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-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.


CONCLUSIONS
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.
Kim
Ji Young
JY
Department of Pharmacology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea.
Kim
Dae Yong
DY
Ro
Jai Youl
JY
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017305:Annexin A1 / Q000096:biosynthesis* / Q000276:immunology; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects* / Q000276:immunology / Q000648:ultrastructure; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D026121:Indole Alkaloids / Q000494:pharmacology; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D009025:Morpholines / Q000494:pharmacology; D020932:Nerve Growth Factor / Q000276:immunology / Q000494:pharmacology*; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000096:biosynthesis* / Q000276:immunology; D034741:RNA, Small Interfering / Q000276:immunology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D000077191:Wortmannin
D000730:Androstadienes; D017305:Annexin A1; D002227:Carbazoles; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D026121:Indole Alkaloids; D009025:Morpholines; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D020932:Nerve Growth Factor; C049985:staurosporine aglycone; D011770:Pyruvate Kinase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin