Fer kinase is required for sustained p38 kinase activation and maximal chemotaxis of activated mast cells

Abstract

Mast cells play important roles in inflammation and immunity and express the high-affinity immunoglobulin E receptor (Fc epsilon RI) and the receptor protein-tyrosine kinase Kit. Aggregation of Fc epsilon RI via antigen binding elicits signals leading to the release of preformed inflammatory mediators as well as de novo-synthesized lipid mediators and cytokines and to elevated cell adhesion and migration. Here, we report that in mouse bone marrow-derived mast cells, Fer kinase is activated downstream of activated Fc epsilon RI and activated Kit receptor, and this activation is abolished in cells homozygous for a kinase-inactivating mutation in Fer (fer(DR/DR)). Interestingly, the highly related Fps/Fes kinase is also activated upon Fc epsilon RI aggregation. This report represents the first description of a common signaling pathway activating Fer and Fps/Fes. While Fer-deficient cells showed similar activation of the Erk mitogen-activated protein (MAP) kinases, p38 MAP kinase activation was less sustained than that in wild-type cells. Although no major defects were observed in degranulation, leukotriene biosynthesis, and cytokine secretion, Fer-deficient cells displayed increased adhesion and decreased motility upon activation of Fc epsilon RI and the Kit receptor. The restoration of Fer kinase activity in fer(DR/DR) mast cells resulted in prolonged p38 kinase activation and increased antigen-mediated cell migration of sensitized mast cells. Thus, Fer is required for maximal p38 kinase activation to promote the chemotaxis of activated mast cells. Further studies with mast cells derived from fps/fes-deficient mice will be required to provide insight into the role of Fps/Fes in mast cell activation.

FIG. 1.

Maturation of BMMC cultures. Bone marrow cells from wild-type or fer^DR/DR mice were grown in the presence of IL-3 and maintained as BMMC cultures as described in Materials and Methods. (A) Surface expression of FcɛRI was assessed by flow cytometry by preincubating BMMCs with α-DNP-IgE, followed by staining with FITC-conjugated α-IgE or isotype control antibodies. (B) Surface expression of Kit was analyzed by flow cytometry after staining of BMMCs with FITC-conjugated α-Kit or isotype control antibodies. (C) BMMC morphology was assessed after cytospinning and staining with DiffQuik (Baxter) and was captured by using an RT Color SPOT camera (Diagnostics Instruments Inc.) with an inverted microscope (Leitz Labovert FS; ≈300-fold magnification).

FIG. 2.

Increased phosphorylation of Fer and Fps kinases upon aggregation of FcɛRI. BMMCs were starved, sensitized with α-DNP-IgE, and challenged or not challenged with DNP-HSA (100 ng/ml) for various times. (A) SCLs were immunoblotted with α-pY and α-Fer antibodies. Proteins displaying reduced phosphorylation in Fer-deficient BMMCs are marked with an arrowhead on the right. (B) Lysates were subjected to immunoprecipitation (IP) with α-Fer serum and immunoblotted with both α-pY and α-Fer antibodies. (C) Lysates were subjected to immunoprecipitation with α-Fps/Fer serum and immunoblotted with both α-pY and α-Fps/Fer antibodies. The positions of p94 Fer and p92 Fps are indicated by arrows on the left. The relative positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 3.

Increased Fer phosphorylation downstream of the activated Kit receptor. BMMCs were starved and challenged or not challenged with SCF (10 ng/ml) for the indicated times. (A) Lysates were prepared and immunoblotted with α-pY and α-Fer antibodies. (B) Immunoprecipitation was carried out with α-Fer serum, and immunoblotting was done with α-pY and α-Fer antibodies. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 4.

Fer kinase is required for maximal p38 MAP kinase activation downstream of the activated Kit receptor. BMMCs were starved and challenged or not challenged with SCF (10 ng/ml) for the indicated times. SCLs were immunoblotted with α-pY, α-phosphorylated Erk1/Erk2 (α-pErk), α-Erk1/Erk2 (α-Erk), α-pp38, and α-p38 antibodies. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 5.

Fer kinase is required for sustained FcɛRI signaling to p38 MAP kinase. BMMCs were starved, sensitized with α-DNP-IgE, and challenged or not challenged with DNP-HSA (100 ng/ml) for the indicated times. SCLs were prepared and subjected to immunoblotting with α-pY, α-pErk, α-pp38, and α-pMkk3/6 antibodies as well as control antibodies to Erk, p38, and Mkk3. The position of a 70-kDa protein that is hypophosphorylated in fer^DR/DR cells is indicated by an arrow on the right. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 6.

Fer kinase is not required for degranulation and secretion of leukotrienes and cytokines. (A) BMMCs (5 × 10⁵/condition) were starved, sensitized with α-DNP-IgE, and challenged or not challenged with DNP-HSA (100 ng/ml) or with the calcium ionophore A23187 (0.5 μM) for 30 min. BMMCs were pelleted, and the amounts of β-hexosaminidase in the supernatant and in the solubilized pellet were measured. Percent degranulation was calculcated as (released activity/total activity) × 100 for three separate experiments with samples measured in triplicate. Error bars indicate standard deviations. (B) An ELISA was used to measure LTC₄ levels in cell-free supernatants from BMMCs (10⁷) that were starved, sensitized with α-DNP-IgE, and stimulated with DNP-HSA (100 ng/ml) for 2 h. Values given are means and standard deviations for three separate experiments. (C) An ELISA was used to measure the secretion of TNF-α in cell-free supernatants from BMMCs (10⁷) that were starved, sensitized with α-DNP-IgE, and stimulated with DNP-HSA (100 ng/ml) for 2 h. Values given are means and standard deviations for three separate experiments.

FIG. 7.

Elevated cell adhesion and reduced chemotaxis in Fer-deficient BMMCs. (A) BMMCs were starved, sensitized with α-DNP-IgE, and labeled with a fluorescent cell-permeating stain (Cell Tracker Green). Cells were then stimulated with DNP-HSA (100 ng/ml) or SCF (20 ng/ml) and placed in triplicate fibronectin-coated wells of a 96-well plate for 30 min. Nonadherent cells were removed, wells were washed, and bound fluorescence was measured by fluorimetry. Values given are relative fluorescence units (RFU) and standard errors of the mean for triplicate samples. (B) BMMCs were starved, sensitized with α-DNP-IgE, and placed in the upper well of a Transwell chamber (8-μm-pore size). Lower wells contained medium with or without DNP-HSA (100 ng/ml) or SCF (20 ng/ml). Cells were incubated at 37°C for 4 h, and cells that migrated were counted by using a Coulter Counter. Values given are means and standard deviations for triplicate samples.

FIG. 8.

Rescue of p38 MAP kinase activation and chemotaxis of fer^DR/DR BMMCs. (A) Schematic representation of the parental retroviral vector (control) and a retroviral construct designed to express a catalytically active Myc-epitope-tagged Fer kinase (rescue). LTR, long terminal repeat. (B) fer^DR/DR BMMCs were infected with control and rescue viruses, and selection was performed with puromycin as described in Materials and Methods. BMMCs were sensitized withα-DNP-IgE and stimulated with DNP-HSA (100 ng/ml) for the indicated times. SCLs were immunoblotted with α-pY, α-Fer, α-pp38, α-p38, α-pMkk3/6, and α-Mkk3 antibodies. The positions of several hyperphosphorylated proteins in the rescue cells are indicated by arrows on the right. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (C) The migration of DNP-sensitized control and rescue BMMCs was measured in the absence and presence of DNP-HSA (100 ng/ml) in the lower wells of a Transwell chamber. Mean values for cells present in the lower wells after 4 h of incubation at 37°C were plotted for quadriplicate samples. Error bars indicate standard deviations.

FIG. 9.

Model for Fer and Fps kinase signaling in mast cells. Dimeric FcɛRI is depicted with bound IgE and antigen, which triggers Lyn activation and the recruitment and activation of Syk. These events may precede Fer and Fps activation. Targets of Fps in mast cells remain unknown. Fer plays an amplifying role in FcɛRI-induced p38 MAP kinase activation and chemotaxis, likely acting upstream of Mkk3 and Mkk6. Binding of dimeric SCF to the Kit receptor results in dimerization and activation by transphosphorylation, leading to recruitment and activation of PI3-K and Src family kinase (SFK). SFK and PI3-K activation plays a role in Kit signaling to p38 and chemotaxis. Fer activation may occur downstream of SFK, based on previous results obtained with other cell types. In addition to promoting chemotaxis, p38 MAP kinase regulates transcription and mRNA translation.