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Originally published In Press as doi:10.1074/jbc.M407026200 on September 16, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48923-48929, November 19, 2004
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MITF Is Necessary for Generation of Prostaglandin D2 in Mouse Mast Cells*

Eiichi Morii{ddagger} and Keisuke Oboki§

From the Department of Pathology, Osaka University Medical School, Suita, Osaka 565-0871, Japan

Received for publication, June 23, 2004 , and in revised form, September 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. A basic-helix-loop-helix leucine zipper transcription factor termed MITF is essential for the development of mast cells. Although other substances also linked to inflammatory reactions (such as various proteases and serotonin) require MITF for their expression, the role of MITF in eicosanoid generation has not been studied. We examined eicosanoid generation in bone marrow-derived mast cells (BMMCs) of tg/tg mice that lack MITF. Most eicosanoids generated by BMMCs are either prostaglandin (PG) D2 or leukotriene C4. The former is synthesized via the cyclooxygenase pathway, whereas the latter is synthesized via the 5-lipoxygenase pathway. In response to stimulation with IgE and antigens, BMMCs of tg/tg mice synthesized leukotriene C4 normally. However, neither immediate nor delayed PGD2 production was detected in these BMMCs. This indicates that MITF is a transcription factor that specifically activates the cyclooxygenase pathway, but not the 5-lipoxygenase pathway. Significant decreases in expression of hematopoietic PGD2 synthase (hPGDS, a terminal synthase for PGD2) were observed at both mRNA and protein levels in tg/tg BMMCs. MITF transactivated the hPGDS gene via a CACCTG motif located in the promoter region. MITF appeared to be essential for generation of PGD2 by enhancing expression of the hPGDS gene in BMMCs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells function as effectors in inflammatory reactions by secreting various chemical mediators, such as histamines, serotonin, proteases, and eicosanoids (1, 2). Eicosanoids are known to regulate allergic and inflammatory responses, including recruitment of eosinophils, Th2 cells, and basophils, induction of bronchoconstriction, and relaxation of smooth muscle contraction (3, 4). Mast cell development is regulated by a basic helix-loop-helix leucine zipper transcription factor named MITF (5–8). Bone marrow-derived mast cells (BMMCs)1 derived from transgene-insertional Mitf mi-vga9/Mitf mi-vga9 mutant mice (hereafter called tg/tg mice), which do not express MITF owing to an insertion in the promoter region of the gene (5), showed abnormal phenotypes including deficiencies in expression of mouse mast cell protease-4 (9), -5 (10), -6 (11), -7 (12), transmembrane-type tryptase (13), granzyme B (14), and tryptophan hydroxylase (14), the rate-limiting enzyme for serotonin synthesis. Although the importance of MITF in the expression of proteases and serotonin-synthesizing enzyme has been demonstrated, no study has investigated the role of MITF in the generation of eicosanoids in mast cells.

Stimulation of BMMCs by cross-linking of Fc{epsilon}RI with IgE and antigens elicits secretory granule exocytosis and immediate eicosanoid generation (15, 16). BMMCs generate mainly prostaglandin D2 (PGD2) and LTC4 (16). The former is generated via the cyclooxygenase pathway, whereas the latter is generated via the 5-lipoxygenase pathway. The immediate generation of PGD2 and LTC4 is completed within 10 min of stimulation. When BMMCs are cultured with Kit ligand (KitL), IL-1{beta}- and IL-10-delayed synthesis of PGD2, but not of LTC4, occurs 2–8 h after stimulation with IgE and antigen (17–20). The immediate synthesis of PGD2 is mediated by constitutively expressed PG endoperoxide H synthase (PGHS)-1, whereas the delayed synthesis of PGD2 is mediated by inductively expressed PGHS-2. PGH2 is generated in BMMCs by both PGHS-1 and -2 and is unstable, quickly being metabolized to PGD2 by hematopoietic PGD2 synthase (hPGDS) (21). Recently, Stevens and coworkers (22) demonstrated that Ras guanine nucleotide-releasing protein (RasGRP) 4 regulates expression of the hPGDS gene and that mast cell lines with low RasGRP4 expression levels show defective generation of PGD2 but normal generation of LTC4. RasGRP4 appeared to be involved in the cyclooxygenase pathway, but not in the 5-lipoxygenase pathway. In our study we examined eicosanoid generation in BMMCs of tg/tg mice and found that PGD2, but not LTC4, synthesis requires MITF. MITF appears to be involved in the cyclooxygenase pathway but not in the 5-lipoxygenase pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Cells—The original stock of tg/tg mice, in which the mouse vasopressin-Escherichia coli {beta}-galactosidase transgene was integrated at the promoter region of the MITF gene, was provided by Dr. H. Arnheiter (National Institutes of Health, Bethesda, MD) (5). The tg/tg mice were maintained by consecutive backcrosses to our own C57BL/6 (B6) and WB inbred colonies for more than 15 generations. Female B6-tg/+ and male WB-tg/tg mice were crossed, and the resulting (WB x B6) F1 (WBB6F1)-tg/tg mice were selected by their white coat color. WBB6F1-+/+ mice were purchased from Japan SLC (Hamamatsu, Japan). WBB6F1-+/+ and -tg/tg mice were termed +/+ and tg/tg mice, respectively. BMMCs were established from 4–6-week-old +/+ or tg/tg mice by culturing bone marrow cells with {alpha}-minimal essential medium (ICN Biomedicals, Costa Mesa, CA) containing 10 ng/ml recombinant mouse (rm) interleukin (IL)-3 (R&D, Minneapolis, MN) for ~4–6 weeks. In some experiments, BMMCs established in the medium containing IL-3 were stimulated with {alpha}-minimal essential medium containing rmIL-10 (10 ng/ml; R&D), rmIL-1{beta} (5 ng/ml; R&D) and rmKitL (50 ng/ml; R&D) for 2 h.

Stimulation of BMMCs—BMMCs were suspended at a concentration of 1 x 107 cells/ml in medium containing cytokines for eicosanoid generation or in Tyrode's buffer containing 1.8 mM Ca2+, 0.2 mM Mg2+, 0.4% (w/v) bovine serum albumin (type V; Sigma), and 10 mM Hepes (pH 7.2) for {beta}-hexosaminidase release. The suspended cells were sensitized with 1 µg/ml anti-dinitrophenyl (DNP) IgE (Sigma) for 2 h, washed, and then elicited with various concentrations of DNP-conjugated human serum albumin (HSA; Sigma).

Measurement of {beta}-Hexosaminidase Release and Generation of Eicosanoids—The assay for {beta}-hexosaminidase release was performed 30 min after elicitation with DNP-HSA. BMMCs were precipitated, and {beta}-hexosaminidase activity in the supernatant and in the cell pellets (after lysis by freeze-thawing) was quantitated by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-{beta}-D-2-acetamido-e-deoxyglucopyranoside (Sigma). The percent release of {beta}-hexosaminidase was calculated by the formula [S/(S + P)], where S and P are the {beta}-hexosaminidase contents of equal portions of supernatant and cell pellet, respectively. PGD2 and LTC4 levels were quantified in the supernatant of elicited BMMCs using enzyme immunoassay kits according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI). In some experiments, the amount of PGD2 generated was measured in BMMCs preincubated with 1 µg/ml indomethacin (Sigma). In these cases, BMMCs were sensitized with anti-DNP IgE in the presence of indomethacin, washed, and then elicited with DNP-HSA.

Transcript Analysis of Stimulated BMMCs of +/+ or tg/tg Mice—The expression profiles of genes in +/+ or tg/tg BMMCs stimulated with anti-DNP IgE alone or stimulated with anti-DNP IgE and then elicited with DNP-HSA for 30 min were examined with a CodeLink UniSet Mouse 20K I bioarray (Amersham Biosciences) using 2 µg of total RNA extracted with an RNeasy column (Qiagen, Valencia, CA). Experimental procedures, including the synthesis of double-stranded cDNA and biotin-labeled cRNA target, and the analysis of results were performed by Kurabo Co. Ltd. (Osaka, Japan).

Quantification of mRNA Levels by Real-time RT-PCR—RNA was extracted from BMMCs using an RNeasy kit (Qiagen) with DNase I treatment. BMMCs cultured under the following four conditions were used: unstimulated, sensitized with anti-DNP IgE alone, sensitized with anti-DNP IgE and elicited with DNP-HSA for 30 min, or sensitized with anti-DNP IgE and elicited with DNP-HSA for 120 min. The mRNA levels for RasGRP4, PGHS-1, PGHS-2, hPGDS, and glyceraldehyde-3-phosphate dehydrogenase genes were verified using a TaqMan Universal PCR Master Mix and Assays-on-Demand primers from Applied Biosystems (Foster City, CA). The primers and probes used for cell cycle regulators were Assays-on-Demand gene products. The mRNA levels for each gene were normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA.

Immunoblot Analysis—BMMCs were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. The resulting lysates were separated on 10% SDS-polyacrylamide gels, transferred to Immobilon (Millipore, Bedford, MA), and reacted with anti-hPGDS, anti-PGHS-1, and anti-PGHS-2 (Cayman) and with an anti-actin (Sigma). After washing, the blots were incubated with an appropriate peroxidase-labeled secondary antibody and then reacted with Renaissance reagents (PerkinElmer Life Sciences) before exposure.

Nuclear Run-on Assay—A nuclear run-on assay was used to measure gene transcription rates. BMMCs (2 x 107) were washed twice in phosphate-buffered saline, lysed in buffer containing 10 mM HEPES (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40, and incubated for 7 min on ice. Nuclei were isolated by centrifugation at 600 x g for 5 min, resuspended in buffer containing 20 mM HEPES (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA, and 40% glycerol, and stored until use. Frozen nuclei (100 µl) were added to 100 µl of buffer containing 20 mM HEPES-KOH (pH 8.0), 25% glycerol, 10 mM MgCl2, 0.2 mM KCl, 1.2 mM ATP, 0.6 mM CTP, and 0.6 mM GTP. After the addition of 40 units/ml RNase inhibitor and 100 µCi of [{alpha}-32P]UTP, the mixture was incubated at room temperature for 45 min. The labeled RNA was passed through a QIAshredder column (Qiagen) and purified with a Qiagen RNeasy kit according to the manufacturer's protocol. The labeled RNA from +/+ and tg/tg BMMCs was hybridized to dot blots containing 2 µg of purified fragments of hPGDS cDNA, {beta}-actin cDNA (as a positive control), or pEF-BOS expression vector (as a negative control) immobilized onto nylon filters. Blots were hybridized for 48 h in Church buffer (7% SDS, 0.5 M phosphate buffer (pH 7.5), 1% bovine serum albumin). The blots were then washed twice in 2x SSC, 0.1% SDS and once in 0.1x SSC, 0.1% SDS. The membranes were then exposed to x-ray films.

Luciferase Assay—The DNA fragment containing the promoter and 5'-untranslated region of the hPGDS gene (nt –1500 to +200, where +1 is a transcription initiation site) was amplified by PCR from genomic DNA of +/+ BMMCs with LA-Taq DNA polymerase (Takara, Kyoto, Japan). This fragment was cloned upstream of the luciferase gene, and the reporter plasmid was constructed. Reporter plasmids sequentially deleted in the promoter region or mutated at the CACCTG motif were also constructed by PCR. The sequence of all constructs was verified using an ABI 3100 sequencer (Applied Biosystems). 10 µg of a reporter, 1 µg of a pEF-BOS expression vector containing MITF cDNA or containing no insert, and 1 µg of an expression vector containing the {beta}-galactosidase gene were cotransfected into tg/tg BMMCs by electroporation (350 V, 500 µF). In each transfection, 5 x 106 tg/tg BMMCs were used. The cells were harvested 48 h after transfection, and the soluble extracts were assayed for luciferase and {beta}-galactosidase activity. The normalized value by {beta}-galactosidase activity was expressed as relative luciferase activity. The effect of MITF on the reporter plasmid was demonstrated by the ratio of the relative luciferase activity with MITF to the relative luciferase activity without MITF (-fold activation).

Electrophoretic Gel Mobility Shift Assay—Production of the fusion protein containing glutathione S-transferase (GST) and MITF was as previously described (23). To examine whether MITF bound to the CACCTG motif mediating the transactivation ability, an oligonucleotide containing this motif was used as a probe. The sequence of the oligonucleotide was 5'-CACACAAGCACCTGTGACTGCGACTT (the CACCTG motif is underlined). The oligonucleotide was labeled with [{alpha}-32P]dCTP by filling 5'-overhangs and used as a probe for electrophoretic gel mobility shift assay. DNA binding assays were performed in a 20-µl reaction mixture containing 10 mmol/liter Tris-HCl (pH 8.0), 1 mmol/liter ethylenediaminetetraacetic acid (EDTA), 75 mmol/liter KCl, 1 mmol/liter dithiothreitol, 4% Ficoll type 400, 50 ng of poly(dI-dC), 25 ng of labeled DNA probe, and 3.5 µg of GST-MITF fusion protein. After incubation at 37 °C for 15 min, the reaction mixture was subjected to electrophoresis at 14 volt/cm at 4 °C on a 5% polyacrylamide gel in 0.25x TBE buffer (1x TBE is 90 mmol/liter Tris-HCl, 64.6 mmol/liter boric acid, and 2.5 mmol/liter EDTA, pH 8.3). In some experiments, GST was used as a protein instead of GST-MITF. Non-labeled oligonucleotides containing the CACCTG motif or non-labeled oligonucleotides mutated from CACCTG to CTCCAG were added in a competition assay. Polyacrylamide gels were dried on Whatman 3MM chromatography paper and subjected to autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of MITF on the Response of BMMCs Stimulated with IgE and Antigens—The effect of MITF on the response of BMMCs after stimulation with IgE and antigens had not previously been investigated. We examined this by measuring {beta}-hexosaminidase release from BMMCs of tg/tg mice that effectively lacked MITF (5, 23) in response to IgE and antigens. BMMCs were sensitized with anti-DNP IgE, washed, and elicited by various concentrations of DNP-HSA. The released {beta}-hexosaminidase was measured 30 min after elicitation. Levels of released {beta}-hexosaminidase increased in a dose-dependent manner from 5 to 100 ng/ml DNP-HSA, thereafter reaching a plateau (Fig. 1). The +/+ and tg/tg BMMCs showed comparable patterns of {beta}-hexosaminidase release after stimulation with IgE and antigens (Fig. 1).



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  		FIG. 1.
Effect of the concentration of antigen on {beta}-hexosaminidase release in +/+ and tg/tg BMMCs. BMMCs derived from +/+ and tg/tg mice were established in rmIL-3, sensitized with 1 µg/ml anti-DNP IgE for 2 h, washed, and then elicited using various concentrations of DNP-HSA. The concentration of {beta}-hexosaminidase in the supernatants and the cell pellets was measured 30 min after elicitation. The percent release of {beta}-hexosaminidase is shown. The values represent the mean ± S.E. of five experiments.

 
Production of Eicosanoids in BMMCs Derived from tg/tg Mice—We next examined the generation of eicosanoids in BMMCs of tg/tg mice. BMMCs sensitized with anti-DNP IgE were elicited with 100 ng/ml DNP-HSA, at which concentration the effect of DNP-HSA reached a plateau (Fig. 1). The concentrations of generated PGD2 and LTC4 (the major synthesized eicosanoids in BMMCs) were measured 30 min after elicitation. The amount of generated PGD2 was significantly lower in tg/tg BMMCs than in +/+ BMMCs (Fig. 2). In contrast, the amount of generated LTC4 was comparable between +/+ and tg/tg BMMCs (Fig. 2).



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  		FIG. 2.
Amounts of generated PGD2 and LTC4 following stimulation by IgE and antigens in +/+ and tg/tg BMMCs. BMMCs were sensitized with 1 µg/ml anti-DNP IgE for 2 h, washed, and elicited with 100 ng/ml DNP-HSA. The quantities of PGD2 and LTC4 generated were measured 30 min after elicitation. The amounts of PGD2 and LTC4 in BMMCs that had not been elicited were also measured. The values represent the mean ± S.E. of three experiments. In some cases, the S.E. was too small to be shown by bars. *, p <.01 by t test when compared with the amount in non-elicited BMMCs.

 
Because the generation of PGD2, but not of LTC4, was defective in tg/tg BMMCs, we subsequently concentrated our study on PGD2 production. We examined the process of PGD2 generation elicited by 100 ng/ml DNP-HSA (Fig. 2). Because there was a possibility that tg/tg BMMCs responded at a higher concentration of antigen, we examined generation of PGD2 at various concentrations of DNP-HSA. At all examined concentrations (even 1 µg/ml DNP-HSA), PGD2 generation was barely detectable in tg/tg BMMCs (Fig. 3A).



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  		FIG. 3.
Generation of PGD2 in +/+ and tg/tg BMMCs. A, effect of antigen concentration on the generation of PGD2 in +/+ and tg/tg BMMCs. BMMCs were sensitized with 1 µg/ml anti-DNP IgE for 2 h, washed, and then elicited with various concentrations of DNP-HSA. The amount of PGD2 generated was measured 30 min after elicitation. Values represent the mean ± S.E. of three experiments. In some cases, the S.E. was too small to be shown by bars. *, p < 0.01 by t test when compared with the amount in tg/tg BMMCs. B, time course of PGD2 generation in +/+ and tg/tg BMMCs maintained in IL-3. BMMCs established in rmIL-3 were sensitized with 1 µg/ml anti-DNP IgE for 2 h, washed, and then elicited by 100 ng/ml DNP-HSA. The values represent the mean ± S.E. of three experiments. In some cases, the S.E. was too small to be shown by bars. *, p < 0.01 by t test when compared with the amount in tg/tg BMMCs. C, time course of delayed phase of PGD2 generation in +/+ and tg/tg BMMCs stimulated with IL-10, IL-1{beta}, and KitL. BMMCs established in rmIL-3 were washed and maintained in medium containing rmIL-10 (10 ng/ml), rmIL-1{beta} (5 ng/ml), rmKitL (50 ng/ml), and anti-DNP IgE (1 µg/ml) for 2 h. Cells were then washed and elicited using 100 ng/ml DNP-HSA. Values represent the mean ± S.E. of three experiments. In some cases, the S.E. was too small to be shown by bars. *, p < 0.01 by t test when compared with the amount in tg/tg BMMCs. D, PGD2 generation in BMMCs preincubated with indomethacin. BMMCs were preincubated with indomethacin (1 µg/ml), rmIL-10 (10 ng/ml), rmIL-1{beta} (5 ng/ml), rmKitL (50 ng/ml), and anti-DNP IgE (1 µg/ml) for 2 h, washed, and then elicited with 100 ng/ml DNP-HSA. The amount of PGD2 generated was measured 8 h after elicitation. Values represent the mean ± S.E. of three experiments. *, p < 0.01 by t test when compared with the amount in +/+ BMMCs.

 
The time-dependent pattern of PGD2 generation elicited with 100 ng/ml DNP-HSA was compared between +/+ and tg/tg BMMCs. In +/+ BMMCs, PGD2 generation was maximal within the first 15 min, and the amount of generated PGD2 did not change over the next 8 h (Fig. 3B). In contrast, PGD2 generation was barely detectable in tg/tg BMMCs at any time during the period of examination (Fig. 3B).

Delayed PGD2 Production in tg/tg BMMCs—Next, we examined delayed phase of PGD2 generation in tg/tg BMMCs. Murakami et al. (17) reported that a delayed phase of PGD2 generation was hardly detectable in BMMCs cultured in medium containing IL-3 alone. Their observations are consistent with the result shown in Fig. 3B in which BMMCs cultured with IL-3 alone were used. We changed the medium for +/+ and tg/tg BMMCs to one containing IL-10, IL-1{beta}, and KitL, in which +/+ BMMCs had been reported to show delayed phase generation of PGD2 (17–20). After culturing BMMCs in this medium for 2 h with anti-DNP IgE, 100 ng/ml DNP-HSA was added. As previously reported (17), the presence of IL-10, IL-1{beta}, and KitL induced generation of PGD2 in +/+ BMMCs without the need for elicitation by DNP-HSA, although in relatively small amounts (0.52 ± 0.02 ng/106 cells, Fig. 3C). Elicited +/+ BMMCs generated PGD2 during the first 15 min, followed by a gradual increase of PGD2 generation over the next 8 h (Fig. 3C). Such patterns of PGD2 generation were not detected in tg/tg BMMCs (Fig. 3C).

To reveal only the delayed phase of PGD2 generation, the immediate phase was eliminated by preincubation of BMMCs with 1 µg/ml indomethacin for 2 h. This reagent inactivates PGHS-1, which is necessary for the immediate phase of PGD2 generation; the PGD2 synthesized in BMMCs preincubated with this reagent reflects the delayed phase of PGD2 generation (18). BMMCs were preincubated with indomethacin in the presence of IL-10, IL-1{beta}, and KitL, sensitized with anti-DNP IgE, washed, and then elicited with DNP-HSA. The +/+ BMMCs synthesized ~2 ng of PGD2/106 cells 8 h after elicitation with DNP-HSA (Fig. 3D). A similar delayed phase of PGD2 generation was barely detectable in tg/tg BMMCs (Fig. 3D).

Deficient Expression of hPGDS Gene in tg/tg BMMCs—The deficient PGD2 generation observed in both immediate and delayed phases suggests that the expression of some gene(s) related to the synthesis of PGD2 might be defective in tg/tg BMMCs. We examined the expression of genes related to PGD2 synthesis using BMMCs cultured in IL-3. The expression levels of genes participating in eicosanoid generation between +/+ and tg/tg BMMCs were compared using CodeLink UniSet mouse expression bioarrays. BMMCs were examined under the following two conditions, BMMCs sensitized with anti-DNP IgE or BMMCs sensitized with anti-DNP IgE and then elicited with DNP-HSA for 30 min. Under both conditions, the levels of expression of hPGDS mRNA were significantly lower in tg/tg BMMCs than in +/+ BMMCs (Table I). Other genes had comparable expression levels between +/+ and tg/tg BMMCs under both conditions.


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  		TABLE I
Comparative expression of the transcripts that encode 16 proteins participating in eicosanoid generation in BMMCs

 
We used real-time RT-PCR to confirm the apparently defective hPGDS expression in tg/tg BMMCs. We also quantified the levels of mRNAs from three genes related to PGD2 generation, RasGRP4, PGHS-1, and PGHS-2. We examined BMMCs under the following four conditions, BMMCs without any stimulation, BMMCs sensitized with anti-DNP IgE, BMMCs sensitized with anti-DNP IgE and then elicited with DNP-HSA for 30 min, or BMMCs sensitized with anti-DNP IgE and then elicited with DNP-HSA for 120 min. Under all conditions examined, the hPGDS gene was expressed in tg/tg BMMCs at levels less than one tenth those observed in +/+ BMMCs (Fig. 4). In contrast, the expression levels of RasGRP4 and PGHS-2 genes were comparable between +/+ and tg/tg BMMCs under all conditions. The expression level of the PGHS-1 gene in tg/tg BMMCs was approximately one third that found in +/+ BMMCs, but this magnitude of reduction was small compared with that observed for the hPGDS gene (Fig. 4).



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  		FIG. 4.
Quantification of hPGDS, RasGRP4, PGHS-1, and PGHS-2 transcripts in +/+ and tg/tg BMMCs. The mRNA levels of hPGDS, RasGRP4, and PGHS-1 and -2 genes were quantified by real-time PCR. BMMCs were unstimulated (designated by IgE– Ag–), sensitized with 1 µg/ml anti-DNP IgE (IgE+ Ag–), sensitized with 1 µg/ml anti-DNP IgE, and elicited with 100 ng/ml DNP-HSA for 30 min (IgE+ Ag30) or sensitized with 1 µg/ml anti-DNP IgE and elicited with 100 ng/ml DNP-HSA for 120 min (IgE+ Ag120). Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous reference mRNA to standardize mRNA concentrations. The amount of each mRNA was divided by the amount found in unstimulated +/+ BMMCs, and the resultant value is shown as a relative quantity of mRNA. Values represent the mean ± S.E. of three experiments. In some cases, the S.E. was too small to be shown by bars.

 
The expression levels of hPGDS, RasGRP4, and PGHS-1 genes were unchanged under the four experimental conditions, whereas the expression level of the PGHS-2 gene increased after stimulation with anti-DNP IgE (Fig. 4). The PGHS-2 expression level further increased following elicitation by DNP-HSA. The magnitude of change in PGHS-2 expression levels induced by stimulation with IgE and antigens was comparable between +/+ and tg/tg BMMCs (Fig. 4).

Expression of hPGDS, PGHS-1, and PGHS-2 genes was examined in terms of protein concentrations using BMMCs without any stimulation. As in the case of mRNA expression, hPGDS protein was detected in +/+ BMMCs but not in tg/tg BMMCs (Fig. 5). The amount of PGHS-1 protein detected was comparable between +/+ and tg/tg BMMCs, and no PGHS-2 protein was detected in either BMMC type (Fig. 5).



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  		FIG. 5.
Immunoblot of hPGDS, PGHS-1, and PGHS-2 in BMMCs of +/+ and tg/tg mice. Whole cell extracts of BMMCs established with rmIL-3 were immunoblotted with anti-hPGDS, anti-PGHS-1, or anti-PGHS-2 antibody. Three independent experiments were performed, and a representative result is shown.

 
Time-dependent changes in the amounts of hPGDS, PGHS-1, and PGHS-2 proteins produced after elicitation were examined in +/+ and tg/tg BMMCs sensitized with anti-DNP IgE. The abundant hPGDS protein was detected in +/+ BMMCs, and its concentration increased transiently ~2 h after elicitation (Fig. 6). In contrast, production of hPGDS could not be detected in tg/tg BMMCs during the period examined (Fig. 6). The protein concentrations of PGHS-1 were unchanged after elicitation in both +/+ and tg/tg BMMCs (Fig. 6). As previously reported (18), PGHS-2 production was barely detectable in +/+ BMMCs. Unexpectedly, low levels of PGHS-2 production were detected in tg/tg BMMCs during the examined period, peaking 2 h after elicitation (Fig. 6).



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  		FIG. 6.
Time-dependent changes in amounts of hPGDS, PGHS-1, and PGHS-2 proteins in BMMCs of +/+ and tg/tg mice. Whole cell extracts of BMMCs established with rmIL-3 were immunoblotted. BMMCs sensitized with 1 µg/ml anti-DNP IgE (shown at time 0) or sensitized with 1 µg/ml anti-DNP IgE and elicited with 100 ng/ml DNP-HSA for various periods (from 1 min to 8 h) were examined. Two independent experiments were performed, and a representative result is shown.

 
Enhancement of hPGDS Transcription by MITF—Because real-time RT-PCR and immunoblotting indicated the steady state amounts of hPGDS mRNA and protein, we examined directly whether transcription of the hPGDS gene was defective in tg/tg BMMCs using a nuclear run-on assay. The transcription rate of the hPGDS gene was significantly higher in +/+ BMMCs than in tg/tg BMMCs (Fig. 7). Then we examined the motif mediating the transactivation effect of MITF. The reporter plasmid containing the promoter region of the hPGDS gene (–1500 to +200) was transfected with or without MITF. Luciferase activity increased ~5-fold when transfected with MITF as compared with transfection without MITF (Fig. 8A). This MITF-related increase in luciferase activity was detected even in the reporter plasmid that contained the promoter region starting at –200 (Fig. 8A). MITF recognizes a CANNTG (N is any nucleotide) motif. Only one CANNTG motif was present in the promoter region starting at –200 (CACCTG between –22 and –17). To examine whether MITF transactivated the hPGDS promoter via this motif, we constructed a reporter plasmid mutated from CACCTG to CTCCAG (the mutated nucleotides are underlined). The mutated reporter plasmid did not show increased luciferase activity mediated by MITF (Fig. 8A).



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  		FIG. 7.
Transcriptional defects in the hPGDS gene in tg/tg BMMCs. Nuclei were isolated from +/+ and tg/tg BMMCs, and transcripts were labeled. The labeled RNA from +/+ and tg/tg BMMCs was hybridized to dot blots containing purified fragments of hPGDS cDNA, {beta}-actin cDNA (as a positive control), or pEF-BOS expression vector (as a negative control) immobilized onto nylon filters.

 



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  		FIG. 8.
Transactivation by MITF through the CACCTG motif in the promoter region of the hPGDS gene. A, luciferase assay using reporter plasmids containing sequentially deleted or mutated versions of the hPGDS promoter. The reporter plasmid and the effector plasmid (expression plasmid containing MITF cDNA or expression vector alone) were transfected, and the relative luciferase activity in the presence of MITF was divided by the relative luciferase activity in the absence of MITF. The divided value is shown as -fold activation. The box in the hPGDS promoter region indicates the CACCTG motif. B, electrophoretic gel mobility shift assay showing that MITF can bind specifically to the CACCTG motif. An oligonucleotide containing the CACCTG motif was labeled. A retarded band formed with the addition of GST-MITF, but not with the addition of GST alone. Excess cold oligonucleotides with the CACCTG motif inhibited retarded band formation, but excess cold oligonucleotides mutated at the CACCTG motif did not.

 
The binding of MITF to the CACCTG motif was examined by electrophoretic gel mobility shift assay. When a GST-MITF fusion protein was added to labeled oligonucleotide containing a CACCTG motif, a retarded band was detected (Fig. 8B). This retarded band was not found when GST protein alone was added (Fig. 8B). The intensity of this retarded band was gradually weakened by additions of cold oligonucleotide with the CACCTG motif (10-, 50-, and 100-fold molar excess), but not by adding cold oligonucleotide mutated at the CACCTG motif (Fig. 8B). These results demonstrate the specific binding of MITF to the CACCTG motif.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BMMCs generate PGD2 immediately after stimulation with IgE and antigens. When BMMCs are cultured in a medium containing IL-10, IL-1{beta}, and KitL, the amount of generated PGD2 increases further up to 8 h after stimulation. These immediate and delayed phases of PGD2 generation were not observed in BMMCs derived from tg/tg mice, indicating that MITF is essential for both phases of PGD2 generation. Among the genes related to eicosanoid generation, hPGDS showed significant decreases in expression level in tg/tg BMMCs. The hPGDS protein converts unstable PGH2 generated by PGHS-1 and -2 into PGD2. As detected by real-time PCR, the amount of hPGDS mRNA in tg/tg BMMCs was approximately one tenth that observed in +/+ BMMCs. The hPGDS protein was barely detected in tg/tg BMMCs. A nuclear run-on assay revealed that transcription of the hPGDS gene was defective in tg/tg BMMCs. MITF appeared to transactivate the hPGDS promoter by binding to the CACCTG motif. Urade and coworkers (21, 24) reported that mice with a disrupted hPGDS gene were defective in both the immediate and delayed phases of PGD2 generation. This indicates that hPGDS is a key enzyme for PGD2 generation. The defects in PGD2 generation observed in tg/tg BMMCs may be attributable to decreased expression of the hPGDS gene.

Stevens and coworkers (22) recently reported that RasGRP4 regulated the generation of PGD2. RasGRP4 is a mast cell-specific guanine nucleotide exchange factor and is thought to act downstream of KIT, a receptor for KitL (25). Overexpression of RasGRP4 in a human mastocytoma cell line increases hPGDS mRNA levels and the amount of generated PGD2. Inhibition of RasGRP4 expression with siRNA in a rat mastocytoma cell line decreases hPGDS protein levels. RasGRP4 appears to act as an upstream regulator for hPGDS transcription. In tg/tg BMMCs, RasGRP4 expression levels were comparable with those observed in +/+ BMMCs. However, we have not examined the expression level of RasGRP4 at the protein level, and the possibility that MITF regulates RasGRP4 protein expression remains. Further analyses using an anti-RasGRP4 antibody are needed to address this possibility. An alternative is that MITF acts downstream of RasGRP4. RasGRP4 acts downstream from KIT, and Fisher and colleagues (26) reported that MITF was also a downstream molecule of KIT. The signal from RasGRP4 may activate MITF and subsequently activate expression of hPGDS. RasGRP4 is known to activate the cyclooxygenase pathway, but not the 5-lipoxygenase pathway (22). MITF also activates the cyclooxygenase pathway alone, which supports the hypothesis that MITF is a downstream molecule for RasGRP4.

Murakami et al. (17, 18) reported that elicitation with IgE and antigens increased the amount of PGHS-2 mRNA in BMMCs. In fact, the addition of IgE to BMMCs increased the expression level of PGHS-2 mRNA, and elicitation with the antigen resulted in a further increase. The magnitude of the increase in PGHS-2 mRNA levels was comparable between +/+ and tg/tg BMMCs. Recently, Inoue et al. (27) reported that a metabolite of PGD2 named 15-deoxy-{Delta} (12, 14)-PGJ2 bound the nuclear peroxisome proliferator-activated receptor (PPAR) {gamma} and that PPAR{gamma} negatively regulated the expression of PGHS-2 in macrophage cell lines. Because tg/tg BMMCs defective in PGD2 generation normally enhanced the mRNA level of the PGHS-2 gene after stimulation with IgE and antigens, the negative feedback loop between PGD2 and PGHS-2 observed in macrophage cell lines does not appear to apply to BMMCs.

In contrast to the changes in PGHS-2 mRNA levels, the changes in PGHS-2 protein levels in tg/tg BMMCs differed from those observed for +/+ BMMCs. The PGHS-2 protein was barely detected in +/+ BMMCs during the examined period (for 8 h after elicitation). This was consistent with a previous report by Murakami et al. (17) that +/+ BMMCs cultured in IL-3 did not express PGHS-2 protein. In contrast to the case of +/+ BMMCs, PGHS-2 protein was unexpectedly detected in tg/tg BMMCs, with levels peaking 2 h after elicitation. Expression of the PGHS-2 gene might be regulated at a translational level, and the mechanism negatively regulating PGHS-2 translation might be defective in tg/tg BMMCs. Several prostanoids, such as PGE2, are known to augment the induction of PGHS-2 (24). Another possibility is that tg/tg BMMCs might be more sensitive to such prostanoids than +/+ BMMCs.

KitL induces the expression of PGHS-2 protein (17). We recently reported that the expression of KIT and the response to KitL were partially impaired in tg/tg BMMCs (28). However, the addition of KitL induced the expression of PGHS-2 protein to comparable levels in +/+ and tg/tg BMMCs (data not shown). The partially deficient signal from KIT appeared to be sufficient for induction of PGHS-2 protein in tg/tg BMMCs.

PGD2 is known to recruit eosinophils (3, 4). Recently we found defective eosinophil recruitment in tg/tg mice (29), suggesting that MITF may play an important role in recruitment of eosinophils through production of PGD2. MITF appears to be a key transcription factor regulating the function of mast cells.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and the Osaka Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Research Unit for Allergy Transcriptome, RIKEN Yokohame Institute, Yokohame, Kanagawa 230-0045, Japan. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pathology, Rm. C2, Osaka University Medical School, Yamada-oka 2–2, Suita 565-0871, Japan. Tel.: 81-6-6879-3721; Fax: 81-6-6879-3729; E-mail: morii{at}patho.med.osaka-u.ac.jp.

1 The abbreviations used are: BMMC, bone marrow-derived mast cell; PGD2, prostaglandin D2; PGHS, PG endoperoxide H synthase; hPGD, hematopoietic PGD2 synthase; LTC4, leukotriene C4; KitL, Kit ligand; IL, interleukin; RasGRP, Ras guanine nucleotide-releasing protein; rm, recombinant mouse; DNP, dinitrophenyl; HSA, human serum albumin; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Professor Y. Kitamura for helpful discussions and C. Murakami, K. Hashimoto, M. Kohara, and T. Sawamura for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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