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HEMATOPOIESIS
From the Department of Pathology, Osaka University
Medical School, Suita, Japan; Department of Oriental Pharmacy, Wonkwang
University, Iksan, Korea; Department of Pharmacology, Ehime University
Medical School, Ehime, Japan; and the Department of Veterinary
Pathobiology, Texas A&M University, College Station, TX.
The mi transcription factor (MITF) is a basic
helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is
important for the development of mast cells. Mast cells of
mi/mi genotype express normal amount of abnormal MITF
(mi-MITF), whereas mast cells of tg/tg genotype
do not express any MITFs. Mast cells of mi/mi mice show
more severe abnormalities than those of tg/tg mice,
indicating that the mi-MITF possesses the inhibitory
function. The MITF encoded by the mice mutant
allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain using
mice/mice mice. The amounts of
c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH)
messenger RNAs decreased in mast cells of
mice/mice mice to levels comparable
to those of tg/tg mice, and the amounts were intermediate
between those of +/+ mice and those of mi/mi mice. Gr B
mediates the cytotoxic activity of mast cells, and TPH is a
rate-limiting enzyme for the synthesis of serotonin. The cytotoxic
activity and serotonin content of
mice/mice mast cells were
comparable to those of tg/tg mast cells and were significantly higher than those of mi/mi mast cells. The
phenotype of mice/mice mast cells
was similar to that of tg/tg mast cells rather than to that
of mi/mi mast cells, suggesting that the
ce-MITF had no functions. The Zip domain of MITF appeared
to be important for the development of mast cells.
(Blood. 2001;97:2038-2044) The mi locus of mice encodes a member of
the basic helix-loop-helix leucine zipper (bHLH-Zip) protein family of
transcription factors (hereafter called MITF, for mi
transcription factor).1,2 Spontaneous chemical, radiation,
and insertional mutageneses provide abundant mutant alleles at the
mi locus,3,4 which are useful for the analysis
of the relationship between the structure and function of
MITF.5-8 The mutant allele that has been studied most
intensively is mi. The mi/mi mice show depletion
of pigment in both hair and eyes, microphthalmia, osteopetrosis, and
deficient natural killer activity.3,4 In addition, the
number of mast cells decreases and their phenotype is abnormal in
mi/mi mice.9-15 Although most mast cells in the
skin of normal (+/+) mice are stained with berberine sulfate that binds
heparin proteoglycan, few mast cells are berberine sulfate+
in the skin of mi/mi mice.13,16-19 Cultured
mast cells (CMCs) derived from the spleen of mi/mi mice are
deficient in the expression of various genes, such as the mouse mast
cell protease (MMCP)-4,20 MMCP-5,21
MMCP-6,22 c-kit,23 p75 nerve
growth factor receptor,24 granzyme B (Gr
B),25 tryptophan hydroxylase (TPH),25
integrin The tg is another mutant allele of the mi
locus.1,30 The tg/tg mice possess the
insertional mutation at the promoter region of mi gene and
do not express any MITFs.1,31 The tg/tg and mi/mi mice share several phenotypic features, but the
phenotypic abnormality of tg/tg mice is apparently mild
compared with that of mi/mi mice. The transcription of
c-kit, Gr B, and TPH genes was significantly reduced in
mi/mi CMCs, but the reduction was moderate in
tg/tg CMCs.32 This indicated that the presence
of mi-MITF caused more severe abnormalities than the absence
of normal (+) MITF. In addition to the loss of transactivation ability, the mi-MITF possesses an inhibitory effect on the
transcription of some particular genes in mast cells.32
Because the tg is considered to be a null mutant allele, the
tg/tg mice may be useful for evaluating the function of
other mutant MITFs. When a homozygous mouse at a certain mutant
mi allele shows more severe phenotype than that of the
tg/tg mouse, the MITF encoded by the mutant mi allele may possess an inhibitory function. When a homozygous mouse at
another mutant mi allele shows a phenotype comparable to
that of tg/tg mouse, the MITF encoded by the mutant
mi allele may not possess any functions.
MITF encoded by the mice mutant allele
(ce-MITF) lacks the Zip domain because of a stop codon
between HLH and Zip.6 To our knowledge, the
mice is the only available mutant of
genes encoding bHLH-Zip proteins lacking the Zip domain. In the present
study, we compared the phenotype of mast cells of
mice/mice mice with that of
mi/mi or tg/tg mice to clarify the importance of
the Zip domain of MITF for development of mast cells. The
phenotype of mice/mice mast cells
was similar to that of tg/tg mast cells rather than to that
of mi/mi mast cells, indicating that the ce-MITF
had no functions.
Mice
Cells
Staining and counting of mast cells Mice 20 days of age were killed by decapitation after ether anesthesia. Pieces of dorsal skin were removed, smoothed onto a piece of the filter paper to keep them flat, fixed in Carnoy's solution, and embedded in paraffin. Sections of skin pieces were stained with alcian blue or with berberine sulfate. The staining method with alcian blue or berberine sulfate has been described previously.11,16-18 Mast cells between epithelium and panniculus carnosus were counted under the microscope, and the number was expressed as mast cells per centimeter of skin. Berberine sulfate+ mast cells were counted under the fluorescent microscope, and the proportion of berberine sulfate+ cells to alcian blue+ cells was calculated.In situ hybridization Skin pieces were removed from the back of 20-day-old mice, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and embedded in paraffin. The technique of in situ hybridization has been described in detail.35 To obtain the MMCP-4,36 MMCP-5,37 MMCP-6,38 and mast cell carboxypeptidase A (MC-CPA)39 probes, single-stranded complementary DNA (cDNA) was generated from total RNA extracted from CMCs of +/+ mouse origin by lithium chloride-urea method.40 The specific cDNA of proteases was then amplified with specific primers for each protease by polymerase chain reaction (PCR).15 The cDNAs were subcloned into the EcoRV site of pBluescript KS plasmid (pBS;
Stratagene, La Jolla, CA) that contains T3 and T7 promoters to
generate probes.
After hybridization with the antisense probe, the cells possessing signals that were stronger than those obtained with the sense probe were considered to be messenger RNA+ (mRNA+). We counted the number of mRNA+ cells per centimeter of skin. In the adjacent section, the number of alcian blue+ cells per centimeter of skin was counted. Then, the proportion of various protease mRNA+ cells to alcian blue+ cells was calculated. Northern blot analysis Each RNA sample was prepared from 1 × 107 CMCs by the lithium chloride-urea method.40 Northern blot analysis was performed using c-kit,41 MMCP-4,36 MMCP-5,37 MMCP-6,38 MC-CPA,39 Gr B,25 TPH,25 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)42 cDNAs labeled with -[32P]deoxycytidine triphosphate (370 MBq/mL; NEN Life Science Products, Boston, MA) by
random oligonucleotide priming. After hybridization at 42°C, blots
were washed to a final stringency of 0.2 × SSC (1 × SSC is 150 mM
NaCl and 15 mM trisodium citrate, pH 7.4) and subjected to autoradiography.
Cytotoxicity assay Mast cell cytotoxicity was measured using a 51Cr release assay according to the procedure described by Bissonnete and Befus.43 As a positive control, spleen cells were freshly prepared from 8-week-old +/+ mice. The target was YAC-1 cells, which were obtained from American Type Culture Collection (Bethesda, MD). The cells were maintained in-MEM supplemented with 10% FCS. CMCs
derived from +/+, mi/mi,
mice/mice, and tg/tg mice
and spleen cells of +/+ mice were washed, suspended in -MEM
supplemented with 10% FCS, and distributed at different concentrations
(0.5, 1.0, and 2.5 × 106 cells) in triplicate into
96-well microtiter plates with round bottoms. YAC-1 cells
(5 × 106) were labeled with 3.7 MBq µCi
[51Cr]Na2CrO4 (Amersham-Pharmacia
Biotech, Amersham Place, UK) for 2 hours, washed 3 times, and
resuspended in -MEM supplemented with 10% FCS. Labeled YAC-1 cells
(1.0 × 104) were mixed with various numbers of CMCs in a
total volume of 200 µL/well. Plates were incubated at 37°C for 18 hours in a CO2 incubator and spun at 150g for 10 minutes, and the radioactivity was determined in 100 µL samples of
cell-free supernatants. The radioactivity released in the well
containing YAC-1 cells alone was designated spontaneous release (SR).
Total 51Cr release (TR) was measured by adding 0.01%
Triton X-100 to the well containing YAC-1 cells alone. The percentage
of specific 51Cr release was calculated using the following
formula: (cpm in the presence of CMCs SR)/(TR SR) × 100.
Concentration of serotonin The concentration of serotonin was measured using high performance liquid chromatography (HPLC) with electrochemical detection.44 Briefly, CMCs were collected, washed with phosphate-buffered saline (PBS), counted, and sonicated for 20 seconds in a sonicator (Tomy, Tokyo, Japan) in 1 mL ice-cold 3% perchloric acid containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM sodium metabisulfate. The homogenate was centrifuged at 10 000g for 15 minutes at 4°C, and the supernatant was applied directly to the HPLC column. The concentration of serotonin per 1.0 × 106 cells was calculated.Electrophoretic gel mobility shift assay (EGMSA) The production of the fusion protein containing glutathione-S-transferase (GST) and MITF was described previously.28 To examine the DNA binding ability of MITF, an oligonucleotide that is a part of MMCP-6 promoter was used as a probe.28 The sequence of the oligonucleotide is 5'-TGGTGGGGACACATGTTACATGGA (the sequence recognized by +-MITF is underlined). The oligonucleotide was labeled with-[32P]deoxycytidine triphosphate by filling 5'
overhangs and used as probes of EGMSA. DNA-binding assays were
performed in a 20 µL reaction mixture containing 10 mM Tris-HCl (pH
8.0), 1 mM EDTA, 75 mM KCl, 1 mM dithiothreitol (DTT), 4% Ficoll 400, 50 ng poly(dI-dC), 25 ng labeled DNA probe, and 3.5 µg GST-MITF
fusion protein. After the incubation at room temperature for 15 minutes, the reaction mixture was subjected to electrophoresis at 14 V/cm at 4°C on a 5% polyacrylamide gel in 0.25 × Tris-borate-EDTA
(TBE) buffer (1 × TBE is 90 mM Tris-HCl, 64.6 mM boric acid, and 2.5 mM EDTA, pH 8.3). The polyacrylamide gels were dried on Whatman 3MM
chromatography paper and subjected to autoradiography.
Construction of expression plasmids and immunocytochemistry The pBS containing the whole coding region of +-MITF, ce-MITF, or mi-MITF was constructed in our laboratory (hereafter called pBS-+-MITF, pBS-ce-MITF, and pBS-mi-MITF, respectively). To generate the Myc-tagged MITF construct, we subcloned the SmaI-HincII fragment of pBS-+-MITF, pBS-ce-MITF, or pBS-mi-MITF into the StuI site of the CS2+MT expression vector that provides 6 copies of the Myc epitope tag at the N-terminal end of the protein (a gift from Dr I. Matsumura, Osaka University, Osaka, Japan).45 The resultant chimeric gene was subcloned into pEF-BOS expression vector kindly provided by Dr S. Nagata (Osaka University, Osaka, Japan).46 The expression plasmid was transfected into NIH3T3 cells, and the overexpressed MITF protein was detected by anti-Myc antibody as described previously.29 Briefly, the cells were fixed with 100% methanol, permealized by treatment with 0.2% Triton X-100 in PBS, and incubated with the mouse monoclonal anti-Myc antibody (9E10; Pharmingen, San Diego, CA). Immunoreacted cells were detected by direct immunofluorescence with goat antimouse immunoglobulin G antibody conjugated with fluorescein isothiocyanate (MBL, Nagoya, Japan).ce-MITF cDNA containing the nuclear localization The nuclear localization signal (NLS) of SV40 large-T antigen (PKKKRKV)47 was inserted into the N-terminus of ce-MITF by PCR. The amplified product was verified by sequencing and cloned into CS2+MT expression vector. Then, the resultant chimeric gene was subcloned into pEF-BOS expression vector. The subcellular localization of the ce-MITF with NLS of SV40 large-T antigen was examined by immunocytochemistry as described above.Immunoblotting for the nuclear and cytoplasmic extracts Nuclear and cytoplasmic extracts of NIH3T3 cells transfected with expression plasmid of Myc-tagged +-MITF, ce-MITF, or mi-MITF were prepared as described before.29 Briefly, transfected cells were washed with PBS twice and resuspended in ice-cold buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsufonyl fluoride (PMSF). Then, Nonidet P-40 was added to a final concentration of 0.1%. After vigorous vortexing, the homogenate was centrifuged at 3000 rpm for 3 minutes. The supernatant was used as the cytoplasmic fraction. The pellet was resuspended in ice-cold buffer containing 400 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 7.4), and 1 mM PMSF, kept on ice for 1 hour, and centrifuged at 15 000 rpm. The supernatant was used as the nuclear fraction. Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Immobilon P, Millipore, Bedford, MA). The blots were incubated with 5% skim milk in Tris-buffered saline (20 mM Tris-HCl [pH 7.4], 150 mM NaCl). Then, the blots were incubated with Tris-buffered saline containing 5% skim milk with the anti-Myc antibody. The membrane was incubated with peroxidase-conjugated goat antimouse immunoglobulin G antibody, and the immune complexes were visualized with Western blot chemiluminescence reagent (NEN Life Science Products).Transient cotransfection assay The reporter plasmid that contained Gr B promoter starting from nt910 (+1 shows a transcription initiation site) was previously reported.25,32 As the expression plasmids, the
SmaI-HincII fragment of pBS-ce-MITF,
or pBS-mi-MITF, was introduced into the blunted
XbaI site of pEF-BOS, and the PCR-amplified fragment of ce-MITF with the NLS of SV40 large-T antigen was also cloned
into the blunted XbaI site of pEF-BOS. A total of 10 µg of
a reporter, 2 µg of an expression plasmid, and 3 µg of an
expression vector containing  -galactosidase gene were cotransfected
into P815 cells by electroporation. The expression vector containing
-galactosidase gene was used as an internal control. The cells were
harvested 48 hours after the transfection and lysed with 0.1 M
potassium phosphate buffer (pH 7.4) containing 1% Triton X-100.
Soluble extracts were then assayed for luciferase activity with a
luminometer LB96P (Berthold, Wildbad, Germany) and for
-galactosidase activity. The luciferase activity was normalized by
the -galactosidase activity and total protein concentration
according to the method described by Yasumoto et al.48 The
normalized value was expressed as the relative luciferase activity.
The number of mast cells was examined in the skin of
mice/mice mice. Histologic sections
of the skin pieces of mice/mice mice
were stained with alcian blue. The number of mast cells in the
mice/mice mice decreased to one
third that of the control +/+ mice and was comparable to that of
mi/mi and tg/tg mice (Table
1). In +/+ mice, most of skin mast cells
were berberine sulfate+, indicating that they contained
heparin.16 The proportion of berberine
sulfate+ mast cells in the skin of
mice/mice mice was comparable to
that of +/+ mice and was also comparable to that of tg/tg
mice (Table 1). In contrast, the proportion of berberine
sulfate+ mast cells was 3% in the skin of mi/mi
mice, as reported previously.8,11
The expression of mast cell-specific protease genes was analyzed by in
situ hybridization in mast cells of the skin. The proportion of MMCP-4
mRNA+ and MMCP-6 mRNA+ mast cells decreased
remarkably in the skin of mice/mice
mice, but that of MMCP-5 mRNA+ and MC-CPA mRNA+
mast cells did not (Table 2). No
significant differences were detectable in the expression of mast cell
proteases among skin mast cells of
mice/mice, tg/tg, and
mi/mi mice.
The expression of genes that had been demonstrated to be affected by
MITF was examined in CMCs derived from the spleen of mice/mice, tg/tg, and
mi/mi mice using Northern blot. The amount of
c-kit mRNA reduced in mi/mi CMCs, as reported
previously.23 The amount of c-kit mRNA of
mice/mice CMCs was comparable to
that of tg/tg CMCs and was intermediate between the amount
of +/+ CMCs and that of mi/mi CMCs (Figure 1).
The amounts of MMCP-4, MMCP-5, and MMCP-6 mRNAs in mice/mice CMCs reduced to the levels comparable to those of mi/mi and tg/tg CMCs (Figure 1). In contrast to the reduced expression of MMCP-5 mRNA in mice/mice CMCs, the proportion of MMCP-5 mRNA+ mast cells was not reduced in the skin of mice/mice mice (Table 2). We previously reported that the addition of stem cell factor (SCF) significantly increased the amount of MMCP-5 mRNA in mi/mi CMCs and speculated that SCF synthesized by skin fibroblasts may induce the MMCP-5 expression in mi/mi skin mast cells.21 The different pattern of MMCP-5 expression between CMCs and skin mast cells of mice/mice, tg/tg, and mi/mi mice may be attributable to the different concentration of SCF surrounding mast cells. The amount of Gr B mRNA reduced in
mice/mice CMCs to a level comparable
to that of tg/tg CMCs, but their magnitude of reduction was
significantly smaller than that of mi/mi CMCs (Figure 1). We
compared the cytotoxic activity of various CMCs, because Gr B mediates
the cytotoxic activity of mast cells against YAC-1 cells.32 CMCs of +/+, mi/mi,
mice/mice, or tg/tg
genotype were cultured together with 51Cr-labeled YAC-1
cells, and the 51Cr release from YAC-1 cells was measured
after 18 hours. At an effector:target (E:T) ratio of 50, neither +/+,
mi/mi, mice/mice, nor
tg/tg CMCs showed any cytotoxic activity (Table
3). At an increased E:T ratio of 100 or
250, a remarkable cytotoxic activity of
mice/mice CMCs was detected as in
the case of +/+ or tg/tg CMCs, but no cytotoxic activity was
observed in mi/mi CMCs (Table 3).
The amount of TPH mRNA also reduced in
mice/mice CMCs to a level comparable
to that of tg/tg CMCs, but the magnitude of reduction was
significantly smaller than that of mi/mi CMCs (Figure 1). The serotonin content of various CMCs was compared, because TPH is the
rate-limiting enzyme of the serotonin synthesis.49 The serotonin content of mice/mice CMCs
was comparable to that of tg/tg CMCs. Both values were
smaller than the value of +/+ CMCs but were larger than the value
of mi/mi CMCs (Table
4).
We then examined functions of ce-MITF. First, the DNA
binding ability of ce-MITF was examined by EGMSA. A part of
the MMCP-6 promoter containing the MITF binding motif, CACATG, was used
as a probe. The specific binding of ce-MITF was not
detectable as in the case of mi-MITF (Figure
2). Second, the subcellular localization of ce-MITF was examined by immunocytochemistry. Because the
ce-MITF lacks the carboxy-terminal region that is recognized
by anti-MITF antibody used in our previous studies, we detected the
localization of various epitope-tagged MITFs in the present
study.8 The expression vector that contained +-MITF,
ce-MITF, or mi-MITF cDNA downstream from the
sequence of Myc-epitope was transfected into NIH3T3 cells. The
localization of MITF was detected with anti-Myc antibody. A strong
signal was detected only in the nucleus of the NIH3T3 cells
overexpressing Myc-tagged +-MITF (Figure
3A). In contrast, signals were detected
in both nucleus and cytoplasm of the NIH3T3 cells overexpressing
Myc-tagged ce-MITF (Figure 3A). Signals were detected in
both nucleus and cytoplasm as well in the NIH3T3 cells overexpressing
Myc-tagged mi-MITF (Figure 3A). When the modified
ce-MITF possessing the NLS of SV40 large-T antigen was
overexpressed, signals were detected only in the nucleus (Figure 3A).
Subcellular localization of +-MITF, ce-MITF, and mi-MITF was also examined by immunoblotting analysis. A strong signal was detected in the nuclear fraction prepared from the NIH3T3 cells transfected with Myc-tagged +-MITF cDNA but not in the cytoplasmic fraction. In contrast, moderate signals were detected in both nuclear and cytoplasmic fractions of the NIH3T3 cells transfected with Myc-tagged ce-MITF or Myc-tagged mi-MITF cDNA (Figure 3B). The size of the immunoreactive protein in NIH3T3 cells transfected with Myc-tagged ce-MITF cDNA was smaller than that of NIH3T3 cells transfected with Myc-tagged +-MITF cDNA or Myc-tagged mi-MITF cDNA because of the truncation of the C-terminal region of ce-MITF (Figure 3B). We examined the effect of ce-MITF on the transactivation of
the Gr B promoter using the transient cotransfection assay (Figure 4). The 5' flanking sequence of the Gr B
gene (nt
We examined the importance of Zip domain of MITF by comparing the mast cell abnormalities of mice/mice mice with those of tg/tg and mi/mi mice. First, we examined the abnormalities of skin mast cells. In mice/mice mice, the number of skin mast cells decreased to a level comparable to that of mi/mi and tg/tg mice. Although the proportion of berberine sulfate+ mast cells decreased in mi/mi mice, the proportion was normal in mice/mice mice as in the case of tg/tg mice. The proportions of MMCP-4 mRNA+ and MMCP-6 mRNA+ skin mast cells reduced in mice/mice mice to levels comparable to those of tg/tg and mi/mi mice. The abnormalities of skin mast cells of mice/mice mice were similar to those of tg/tg mice rather than to those of mi/mi mice. Next, we compared the phenotype of mice/mice CMCs to that of tg/tg and mi/mi CMCs. The amounts of MMCP-4, MMCP-5, and MMCP-6 mRNAs reduced in mice/mice CMCs to levels comparable to those of tg/tg and mi/mi CMCs. The amounts of c-kit, Gr B, and TPH mRNAs in mice/mice CMCs were comparable to those of tg/tg CMCs and were intermediate between the amounts of +/+ and those of mi/mi CMCs. The phenotype of mice/mice CMCs was similar to that of tg/tg CMCs rather than to that of mi/mi CMCs. The level of c-kit mRNA expression in mice/mice and tg/tg CMCs was higher than that of mi/mi CMCs. In contrast, numbers of mast cells in the skin of mice/mice and tg/tg mice were comparable to those of mi/mi mice. The expression level of c-kit in CMCs was not necessarily proportional to the number of mast cells in the skin. The mechanisms remain to be clarified. There was a possibility that the decreased mast cell number in the skin of 20-day-old mi/mi, mice/mice, and tg/tg mice might be a consequence of delayed homing or development of the mutant mast cells. If the decrease in 20-day-old mice was due to the delay of homing or development, the number of mast cells may be corrected in older mice. We examined the number of mast cells in the skin of approximately 60-day-old tg/tg mice, but the number of mast cells was comparable to that of 20-day-old tg/tg mice (unpublished data). We considered that the homing or development of mast cells was completed in 20-day-old mutant mice. CMCs of mice/mice genotype killed YAC-1 cells as effectively as +/+ or tg/tg CMCs, but the cytotoxic activity of mi/mi CMCs was deficient. This was partly consistent with the expression level of Gr B mRNA demonstrated by the Northern analysis. Probably the expression level of Gr B observed in mice/mice and tg/tg CMCs may be enough for the cytotoxic activity. Serotonin contents of +/+, mice/mice, tg/tg, or mi/mi CMCs were well correlated with the expression levels of the TPH gene in mice of each genotype. Because we have not determined the amount of Gr B or TPH proteins, we were not able to indicate the direct correlation between mRNA and protein levels. Determination of the amounts of Gr B and TPH proteins will clarify this point. We examined the function of ce-MITF by the luciferase assay using the Gr B promoter. As previously reported, the expression of +-MITF increased the activity of Gr B promoter significantly, whereas the expression of mi-MITF reduced it.32 The expression of ce-MITF showed a promoter activity comparable to the value obtained by the expression of vector alone, suggesting that the ce-MITF lacked not only the transactivation ability but also the inhibitory effect on transcription. This was consistent with the fact that the phenotype of mice/mice mast cells was similar to that of tg/tg mast cells. The mi-MITF showing the inhibitory effect possessed the mutated basic domain and the intact Zip domain. Because the ce-MITF did not show such an inhibitory effect, the Zip domain may be necessary for the inhibitory effect of MITF. This was consistent with the result of Krylov et al that mutants of various bHLH-Zip proteins showing inhibitory effects possessed the abnormal basic domain and the normal Zip domain.50 The effect of Zip domain on DNA binding ability has been reported in various bHLH-Zip proteins. Mutant upstream stimulatory factor (USF) and Max lacking the Zip domain bind DNA.51,52 On the other hand, TFE3 lacking Zip domain did not bind it.53 Fisher and his colleagues reported that the ce-MITF synthesized by reticulocyte lysates did not bind DNA.7 Here, we obtained the same result using recombinant GST-ce-MITF fusion protein. The Zip domain of MITF and TFE3 appeared to be indispensable for DNA binding, whereas the Zip domain of USF and Max did not. The different attitude of Zip domain between MITF/TFE3 and USF/Max may be related to the fact that MITF forms a heterodimer with TFE3 but not with USF or Max.7 The ce-MITF was detected both in the nucleus and cytoplasm as in the case of mi-MITF.8,29 Small molecules less than 50 kd are able to pass through the nuclear pore by passive diffusion.54 Because the molecular mass of monomeric Myc-tagged ce-MITF was approximately 50 kd, the monomeric form of ce-MITF may diffuse passively through the nuclear pore. The inability to dimerize and the reduction of the molecular mass due to truncation of Zip domain might cause passive diffusion of ce-MITF. The second explanation is that the Zip domain of MITF possesses an NLS. Recently, Nagoshi et al reported that the NLS located in the Zip domain of sterol regulatory element binding protein 2 (SREBP2), another bHLH-Zip protein.55 The truncation of Zip domain abolishes the nuclear localization of SREBP2. Further biochemical studies may clarify whether the NLS is present in the Zip domain of MITF. There was a possibility that the abnormality of ce-MITF was simply due to its inability to translocate into the nucleus. To examine this possibility, we constructed the modified ce-MITF that possessed the NLS of SV40 large-T antigen. The expression of the modified ce-MITF did not show any promoter activities as that of the original ce-MITF. This indicated that the abnormality of ce-MITF was not due to a defect in the nuclear localization. Taken together, the Zip domain was important for the function of MITF. The mice/mice mice are useful for clarifying the function of MITFs.
The authors thank Dr S. Nagata of Osaka University for pEF-BOS and Dr I. Matsumura of Osaka University for CS2+MT.
Submitted July 11, 2000; accepted December 7, 2000.
Supported by grants from the Ministry of Education, Science and Culture; the Ministry of Health and Welfare; the Organization of Pharmaceutical Safety and Research; the Welfide Medicinal Research Foundation; and NIH grant EY-10223 to M.L.L.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eiichi Morii, Dept of Pathology, Osaka University Medical School, Yamada-oka 2-2, Suita 565-0871, Japan; e-mail: morii{at}patho.med.osaka-u.ac.jp.
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© 2001 by The American Society of Hematology.
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  N. A. Meadows, S. M. Sharma, G. J. Faulkner, M. C. Ostrowski, D. A. Hume, and A. I. Cassady The Expression of Clcn7 and Ostm1 in Osteoclasts Is Coregulated by Microphthalmia Transcription Factor J. Biol. Chem., January 19, 2007; 282(3): 1891 - 1904. [Abstract] [Full Text] [PDF]
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E. Morii and K. Oboki MITF Is Necessary for Generation of Prostaglandin D2 in Mouse Mast Cells J. Biol. Chem., November 19, 2004; 279(47): 48923 - 48929. [Abstract] [Full Text] [PDF]
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 K. Oboki, E. Morii, and Y. Kitamura Deficient Eosinophil Chemotaxis-Promoting Activity of Genetically Normal Mast Cells Transplanted into Subcutaneous Tissue of Mitfmi-vga9/Mitfmi-vga9 Mice: Comparison of the Activity and Mast Cell Distribution Pattern with KitW/KitW-vMice Am. J. Pathol., October 1, 2004; 165(4): 1141 - 1150. [Abstract] [Full Text] [PDF]
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 E. Morii, K. Oboki, K. Ishihara, T. Jippo, T. Hirano, and Y. Kitamura Roles of MITF for development of mast cells in mice: effects on both precursors and tissue environments Blood, September 15, 2004; 104(6): 1656 - 1661. [Abstract] [Full Text] [PDF]
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E. Morii, A. Ito, T. Jippo, Y.-i. Koma, K. Oboki, T. Wakayama, S. Iseki, M. L. Lamoreux, and Y. Kitamura Number of Mast Cells in the Peritoneal Cavity of Mice: Influence of Microphthalmia Transcription Factor through Transcription of Newly Found Mast Cell Adhesion Molecule, Spermatogenic Immunoglobulin Superfamily Am. J. Pathol., August 1, 2004; 165(2): 491 - 499. [Abstract] [Full Text] [PDF]
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 T. Jippo, E. Morii, A. Ito, and Y. Kitamura Effect of Anatomical Distribution of Mast Cells on Their Defense Function against Bacterial Infections: Demonstration Using Partially Mast Cell-deficient tg/tg Mice J. Exp. Med., June 2, 2003; 197(11): 1417 - 1425. [Abstract] [Full Text] [PDF]
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E. Morii, K. Oboki, T. Jippo, and Y. Kitamura Additive effect of mouse genetic background and mutation of MITF gene on decrease of skin mast cells Blood, February 15, 2003; 101(4): 1344 - 1350. [Abstract] [Full Text] [PDF]
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A. R. Migliaccio, R. A. Rana, M. Sanchez, R. Lorenzini, L. Centurione, L. Bianchi, A. M. Vannucchi, G. Migliaccio, and S. H. Orkin GATA-1 as a Regulator of Mast Cell Differentiation Revealed by the Phenotype of the GATA-1low Mouse Mutant J. Exp. Med., February 3, 2003; 197(3): 281 - 296. [Abstract] [Full Text] [PDF]
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E. Morii, K. Oboki, T. R. Kataoka, K. Igarashi, and Y. Kitamura Interaction and Cooperation of mi Transcription Factor (MITF) and Myc-associated Zinc-finger Protein-related Factor (MAZR) for Transcription of Mouse Mast Cell Protease 6 Gene J. Biol. Chem., March 1, 2002; 277(10): 8566 - 8571. [Abstract] [Full Text] [PDF]
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K. Nishida, L. Wang, E. Morii, S. J. Park, M. Narimatsu, S. Itoh, S. Yamasaki, M. Fujishima, K. Ishihara, M. Hibi, et al. Requirement of Gab2 for mast cell development and KitL/c-Kit signaling Blood, March 1, 2002; 99(5): 1866 - 1869. [Abstract] [Full Text] [PDF]
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E. Morii, H. Ogihara, K. Oboki, T. R. Kataoka, K. Maeyama, D. E. Fisher, M. L. Lamoreux, and Y. Kitamura Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells Blood, October 15, 2001; 98(8): 2577 - 2579. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
.
J Immunol.
1990;145:3385-3390