siRNA Screen for Phosphatases Involved in IgE-mediated Mast Cell Degranulation

摘要

Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding on the functions played by phosphatases in IgE-mediated mast cell activation, a siRNA library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Out of 198 targets, 10 enhanced and 7 inhibited high affinity IgE receptor (FcεRI) induced degranulation. For 7 of the strongest hits, four different siRNAs per target were tested, and at least 2 out of the 4 single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow derived mast cells from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAP kinases and the phosphorylation of PKD/PKCμ and PKCδ, which are involved in FcεRI signaling. The screen therefore, has identified several new molecules that are critical for FcεRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The result also indicates that the system used is efficient for searching molecules implicated in complex receptor-induced signaling.