，而显性负性Tiam1突变体显著减弱了S1P介导的Rac1激活。综上所述，这些研究确定G蛋白βγ亚基、Src激酶和GEF Tiam1是S1P介导的Rac1激活的上游调节因子，并确立了Rac1在血管内皮细胞中S1P介导的PI 3激酶/Akt/内皮型一氧化氮合酶信号激活中的核心作用。
