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.
Subject(s)
DNA-Binding Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , Mast Cells/cytology , Nuclear Proteins/physiology , Proto-Oncogene Proteins c-kit/metabolism , Actins/metabolism , Animals , Calcium/metabolism , Cell Adhesion , Cell Differentiation , Cell Membrane/metabolism , Cell Movement , Cell Proliferation , Cell Survival , Cells, Cultured , Cytoplasm/metabolism , Cytoskeleton/metabolism , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibronectins/metabolism , Flavonoids/pharmacology , Glutathione Transferase/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Mice , Mice, Knockout , Minor Histocompatibility Antigens , Mutation , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction , Stem Cell Factor/metabolism , Subcellular Fractions/metabolism , Time Factors , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , RAC2 GTP-Binding ProteinABSTRACT
Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.
