The Transcription Factor Ehf Is Involved in TGF-β–Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells

Mast cells are widely recognized as the major effector cells in IgE-dependent immediate hypersensitivity. They are characterized by expression of FcεRI and the stem cell factor (SCF) receptor c-Kit on the cell surface. FcεRI, which is composed of one α, one β, and two γ subunits, plays a critical role in IgE-mediated hypersensitivity reactions. The expression of FcεRI is regulated by multiple transcription factors in mast cells. Previous studies have shown that the transcription of the FcεRIα subunit–encoded gene Fcer1a is positively regulated by the transcription factors PU.1 and GATA-1 in both human and mouse mast cells, and that the transcription of the FcεRIβ subunit–encoded gene Ms4a2 in humans and mice is positively regulated by GATA-2 and GATA-1, respectively (1–7). Alternatively, c-Kit is essential for the development of mast cells (8–10). GATA-2 is also involved in the transcription of the c-Kit–encoding gene Kit in mast cells (11). Several studies have demonstrated that TGF-β1 suppresses mast cell FcεRI and c-Kit expression, and also mast cell activation induced by cross-linking of FcεRI (12–14). TGF-β1 acts as a negative regulator of mast cell function, but it induces the expression of mouse mast cell protease (mMCP)-1 and -2 and integrin α_E (CD103), markers of mucosal mast cells (15–19). TGF-β signaling is mediated by Smads, which accumulate in the nucleus to transcriptionally regulate TGF-β target genes. The promoter and enhancer element of Itgae, encoding CD103, are known to have Smad-binding elements (20). Thus, CD103 expression is directly regulated by TGF-β/Smad signaling. However, the molecular mechanism by which TGF-β suppresses FcεRI and c-Kit expression, in addition to FcεRI-mediated activation in mast cells, is poorly understood.

In this study, we found that the expression of Ets homologous factor (Ehf) is significantly upregulated by TGF-β1 in mouse bone marrow–derived mast cells (BMMCs). Ehf, also known as Ese-3, is a member of the Ets family of transcription factors that play a crucial role in development (21, 22). Ehf is expressed in various epithelial cells and tumor cells, and it is involved in the control of epithelial differentiation or tumor development (23–28). Functionally, Ehf is a transcriptional repressor of several genes regulated by MAPK signaling with a dependency on an Ets binding site (29). Recently, Ehf has been reported to be involved in the development of monocyte-derived dendritic cells (30–32).

In this study, we show that the transcription factor Ehf contributes to TGF-β–induced suppression of mast cell FcεRI and c-Kit expression through inhibition of the transcriptional activity of the promoters of genes encoding FcεRIα, FcεRIβ, and c-Kit, as well as downregulation of GATA-1 and GATA-2 expression. Additionally, our results also indicate that Ehf is probably involved in TGF-β–induced suppression of FcεRI-mediated mast cell activation through downregulation of FcεRI and Stat5 expression.

BMMCs were generated from femoral bone marrow cells of BALB/c mice as described previously (33). Cells were cultured for 2–4 wk in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated FCS (Life Technologies, Carlsbad, CA), 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM 2-ME, 10 mM sodium pyruvate, and 10 μM MEM nonessential amino acid solution (Life Technologies) in the presence of 10 ng/ml recombinant murine IL-3 (Wako Pure Chemical Industries, Osaka, Japan) and 10 ng/ml recombinant murine SCF (Wako Pure Chemical Industries) at 37°C in a humidified atmosphere in the presence of 5% CO₂. Mast cells were identified by flow cytometric analysis of cell surface expression of c-Kit and the FcεRI α-chain.

Two-week cultured bone marrow cells were cultured in BMMC medium in the presence of 10 ng/ml IL-3, 10 ng/ml SCF, and 5 ng/ml recombinant murine IL-9 (PeproTech, Rocky Hill, NJ) with or without 1 or 5 ng/ml recombinant human TGF-β1 (Wako Pure Chemical Industries).

The full-length mouse Ehf (NM_007914.3) cDNA was subcloned into the retrovirus vector pMXs-puro, which was a gift from Dr. Toshio Kitamura (University of Tokyo). A retrovirus-packing cell line, PLAT-E, was transfected with a retroviral vector encoding mouse Ehf or mock vector using FuGENE 6 (Promega, Madison, WI) to generate recombinant retroviruses. The nucleotide sequences of the primer set for mouse Ehf cDNA are listed in Supplemental Table I. Forty-eight hours after transfection, the virus-containing medium was collected and passed through a 0.45-μm filter membrane. The supernatants were incubated for 6 h at 37°C in RetroNectin (Takara Bio, Shiga, Japan)–coated plates to attach the virus particles onto the RetroNectin. After removing the supernatants, BMMCs were plated onto the virus-attached plates. The transduced cells were selected by additional culture in the presence of 1.6 μg/ml puromycin for 14 d.

Cells were collected and lysed by the direct addition of sample buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 0.1 mg/ml bromophenol blue dye, and 10% 2-ME). The cell lysates were electrophoretically resolved on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). Abs against FLAG (M2) and Ehf, which were purchased from Sigma-Aldrich, were used as primary Abs. Alexa Fluor 680– or IRDye 800–conjugated anti-mouse or anti-rabbit IgG Abs (Life Technologies) were used as secondary Abs. Infrared fluorescence on the membrane was detected by the infrared imaging system Odyssey (LI-COR Biosciences, Lincoln, NE).

BMMCs were stained with FITC-conjugated anti-mouse FcεRIα mAb (MAR-1; eBioscience, San Diego, CA), PerCP (PerCP complex)/Cy5.5 or PE-conjugated anti-mouse c-Kit mAb (2B8; BioLegend, San Diego, CA), and PE-conjugated anti-mouse CD103 mAb (2E7; BioLegend) after blocking Fc receptors with anti-mouse CD16/CD32 mAb (2.4G2). The expression of cell surface markers was analyzed on a FACSCalibur (BD Biosciences, San Jose, CA).

Total cellular RNA was purified from cells using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 500 ng total RNA using a ReverTra Ace quantitative real-time PCR (qPCR) RT kit (Toyobo, Osaka, Japan). Real-time quantitative PCR (real-time PCR) was performed with the Eco real-time PCR system (Illumina, San Diego, CA) using TaqMan Universal PCR master mix and Assays-on-Demand gene expression products for Ehf (Mm00468193_m1), Fcer1a (Mm00438867_m1), Ms4a2 (Mm00442778_m1), Fcer1g (Mm02343757_m1), Kit (Mm00445212_m1), Itgae (Mm00434443_m1), Gata1 (Mm01352636_m1), Gata2 (Mm00492301_m1), and Spi1 (Mm00488142_m1), which were purchased from Life Technologies. The mRNA expression levels were quantified with the comparative method using Eco software and normalized against the housekeeping gene Gapdh.

Transient silencing of the Smad4 gene was achieved using small interfering RNAs (siRNA) targeted against Smad4 (no. 1, Stealth RNA interference siRNA MSS206435; no. 2, MSS206437), which were purchased from Life Technologies. A nontargeting siRNA was used as a negative control. Cells (2 × 10⁶) were transfected with 500 nM siRNA using a mouse macrophage Nucleofector kit (Lonza, Basel, Switzerland) according to the manufacturer’s instructions with a Nucleofector II device (Lonza).

Fcer1a and Fcer1g promoter regions were amplified from BALB/c mouse genomic DNA by using PCR and inserted into the multicloning site of pGL4.10 (Promega) to generate luciferase reporter plasmids. The nucleotide sequences of primer sets are listed in Supplemental Table I. The luciferase reporter plasmids pGL3-Basic (Promega) encoding Ms4a2 promoter region −763 to +103 relative to the transcription start site (+1) (3, 5) and Kit promoter region −622 to +22 (11) were generated as in our previous studies. The expression plasmid pCR3.1 (Life Technologies) encoding FLAG-tagged mouse Ehf cDNA and its empty plasmid were used in this study. BMMCs (4 × 10⁵ cells) were transfected with 1.6 μg reporter plasmid, 16 ng pRL-null vector (Promega), and 10 ng expression plasmid with a Neon 10 μl kit using a Neon transfection system (Life Technologies) set at program 5. Twenty hours after transfection, luciferase activity was determined using MicroLumat Plus (Berthold Technologies, Bad Wildbad, Germany) and a Dual-Luciferase assay kit (Promega) as described previously (5). Firefly luciferase activity derived from the reporter plasmid was normalized to Renilla luciferase activity derived from pRL-null.

Chromatin immunoprecipitation (ChIP) assays were performed as described in our previous reports (5, 34). Anti-FLAG tag mAb (M2) purchased from Sigma-Aldrich was used for immunoprecipitation, and mouse IgG (Life Technologies) was used as a control Ab. The amount of chromosomal DNA, including promoters of Fcer1a, Ms4a2, and Kit genes, were determined by the TaqMan system using the primers and TaqMan probes listed in Supplemental Table II.

Cells were sensitized with mouse IgE as described previously (35). To assess cytokine production, IgE-sensitized cells were incubated at 1 × 10⁶ cells/ml in the presence or absence of 1 μg/ml anti-mouse IgE mAb (R35-72; BD Biosciences) for 6 h. Cytokine concentrations in culture supernatants were quantified using a corresponding ELISA kit (R&D Systems, Minneapolis, MN).

The β-hexosaminidase release assay was performed as described previously (36). Briefly, BMMCs (4 × 10⁶ cells/ml) were incubated for 1 h at 4°C with 1 μg/ml mouse IgE mAb (BD Biosciences) and then were stimulated for 40 min at 37°C with 1 μg/ml anti-mouse IgE (BD Biosciences). After centrifugation, the supernatants were collected and total cell lysates were obtained using 1% Triton X-100. The percentage of β-hexosaminidase release was calculated as described previously (36).

The unpaired Student t test was used as appropriate for parametric differences. One-way ANOVA or two-way ANOVA with the Dunnett method was used for multiple testing of data. A p value <0.05 was considered significant.

TGF-β1 suppresses the expression of FcεRI and c-Kit and promotes the expression of CD103 in human and mouse mast cells (12–14, 17, 19, 37). We previously demonstrated that the transcription of genes encoding FcεRIα and FcεRIβ is positively regulated by the transcription factors PU.1, GATA-1, and GATA-2 in mast cells (3, 5, 7, 38–41). Additionally, GATA-2 also plays an important role in the transcription of the Kit gene (11). Therefore, we first investigated the expression of those transcription factors in BMMCs cultured in media supplemented with IL-3 plus SCF, IL-3 plus SCF plus IL-9, or IL-3 plus SCF plus IL-9 plus TGF-β1 for 14 d. These systems have been widely used to study mast cell development (17, 18). Cell surface expression of FcεRI and c-Kit was downregulated in BMMCs cultured in the presence of TGF-β1 (Fig. 1A), whereas expression of CD103 on the cell surface was upregulated by TGF-β1 (Fig. 1B). qPCR assays revealed that the mRNA levels of FcεRI subunits Fcer1a (encoding FcεRIα), Ms4a2 (encoding FcεRIβ), and Fcer1g (encoding FcεRIγ), as well as Kit (encoding c-Kit), were significantly downregulated, and the mRNA level of Itgae (encoding CD103) was markedly upregulated by TGF-β1 (Fig. 1C). Mokrani et al. (20) reported that CD103 expression is induced by signaling through Smad and NFAT pathways in human CD8 T cells. However, the mechanism by which TGF-β1 suppresses the expression of FcεRI and c-Kit on the mast cell surface is unknown. The present results of gene expression showed that the transcription levels of Gata1 (encoding GATA-1), Gata2 (encoding GATA-2), and Spi1 (encoding PU.1) were significantly lower in BMMCs cultured in the presence of TGF-β1 compared with BMMCs cultured in the absence of TGF-β1 (Fig. 1D). These results suggest that a decrease in expression levels of GATA-1, GATA-2, and PU.1 contributes to the TGF-β1–induced downregulation of FcεRI and c-Kit expression in mast cells.

Ehf is a member of the Ets family of transcription factors. In this study, we found that the level of Ehf mRNA was markedly higher in BMMCs cultured for 14 d in the presence of TGF-β1 compared with cells cultured in the absence of TGF-β1 (Fig. 2A). A significant increase in the level of Ehf mRNA was observed at 6 h after TGF-β1 stimulation (Fig. 2B). TGF-β1 signaling is transmitted to the nucleus to activate or repress gene expression by activated intracellular messengers Smad2 and Smad3, which form heteromeric complexes with Smad4. Therefore, to demonstrate that Ehf expression is induced in BMMCs by TGF-β1 signaling, we blocked the TGF-β1/Smad signaling pathway using Smad4-specific siRNAs. In this experiment, two siRNAs for Smad4 were used to exclude the possibility of any off-target effects of siRNAs. The expression of Smad4 mRNA in BMMCs transfected with siRNA nos. 1 and 2 was reduced by ∼32 and 76%, respectively (Fig. 2C). Knockdown of Smad4 significantly reduced the levels of Ehf and Itgae mRNAs in TGF-β1–treated cells (Fig. 2C). The mRNA levels of Ehf and Itgae in BMMCs treated with TGF-β1 are most likely to be proportional to the level of Smad4 expression. Smads have been shown to directly activate transcription of the Itgae gene (20). Alternatively, it is unclear whether Smads directly bind to the Ehf promoter. Our data indicate that TGF-β1/Smad signaling directly or indirectly upregulates the expression of Ehf in mast cells.

Previous studies have demonstrated that some Ets family transcription factors, such as PU.1 and Elf-1, regulate expression of FcεRI in mast cells (6, 7, 42). Although Ehf is also a member of the Ets family of transcription factors, the role of Ehf in mast cells has been unknown. In this study, we hypothesized that Ehf contributes to the suppression of FcεRI and c-Kit expression induced by TGF-β1 in mast cells. To test our hypothesis, we generated BMMCs stably expressing Ehf. A retroviral vector encoding FLAG-tagged mouse Ehf or the empty vector (mock) was transfected into bone marrow cells cultured for 2 wk in medium supplemented with IL-3 and SCF, and then transfectants were selected by culturing for 2 wk in the presence of puromycin. The expression of FLAG-tagged Ehf protein was observed in Ehf-transfected BMMCs in the absence of TGF-β1 (Fig. 3A, 3B). The cell surface expressions of FcεRI and c-Kit were markedly suppressed in BMMCs stably expressing Ehf compared with mock-transfected BMMCs (Fig. 3C). Forced expression of Ehf significantly decreased the levels of mRNA for Fcer1a and Ms4a2, and for Kit in BMMCs, but it did not affect the mRNA level of Fcer1g (Fig. 3E). Although CD103 expression is upregulated by TGF-β1 treatment, the expression levels of protein and mRNA were decreased in BMMCs stably expressing Ehf compared with mock-transfected BMMCs (Fig. 3D, 3E). These results suggest that Ehf contributes to TGF-β1–induced downregulation of FcεRIα, FcεRIβ, and c-Kit, but not upregulation of CD103, in BMMCs.

Our previous studies have shown that the transcriptional activity of the Fcer1a gene promoter is upregulated by binding of PU.1, an Ets family transcription factor, to an Ets binding site on the promoter region (2, 6, 7). Ms4a2 and Kit genes also have some canonical Ets binding sites on their promoter regions (data not shown). Thus, to examine the mechanism by which the transcription of Fcer1a, Ms4a2, and Kit genes is selectively repressed by forced expression of Ehf, an Ets family transcription factor, we analyzed the influence of Ehf on their promoter activities in BMMCs. First, the promoter activity of the genes encoding FcεRI subunits and c-Kit were measured by luciferase reporter assays in BMMCs. The luciferase activity of reporter plasmids containing the promoter segments of Fcer1a, Ms4a2, Fcer1g, or Kit genes was significantly higher than that of a promoterless reporter plasmid (Fig. 4A). The luciferase activities driven by Fcer1a, Ms4a2, and Kit promoters were strongly repressed by transient expression of Ehf, whereas that of Fcer1g was only modestly decreased by Ehf expression (Fig. 4A). These results indicate that Ehf represses the transcription of Fcer1a, Ms4a2, and Kit genes, but not the Fcer1g gene, in BMMCs through downregulation of these promoter activities.

We next used ChIP assays to confirm association of Ehf with the promoter regions of Fcer1a, Ms4a2, and Kit genes. A significantly higher amount of chromatin containing the promoter segment of these genes was immunoprecipitated by anti-FLAG mAb compared with isotype control IgG in BMMCs expressing FLAG-tagged Ehf (Fig. 4B), demonstrating that Ehf represses the transcriptional activities of Fcer1a, Ms4a2, and Kit genes by directly binding to these promoter regions. It therefore follows that Ehf acts as a transcriptional repressor of the Fcer1a, Ms4a2, and Kit genes.

Interestingly, forced expression of Ehf significantly suppressed the transcription levels of Gata1 and Gata2 in BMMCs but did not affect the transcription of Spi1 (Fig. 5). Previously, we demonstrated that knockdown of GATA-1 and/or GATA-2 by siRNA suppresses the transcription of Fcer1a and Ms4a2, but not Fcer1g, in mast cells (7). Additionally, knockdown of GATA-2 also suppresses the transcription of Kit in mast cells (11). The data shown in Fig. 5 are consistent with the previous results. Therefore, the downregulation of GATA-1 and GATA-2 expression may also contribute to the suppression of FcεRI and c-Kit expression on BMMCs expressing Ehf. However, the detailed molecular mechanism of the downregulation of transcription of Gata1 and Gata2 by Ehf remains unknown, because, in this study, we were not able to identify the functional promoter regions of Gata1 and Gata2 genes in BMMCs.

Mast cell activation by cross-linking of FcεRI results in degranulation and de novo synthesized cytokines. TGF-β1 has been shown to inhibit FcεRI-mediated degranulation and synthesis of cytokines in mast cells (12, 14). To investigate the role of Ehf in FcεRI-mediated mast cell activation, we measured the FcεRI-mediated release of β-hexosaminidase as an index of degranulation and production of cytokines IL-6 and IL-13 by BMMCs stably expressing Ehf. The β-hexosaminidase release by BMMCs treated with TGF-β1 and BMMCs stably expressing Ehf was reduced by ∼60 and 58% compared with that by control cells cultured with IL-3 and SCF, respectively (Fig. 6A). The low levels of degranulation in these BMMCs may be explained by reduced expression of FcεRI (Figs. 1A, 3C). Additionally, FcεRI-mediated productions of IL-6 and IL-13 were reduced by >70% in both BMMCs treated with TGF-β1 and those stably expressing Ehf compared with control cells cultured with IL-3 and SCF (Fig. 6B). However, the marked reduction in FcεRI-mediated cytokine production cannot be explained only by reduced expression of FcεRI. Fernando et al. (14) showed that TGF-β1 reduces the expression of Stat5, which is a critical regulator of cytokine induction, in mast cells. The expression level of Stat5b mRNA in BMMCs treated with TGF-β1 and in BMMCs stably expressing Ehf was reduced by ∼45 and 62% compared with that in control cells cultured with IL-3 and SCF, respectively (Fig. 6C). Thus, reduced expression of FcεRI and Stat5 may contribute to the inhibition of FcεRI-mediated cytokine production by treatment with TGF-β1 or forced expression of Ehf. These results suggest that upregulation of Ehf is involved in TGF-β1–induced inhibition of FcεRI-mediated mast cell activation.

Short-term exposure to TGF-β inhibits mast cell FcεRI expression through a reversible pathway that diminishes protein but not mRNA expression of α, β, and γ subunits (12). In contrast, long-term exposure to TGF-β during development of mast cells inhibits not only FcεRI expression but also c-Kit expression (13). Additionally, TGF-β is considered to be a key cytokine in mucosal mast cell differentiation because it induces expression of mMCP-1, mMCP-2, and CD103, marker molecules of mature mucosal mast cells, in mice (15–19). Indeed, the levels of cell surface FcεRI and c-Kit expression are lower in mucosal mast cells than in connective tissue-type mast cells (43). We showed that the mRNA levels of c-Kit and each subunit composed of FcεRI were lower in BMMCs cultured in the presence of TGF-β1 for 14 d than in BMMCs cultured in the absence of TGF-β1 during the same period (Fig. 1A). The decreased expression of FcεRI and c-Kit may be explained, in part, by the decrease in the transcription factors GATA-1, GATA-2, and PU.1 (Fig. 1D), because these transcription factors play a crucial role in mast cell FcεRI and c-Kit expression. In particular, mouse FcεRI α and β subunits are positively regulated by GATA-1/PU.1 and GATA-1, respectively, and mouse c-Kit is positively regulated by GATA-2 (3, 5, 6). However, the molecular mechanism by which TGF-β1 inhibits the expression of these transcription factors in mast cells is not clear. Moreover, an unknown mechanism to directly repress the expression of genes encoding FcεRI subunits and c-Kit may be involved.

In this study, we found that TGF-β1 induces upregulation of Ehf in BMMCs (Fig. 2A, 2B). The upregulation of Ehf was significantly suppressed by knockdown of Smad4 using specific siRNA (Fig. 2C), and this almost certainly depends on TGF-β/Smad signaling in mast cells. However, it remains unclear whether the transcription of the Ehf gene is directly regulated by Smads, because there is insufficient information regarding the promoter element of the Ehf gene. Ehf is an Ets family transcription factor, a family that includes PU.1. Although PU.1 plays an important role in the activation of the Fcer1a gene promoter in mast cells through binding to Ets binding sites, the function of Ehf in mast cells has been unknown. In this study, we showed that forced expression of Ehf suppressed the cell surface expression of FcεRI and c-Kit on BMMCs (Fig. 3C). The levels of mRNA encoding c-Kit and FcεRI subunits α and β, but not γ, were lower in BMMCs stably expressing Ehf than in the control cells (Fig. 3E). Ehf functions as a transcriptional repressor of several genes through Ets binding sites (29). Ets binding motifs are present not only in the Fcer1a gene promoter but also in Ms4a2 and Kit gene promoters. Luciferase reporter assays and ChIP assays showed that the promoter activities of Fcer1a, Ms4a2, and Kit genes were directly downregulated by binding of Ehf to these gene promoter regions (Fig. 4). These results demonstrate that Ehf functions as a transcriptional repressor of Fcer1a, Ms4a2, and Kit genes in mast cells. Moreover, forced expression of Ehf in BMMCs resulted in a decrease in mRNA expression of Gata1 and Gata2, which act as transcriptional activators of Fcer1a, Ms4a2, and/or Kit genes (Fig. 5). Therefore, Ehf not only directly represses the transcription of Fcer1a, Ms4a2, and Kit genes as a transcriptional repressor, but it may also indirectly repress them by downregulating the expression of GATA-1 and GATA-2. Alternatively, the transcriptions of Fcer1g and Spi1 genes were not repressed in BMMCs stably expressing Ehf (Figs. 3E, 4), although they were repressed in BMMCs treated with TGF-β1 (Fig. 1C, 1D). Thus, the repressions of FcεRIγ and PU.1 expression were probably induced by a mechanism that is not mediated by Ehf in BMMCs treated with TGF-β1.

The reduction of FcεRI expression leads to the inhibition of FcεRI-mediated mast cell activation. Our data showed that treatment with TGF-β or forced expression of Ehf downregulates FcεRI-mediated degranulation and markedly downregulates FcεRI-mediated cytokine production (Fig. 6A, 6B). Recently, the Fyn–Stat5B pathway has been reported to be a novel mechanism for FcεRI-mediated cytokine production (44). The marked suppression of FcεRI-mediated cytokine production may be due not only to reduced FcεRI expression, but also to reduced transcription factor Stat5B expression (Fig. 6C). These findings indicate that the upregulation of Ehf is probably involved in the downregulation of GATA-1, GATA-2, and Stat5B in BMMCs, although it remains unclear whether Ehf acts as a direct transcriptional repressor of these genes. Further investigation is needed to understand the function of Ehf in mast cells. Additionally, the generation and phenotypic analysis of Ehf knockout mice will be of use in evaluating the biological significance of Ehf in mast cells.

In this study, some mast cell responses to TGF-β1 were mimicked by forced expression of Ehf. Therefore, we suggest that Ehf is involved in the suppression of FcεRI and c-Kit expression and FcεRI-mediated mast cell activation caused by TGF-β1/Smad signaling. These findings provide mechanistic insights into TGF-β1–mediated mast cell responses. This novel insight may contribute to understanding the mechanisms of mucosal mast cell development and regulation of mast cell functions.

We thank Prof. Toshio Kitamura for providing retrovirus vectors and PLAT-E cells. We are grateful to the members of the Atopy (Allergy) Research Center and the Department of Immunology of the Juntendo University Graduate School of Medicine for comments, encouragement, and technical assistance and Michiyo Matsumoto for secretarial assistance.

The authors have no financial conflicts of interest.