Small interfering RNA screen for phosphatases involved in IgE-mediated mast cell degranulation.
Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phosphatases 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 FcepsilonRI-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 Cmu and protein kinase Cdelta, which are involved in FcepsilonRI signaling. The screen, therefore, has identified several new molecules that are critical for FcepsilonRI-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.
Zhang
Juan
J
Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Mendoza
Mary
M
Guiraldelli
Michel F
MF
Barbu
Emilia Alina
EA
Siraganian
Reuben P
RP
D000818:Animals; D015550:Cell Degranulation / Q000502:physiology*; D002460:Cell Line; D015723:Gene Library; D005821:Genetic Techniques; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism*; D008407:Mast Cells / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D034741:RNA, Small Interfering; D007073:Immunoglobulin E; D010744:Phosphoric Monoester Hydrolases