Involvement of Fyn kinase in Kit and integrin-mediated Rac activation, cytoskeletal reorganization, and chemotaxis of mast cells - PubMed (original) (raw)

Comparative Study

Involvement of Fyn kinase in Kit and integrin-mediated Rac activation, cytoskeletal reorganization, and chemotaxis of mast cells

Lionel A Samayawardhena et al. Blood. 2007.

Abstract

Kit receptor and its ligand stem cell factor (SCF) are critical regulators of mast cell production, proliferation, degranulation, and chemotaxis. In this study, we investigated how Fyn kinase regulates chemotaxis of mast cells toward SCF. On beta1-integrin engagement, Fyn-deficient (fyn(-/-)) mast cells displayed a striking defect in cell spreading and lamellipodia formation compared to wild-type mast cells. The hematopoietic-specific Src family kinases (Lyn/Fgr/Hck) were not required for initial SCF-induced cell spreading. Reduced SCF-induced activation of Rac1 and Rac2 GTPases, p38 mitogen-activated protein kinase, and filamentous actin polymerization was observed in fyn(-/-) mast cells compared to wild-type mast cells. Retroviral-mediated expression of Fyn, constitutively active forms of Rac2 or phosphatidylinositol 3-kinase (PI3K) in fyn(-/-) mast cells rescued defects in SCF-induced cell polarization and chemotaxis of Fyn-deficient mast cells. Thus, we conclude that Fyn kinase plays a unique role upstream of PI3K and Rac GTPases to promote the reorganization of the cytoskeleton during mast cell spreading and chemotaxis.

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Figures

Figure 1

Defective SCF-induced cell spreading of _fyn_−/− BMMCs on fibronectin. (A) fyn+/+, _fyn_−/− BMMCs, and _fyn_−/− BMMCs transduced with MSCV-Fyn were plated on fibronectin in the presence of SCF (20 ng/mL) for 15 or 45 minutes prior to fixation and F-actin staining as described in “Materials and methods.” Representative images obtained by fluorescence microscopy are shown. (B) Cell spreading and lamellipodia formation of SCF-treated BMMCs (20 ng/mL for 45 minutes) plated on fibronectin-coated wells were analyzed by light microscopy. Significant differences in cell spreading and lamellipodia formation in _fyn_−/− BMMCs (indicated by an asterisk, P < .01, n > 100; error bars indicate standard deviations [SD]) with those of fyn+/+ BMMCs. (C) _fyn_−/− BMMCs showed significantly lower total cell spread area compared to wild-type (indicated by an asterisk; P < .01, n = 50/genotype; mean ± SD), as quantified using Image Pro Plus software (Media Cybernetics, Silver Spring, MD).

Figure 2

SCF-induced cell spreading of BMMCs on fibronectin is independent of Lyn/Fgr/Hck kinases. (A) BMMCs from lyn+/+, lyn_−/−, lyn/fgr/hck_−/− mice were plated on fibronectin-coated coverslips in the presence of SCF (20 ng/mL) for 15 or 45 minutes. Cells were fixed, permeabilized, and stained with TRITC-phalloidin. Representative fluorescence microscopy images are shown. (B) Cell spreading and lamellipodia formation of SCF-treated BMMCs (20 ng/mL for 45 minutes; mean ± SD; n = 100) plated on fibronectin-coated wells were analyzed by light microscopy.

Figure 3

SCF-induced activation of Rac1 and Rac2 GTPases and p38 MAPK are coordinated through Fyn kinase. (A) fyn+/+ and _fyn_−/− BMMCs were activated with SCF (50 ng/mL) for 0, 5, 15, or 30 minutes. CRIB pull-down assays were used to detect activated Rac GTPases (RacGTP), as described in “Materials and methods.” SCLs and pull-down samples were immunoblotted (IB) with anti-Rac1 and anti-Rac2 antibodies. In all experiments, 10% of input was used for loading of GTPases with excess GDP or GTPγS to serve as controls. (B) _fyn_−/− BMMCs transduced with MSCV or MSCV-Fyn were analyzed as described. (C) fyn+/+ and _fyn_−/− BMMCs were plated on fibronectin-coated wells and treated with SCF (50 ng/mL) for 0, 5, or 15 minutes. SCLs were prepared and IB with: antiphosphotyrosine (pY), antiphospho-Akt (pAkt), anti-Akt, antiphospho-p38 (pp38), or anti-p38. Arrow at right indicates a phosphoprotein that appears to be elevated in _fyn_−/− BMMCs.

Figure 4

Involvement of Fyn, PI3K, and Rac2 in F-actin remodeling in response to SCF-induced integrin engagement. fyn+/+, _fyn_−/−, and _fyn_−/− transduced with either MSCV-Fyn, MSCV-Rac2V12, or MSCV-p110CAAX BMMCs were plated on glass coverslips, previously coated with fibronectin. Cells were then activated with SCF for 1 minute at 37°C, prior to fixation and Rac2 and F-actin staining, as described in “Materials and methods.” Images were acquired by confocal microscopy, and representative images are shown.

Figure 5

Effects of constitutively active PI3K and Rac2 on SCF-induced Rac activation. (A) _fyn_−/−MSCV and _fyn_−/−MSCV-Rac2V12 BMMCs were activated with SCF for 0 or 5 minutes. CRIB pull-down assays were carried out to detect Rac2GTP (endogenous and Myc-tagged) and total Rac2 in SCLs. Position of Myc-Rac2V12 is indicated by an open arrow on the left. (B) fyn+/+ and _fyn_−/− transduced with either MSCV or MSCV-p110CAAX BMMCs were subjected to CRIB pull-down assays as described. Position of Rac2 is indicated on the left with an arrow.

Figure 6

Constitutively active PI3K or Rac2 can compensate for Fyn kinase during SCF-induced polarization and chemotaxis of mast cells. (A) Status of F-actin and microtubule reorganization during chemotaxis of SCF-treated (20 ng/mL) BMMCs (fyn+/+ and _fyn_−/− transduced with either MSCV or MSCV-Rac2V12) plated on fibronectin-coated coverslips. Representative confocal images for tubulin immunostaining/TRITC-phalloidin staining are shown. (B) SCF-treated (20 ng/mL) BMMCs from fyn+/+ and _fyn_−/− transduced with either MSCV or MSCV-Rac2V12 were plated on fibronectin-coated tissue culture plates for 45 minutes. The percentages of polarized cells are depicted for 3 separate fields of view (n = 50; mean ± SD). Fyn-deficient cells displayed a significant decrease in polarization (P < .05) that was rescued by Rac2V12 expression (P < .01). (C) BMMCs from fyn+/+ and _fyn_−/− transduced with either MSCV, MSCV-Rac2V12, or MSCV-p110CAAX BMMCs were subjected to Transwell chemotaxis assays as described in “Materials and methods.” Numbers of BMMCs that had migrated to the bottom chamber were counted. Migration of BMMCs from the various genotypes was compared to numbers of fyn+/+ BMMCs that had migrated (set at 100%). Results from 3 independent experiments are shown (mean ± SD). Asterisk indicates a significant difference (P < .05) between _fyn_−/− and fyn+/+ and _fyn_−/− transduced with MSCV-Rac2V12 or MSCV-p110CAAX BMMCs.

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Involvement of Fyn kinase in Kit and integrin-mediated Rac activation, cytoskeletal reorganization, and chemotaxis of mast cells - PubMed (original) (raw)