Lung-specific MCEMP1 functions as an adaptor for KIT to promote SCF-mediated mast cell proliferation

Abstract

Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.

Fig. 1. MCEMP1 has an ITAM-dependent signal-transducing activity in mast cell.

a Schematic diagram of MCEMP1 structure. ITAM motif, YENI; TM, transmembrane. b MCEMP1 tyrosine phosphorylation and its interaction with Grb2 and SOS1. C57 cells expressing vector (VEC), wild-type MCEMP1 (WT), or tyrosine to phenylalanine mutant MCEMP1 (YF) were treated with αFlag for 1 min and cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Immunoprecipitates and whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies. The upper band of MCEMP1 is a glycosylated form. c IP and IB analysis of MCEMP1 phosphorylation and downstream NF-κB signaling in C57 cells. d Mast cell-specific activation of MCEMP1 and downstream MAPK signal transduction in C57, Raw264.7, or DC2.4 cells. e Gene expression of Il4, Il13, Il6, Tnf, and Ifng in C57 cells after αFlag treatment. Mock or αV5 antibody was treated as negative controls. f Intracellular calcium influx upon αFlag-mediated MCEMP1 activation. Calcium Ionophore was used as a positive control. g β-hexosaminidase assay measuring mast cell degranulation. PMA + ionophore (positive control); dinitrophenyl (DNP) + α-DNP-IgE (FcεRI activation). Data are representative of at least two independent experiments in b–f. Data are presented by mean±s.e.m. and p-values were determined by two-way ANOVA with Tukey’s comparison in g (n = 3) and e (n = 3). ns, not significant.

Fig. 2. MCEMP1 deficiency impairs SCF-induced mast cell proliferation.

a Loss of MCEMP1 protein expression in Mcemp1^–/– mice. Lungs were immunoblotted with MCEMP1 antibody. Blots are representative of two independent experiments. b, c Cell growth kinetics of Mcemp1^+/+ and Mcemp1^–/– peritoneal cell (PC, n = 5 per group) or bone marrow cells (BM, n = 4 per group) cultured with SCF and IL-3 for the indicated days. d Cell growth kinetics of Mcemp1^+/+ and Mcemp1^–/– KIT-positive PC isolated by magnetic beads and cultured with SCF and IL-3 for the indicated days. e, f Absolute counts of Mcemp1^+/+ and Mcemp1^–/– PC cultured with either SCF or IL-3 for the indicated days (n = 3 per group). g Schematics of SCF intranasal (i.n.) challenge of Mcemp1^+/+ and Mcemp1^–/– mice. h Representative flow cytometry plots illustrating the gating strategy to identify KIT/FcεRI double-positive mast cells; CD45⁺LIN^–KIT⁺FcεRI⁺. The percentages of KIT⁺FcεRI⁺ mast cells in the lungs of Mcemp1^+/+ or Mcemp1^–/– mice challenged with saline or SCF (n = 6-7 mice per group). Data are presented as violin plot with lines at median and quartiles and p-values were determined by two-way ANOVA with Sidak’s multiple comparison in b, c, d, e, f, h. ns, not significant.

Fig. 3. MCEMP1-KIT interaction amplifies downstream signal transduction.

a Co-immunoprecipitation assay of human and mouse MCEMP1 and KIT interaction in 293 T cells. b Co-immunoprecipitation assay of MCEMP1 interaction with wild-type KIT or enzymatic dead (D794N) mutant in 293 T cells. c Schematic diagram of MCEMP1 and KIT structure and co-immunoprecipitation assay of MCEMP1 interaction with KIT deletion mutants in 293T cells. ΔKI was not expressed. d Immunoprecipitation and Immunoblot analysis of MCEMP1 and KIT phosphorylation in C57 cells expressing vector (VEC), wild-type MCEMP1 (WT), or YF mutant MCEMP1 (YF). C57 cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and WCL were analyzed by IB with the indicated antibodies. Band intensity of Immunoprecipitated KIT and phosphorylated KIT was measured with densitometric analysis by ImageJ and normalized to the intensity of MCEMP1 WT or YF. Data are presented by min to max of box and whiskers and p-values were determined by two-tailed unpaired Student’s t-test (n = 4). e KIT phosphorylation and downstream MAPK signal transduction upon SCF stimulation in C57 cells expressing VEC, WT MCEMP1 or YF mutant MCEMP1. f, g Gene expression of Il6 and Il13 after SCF stimulation in C57 cells expressing VEC, WT MCEMP1 or YF mutant MCEMP1. Data are representative of at least two independent experiments in a–e. Data are presented by mean±s.e.m. and p-values were determined by two-way ANOVA with Sidak’s multiple comparison in f (n = 3) and g (n = 3).

Fig. 4. The effect of MCEMP1 deficiency on gene expression related to cell growth.

a IPA canonical pathways enriched in Mcemp1^–/– PC versus Mcemp1^+/+ PC cultured with SCF and IL-3 for 9 days. The z-scores indicate a predicted inhibition or activation of the indicated pathway. The ratio was calculated by the number of DEGs in the indicated pathway divided by the total number of genes that map to the pathway. The p-value of overlap was calculated by the right-tailed Fisher’s Exact Test. b Heatmaps of differentially expressed genes (DEGs) of Mcemp1^–/– PC versus Mcemp1^+/+ PC. c A dot plot of comparison analysis of upstream regulators repressed in Mcemp1^–/– PC in comparison with Mcemp1^+/+ PC. The color bar indicates z-score of a predicted inhibition in the indicated upstream regulators. The size of each dot corresponds to the p-value of overlap (right-tailed Fisher’s Exact Test). d, e Representative immunofluorescence images of Mcemp1^+/+ or Mcemp1^–/– PC cultured with SCF and IL-3 for the indicated days and stained with anti-phospho-JNK antibody (green), anti-phospho-MITF antibody (red) or Hoechst 33342 (blue). Scale bar, 20 μm. The average percentage of p-JNK or p-MITF positive cells per slide (n = 4–9 slides) were quantified. Data are representative of two independent experiments. f Expressions of Ccne1, Ccna1, Ccnb1, and Ccnb2 in Mcemp1^+/+ or Mcemp1^–/– PC. KIT-positive PC were isolated and cultured with SCF and IL-3 for 6 days and stimulated with 30 ng/ml SCF and further cultured for 12 h (n = 9-18 per group). g Schematic illustration of SCF-KIT and IgE-FcεRI signal transduction in mast cells. The linker for activation of T cells (LAT) is an essential adaptor for FcεRI receptor that induces IgE-mediated mast cell activation. In parallel, MCEMP1 acts as a critical adaptor for KIT receptor that promotes SCF-mediated mast cell proliferation. Data are presented as violin plot with lines at median and quartiles and p-values were determined by two-tailed unpaired t-test in d–f. ns, not significant.

Fig. 5. MCEMP1 deficiency attenuates OVA-induced lung inflammation.

a Schematics of ovalbumin (OVA)-sensitized and challenged chronic asthma model. Mcemp1^+/+ and Mcemp1^–/– mice were sensitized with saline or 50 μg OVA via intraperitoneal (i.p.) injection and challenged with saline or 20 μg OVA via intranasal (i.n.) instillation on the indicated days. b Representative images of hematoxylin and eosin staining for histological analysis of lungs. B, bronchiole; A, alveoli; V, blood capillary. Scale bar, 100 μm. c Percentages and absolute numbers of eosinophils in bronchoalveolar lavage (BAL) were determined by manual counting and by differential counts on cytospin slides (n = 3 for saline group, n = 4 for OVA group). d Percentages and absolute numbers of eosinophils and Ly-6C⁺ inflammatory monocytes in the lungs were determined by flow cytometry (n = 3 per group). e Average numbers of toluidine blue positive mast cells per slide (n = 3 slides per group) were quantified by ImageJ. f Quantification of Masson’s trichrome staining (n = 2 slides per group) by Image-Pro 10. g Schematics of BMMC adoptive transfer and OVA-induced chronic asthma model. h Percentages and/or absolute numbers of BAL eosinophils, lung CD45⁺ leukocytes, eosinophils and neutrophils were determined by flow cytometry (n = 6 for saline group. n = 3-4 for OVA group). Data are presented as violin plots with lines at median and quartiles and p-values were determined by two-way ANOVA with Sidak’s multiple comparison in c, d, e, f or by one-way ANOVA with Tukey’s multiple comparison in h. ns, not significant.

Fig. 6. MCEMP1 deficiency diminishes asthma-associated inflammatory gene expression.

a, b Cytokine and growth factor levels in the BAL and plasma were determined by multiplex analysis (n = 3 for saline group, n = 4 for OVA group). c A dot plot of comparison analysis of OVA-challenged versus saline-treated Mcemp1^+/+ mice in comparison with OVA-challenged versus saline-treated Mcemp1^–/– mice. The color bar indicates z-score of a predicted activation in the indicated pathway. The size of each dot corresponds to the p-value of overlap (right-tailed Fisher’s Exact Test). d Heatmaps of significantly upregulated and downregulated genes in OVA-challenged versus saline-treated Mcemp1^+/+ mice in comparison with OVA-challenged versus saline-treated Mcemp1^–/– mice. Red, white, and blue indicate induction, no change, and repression, respectively. e Gene expression of Il13, Il5, Il10, Ccl2, Ccr5, and Cxcl10 in Mcemp1^+/+ mice or Mcemp1^–/– mice after saline or OVA challenge. Data are presented as violin plots with lines at median and quartiles and p-values were determined by two-way ANOVA with Sidak’s multiple comparison in a, b, e. ns, not significant.

Fig. 7. MCEMP1 deficiency ameliorates lung compliance and airflow in OVA-induced chronic asthma.

a–c Lung function parameters; compliance (Crs), elastance (Ers) of respiratory system, and tissue elastance (H) in response to the indicated concentrations of methacholine were measured 1 day after the eighth intranasal challenge with OVA or saline (n = 3-4 per group). d Representative images of 4DMedical lung scanning for regional ventilation visualization across the lung over a full inhalation. One coronal slice and three axial slices of upper, middle, and lower lungs of Mcemp1^+/+ or Mcemp1^–/– mice challenged with OVA or saline. The color bar indicates the degree of ventilation as low in red and high in blue. e Representative histogram of 4DMedical ventilation frequency distribution. VDP, ventilation deficit percent. f–h Ventilation metrics; VDP, ventilation heterogeneity (VH), and tidal volume (n = 3 per group). Data are presented as mean±s.d. in a, b, c and as violin plots with lines at median and quartiles in f, g, h. P-values were determined by two-way ANOVA with Dunnett’s multiple comparison in a, b, c and by two-way ANOVA with Sidak’s multiple comparison in f, g, h. ^*P < 0.05 (Crs, the p-values for 0, 25, and 100 mg/ml methacholine are 0.0115, 0.034, and 0.0444, respectively; Ers, the p-values for 25 and 100 mg/ml methacholine are 0.0442 and 0.0286, respectively; H, the p-values for 0, 25, 50, and 100 mg/ml methacholine are 0.0361, 0.0425, 0.0346, and 0.0157, respectively). ns, not significant.