The limited contribution of Fyn and Gab2 to the high affinity IgE receptor signaling in mast cells

Abstract

Several studies with mast cells from knock-out mice have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation. To better understand the role of these two molecules and of Syk, we transiently transfected mast cells with small interference RNA (siRNA) targeted to Fyn, Gab2, or Syk to specifically decrease their expression. The siRNA suppression of Gab2 but not Fyn reduced activation of the phosphoinositide-3-kinase (PI3K) pathway as demonstrated by the change in phosphorylation of Akt; this indicates that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 and Fyn had minor effects on degranulation. There were also some minor changes in activation of the NFAT or NFkappaB transcription factors in cells with reduced expression of Fyn or Gab2. Decreased Gab2 but not Fyn reduced the FcepsilonRI-induced activation of the Erk, Jnk, and p38 MAP kinases and the release of TNF-alpha. In contrast, decreased expression of Syk dramatically reduced FcepsilonRI-induced degranulation, activation of NFAT and NFkappaB. Therefore, the reduction in expression of these proteins in mast cells indicates that Syk is the major regulator of FcepsilonRI-mediated reactions, whereas Fyn has minor if any effects and Gab2 regulates primarily late events including MAP kinase activation and release of cytokines.

FIGURE 1.

Decreased expression of Syk but not Fyn or Gab2 reduces FcϵRI-induced degranulation. A, MMC-1 cells were treated with siRNA specific for Syk, Fyn, or Gab2 (indicated by T) or with siCONTROL (indicated as C) and expression of the target proteins was determined by immunoblotting 24, 48, and 72 h after transfection. The fraction of the protein left in the transfected cells compared with the controls was determined by densitometry after normalization for gel loading from blots probed with anti-FcϵRIβ. One representative experiment is shown. B, summary of the change in the expression level of these proteins; results are mean ± S.D., n = 6. C, antigen-induced β-hexosaminidase release in transfected MMC-1 cells. Degranulation was determined at the indicated times in IgE-sensitized cells. Percent of total β-hexosaminidase release in control transfected cells was 39% ± 3% at 24 h; 33% ± 6% at 48 h; 28% ± 5% at 72 h. Results are mean ± S.D. (n = 6). D, antigen dose response of β-hexosaminidase release at 48 h after transfection. IgE-primed MMC-1 cells were stimulated with the indicated concentrations of antigen. β-Hexosaminidase release is presented as percent of total cellular enzyme content (mean ± S.D.; n = 6). The release curves with Syk and Gab2 siRNA were significantly different than the control (p ≤ 0.001 paired two-sided Student's t test).

FIGURE 2.

Decreased expression of Fyn or Gab2 has minor effects on FcϵRI-induced activation of NFkB and NFAT. A, MC9 NFκB-GFP cells or C, MC9 NFAT-GFP cells were treated with siRNA specific for Syk, Fyn, or Gab2 (indicated by T) or with siCONTROL (indicated by C) and expression of the target proteins was determined by Western blotting at the indicated times. The fraction of the protein left in the transfected cells compared with the controls as determined by densitometry is indicated. One representative experiment is shown. B, activity of NFκB (n = 9) and D, the activity of NFAT (n = 10) was determined by FACS analysis at the indicated times after transfection and are expressed as percentage of the control (dotted line). Bars indicate S.D.

FIGURE 3.

Gab2 but not Fyn regulated IgE-dependent Akt phosphorylation. A, Gab2 regulates Akt phosphorylation. MC9 cells were transfected, starved of SCF for 24 h and then stimulated with antigen for the indicated times. Lysates were analyzed by immunoblotting sequentially with antibodies for phospho-Akt Ser-473 (anti-pAkt) and Akt. B, changes in phospho-Akt are expressed as percent of the control (dotted line) in cells transfected with the indicated siRNA. Results are mean ± S.D. (n = 4). C, Fyn-independent phosphorylation of Gab2. MC9 cells were treated with Fyn-specific siRNA or siCONTROL then stimulated with antigen at 48 h after transfection. The resolved proteins were immunoblotted with antibodies for Gab2 phospho-Ser 159, Gab2 phospho-Tyr 452, and Gab2. One representative experiment out of three is shown.

FIGURE 4.

Gab2 but not Fyn regulated IgE-induced MAPK phosphorylation. IgE-primed MC9 cells were stimulated with antigen for the indicated times 48 h after the siRNA treatment. Resolved proteins were blotted as follows: A, with antibodies for phospho-Erk (Thr-202/Tyr-204) and Erk; C, with antibodies for phospho-p38 (Thr-180/Tyr-182) and p-38; E, with antibodies for phospho-JNK (Thr-183/Tyr-185) and JNK. Percent decrease in the phosphorylation levels of Erk, n = 4 (B), or p38, n = 4 (D), and JNK, n = 2 (F) is expressed as percentage of the control (dotted line). Bars indicate S.D.

FIGURE 5.

TNF-α secretion was inhibited by reduced expression of Gab2 but not Fyn. MMC-1 (A) or MC9 cells (C) were transfected with siRNA specific for Fyn or Gab2 or with siCONTROL. After 48 h, cells were stimulated with antigen for 3 h. Amount of released TNF-α is expressed as percent of the release in the control cells (TNF-α release in control treated cells was 1394 ± 182 pg/ml in MMC-1 and 170 ± 76 pg/ml in MC9). Each cell line was tested twice. Representative immunoblots for MMC-1 (B) and MC9 (D) are shown.

FIGURE 6.

IgE-mediated degranulation in BMMC with reduced expression of Syk, Fyn, or Gab2. A, efficient siRNA-induced decrease in expression of Syk, Fyn, and Gab2 in BMMC. Knockdown levels were determined at the indicated times by Western blotting. The protein left in the transfected cells compared with the controls was calculated by densitometry after normalization for gel loading and expressed as percent of controls. Results are mean ± S.E.; for Syk n = 4 (24 and 48 h); for Fyn n = 12 (24 h), n = 18 (48 h); for Gab2 n = 10 (24 h), n = 18 (48 h). B, a representative immunoblot showing decreased expression of target proteins 48 h after transfection. C, Ag-induced β-hexosaminidase release in transfected BMMC. IgE-primed BMMC were stimulated for 45 min with antigen. Percent of total β-hexosaminidase release in control transfected cells was 18% ± 6% at 24 h and 19% ± 7% at 48 h. Results are mean ± S.E.

FIGURE 7.

IgE-mediated release of cytokines in BMMC with reduced expression of Fyn or Gab2. BMMCs were transfected with siRNA specific for Fyn, Gab2, or with siCONTROL, then 48 h later IgE-primed cells were stimulated with antigen for 3 h. Amounts of released TNF-α (A, n = 8), IL-13 (B, n = 8), and CCL-4 (C, n = 7) was determined by ELISA.