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. 2014 May 15;192(10):4859-66.
doi: 10.4049/jimmunol.1301155. Epub 2014 Apr 14.

SHP2 Phosphatase Promotes Mast Cell Chemotaxis Toward Stem Cell Factor via Enhancing Activation of the Lyn/Vav/Rac Signaling Axis

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Free PMC article

SHP2 Phosphatase Promotes Mast Cell Chemotaxis Toward Stem Cell Factor via Enhancing Activation of the Lyn/Vav/Rac Signaling Axis

Namit Sharma et al. J Immunol. .
Free PMC article

Abstract

SHP2 protein-tyrosine phosphatase (encoded by Ptpn11) positively regulates KIT (CD117) signaling in mast cells and is required for mast cell survival and homeostasis in mice. In this study, we uncover a role of SHP2 in promoting chemotaxis of mast cells toward stem cell factor (SCF), the ligand for KIT receptor. Using an inducible SHP2 knockout (KO) bone marrow-derived mast cell (BMMC) model, we observed defects in SCF-induced cell spreading, polarization, and chemotaxis. To address the mechanisms involved, we tested whether SHP2 promotes activation of Lyn kinase that was previously shown to promote mast cell chemotaxis. In SHP2 KO BMMCs, SCF-induced phosphorylation of the inhibitory C-terminal residue (pY507) was elevated compared with control cells, and phosphorylation of activation loop (pY396) was diminished. Because Lyn also was detected by substrate trapping assays, these results are consistent with SHP2 activating Lyn directly by dephosphorylation of pY507. Further analyses revealed a SHP2- and Lyn-dependent pathway leading to phosphorylation of Vav1, Rac activation, and F-actin polymerization in SCF-treated BMMCs. Treatment of BMMCs with a SHP2 inhibitor also led to impaired chemotaxis, consistent with SHP2 promoting SCF-induced chemotaxis of mast cells via a phosphatase-dependent mechanism. Thus, SHP2 inhibitors may be useful to limit SCF/KIT-induced mast cell recruitment to inflamed tissues or the tumor microenvironment.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Defective cell spreading and polarization in SHP2 KO BMMCs treated with SCF. (A) BMMCs obtained from Shp2fl/fl and TgCreER:Shp2fl/fl were treated with 4-TM (200 nM) for 3 d to generate WT and KO BMMCs, respectively. (A) PCR analyses of Shp2 null and flox alleles for genomic DNA isolated from WT and KO BMMCs. Position of DNA size markers (kilobases) are shown on the left. (B) IB analysis for WT and KO BMMC lysates probed with SHP2 and ERK Abs. Positions of relative mass markers (kDa) are shown on the left. (C) WT and KO BMMCs were straved of IL-3 for 6 h and seeded on fibronectin-coated coverslips in the presence of SCF (25 ng/ml for 45 min). Cells were fixed, permeabilized, and stained with TRITC–phalloidin. Representative epiflourescence micrographs are shown for F-actin staining. (D) The graph depicts the percentage of cells that had spread for multiple fields for WT and KO BMMCs (n = 4 > 200 cells; mean ± SD; triplicate samples; *p < 0.05, significant difference between genotypes. (E) Graph depicts the percentage of cells that had polarized for multiple fields for WT and KO BMMCs (n = 4, >200 cells; mean ± SD; triplicate samples; *p < 0.05, significant difference between genotypes.
FIGURE 2
FIGURE 2
SHP2 promotes chemotaxis of BMMCs. (A) Transwell assays were performed to measure WT and KO BMMC chemotaxis toward SCF (25 ng/ml). The graph depicts the number of cells per field (mean ± SD, n = 3 replicates, triplicate samples; *p < 0.05, significant difference between genotypes). (B) Schematic representation of experimental design using live cell imaging of agarose drop assays. WT and KO BMMCs were stained with CellTracker Orange and Green, respectively, and mixed prior to subjecting to SCF-embedded agarose drop chemotaxis assay, as described in Materials and Methods. (C) Representative confocal micrographs showing the positions of WT and KO BMMCs at the indicated times (0–16 h) during the time lapse are shown (margin of agarose drop indicated by dashed white line). White and yellow arrows depict positions of WT and KO BMMCs, respectively, that had migrated under the agarose at 16 h. (D) Cells from multiple fields (n = 5, >30 cells/genotype) were tracked individually to calculate migration distances (micrometers). The graph depicts mean migration distance (± SD) for WT and KO BMMCs (*p < 0.05, significant difference between genotypes). (E) Graph depicts the average velocity (micrometer per minute) for the same set of WT and KO BMMCs described in (D) (*p < 0.05, significant difference between genotypes).
FIGURE 3
FIGURE 3
Lyn C-terminal inhibitory tyrosine residue (pY507) is a direct substrate of SHP2. (A) WT and KO BMMCs were starved of IL-3 for 6 h and stimulated with SCF (50 ng/ml) for indicated times. Lysates were subjected to IB analysis with pY507-Lyn, pYSrc 416 (cross-reacts with pY396 Lyn) and Lyn Abs. (B and C) Line graph depicting the relative phosphotyrosine levels of Lyn at tyrosines 507 and 396, respectively, at indicated time points (mean ± SD; n = 3; *p < 0.05, significant difference between genotypes). (D) Lysates were prepared from SCF-treated Baf/3-KIT SHP2 KD cells (50 ng/ml for 5 min) and incubated with either GST or GST-PTPD/A:Q/A bound to beads overnight at 4°C. Mock reactions were performed with including lysates. IB analyses were carried out with Abs indicated on the right.
FIGURE 4
FIGURE 4
SHP2 and Lyn promote phosphorylation of Vav1 downstream of KIT. (A) Lysates prepared from WT and KO BMMCs were subjected to IP with anti-Vav1 prior to IB analysis using anti-pY (PY99) and anti-Vav1. Positions of relative mass markers (kilodaltons) are shown on the left. (B) Graph depicting the relative total phosphotyrosine levels of Vav1 at indicated time points (mean ± SD; n = 4; *p < 0.05, significant difference between genotypes). (C) Lysates prepared from Lyn+/+ and Lyn−/− BMMCs were subjected to IB analysis with anti-Lyn and anti-actin. (D) Lysates prepared from Lyn+/+ and Lyn−/− BMMCs were subjected IP with anti-Vav1 prior to IB analysis using anti-pY (PY99) and anti-Vav1. Positions of relative mass markers (kilodaltons) are shown on the left.
FIGURE 5
FIGURE 5
Reduced plasma membrane RacGTP and F-actin polymerization in SHP2 KO BMMCs treated with SCF. (A) WT and KO BMMCs were starved of IL-3 for 6 h and plated on fibronectin-coated coverslips in presence of SCF (25 ng/ml for 45 min). Fixed and permeabilized cells were subjected to immunofluorescence staining with anti-active Rac1 (Rac1GTP conformation specific) and anti-Lyn. The TIRF micrograph depicting the ventral surface of the cells staining membrane fraction of active Rac1 and Lyn and the merge image of active Rac1 and Lyn channel. (B) Graph depicting the quantification analysis Mander’s coefficient to measure the degree of colocalization of active Rac1 and Lyn channels between WT and KO genotypes. (mean ± SD; n = 20; *p < 0.05, significant difference between genotypes). (C) Cytokine starved WT and KO BMMCs were stimulated with SCF (100 ng/ml) for indicated times, fixed, and permeablized prior to staining with Alexa Fluor 488–conjugated phalloidin. The graph depicts the percentage change in F-actin mean fluorescence intensity (mean ± SD; triplicate samples) for WT and KO BMMCs compared with 0 min (*p < 0.05, significant difference between genotypes).
FIGURE 6
FIGURE 6
SHP2 phosphatase activity promotes chemotaxis of BMMCs toward SCF. (A) BMMCs grown in IL-3 and SCF for 3 wk were pretreated with vehicle (DMSO) or different concentrations of SHP2 inhibitor (II-B08; 10 or 20 μM) for 1 h. Lysates were subjected to IB analysis with anti-pERK, anti–phospho-Lyn (pY507), ERK, and Lyn Abs. (B) BMMCs grown in IL-3 and SCF for 3 wk were pretreated with DMSO or different concentrations of II-B08 as above for 24 h. The graph depicting the percent cell viability determined with alamar blue assay as described in Materials and Methods (mean ± SD; triplicate samples). (C) Cytokine-starved BMMCs were pretreated for 1 h and maintained with either vehicle (DMSO) or different concentrations of SHP2 inhibitor (II-B08; 10 or 20 μM) prior to plating on fibronectin-coated coverslips containing SCF-embedded agarose drops. After 18 h, cells were fixed and stained with DAPI, and the images were acquired on an epifluorescence microscope. Representative micrographs are shown for DAPI+ cells at the margin of the agarose drop (dashed line) and underneath the agarose drop (below the dashed line). (D) The graph depicting the total number of BMMCs that had migrated under each SCF-embedded agarose drop (n = 3 replicates, triplicate samples; **p < 0.01, statistical significant difference between the genotype). (E) Simplified pathway model relating SHP2 to mast cell chemotaxis. SHP2 promotes Lyn activation downstream of KIT, leading to Vav1 phosphorylation, Rac activation, and F-actin branching and polymerization required for chemotaxis.

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