Supplementary Material for: Granule Formation in NGF-Cultured Mast Cells Is Associated with Expressions of Pyruvate Kinase Type M2 and Annexin I Proteins

Background: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved. Methods: NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grünwald-Giemsa staining, TNF-α by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting. Results: After NGF treatment, granule formation and total amounts of granular mediator, TNF-α increased in both mast cells. This TNF-α was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-α content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin. Conclusion: These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-α as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway.