GATA-1 is required for expression of Fc
RI on mast cells: analysis of mast cells derived from GATA-1 knockdown mouse bone marrow
Chiharu Nishiyama1,
Tomonobu Ito1,
Makoto Nishiyama2,
Shigehiro Masaki1,
Keiko Maeda1,
Nobuhiro Nakano1,
William Ng1,
Kanako Fukuyama2,
Masayuki Yamamoto3,
Ko Okumura1 and
Hideoki Ogawa1
1 Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
2 Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3 Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8577, Japan
Correspondence to: C. Nishiyama; E-mail: chinishi{at}med.juntendo.ac.jp
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Abstract
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The high-affinity receptor for IgE (Fc
RI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the Fc
RI
- and ß-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the
- and ß-chain promoters. To confirm that GATA-1 is involved in the expression of Fc
RI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of Fc
RI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcriptionPCR analysis showed that the level of transcripts not only for GATA-1 but also for both the
- and ß-chains was significantly lower in KD mast cells, whereas that of the Fc
RI
-chain was not affected. Degranulation caused by cross-linking of Fc
RI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates Fc
RI
- and ß-chain expression and therefore is involved in mast cell development.
Keywords: GATA-1, knockdown mouse, mast cells, transcription factor
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Introduction
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The transcription factor GATA-1 is expressed in erythroid cells, megakaryocytes, eosinophils and mast cells of hematopoietic lineage, and is involved in their development and cell type-specific gene regulation (1). Gene-targeting analyses have shown the functional roles of GATA-1 in the development of hematopoietic cells into erythroid and megakaryocyte types (16). In addition, the involvement of GATA-1 in eosinophil development has recently been elucidated (7, 8). On the other hand, the role of GATA-1 in mast cell development remains controversial. GATA-1-negative mast cells derived from fetal liver have histochemical staining profiles similar to those of wild-type (WT) mast cells (9), whereas targeted deletion of the GATA-1 gene promoter decreases the amount of heparin in connective tissue-type mast cells (10). We recently examined the expression mechanism of high-affinity receptor for IgE (Fc
RI) that is specifically expressed on the mast cell surface. Transient reporter assays in those studies found that genes for the
- and ß-chains of the receptor are transactivated by GATA-1 (1113).
In this study, we investigate the role of GATA-1 in mast cell development. We generated mast cells derived from bone marrow of the GATA-1 knockdown (KD) mouse (2), and analyzed the regulation of Fc
RI expression. Our results definitively showed that GATA-1 positively regulates the expression of the Fc
RI
- and ß-chains and plays a role in mast cell development.
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Methods
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Preparation of bone marrow-derived cultured mast cells (BMMCs)
Mast cells were derived from WT (control littermates female mouse) and heterozygous GATA-1 KD female mouse (2) bone marrow cells as described (14) under the same conditions in parallel. Cells were incubated in RPMI 1640 (SigmaAldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated FCS (Biological Industries, Haemek, Israel), 100 U ml1 penicillin, 100 µg ml1 streptomycin, 100 µM 2-mercaptoethanol, 100 µM MEM non-essential amino acids (Invitrogen, Leek, The Netherlands) and 10% pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM) as a source of IL-3 (15).
Reverse transcriptionPCR analysis
Total RNA was prepared from bone marrow-derived cells using TRIzol Reagent (Invitrogen). Messenger RNA (mRNA) was detected by reverse transcription (RT)PCR using 1 µg of total RNA, oligo dT primer (Invitrogen), Superscript II (Invitrogen) and Advantage 2 polymerase (Clontech Laboratories, Palo Alto, CA, USA). Primer sets used to amplify each target cDNA were as follows:
GATA-1, 5'-ATGGATTTTCCTGGTCTAGGGGC-3' and 5'-TCAAGAACTGAGTGGGGCGATCACG-3'; Fc
RI
-chain, 5'-CAGTAAGCACCAGGAGTCCATGAAGAAGA-3' and 5'-CCTTGAGCACAGACGTTTCTATGTATATT-3'; Fc
RI ß-chain, 5'-GAGCAGAGCAGATCTTGCTC-3' and 5'-ATAAAGACGATCATCTGGGA-3'; Fc
RI
-chain, 5'-CAGCTCTGCTATACCTGGATGC-3' and 5'-GGTGGTTTTTCATGCTTCAGAGTC-3'; ß-actin, 5'-GCGCTCGTCGTCGACAACGG-3' and 5'-CATCGGAACCGCTCATTGCC-3'.
Real-time PCR analysis
Real-time PCR analysis was performed to quantify the transcription level of target molecules as described previously (16). In brief, the amount of mRNAs for Fc
RI
-, ß- and
-chains and GATA-1 and GATA-2 were quantified using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with Assays-on-Demand gene expression products (no. Mm00438867_m1 for mouse Fc
RI
, no. Mm00442780_m1 for mouse Fc
RIß, no. Mm00438869_g1 for mouse Fc
RI
, no. Mm00494678_m1 for mouse GATA-1 and no. Mm00492300_m1 for mouse GATA-2). The expression level of the target molecules was evaluated with a ratio to that of GAPDH by calculation of cycle threshold (Ct) values in amplification plots with 7500 SDS software. The expression level of each molecule in KD bone marrow-derived mast cells (BMMCs) was shown as a ratio to that of each control WT BMMCs (=100%) according to the following equations:
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where
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and
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Flow cytometric analysis
Cell-surface FcRs were blocked with 2.4G2 (PharMingen, San Diego, CA, USA) before staining. We used a PE-conjugated anti-mouse c-kit (PharMingen) to stain c-kit. Mouse Fc
RI was stained with a mouse IgE antibody (PharMingen) in conjugation with FITC. The cells were incubated with 1 µg of 2.4G2, 0.5 µg of PE-conjugated anti-c-kit and 0.5 µg of FITC-mouse IgE in 50 µl of PBS for 1 h at 4°C, washed with PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Fc
RI-high and -low fractions were separated and collected by cell sorting using a FACSVantage (Becton Dickinson).
Western blot analysis
Cells (3 x 105) from each fraction were western blotted. Anti-GATA-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the primary antibody to detect GATA-1 protein. Peroxidase-conjugated anti-rat IgG goat antibody (Wako Pure Chemical Industries, Osaka, Japan) was used as the secondary antibody. Membranes were soaked with the ECL plus western blotting detection reagent (Amersham Biosciences corp., Piscataway, NJ, USA), and chemiluminescence was detected using a LAS-1000 plus (Fuji Film, Tokyo, Japan).
Degranulation
The ß-hexosaminidase-releasing assay proceeded as previously described (17). Cells (3 x 105 per 300 µl) were incubated with anti-trinitrophenol (TNP) IgE (PharMingen; 1 µg per 300 µl) for 1 h at 4°C in Tyrode's buffer (10 mM HEPES buffer, pH 7.4, 130 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.1% BSA). These cells were washed with Tyrode's buffer and re-suspended in 300 µl Tyrode's buffer containing TNPovalbumin peptide (OVA) (10 ng ml1), 1 mM CaCl2 and 0.6 mM MgCl2. After 1 h incubation at 37°C, supernatants from TNPOVA/IgE-stimulated cells and total cell lysates solubilized with 1% Nonidet P-40 were collected and ß-hexosaminidase activity in supernatants or lysates was quantified by spectrophotometric analysis (405 nm) after the hydrolysis of p-nitrophenyl-N-acetyl-ß-D-glucopyranoside (SigmaAldrich). We evaluated ß-hexosaminidase release as described (17).
Cytokine measurements
Cells (1 x 106 cells per 300 µl) were incubated at 4°C in complete culture medium with anti-TNP IgE (1 µg per 300 µl) for 1 h, similar to the case of degranulation experiment as described above and then stimulated with TNPOVA (10 ng per 300 µl) for 6 h at 37°C. Concentrations of IL-6 in the culture supernatant were determined by ELISA kits according to the manufacture's instructions (Genzyme Techne, Minneapolis, MN, USA).
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Results
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GATA-1 affected Fc
RI and c-kit expression on surface of BMMCs
Mast cells are generated from mouse bone marrow-derived cells after 45 weeks culture in the presence of PWM-SCM as a source of IL-3 (15). We examined the involvement of GATA-1 in the development of BMMCs as follows. We cultured bone marrow-derived cells of female heterozygous GATA-1 KD and female control littermates, as WT normal mice, because male GATA-1 KD embryos die by 12.5 d.p.c. (2). The surface expression profiles of Fc
RI and c-kit, both of which are expressed at high levels on mast cells in a cell type-specific manner, were analyzed by FACS. The population of Fc
RI+/c-kit+ was decreased in cells from heterozygous mice (Fig. 1) compared with control littermates. The number of cells that were Fc
RI+/c-kit, Fc
RI/c-kit+ and Fc
RI/c-kit was increased in most cases compared with the controls. After 67 weeks of culture, the Fc
RI/c-kit+ fraction decreased to less than 10% in most cases, although the corresponding fraction remained in KD-1 even under the same conditions (33.1%). Thus, the surface expression of Fc
RI and c-kit were decreased in mast cells prepared from GATA-1 KD heterozygous mice. In contrast, no differential frequency of each population was observed between KD-5 and WT-5. Therefore, it should be also noted that the degree of the decrease in surface expression varied individually. When mean fluorescence intensity indicating the expression level of Fc
RI was compared between WTs and KDs, the ratio of WT-1/KD-1 was the highest and that of WT-5/KD-5 was the lowest (Table 1).
Transcription levels of Fc
RI
-, ß- and
-chain genes in GATA-1 KD and WT BMMCs
Expression levels of GATA-1 in heterozygous knockdown female mice always differ among individuals because of random inactivation of the X chromosome, where the GATA-1 gene is localized (2). Therefore, the difference in FACS profiles among KDs in Fig. 1 may have arisen from differences in GATA-1 expression due to random inactivation of the X chromosome of the KD allele. To confirm this notion, we detected GATA-1 mRNA using RTPCR. We also analyzed mRNAs for Fc
RI
-, ß- and
-chains by RTPCR to determine whether the decrease in the surface expression of Fc
RI in KD cells could be attributed to a decrease in the expression of specific chain(s) of Fc
RI because Fc
RI is an oligomeric receptor composed of
-, ß- and
-chains. Figure 2(A) shows ethidium bromide staining of the PCR products corresponding to GATA-1, Fc
RI
-, ß- and
-chains and ß-actin of three pairs of WT and KD; KD-1 showing the severest suppression of Fc
RI, KD-4 expressing moderately decreased level of Fc
RI and KD-5 expressing normal level of Fc
RI. When the expression levels of GATA-1 in BMMCs after 5 weeks cultivation were compared between WTs and KDs, the most striking difference was observed between WT-1 and KD-1 and a difference was also apparent between WT-4 and KD-4, whereas the GATA-1 level was not affected in KD-5 cells. The profile of
-chain expression was similar. In brief, differences between WT and KD were in the following order: WT-1/KD-1 > WT-4/KD-4 > WT-5/KD-5 at 5 weeks; WT-1/KD-1, WT-4/KD-4 > WT-5/KD-5 at 2 weeks. To compare the transcription level quantitatively, mRNA level of Fc
RI
-, ß- and
-chains and GATA-1 and GATA-2 in BMMCs after 5 weeks cultivation was analyzed by real-time PCR (Fig. 2B). The suppression level of Fc
RI
-chain, ß-chain and GATA-1 in KD BMMCs was similar to that observed in RTPCR. These results closely coincided with the FACS profile shown in Fig. 1, in which apparent Fc
RI-negative fraction was present in KD-1 even at 67 weeks and number of Fc
RI-positive cells in KD-5 was increased at a ratio that was similar to those of other WTs, whereas the Fc
RI-negative fraction of KD-4 disappeared after culture for 47 weeks. These observations indicated that the expression level of GATA-1 varies in KD cells but closely correlates with both the frequency of Fc
RI-positive cells and the transcription level of
-chain. We also detected a decrease in ß-chain mRNA in KD-1 and KD-4 but not in KD-5 (Fig. 2), although the sensitivity of ß-chain detection was not so high in this experiment. On the other hand,
-chain and ß-actin (control) expression did not differ between WTs and KDs. In addition, GATA-2 expression level in the 5-week KD BMMCs was comparable to that of WT BMMCs (Fig. 2B, e), suggesting that suppression of Fc
RI
- and ß-chain transcription is directly affected by GATA-1 but not GATA-2. These results indicated that the decrease in GATA-1 expression is responsible for the decrease in the expression of Fc
RI
- and ß-chains in KD cells, which subsequently increased the Fc
RI-low/negative fraction.

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Fig. 2. Transcription of GATA-1, Fc RI -, ß- and -chains and ß-actin in BMMCs. (A) RTPCR analysis. Numbers 1, 4 and 5 at the top indicate each pair of WT-1/KD-1, WT-4/KD-4 and WT-5/KD-5. Lanes 1, 2 and 3 in each panel are WT, heterozygous KD and negative controls without template, respectively. PCR products obtained at various cycle numbers were applied onto agarose gel and detected by ethidium bromide staining as shown in Fig. 3, and one selected profile showing the difference is presented in each panel: 30 cycles (GATA-1), 25 cycles ( -chain), 30 cycles (ß-chain), 20 cycles ( -chain) and 20 cycles (ß-actin). (B) Quantitative analysis by real-time PCR. The expression level of target molecules (target molecule/GAPDH) of each KD is represented as the ratio to that of a pair of WT by setting the expression level in WT at 100%.
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Comparison between Fc
RI-high and -low/negative fractions
The number of Fc
RI-negative and/or c-kit-low cells was decreased as the cultivation period was prolonged. However, these cells were detected in KD cells (KD-1, 2, 3 and 4) cultured for 4 weeks and a significant portion of KD-1 cells still showed the phenotype even after 67 weeks of culture. In contrast to KD cells, cells positive for both markers constituted the majority of WT cells (
90% after 67 weeks in culture) (Fig. 1). These results suggested that fractions expressing low levels of markers originated from the KD cells expressing low levels of GATA-1. To clarify this, we sorted KD-1 cells into Fc
RI-high and Fc
RI-low/negative fractions (Fig. 3A), and compared Fc
RI and GATA-1 expression levels by semi-quantitative RTPCR. The amounts of GATA-1,
-chain and ß-chain transcripts in the Fc
RI-low/negative fraction were decreased, and even in the Fc
RI-high fraction, the transcription levels of GATA-1 and
-chain were slightly lower than those of WT (Fig. 3B). Consistent with these findings, western blotting using anti-GATA-1 antibodies revealed that the expression of GATA-1 was obviously decreased in Fc
RI-low/negative cells in KD-1 (Fig. 3C, left). Similar decreases in both the transcripts of GATA-1,
-chain and ß-chain (data not shown) and in GATA-1 protein production (Fig. 3C, right) were also detected in KD-2 cells, which were separated into Fc
RI-high and -low fractions, after 5 weeks of culture. These results indicated that cells expressing the GATA-1 KD allele indeed expressed both the GATA-1 gene and its product at lower levels, which subsequently caused the transcription of Fc
RI
- and ß-chains to decrease.
Comparison of stimulation via Fc
RI between GATA-1 KD and WT BMMCs
Fc
RI is a key receptor for the antigen/IgE-mediated activation of mast cells. A previous report described that increase of Fc
RI expression on mouse mast cell surface affects degranulation of the cells and release of the cytokines, IL-6 and IL-4 (18). Therefore, decreased surface expression of Fc
RI on mast cells that were derived from the bone marrow of KD mice would result in a decreased response to antigen/IgE stimulation. We treated the cells with specific antigen (TNPOVA) after incubation with anti-TNP mouse monoclonal IgE and measured the activity of released ß-hexosaminidase which is used as a general marker of mast cell degranulation. Antigen/IgE stimulation caused the release of ß-hexosaminidase in all the KD cells (KD-1, 3, 4 and 5) (Fig. 4). However, KD cells released less ß-hexosaminidase than the WT. This observation was consistent with the FACS profiles of Fc
RI+/c-kit+ cells (Fig. 1). KD-4 cells were degranulated to a lesser extent (
10%) after 5 weeks of culture but this increased (to
40%) after 7 weeks of culture. These findings corresponded with the fact that the expression of both Fc
RI and c-kit increased to a level comparable to that of WT after longer cultivation (Fig. 1). When KD cells were treated with phorbol myristate acetate (PMA) plus ionomycin, which stimulated mast cells in an IgE-independent manner, significant amount of ß-hexosaminidase was released. Considering that the release of ß-hexosaminidase in response to antigen/IgE stimulation was much more significantly suppressed in KD cells, we presume that the degranulation of KD cells was mainly suppressed through low expression of Fc
RI. It should be noted that the release of ß-hexosaminidase was moderately decreased (KD-1, KD-3 and KD-4 after 5 weeks) or comparable to that of WT (KD-4 after 7 weeks, and KD-5). The observation that moderately decreased degranulation was also observed in PMA plus ionomycin-stimulated KD cells suggests that the expression of signaling intermediates, which are involved in the release of ß-hexosaminidase, might be also regulated by GATA-1.

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Fig. 4. Activity of ß-hexosaminidase released from BMMCs upon antigen/IgE stimulation. Pairs of WT-1/KD-1 (a), WT-3/KD-3 (b), WT-4/KD-4 (c) and WT-5/KD-5 (e) were stimulated after 5 weeks of culture. The WT-4/KD-4 pair was stimulated after 7 weeks of culture (d). Cells exposed to anti-TNP mouse IgE antibodies were incubated with (Ag) or without () TNPOVA. PMA plus ionomycin (P/I) was also used for IgE-independent stimulation of degranulation of mast cells. The results are expressed as the mean + SD of triplicate samples.
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In response to antigen/IgE stimulation, mast cells are known to produce and release cytokines including IL-6. This suggests that a signal by the antigen/IgE stimulation is transmitted through Fc
RI. As described above, lower expression of GATA-1 caused the apparent decrease in degranulation in KD cells through the decreased expression of Fc
RI. Then, we measured IL-6 released from mast cells after stimulation. Unexpectedly, most KDs showed no apparent reduction in IL-6 production, although only KD-1 exhibited decreased production of IL-6 (Fig. 5). This suggests that IL-6 production level does not always correlate with the expression level of Fc
RI.

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Fig. 5. IL-6 production form BMMCs upon antigen/IgE stimulation. Pairs of WT-1/KD-1 (a), WT-3/KD-3 (b) and WT-4/KD-4 (c) were cultured for 5 or 6 weeks, and the cells were exposed to anti-TNP mouse IgE antibodies and stimulated with (Ag) or without () the antigen, TNPOVA.
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Effect of stem cell factor on expression of Fc
RI and c-kit on surface of GATA-1 KD cells
Stem cell factor (SCF) is a c-kit ligand that promotes the differentiation of mast cells and is often used with IL-3 to generate BMMC. We investigated the effect of GATA-1 knockdown on the response of Fc
RI and c-kit expression to SCF stimulation as follows. We performed FACS analysis of GATA-1 knockdown mast cells after incubation in medium containing recombinant mouse SCF (Fig. 6). About 90% of the total WT cells derived from bone marrow were Fc
RI+/c-kit+ when cultured for 4 weeks in medium without SCF (Fig. 6a and e), and SCF slightly increased the population of these cells (Fig. 6c and g). On the other hand, only 63.4% of KD-2 and 37.0% of KD-4 cells were double positive under the same culture conditions (Fig. 6b and f). After 35 days of culture with SCF, the number of c-kit-negative cells significantly decreased from 29.2 to 2.0% (KD-2) and from 36.1 to 4.8% (KD-4). Thus, the number of cells positive for the two surface markers was clearly increased (89.8% of KD-2 and 72.5% of KD-4). On the other hand, the fraction of Fc
RI/c-kit+ cells was not affected by SCF (from 7.4 to 8.2% in KD-2 and from 26.9 to 22.7% in KD-4). We concluded that SCF increases Fc
RI/c-kit-double positive cells by increasing the functional expression of c-kit and that GATA-1 is not involved in this cellular process.

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Fig. 6. Effect of SCF on expression of Fc RI and c-kit. BMMCs of WT-2/KD-2 (A) and WT-4/KD-4 (B) were cultured for 4 weeks in medium containing PWM-SCM and then stimulated with 5 ng ml1 of SCF for 3 and 5 days, respectively. Numbers indicate frequency of cells in each quadrant.
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Discussion
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We analyzed the transcriptional regulation of the
- and ß-chains of Fc
RI (1113, 1923), a mast cell marker, and found that exogenously expressed GATA-1 up-regulates the promoters of
-chain and ß-chain through binding to GATA motifs in their promoter regions (1113, 23). Functional roles of GATA-1 in the development of erythroid, megakaryocytes and eosinophils have been analyzed by using GATA-1 gene mutant mice. However, there are few studies analyzing the role of GATA-1 in mast cells prepared from GATA-1 mutant mice. To further clarify the role of GATA-1 in Fc
RI expression, the present study analyzed the expression of Fc
RI in mast cells derived from GATA-1 KD mouse bone marrow cells. Expression of Fc
RI
-chain and ß-chain but not
-chain was lower in mast cells derived from GATA-1 KD heterozygous mouse bone marrow cells, when compared with those from WT control littermates. Consistent with the decreased expression of the genes encoding the
- and ß-chains of the receptor, the response of KD mast cells to antigen/IgE stimulation via Fc
RI was decreased. The frequency of Fc
RI-positive cells and the transcription level of Fc
RI
- and ß-chains in cells from KD mice were decreased but to different extents. GATA-1 KD heterozygous mice contain both kinds of mast cells, one producing normal levels of GATA-1 and the other producing lower levels by lionization. Since both Fc
RI expression and
- and ß-chain transcription closely correlated with GATA-1 expression, we assumed that GATA-1 KD causes the reduction in Fc
RI-expressing cells and therefore Fc
RI expression in the mass depends on the degree of inactivation of GATA-1 gene expression on the X chromosome. These results showed that Fc
RI expression is, in fact, under the control of GATA-1.
The expression of c-kit, another mast cell marker, was also low in mast cells derived from KD mice (Fig. 1). The GATA motif has not been found in the c-kit promoter, but a GATA factor might be involved in c-kit expression by forming a complex with other transcription factors such as SCL, LMO2, E2A and Ldb-1, and the complex binds to the transcription factor Sp1 that recognizes the Sp1 motif in the c-kit promoter (24). The present study showed that SCF stimulation rescued this phenotype (Fig. 5). These results indicated that the signaling from c-kit by binding SCF to induce self-expression could compensate for the decreased GATA-1 function in c-kit expression. This suggests that the SCF stimulation signal required to activate c-kit expression might be mediated by other transcription factors. Consistent with this, SCF stimulation did not up-regulate GATA-1 expression (data not shown). In addition, the expression of Fc
RI that is regulated by GATA-1 directly binding to the promoters for Fc
RI
- and ß-chain genes was not affected by SCF stimulation. However, transcription level of c-kit in response to SCF stimulation has not been examined in this study and the possible involvement of other regulatory mechanisms, such as translation and post-translational event, in high c-kit expression through the SCF stimulation could not be excluded. Further detailed analysis will be required to clarify this point.
Considering that apparent differences were not observed between KD cells and control cells in May-GrünwaldGiemsa's and toluidine blue staining profiles nor in total ß-hexosaminidase activity (data not shown), GATA-1 might not affect granule formation in BMMC. This observation is consistent with the study using a male hemizygous
dblGATA mutant mouse BMMCs, which were stained by GrünwaldGiemsa and toluidine blue (8). We think that further detailed morphological analysis should be performed to clarify the involvement of GATA-1 in the development of mast cells.
In the culture of BMMC, mast cells derived from GATA-1 KD mice showed better growth in most cases except for WT-5/KD-5 (data not shown). Considering that GATA-1-negative megakaryocytes exhibit enhanced proliferation (46), we assume that low levels of GATA-1 production caused hyper-proliferation of mast cells as well. On this point, further detailed analysis is required to clarify the role of GATA-1 in proliferation of mast cells.
By the comparison of transcription level by real-time PCR, we found that the transcription of GATA-2 in KD cells were comparable to that of WT cells after 5 weeks culture. This observation indicates that the suppressed transcription of Fc
RI
- and ß-chains is directly affected by GATA-1 but not GATA-2, although mast cells express both GATA family proteins. However, we cannot exclude the possible involvement of GATA-2 on Fc
RI expression because the expression level of GATA-1 and GATA-2 varies depending on the developmental stage of mast cells; GATA-2 is abundantly expressed in mast cell progenitors and GATA-1 is abundant in maturated mast cells (10). Further detailed analysis of KD mast cells at various development stages is required to clarify this issue.
Recently, Migliaccio et al. analyzed surface expression profile of Fc
RI on mast cells derived from bone marrow of GATAlow mice, and reported the slight decrease in frequency of Fc
RI+ cells and somewhat less expression of Fc
RI on the surface even in Fc
RI-producing cells (25). In this study, we definitively demonstrated that a GATA-1 KD caused the marked decrease in Fc
RI and c-kit expression in mast cells. On the contrary, Yu et al. showed that the expression of c-kit and Fc
RI on mast cells derived from
dblGATA mice bone marrow was not greatly affected by the mutation in the GATA-1 promoter (8). The difference in the effect of these GATA-1 knockdown experiments were probably due to the degree of GATA-1 promoter inactivation in each condition. To create the GATA-1 KD mouse used in this study, a neogene was inserted between the double GATA motifs and the transcription start site of the GATA-1 promoter. In the case of GATAlow mice, a region including a distal promoter and a DNase I hypersensitive region was replaced with a neogene. Thus, in both cases, a decrease in the transcript for GATA-1 is expected. In the mutant mice
dblGATA, on the other hand, double GATA motifs in the GATA-1 promoter were replaced with a loxP sequence. As the authors described, eosinophil-specific regulation would be lost by the mutation, but the mutation would not have an effect of GATA-1 on Fc
RI and c-kit expression in mast cells (8). The populations of Fc
RI+/c-kit+ cells after 7 weeks of culture were 89.4 and 58.8% for hemizygous
dblGATA (8) and heterologous knockdown cells used in this study (Fig. 1C), respectively. The frequency of Fc
RI cells in c-kit+ mast cells derived from GATAlow bone marrow is lower than those of most KD cells (25) (Fig. 1). It is therefore obvious that the effect of the mutation in the GATA-1 KD mouse is more serious than those of
dblGATA and GATAlow to the GATA-1 promoter function in mast cells. To understand the differences in the effect of GATA-1 promoter mutation precisely, quantitative comparison of GATA-1 transcript prepared from BMMCs in the same manner is required.
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Acknowledgements
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We thank members of Atopy (Allergy) Research Center and Department of Immunology for their helpful discussions. We are grateful to T. Sakanishi for operating the cell-sorting system as well as to R. Shimizu (University of Tsukuba), A. Nakao (University of Yamanashi), H. Yagita, T. Kuhara, N. Tada and H. Ushio for critical advice. We commend H. Kawada, A. Takagi, S. kanada, Y. Niwa, and T. Tokura for technical assistance, and M. Matsumoto and E. Kawasaki for secretarial assistance. This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.N.).
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Abbreviations
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BMMC | bone marrow-derived mast cells |
Ct | cycle threshold |
KD | knockdown |
mRNA | messenger RNA |
OVA | ovalbumin peptide |
PMA | phorbol myristate acetate |
PWM-SCM | pokeweed mitogen-stimulated spleen-conditioned medium |
SCF | stem cell factor |
TNP | trinitrophenol |
WT | wild type |
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Notes
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Transmitting editor: S. Koyasu
Received 9 March 2004,
accepted 8 April 2005.
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