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Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

ABSTRACT: Dectin-1 recognizes ?-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the ?-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-?. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by Fc?RI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity.

SUBMITTER: Kimura Y

PROVIDER: S-EPMC4223353 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

Kimura Yukihiro Y Chihara Kazuyasu K Honjoh Chisato C Takeuchi Kenji K Yamauchi Shota S Yoshiki Hatsumi H Fujieda Shigeharu S Sada Kiyonao K

The Journal of biological chemistry 20140922 45

Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1 ...[more]

PMID: 25246527

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Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.

Dataset Information

Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

Publications

Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

Similar Datasets

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