Degranulation MastPhosphatases Involved in IgE - Mediated Small Interfering RNA Screen for

Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phos-phatases in IgE-mediated mast cell activation, a small interfering RNA (siRNA) library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Of 198 targets, 10 enhanced and 7 inhibited Fc«RI-induced degranulation. For seven of the strongest hits, four different siRNAs per target were tested, and at least two out of the four 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 MAPKs and the phosphorylation of protein kinase D/ protein kinase Cm and protein kinase Cd, which are involved in Fc«RI signaling. The screen, therefore, has identified several new molecules that are critical for Fc«RI-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. M ast cells play an important role in allergic reactions and IgE-associated immune responses. Aggregation of FcεRI on these cells initiates a biochemical cascade, which eventually results in mast cell functional responses, such as de-granulation and release of inflammatory mediators (1–5). The activation pathway leading from FcεRI stimulation to degranulation is a complex one that involves many molecules, some of which are directly involved in the release of granules, whereas others regulate this process. The role of several molecules has been established by using cell lines that are deficient in one of these molecules. Such cells are usually derived from either in vitro selection of established cell lines or from genetically modified mice that lack those molecules (6–16). However, the development of small interfering RNA (siRNA) technology has made it possible to perform genetic screens in mammalian cell lines, although such screening has not been commonly applied to such complex pathways. Protein phosphorylation is one of the earliest detectable events after FcεRI aggregation and plays an essential role in this signal transduction pathway. The extent of FcεRI-induced phosphor-ylation is regulated by the balance between protein kinases and phosphatases. Significant advances have been made recently in understanding the functions …