Gab2 antisense oligonucleotide blocks rat basophilic leukemic cell functions.
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.
Chan
Jasmine H P
JH
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Liao
Wupeng
W
Lau
H Y Alaster
HY
Wong
W S Fred
WS
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001491:Basophils / Q000276:immunology*; D002448:Cell Adhesion / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D018390:Gene Targeting; D006636:Histamine Release / Q000235:genetics / Q000276:immunology; D015471:Leukemia, Basophilic, Acute / Q000188:drug therapy* / Q000276:immunology*; D008407:Mast Cells / Q000276:immunology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000037:antagonists & inhibitors / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000276:immunology
D048868:Adaptor Proteins, Signal Transducing; D016207:Cytokines; D005353:Fibronectins; C490893:Gab2 protein, rat; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D012333:RNA, Messenger; D017455:Receptors, IgE; D017346:Protein Serine-Threonine Kinases; D001619:beta-N-Acetylhexosaminidases