Deficiency in steroid receptor coactivator 3 enhances cytokine production in IgE-stimulated mast cells and passive systemic anaphylaxis in mice

Abstract

Background: Steroid receptor coactivator 3 (SRC-3) is a multifunctional protein that plays an important role in malignancy of several cancers and in regulation of bacterial LPS-induced inflammation. However, the involvement of SRC-3 in allergic response remains unclear. Herein we used passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models to assess the role of SRC-3 in allergic response.

Results: SRC-3-deficient mice exhibited more severe allergic response as demonstrated by a significant drop in body temperature and a delayed recovery period compared to wild-type mice in PSA mouse model, whereas no significant difference was observed between two kinds of mice in PCA mouse models. Mast cells play a pivotal role in IgE-mediated allergic response. Antigen-induced aggregation of IgE receptor (FcϵRI) on the surface of mast cell activates a cascade of signaling events leading to the degranulation and cytokine production in mast cells. SRC-3-deficient bone marrow derived mast cells (BMMCs) developed normally but secreted more proinflammatory cytokines such as TNF-α and IL-6 than wild-type cells after antigen stimulation, whereas there was no significant difference in degranulation between two kinds of mast cells. Further studies showed that SRC-3 inhibited the activation of nuclear factor NF-κB pathway and MAPKs including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in antigen-stimulated mast cells.

Conclusions: Our data demonstrate that SRC-3 suppresses cytokine production in antigen-stimulated mast cells as well as PSA in mice at least in part through inhibiting NF-κB and MAPK signaling pathways. Therefore, SRC-3 plays a protective role in PSA and it may become a drug target for anaphylactic diseases.

Keywords: Mast cell; Passive cutaneous anaphylaxis; Passive systemic anaphylaxis; SRC-3.

Figure 1

Passive systemic anaphylaxis in wild-type and SRC-3^-/- mice. SRC-3^+/+ (n = 5) and SRC-3^-/- mice (n = 5) were sensitized with anti-DNP IgE and DNP-HSA to induced systemic anaphylaxis as described in methods. Passive systemic anaphylaxis was monitored by measuring rectal temperatures after DNP-HSA challenge. Data represent the mean rectal temperature ± SD. *p<0.05 versus SRC-3^+/+ mice by t-test.

Figure 2

Passive cutaneous anaphylaxis in SRC-3^+/+ and SRC-3^-/- mice. SRC-3^+/+ (n = 6) and SRC-3^-/- mice (n = 6) were sensitized with anti-DNP IgE and DNP-HSA to induce cutaneous anaphylaxis as described in methods (A-D). A, dye extravasation was observed after DNP-HSA injection at the injection sites in the ears. Photographs of the mice were taken 90 min after DNP-HSA administration. Representative images are shown. B, Extravasation of Evan’s blue was quantified as described in methods. Values are expressed as means + SD from three independent experiments. C, Toludine blue staining of mast cells in the ear skin of SRC-3^+/+ and SRC-3^-/- mice after antigen challenge. Representative images are shown; arrows indicate degranulated tissue mast cells. D, mast cells were quantified, values are expressed as means + SD from three independent experiments.

Figure 3

The degranulation of SRC-3^+/+ and SRC-3^-/- BMMCs. (A) Identification of BMMCs. Bone marrow cells were obtained from BALB/c mice and cultured in BMMC-complete medium. After 5 weeks, cells were identified by flow cytometric analysis for FcϵRI and c-kit expression. The experiment was repeated for 3 ~ 5 times. Representative results are shown from three independent experiments. (B) BMMCs were stimulated with IgE and different concentration of DNP-HSA. Degranulation was measured by assessing hexosaminidase activity in the media or cell lysates. Values are shown as the mean + SD from three independent experiments.

Figure 4

Increased antigen-stimulated IL-6 and TNF-α expression from SRC-3^-/- BMMCs compared with SRC-3^+/+ BMMCs. BMMCs were pretreated with 1 μg/ml anti-DNP IgE for 4 h and then were stimulated with different concentration of DNP-HSA overnight at 37°C in 5% CO₂. (A and B) The amounts of IL-6 and TNF-α in the medium were measured using ELISA assay kits. C and D, The mRNA levels of IL-6 and TNF-α were measured by real-time PCR. Values are shown as the mean + SD from three independent experiments. *p < 0.05.

Figure 5

SRC-3 deficiency has no effect on activation of Syk and PLCγ. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for Syk, p-Syk, PLCγ and p-PLCγ. Representative images are shown from three independent experiments.

Figure 6

SRC-3 deficiency leads to increased activation of IKK-IκB-NF-κB pathway. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for IKK, p-IKK, IκB, p-IκB, p65 and p-p65. Representative images are shown from three independent experiments.

Figure 7

SRC-3 deficiency leads to increased MAPK pathway activation. BMMCs were stimulated with anti-DNP IgE 1 μg/ml for 4 h, and then treated with DNP-HSA 10 ng/ml for various times. Whole cell lysates were analyzed by western-blotting for ERK, p-ERK, JNK, p-JNK, p38 and p-p38. Representative images are shown from three independent experiments.