Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability
Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability
复制标题
DOI：
10.1182/blood.v98.7.2014
复制
发表时间：
2001-10-01
期刊：
影响因子：
20.3
通讯作者：
Bertics, PJ
中科院分区：
文献类型：
--
作者：
Hall, DJ;Cui, J;Bertics, PJ
通过暴露于白细胞介素-5（IL-5）抑制嗜酸性粒细胞凋亡与组织嗜酸性粒细胞的发展有关，并可能导致哮喘的炎症特征。与此过程相关的信号事件的分析已经受到阻碍，不能有效地操纵嗜酸性粒细胞通过引入活性或抑制性效应分子。使用显性负性N17 H-Ras蛋白（dn-H-Ras）和MEK抑制剂U 0126提供的证据表明，Ras-Raf-MEK-ERK途径的激活在IL-5延长嗜酸性粒细胞存活中起决定性作用。对于这些研究，将人免疫缺陷病毒达特蛋白的一个小区域（已知有效进入哺乳动物细胞的蛋白转导结构域）融合到dn-H-Ras的N-末端。由该构建体产生的Tat-dn-H-Ras蛋白以超过95%的效率转导分离的人血嗜酸性粒细胞。当用IL-5处理Tat-dn-H-Ras转导的嗜酸性粒细胞时，它们表现出细胞外调节激酶1和2激活的时间和剂量依赖性降低，以及p90 Rsk 1磷酸化和IL-5介导的嗜酸性粒细胞存活的抑制。相反，Tat-dn-H-Ras不抑制CD 11b上调或STAT 5酪氨酸磷酸化。这些数据表明，达特显性负性蛋白转导可以作为一个重要的和新的工具，在研究初级髓细胞的信号转导在原代白细胞，并可以牵连的Ras-Raf-MEK-ERK途径在IL-5启动的嗜酸性粒细胞的生存。(C)2001年，美国血液学会。
Inhibition of eosinophil apoptosis by exposure to interleukin-5 (IL-5) is associated with the development of tissue eosinophilla and may contribute to the inflammation characteristic of asthma. Analysis of the signaling events associated with this process has been hampered by the inability to efficiently manipulate eosinophils by the introduction of active or inhibitory effector molecules. Evidence is provided, using a dominant-negative N17 H-Ras protein (dn-H-Ras) and MEK inhibitor U0126, that activation of the Ras-Raf-MEK-ERK pathway plays a determining role In the prolongation of eosinophil survival by IL-5. For these studies, a small region of the human immunodeficiency virus Tat protein, a protein transduction domain known to enter mammalian cells efficiently, was fused to the N-terminus of dn-H-Ras. The Tat-dn-H-Ras protein generated from this construct transduced isolated human blood eosinophils at more than 95% efficiency. When Tat-dn-H-Ras-transduced eosinophils were treated with IL-5, they exhibited a time- and dosage-dependent reduction in extracellular regulated kinase 1 and 2 activation and an Inhibition of p90 Rsk1 phosphorylation and IL-5-mediated eosinophil survival in vitro. In contrast, Tat-dn-H-Ras did not inhibit CD11b upregulation or STAT5 tyrosine phosphorylation. These data demonstrate that Tat dominant-negative protein transduction can serve as an important and novel tool in studying primary myeloid cell signal transduction in primary leukocytes and can implicate the Ras-Raf-MEK-ERK pathway in IL-5-initiated eosinophil survival. (C) 2001 by The American Society of Hematology.