Inhibition of the Antigen-Induced Activation of RBL-2H3 Cells by Gab2 siRNA

Abstract

Gab2 plays an important role in FcepsilonRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 (scaffolding adapter Grb2-associated binder 2) siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2-mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl (DNP)-specific IgE overnight and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of beta-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine, reduced the production of IL-4 and TNF-alpha and inhibited the phosphorylation of Akt, PKCdelta and p38 mitogen-activated protein kinase (MAPK). Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the FcepsilonRI-mediated Ca(2+)-independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated.