Nuclear receptor 4a3 (nr4a3) regulates murine mast cell responses and granule content

Abstract

Nuclear receptor 4a3 (Nr4a3) is a transcription factor implicated in various settings such as vascular biology and inflammation. We have recently shown that mast cells dramatically upregulate Nuclear receptor 4a3 upon activation, and here we investigated the functional impact of Nuclear receptor 4a3 on mast cell responses. We show that Nuclear receptor 4a3 is involved in the regulation of cytokine/chemokine secretion in mast cells following activation via the high affinity IgE receptor. Moreover, Nuclear receptor 4a3 negatively affects the transcript and protein levels of mast cell tryptase as well as the mast cell's responsiveness to allergen. Together, these findings identify Nuclear receptor 4a3 as a novel regulator of mast cell function.

Figure 1. Nr4a3 does not affect mast cell development.

(A, B) Toluidine blue staining of WT (A) and Nr4a3^−/− (B) mast cells after 4 weeks of culture. Note that the absence of Nr4a3 does not affect the morphology or granular staining of the cells.

Figure 2. Nr4a3 affects cytokine/chemokine secretion in response to FcεeRI cross-linking.

(A–D) WT and Nr4a3^−/− mast cells were incubated with TNP-specific IgE over-night followed by activation for 4 hours or 24 hours with TNP-OVA as indicated. Supernatants were analyzed for levels of MCP-1 (A), TNFα (B), IL-6 (C) and IL-13 (D) by ELISA (T-test, n = 4; *p≤0.05). The lack of Nr4a3 is associated with a reduction in cytokine release following FcεRI cross-linking.

Figure 3. Nr4a3 modulates signaling pathways involved in mast cell degranulation (A–F).

(A–B) Whole cell lysates from resting WT and Nr4a3^−/− mast cells were prepared and analyzed by Western blot for the Src-kinases Fyn (A) and Lyn (B). The signal was compared against β-actin followed by normalization using the average signal from WT samples set to 1. (C–D) Graphical representation of the densitometric analysis for Fyn (C) and Lyn (D) (T-test, n = 4; *p≤0.05). (E) WT and Nr4a3^−/− mast cells were incubated with TNP-specific IgE over-night followed by activation for 30 minutes with increasing amounts of TNP-OVA. Supernatants were collected and were analyzed for β-hexosaminidase activity as a measure of the extent of degranulation. (ANOVA, n = 4; *p≤0.05). (F) Whole cell lysates were prepared and analyzed for their β-hexosaminidase content.

Figure 4. (A–C) Nr4a3-deficient mast cells are more susceptible than WT cells to cell death induced by granule disruption.

WT and Nr4a3^−/− mast cells were treated with 200 mM LLME (A), 5 µg/ml Cycloheximide (B) or 0.75 mM H₂O₂ (C). The viability was determined at the indicated time-points using CellTiter-Blue reagent (ANOVA, N = 6, *p≤0.05).

Figure 5. Nr4a3 regulates the transcription of mast cell granule components.

Total RNA was extracted from WT and Nr4a3^−/− mast cells followed by qPCR analysis for levels of mRNA encoded by Srgn (A), Mcpt4 (B), Tpsb2 (C) and Cpa3 (D). Hprt was used as house-keeping gene; transcript data were calculated according to DDCT relative to the expression in WT cells (T-test, n = 4; *p≤0.05).

Figure 6. Nr4a3 suppresses the levels of tryptase protein and enzymatic activity.

(A) Whole cell lysates from WT and Nr4a3^−/− mast cells were prepared and analyzed by Western blot for mMCP-6 and CPA3 as indicated (the upper and lower panels represent individual analyses, performed on the same membrane that was stripped of protein between analyses). The absence of Nr4a3 results in increased levels of mMCP-6 but does not affect CPA3. (B) Quantification of band intensities for mMCP-6 showing a significant increase in mMCP-6 protein in the absence of Nr4a3. (n = 4; *p≤0.05). The level of mMCP-6 and CPA3 proteins were quantified and compared against b-actin followed by normalization using the average signal from WT samples set to 1. (C) Whole cell lysates from WT and Nr4a3^−/− mast cells were prepared and analyzed for trypsin-like activity using the chromogenic substrate S-2288. Note that the absence of Nr4a3 results in increased trypsin-like enzymatic activity. (T-test, n = 4; *p≤0.05).