Ji Young Kim1, Dae Yong Kim, Jai Youl Ro. 1. Department of Pharmacology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea.
Abstract
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-Grunwald-Giemsa staining, TNF-alpha 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-alpha increased in both mast cells. This TNF-alpha 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-alpha 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-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway. Copyright 2008 S. Karger AG, Basel.
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-Grunwald-Giemsa staining, TNF-alpha 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-alpha increased in both mast cells. This TNF-alpha 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-alpha 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-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway. Copyright 2008 S. Karger AG, Basel.
Authors: Eva Bernhart; Manfred Kollroser; Gerald Rechberger; Helga Reicher; Akos Heinemann; Petra Schratl; Seth Hallström; Andrea Wintersperger; Christoph Nusshold; Trevor DeVaney; Klaus Zorn-Pauly; Roland Malli; Wolfgang Graier; Ernst Malle; Wolfgang Sattler Journal: Proteomics Date: 2010-01 Impact factor: 3.984
Authors: Gianni García-Faroldi; Carlos E Rodríguez; José L Urdiales; José M Pérez-Pomares; José C Dávila; Gunnar Pejler; Francisca Sánchez-Jiménez; Ignacio Fajardo Journal: PLoS One Date: 2010-11-30 Impact factor: 3.240