Chan Jasmine H P, Liao Wupeng, Lau H Y Alaster, Wong W S Fred
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Int Immunopharmacol. 2007 Jul;7(7):937-44. doi: 10.1016/j.intimp.2007.03.002. Epub 2007 Apr 2.
Adapter molecule Grb2-associated binder-like protein 2 (Gab2) plays a critical role in FcepsilonRI-induced mast cell degranulation and activation. The present study aimed to investigate the pharmacological effects of an antisense oligonucleotide (ASO) targeted at Gab2 on the immune responses of rat basophilic leukemic (RBL)-2H3 cells. Gab2 ASOs were rationally designed and transfected into RBL-2H3 cells. Gab2 mRNA and protein knockdown was confirmed by real-time RT-PCR and immunoblotting, respectively. Effects of Gab2 ASO on FcepsilonRI-induced release of histamine and beta-hexosaminidase was measured by EIA and an enzymatic assay, respectively; signaling events by immunoblotting; and cytokine mRNA expression by RT-PCR. Effects of Gab2 ASO on cell adhesion and migration were performed on fibronectin-coated 96-well plate and transwells cell culture chambers, respectively. We have characterized a phosphorothioate-modified ASO targeted at Gab2 mRNA that was able to knockdown Gab2 mRNA and protein in RBL-2H3 cells. Gab2 ASO significantly blocked IgE-mediated mast cell release of beta-hexosaminidase and histamine; phosphorylation of Akt, p38 mitogen-activated protein kinase and PKCdelta; and up-regulation of cytokine mRNA levels (e.g. IL-4, -6, -9 and -13, and TNF-alpha). In addition, Gab2 ASO markedly prevented mast cell adhesion to fibronectin-coated plates and restrained random migration of RBL-2H3 cells in cell culture chambers. Our findings show that Gab2 knockdown in RBL-2H3 cells by ASO strategy can suppress many aspects of the mast cell functions and, therefore, a selective Gab2 ASO may have therapeutic potential for mast cell-dependent allergic disorders.
衔接分子Grb2相关结合样蛋白2（Gab2）在FcepsilonRI诱导的肥大细胞脱颗粒和激活过程中起关键作用。本研究旨在探讨靶向Gab2的反义寡核苷酸（ASO）对大鼠嗜碱性白血病（RBL）-2H3细胞免疫反应的药理作用。合理设计Gab2 ASO并将其转染到RBL-2H3细胞中。分别通过实时RT-PCR和免疫印迹法确认Gab2 mRNA和蛋白表达的降低。通过酶免疫测定法（EIA）和酶促测定法分别检测Gab2 ASO对FcepsilonRI诱导的组胺和β-己糖胺酶释放的影响；通过免疫印迹法检测信号转导事件；通过RT-PCR检测细胞因子mRNA表达。分别在纤连蛋白包被的96孔板和Transwell细胞培养小室中检测Gab2 ASO对细胞黏附和迁移的影响。我们鉴定了一种靶向Gab2 mRNA的硫代磷酸酯修饰的ASO，它能够降低RBL-2H3细胞中Gab2 mRNA和蛋白的表达。Gab2 ASO显著阻断IgE介导的肥大细胞β-己糖胺酶和组胺的释放；Akt、p38丝裂原活化蛋白激酶和PKCdelta的磷酸化；以及细胞因子mRNA水平（如IL-4、-6、-9和-13以及TNF-α）的上调。此外，Gab2 ASO显著抑制肥大细胞与纤连蛋白包被板的黏附，并抑制RBL-2H3细胞在细胞培养小室中的随机迁移。我们的研究结果表明，通过ASO策略敲低RBL-2H3细胞中的Gab2可以抑制肥大细胞功能的多个方面，因此，选择性Gab2 ASO可能对肥大细胞依赖性过敏性疾病具有治疗潜力。
