SWAP-70 regulates c-kit-induced mast cell activation, cell-cell adhesion, and migration

Abstract

SWAP-70, an unusual phosphatidylinositol-3-kinase-dependent protein that interacts with the RhoGTPase Rac, is highly expressed in mast cells. Cultured bone marrow mast cells (BMMC) from SWAP-70(-/-) mice are reduced in FcepsilonRI-triggered degranulation. This report describes the hitherto-unknown role of SWAP-70 in c-kit receptor signaling, a key proliferation and differentiation pathway in mast cells. Consistent with the role of Rac in cell motility and regulation of the actin cytoskeleton, mutant cells show abnormal actin rearrangements and are deficient in migration in vitro and in vivo. SWAP-70(-/-) BMMC are impaired in calcium flux, in proper translocation and activity of Akt kinase (required for mast cell activation and survival), and in translocation of Rac1 and Rac2 upon c-kit stimulation. Adhesion to fibronectin is reduced, but homotypic cell association induced through c-kit is strongly increased in SWAP-70(-/-) BMMC. Homotypic association requires extracellular Ca(2+) and depends on the integrin alpha(L)beta(2) (LFA-1). ERK is hyperactivated upon c-kit signaling in adherent and dispersed mutant cells. Together, we suggest that SWAP-70 is an important regulator of specific effector pathways in c-kit signaling, including mast cell activation, migration, and cell adhesion.

FIG. 1.

SWAP-70^−/− BMMC are impaired in migration. (A) Transwell assay. Migration of wild-type or mutant BMMC toward the indicated concentrations of SCF, placed in the lower chamber, was assessed by cell counting after 4 h of incubation. The percentage of cells observed in the lower chamber is indicated. (B to D) In vivo migration of BMMC. Wild-type or mutant cells labeled with Cell Tracker Green were injected into the tail vein of 129SvEMS mice (B and C) or of wild-type or W/W-v mice (D). The percentage of green cells found after 24 h in the peritoneum (B and D) or in the blood (C) is shown. The background is given as “Control.”

FIG. 2.

Altered F-actin dynamics in SWAP-70^−/− BMMC. (A) Total F-actin content was measured by FACS after TRITC-phalloidin staining of the cells. Unstained cells served as controls, and measurements were taken immediately before and at the indicated time points after SCF (6 ng/ml) stimulation. The indicated gate shows the percentage of cells within the F-actin-positive (high) population. (B) Staining of BMMC by TRITC-phalloidin and imaging by fluorescence microscopy revealed filopodia-like protrusions on mutant BMMC, which are best visible at the 5- and 10-min time points. +/+, wild-type BMMC; −/−, SWAP-70^−/− BMMC.

FIG. 3.

Impaired Rac activation in SWAP-70^−/− BMMC. (A) Total amounts of Rac1 or Rac2 in wild-type or mutant BMMC. GST-tagged Rac1 or Rac2 was loaded for controls. (B) Translocation of Rac1 or Rac2 into the detergent-resistant cytoskeletal fraction. The cytoskeletal fraction was analyzed by immunoblotting and probing with antibodies specific for either Rac protein. Actin probing served as the loading control (C) Immunofluorescence analysis of Rac translocation. Starved wild-type or SWAP-70^−/− cells were stimulated by SCF (6 ng/ml) for the indicated time periods and stained for actin by rhodamine-phalloidine and for either Rac1 or Rac2 through FITC-labeled secondary antibody. (D) Rac activation measured by a pulldown method that specifically isolates activated Rac, followed by immunoblotting and probing for either Rac1 or Rac2. Rac1-positive controls are very strong, burning out the signal. +/+, wild-type BMMC; −/−, SWAP-70^−/− BMMC.

FIG. 4.

Impaired Ca²⁺ flux in SWAP-70^−/− BMMC. (A) Ca²⁺ flux measured by Indo-1 binding upon SCF stimulation. SCF was added at the 50-s time point. (B) Indo-1 binding of Ca²⁺ triggered by ionomycin (2 μM). The wavelengths 405/485 reflect the ratio of bound to free Ca²⁺.

FIG. 5.

Activation and translocation of Akt in wild-type or SWAP-70^−/− BMMC. (A and B) Generation of Akt phosphorylated at Thr-308 (A) or at Ser-473 (B) at different time points after SCF stimulation, as assessed by immunoblotting of total cell lysates and probing with the respective antibodies. (C and D) Presence of Akt or P-Ser-Akt either in the soluble cytoplasmic fraction (C) or in the NP-40 solubilized membrane fraction (D). (E) Akt kinase activity assay with GSK3α as substrate. The lower image shows the substrate loading control. The kinase activity was quantified by the ImageQuant analysis software (Amersham Biotech) as indicated below the images.

FIG. 6.

Activation of ERK in wild-type or SWAP-70^−/− BMMC. Total cell lysates from wild-type or mutant BMMC, stimulated by SCF for the indicated times, were immunoblotted and probed with antibodies specific for phosphorylated ERK1/ERK2 or for total ERK1/ERK2 as indicated. Probing for actin served as loading control. Cells were starved in 2% FCS (A) or cells were kept in 2% FCS with IL-3 for 16 h before the experiment (B). (C) ERK kinase activity assay with Elk-1 as substrate and P-ERK pulled down from BMMC lysates (starved without FCS). The lower panel shows for control the input substrate (Elk-1-GST) and the amount of anti-P-ERK antibody used. The kinase activity was quantified by the ImageQuant analysis software as indicated below the images.

FIG. 7.

Increased homotypic association of SWAP-70^−/− BMMC. (A) Inhibition of homotypic association induced by 6 ng of SCF/ml (S) by either 5 mM EGTA (E) or by 5 μg of anti-LFA-1 (α_Lβ₂) antibody (αL)/ml. (B) Inhibition of MEK kinase activity by PD98059 (100 μM) or EGTA (5 mM). BMMC, kept at 5% FCS with IL-3, were stimulated with 6 ng of SCF/ml for 2 h, and lysates were prepared and analyzed by immunoblotting and probing with either anti-P-ERK or anti-ERK antibody. +/+, wild-type BMMC; −/−, SWAP-70^−/− BMMC. (C) Adhesion to fibronectin of wild-type (open symbols) or mutant cells (solid symbols) in the presence or absence of SCF, measured at different time points as indicated. (D) ERK phosphorylation of cells (grown in 2% FCS) adherent to fibronectin after 1 h in the presence or absence of SCF. For control nonadherent cells, unstimulated or stimulated are also shown.