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HEMATOPOIESIS
Inhibitory effect of the mi transcription factor encoded by the mutant mi allele on
GA binding protein–mediated transcript expression in mouse mast cells
Eiichi Morii, Hideki Ogihara, Keisuke Oboki, Chika Sawa, Takahiko Sakuma, Shintaro Nomura,
Jeffrey D. Esko, Hiroshi Handa, and Yukihiko Kitamura
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 amounts of abnormal MITF
(mi-MITF), whereas mast cells of tg/tg
genotype do not express any MITFs.
The synthesis of heparin is abnormal in
the skin mast cells of mi/mi mice. Because N-deacetylase/N-sulfotransferase
2 (NDST-2) is essential for the synthesis
of heparin, the amount of NDST-2 messenger RNA (mRNA) was compared among
cultured mast cells (CMCs) of ⴙ/ⴙ, mi/mi,
and tg/tg genotypes. The NDST-2 mRNA
was detected by in situ hybridization in
the skin mast cells of ⴙ/ⴙ and tg/tg mice,
but not in the skin mast cells of mi/mi
mice. The amount of NDST-2 mRNA decreased significantly in CMCs derived
from mi/mi mice when compared to the
values of ⴙ/ⴙ and tg/tg mice, suggesting
that the defective form of MITF inhibited
the expression of the NDST-2 transcript.
The expression of NDST-2 transcript was
mediated by the GGAA motif located in
the 5ⴕ-untranslated region. GA binding
protein (GABP) bound the GGAA motif
and increased the amount of NDST-2 tran-
script. The mi-MITF appeared to inhibit
the ability of GABP to express NDST-2
transcript by disturbing its nuclear localization. This is the first study to show that
expression of an abnormal form of a
bHLH-Zip transcription factor can dramatically alter the intracellular location of
another DNA/RNA binding factor, which
in turn brings about profound and unexpected consequences on transcript
expression. (Blood. 2001;97:3032-3039)
© 2001 by The American Society of Hematology
Introduction
The mi locus of mice encodes a member of the basic-helix-loophelix leucine zipper (bHLH-Zip) protein family of transcription
factors (hereafter called MITF).1,2 The mi/mi mutant mice show
depletion of pigment in both hair and eyes, microphthalmia,
osteopetrosis, and deficient natural killer activity.3,4 In addition, the
development of mast cells is defective in mi/mi mice. The skin mast
cells of mi/mi mice decrease in number and reduce the amount of
mouse mast cell protease-4 (MMCP-4), MMCP-6, and c-kit
messenger RNAs (mRNAs).5-11 Cultured mast cells (CMCs) derived from the spleen of mi/mi mice are deficient in the expression
of various genes, such as MMCP-4,11 MMCP-5,12 MMCP-6,13
c-kit,14 p75 nerve growth factor receptor,15 granzyme B (Gr B),16
tryptophan hydroxylase (TPH),16 integrin-␣4 subunit17 and ␣-melanocyte-stimulating hormone receptor genes.18
MITF encoded by the mi mutant allele (mi-MITF) deletes 1 of 4
consecutive arginines in the basic domain.1,19,20 The mi-MITF is
defective in the DNA binding ability and the nuclear localization
potential.21,22 The mi-MITF does not appear to transactivate target
genes due to these abnormalities.11-18,22 The tg is another mutant
allele of the mi locus.1,23 The tg/tg mice possess the insertional
mutation at the promoter region of mi gene and do not express any
MITFs.1,24 The tg/tg and mi/mi mice share several phenotypic
features, but the phenotypic abnormality of tg/tg mice is apparently
mild when compared to that of mi/mi mice. The transcription of
c-kit, Gr B, and TPH genes as significantly reduced in mi/mi CMCs,
but the reduction was moderate in tg/tg CMCs.25 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.25
Heparin is a highly sulfated proteoglycan that is abundantly
contained by mast cells in the skin of mice.26-28 Because berberine
sulfate is a dye that binds heparin, most of the skin mast cells of
⫹/⫹ mice are stained with berberine sulfate.29-31 In the skin of
mi/mi mice, the content of heparin decreases, and few mast cells are
stained with berberine sulfate.9 In contrast, most of the skin mast
cells of tg/tg mice are stained with berberine sulfate as in the case
of ⫹/⫹ skin mast cells,32 indicating that the content of heparin
decreases in mi/mi skin mast cells but not in tg/tg skin mast cells.
There is a possibility that mi-MITF may possess the inhibitory
effect on the synthesis of heparin as in the case of serotonin.25 In
fact, serotonin content of mi/mi CMCs decreases due to the poor
transcription of TPH gene.16,25 N-deacetylase/N-sulfotransferase 2
(NDST-2) is essential for the synthesis of heparin, and the
disruption of the NDST-2 gene causes the depletion of heparin in
mast cells.33,34 In the present study, we compared the amount of
From the Department of Pathology, Osaka University Medical School, Suita,
Japan; the Research Function of Biotechnology, Frontier Collaborative
Research Center, Tokyo Institute of Technology, Yokohama, Japan; and the
Division of Cellular and Molecular Medicine, Glycobiology Program, University
of California, San Diego, CA.
Memorial Foundation. GenBank accession number AF293452.
Submitted September 8, 2000; accepted January 17, 2001.
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.
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 the Uehara
3032
Reprints: Eiichi Morii, Dept of Pathology, Osaka University Medical School,
Yamada-oka 2-2, Suita 565-0871, Japan; e-mail: [email protected].
osaka-u.ac.jp.
© 2001 by The American Society of Hematology
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
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BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
INHIBITION OF HEPARIN SYNTHESIS BY MUTANT MITF
3033
NDST-2 mRNA among mast cells of ⫹/⫹, mi/mi, and tg/tg
genotypes and found the inhibitory effect of mi-MITF on the
expression of NDST-2 transcript in mast cells.
otide priming. The NDST-2 cDNA used was same as that used for in situ
hybridization. After hybridization at 42°C, blots were washed to a final
stringency of 0.2 ⫻ standard sodium citrate (SSC; 1 ⫻ SSC is 150 mM
NaCl and 15 mM trisodium citrate, pH 7.4), and subjected to
autoradiography.
Materials and methods
Determination of 5ⴕ-end of cDNA
Mice
The 5⬘-rapid amplification of cDNA end (5⬘-RACE) was performed with
the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Palo
Alto, CA) according to the manufacturer’s instructions. The primer for the
first PCR was 5⬘-AGATTCCCATCCTGTGTCTGCCAATGAG (corresponding to the region between nt 66 and nt 38)37 and the primer for the
nested PCR was 5⬘-CTGGTGGGCTCTCGATAACAAGTGGATG (corresponding to the region between nt 30 and nt 3).37 The total RNA obtained
from MST cells was used as the template.
The original stock of C57BL/6-mi/⫹ mice was purchased from the Jackson
Laboratory (Bar Harbor, ME), and was maintained in our laboratory by
consecutive back-cross with our own inbred C57BL/6 colony (⬎ 15
generations at the time of the present experiment). The original stock of
VGA-9-tg/tg mice, in which the mouse vasopressin-Escherichia coli
␤-galactosidase transgene was integrated at the 5⬘ flanking region of the mi
gene, were kindly given by Dr H. Arnheiter (National Institutes of Health,
Bethesda, MD).1 The integrated transgene was maintained by repeated
back-crosses to our own inbred C57BL/6 colony (⬎ 15 generations at the
time of the present experiment). Female and male mi/⫹ or tg/⫹ mice were
crossed together, and the resulting homozygous mi/mi or tg/tg mice were
selected by their white coat color.3,4 C57BL/6-⫹/⫹ mice raised in our
laboratory were used as a control.
In situ hybridization
Skin pieces were removed from the backs of 20-day-old mice, fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4), and embedded
in paraffin. The technique of in situ hybridization has been described in
detail.35 To obtain the NDST-2 probe, single-stranded complementary DNA
(cDNA) was generated from total RNA extracted from CMCs of ⫹/⫹
mouse origin by lithium chloride-urea method.36 The specific cDNA of
NDST-2 was then amplified by polymerase chain reaction (PCR). The
primers for PCR were 5⬘-CGAGCACATGGAGACCCCATTGCTC (corresponding to the region between nt 2661 and nt 2685, the number represents
the nucleotide numbers of the NDST-2 cDNA reported by Orellana and
colleagues37) and 5⬘-TGCTGGTAAAGAAAGACTGGGCCA (corresponding to the region between nt 3188 and nt 3165). The NDST-2 cDNA was
subcloned into the EcoRV site of Bluescript KS (-) plasmid (pBS;
Stratagene, La Jolla, CA) that contains T3 and T7 promoters to generate
probes. Skin mast cells were identified with alcian blue staining, and the
presence of NDST-2 mRNA was examined in the serial section by
hybridization of antisense probe. The sense probe for NDST-2 cDNA was
used as a negative control.
Cells
Pokeweed mitogen-stimulated spleen cell-conditioned medium (PWMSCM) was prepared according to the method described by Nakahata and
coworkers.38 Mice of mi/mi, tg/tg, and control ⫹/⫹ genotype were used at 2
to 3 weeks of age to obtain CMCs. Mice were killed by decapitation after
ether anesthesia and spleens were removed. Spleen cells were cultured in
␣-minimal essential medium (␣-MEM; ICN Biomedicals, Costa Mesa, CA)
supplemented with 10% PWM-SCM and 10% fetal calf serum (FCS;
Nippon Bio-supp Center, Tokyo, Japan). Half of the medium was replaced
every 5 days. Cells derived from the spleen of each mutant genotype
reached to 1 ⫻ 107 in number within 4 weeks. More than 95% of cells
contained alcian blue–positive granules and were considered to be CMCs 4
weeks after initiation of the culture. The MST cells, a heparin-producing
cell line derived from the Furth murine mastocytoma, were maintained in
␣-MEM supplemented with 10% FCS.39 The NIH/3T3 cells and COS-7
cells were maintained in Dulbecco modified Eagle medium (DMEM, Flow
Laboratories, Irvine, United Kingdom) supplemented with 10% FCS.
Northern blot analysis
Each RNA sample was prepared from 1.0 ⫻ 107 of CMCs or MST cells by
the lithium chloride-urea method.36 Northern blot analysis was performed
using NDST-237 and GAPDH cDNAs40 labeled with ␣-[32P]-dCTP (DuPont/
NEN Research Products, Boston, MA; 10 mCi/mL) by random oligonucle-
Promoter region of the NDST-2 gene
The DNA fragment containing the promoter region of the NDST-2 gene was
isolated with the Mouse Genome Walker Kit (Clontech Laboratories)
according to the manufacturer’s instructions. The primers used were same
as those used in 5⬘-RACE. The isolated fragment was cloned into pBS and
sequenced. The isolated fragment contained the most upstream point of the
5⬘-RACE product, and the remaining upstream sequence was considered to
be the promoter region.
Construction of reporter plasmids
The luciferase gene subcloned into pSP72 (pSPLuc) was generously
provided by Dr K. Nakajima (Osaka City University Medical School,
Osaka, Japan).41 To construct reporter plasmids, a DNA fragment containing a promoter and 5⬘-untranslated region (5⬘-UTR) of the NDST-2 gene
(⫺1300 to ⫹668, ⫹1 shows the transcription initiation site determined by
5⬘-RACE) was cloned into the upstream of luciferase gene in pSPLuc. The
deletion of the promoter or 5⬘-UTR of the NDST-2 gene was produced by
PCR. The mutation was also introduced by PCR. All of the PCR products
were verified by sequencing.
Construction of expression plasmids
The pBS containing the whole coding region of ⫹-MITF or mi-MITF was
constructed in our laboratory (hereafter called pBS-⫹-MITF and pBS-miMITF, respectively). The SmaI-HincII fragment of pBS-⫹-MITF, or
pBS-mi-MITF was introduced into the blunted Xba I site of pEF-BOS
expression vector kindly provided by Dr S. Nagata (Osaka University,
Osaka, Japan).42 To generate the Myc-tagged MITF construct, we subcloned the SmaI-HincII fragment of pBS-⫹-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).43 The resultant chimeric gene
was subcloned into pEF-BOS expression vector. The dominant negative
form of GABP cloned in the pET3d expression vector (Novagen, Madison,
WI) was digested with the BamHI and BglII. The BamHI-BglII fragment of
the pET3d expression plasmid was introduced into the BglII site of
pCAGGS expression vector.
Transfection and luciferase assay
The transfection to MST cells was performed by electroporation. The
transfection to NIH/3T3 cells or COS-7 cells was performed with the
TransFast Transfection Reagent (Promega, Madison, WI) according to the
manufacturer’s instructions. In luciferase assays, 10 ␮g of a reporter and 3
␮g of an expression vector containing ␤-galactosidase gene were cotransfected. The expression vector containing ␤-galactosidase gene was used as
an internal control. In some experiments, 10 ␮g of an expression plasmid
containing effector cDNA was cotransfected with 10 ␮g of reporter
plasmids. 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
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3034
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
MORII et al
plexes were washed 4 times with LIP buffer, resuspended in loading buffer,
boiled, and analyzed by immunoblot with anti-Myc monoclonal Ab (9E10,
Pharmingen, San Diego, CA).
Immunocytochemistry
Figure 1. Expression of NDST-2 mRNA by ⴙ/ⴙ and tg/tg skin mast cells but not
by mi/mi skin mast cells. A section of ⫹/⫹, mi/mi, or tg/tg skin was stained with
alcian blue and nuclear fast red, and the adjacent section was hybridized with the
antisense probe for the NDST-2 gene. Another section of ⫹/⫹, mi/mi, or tg/tg skin was
stained with alcian blue and nuclear fast red, and the adjacent section was hybridized
with the sense probe for the NDST-2 gene. Identical mast cells in the pairing sections
are shown by arrows (original magnification ⫻ 400).
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 and coworkers.44 The normalized value was expressed as the
relative luciferase activity.
Electrophoretic gel mobility shift assay
Nuclear extract of MST cells was obtained as described before.22 The
sequence of oligonucleotide used as a probe was 5⬘-GGAGAAGCGGAAGGGGAAGGGA (the GGAA motifs are underlined). The oligonucleotide was labeled with ␣-[32P]-dCTP by filling 5⬘-overhangs and used as
probes of EGMSA. DNA-binding assays were performed in a 20 ␮L
reaction mixture containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 75
mM KCl, 1 mM dithiothreitol (DTT), 4% Ficoll type 400, 50 ng poly
(dI-dC), and 25 ng labeled DNA probe. After the incubation at 4°C for 15
minutes, the reaction mixture was subjected to electrophoresis at 14 V/cm
on a 5% polyacrylamide gel in 0.25 ⫻ TBE buffer (1 ⫻ TBE is 90 mM
Tris-HCl, 64.6 mM boric acid, and 2.5 mM EDTA, pH 8.3). In the
competition experiment, 10-fold molar excess amount of unlabeled oligonucleotide was added. In the supershift experiment, the antibody (Ab) against
GABP␣, GABP␤, or Ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA)
was added. The polyacrylamide gels were dried on Whatman 3MM
chromatography paper and subjected to autoradiography.
In vitro binding assay
The 35S-labeled ⫹-MITF or mi-MITF protein was synthesized using the
reticulocyte lysate system (TNT system, Promega). The ⫹-MITF or
mi-MITF cDNA in pBluescript was transcribed with T7 RNA polymerase
and translated in the presence of 35S-methionine. For the binding assays, the
35S-labeled ⫹-MITF or mi-MITF protein was incubated for 1 hour at room
temperature with GST-GABP␣, GST-GABP␤, or GST alone immobilized
on glutathione-agarose beads. The beads were washed 4 times. Proteins
retained on the beads were subsequently analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography.
The GABP␣ cDNA or GABP␤ cDNA was transfected alone, or both
GABP␣ and GABP␤ cDNAs were cotransfected to NIH/3T3 cells. The
cells were fixed with 100% methanol and permialized by treatment with
0.2% Triton X-100 in PBS. The cells transfected with GABP␣ cDNA were
incubated with mouse anti-GABP␣ Ab, and the cells transfected with
GABP␤ cDNA were incubated with mouse anti-GABP␤ Ab, respectively.
The cells transfected with both GABP␣ and GABP␤ cDNAs were
incubated with mouse anti-GABP␣ Ab, or with mouse anti-GABP␤ Ab,
independently. Immunoreacted cells were detected with antimouse IgG Ab
conjugated with fluorescein isothiocyanate (FITC) (MBL, Nagoya, Japan).
In a separate experiment, GABP␣ cDNA was cotransfected to NIH/3T3
cells with ⫹-MITF cDNA. Because the anti-GABP␣ Ab was the mouse
Ab47 and the anti-MITF Ab was the rabbit Ab,22 we detected the cells
expressing both GABP␣ and ⫹-MITF using the double-staining method.
The transfected cells were incubated with the mixture of anti-GABP␣ Ab
and anti-MITF Ab, and then with the mixture of the above-mentioned
antimouse IgG Ab conjugated with FITC and the antirabbit IgG Ab
conjugated with rhodamine (MBL). The cells expressing GABP␣ were
detected with the green filter and the cells expressing ⫹-MITF were
detected with the red filter of the fluorescence microscope (Olympus
BX-50, Tokyo, Japan). We observed the cells possessing both the green and
red signals. The GABP␣ cDNA was cotransfected with mi-MITF cDNA.
The GABP␤ cDNA was also cotransfected with either ⫹-MITF cDNA or
mi-MITF cDNA. The cells expressing both GABP␣ and mi-MITF, the cells
expressing both GABP␤ and ⫹-MITF, or the cells expressing both GABP␤
and mi-MITF were detected with the double-staining method, as described above.
Results
The expression of the NDST-2 gene was analyzed in skin mast cells
of ⫹/⫹, mi/mi, and tg/tg mice by in situ hybridization. Signals for
NDST-2 mRNA were detected in skin mast cells of ⫹/⫹ and tg/tg
mice but not in skin mast cells of mi/mi mice (Figure 1). We
compared the amount of NDST-2 mRNA among CMCs derived
from ⫹/⫹, mi/mi, or tg/tg mice using Northern blot. The amount of
NDST-2 mRNA reduced significantly in mi/mi CMCs when
compared to that of ⫹/⫹ or tg/tg CMCs (Figure 2).
We attempted to isolate the promoter region and 5⬘-UTR of the
NDST-2 gene. The sequence of 512 base pair (bp) upstream of the
translation initiation site has been reported by Orellana and
Immunoprecipitation
The Myc-tagged ⫹-MITF or Myc-tagged mi-MITF was coexpressed with
GABP␣ or GABP␤ in COS-7 cells. The whole cell extract was obtained by
the method as described before.22 The whole cell extract was incubated with
LIP buffer (10 mM HEPES, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride) and protein G Sepharose
(Amersham-Pharmacia Boitec, Bucks, United Kingdom) for 1 hour with
gentle rocking and centrifuged at 3000 rpm for 3 minutes. The supernatants
were transferred into new tubes and incubated with protein G Sepharose
and anti-GABP␣ or anti-GABP␤ Ab for 1 hour in LIP buffer. Immunecom-
Figure 2. Expression of the NDST-2 gene in CMCs derived from ⴙ/ⴙ, mi/mi, or
tg/tg mice. The blot was hybridized with 32P-labeled cDNA probe of NDST-2 or of
GAPDH. Three independent experiments were done, and comparable results were
obtained. A representative experiment is shown.
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BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
INHIBITION OF HEPARIN SYNTHESIS BY MUTANT MITF
3035
constructed the reporter plasmid containing the promoter region
and 5⬘-UTR of the NDST-2 gene (⫺1300 to ⫹668). The reporter
plasmid, of which promoter region was deleted, was also constructed (⫺196 to ⫹668). Each reporter plasmid was transfected to
MST cells, and the luciferase activity was measured. The expression of reporter plasmid starting from ⫺1300 and that of reporter
plasmid starting from ⫺196 showed comparable luciferase activity
(Figure 4A). Then, we constructed the reporter plasmid, of which
5⬘-UTR was deleted (⫺196 to ⫹649). The luciferase activity of
reporter plasmid ending at ⫹649 reduced to one fifth that of the
reporter plasmids ending at ⫹668 (Figure 4A).
We examined the region between ⫹649 and ⫹668 in detail.
Between ⫹649 and ⫹668, no CANNTG motif that is recognized
and bound by MITF was present. On the other hand, the region
contained 2 GGAA motifs, which are the consensus sequences
recognized by GA binding protein (GABP) (Figure 4B, named
5⬘-GGAA and 3⬘-GGAA, respectively). GABP is a member of
ets-family transcription factors, and is composed of 2 subunits,
GABP␣ and GABP␤.45,46 The mutation at the 5⬘-GGAA motif
(GGAA to CCAA) reduced the luciferase activity to one fifth, but
the mutation at the 3⬘-GGAA motif did not change the luciferase
activity (Figure 4C).
The EGMSA was done using the nuclear extract of MST cells.
The oligonucleotide containing the 2 GGAA motifs was used as a
probe. Multiple bands were detected by mixing the probe and
nuclear extract, but the addition of excess amount of nonlabeled
oligonucleotide with the same sequence as the probe reduced the
intensity of 2 bands (Figure 5). The intensity of these 2 bands was
not reduced by the addition of the nonlabeled oligonucleotide
mutated at the 5⬘-GGAA motif, but were reduced by the addition of
the nonlabeled oligonucleotide mutated at the 3⬘-GGAA motif
(Figure 5). This indicated that the protein in the nuclear extract of
Figure 3. The result of 5ⴕ-RACE and the sequence of promoter region and
5ⴕ-UTR of NDST-2 gene. (A) 5⬘-RACE using mRNA derived from MST cells. A part of
adaptor used in 5⬘-RACE and 5⬘-end of NDST-2 cDNA are shown. (B) The sequence
of promoter region and 5⬘-UTR of the NDST-2 gene. The transcription initiation site
was indicated as ⫹1. The sequence in the promoter region is shown by lower case,
and that of the 5⬘-UTR is shown by capitals.
colleagues, but further upstream regions have not been cloned.37
First, we carried out 5⬘-RACE to determine the transcription
initiation site of the NDST-2 gene. MST cells containing abundant
NDST-2 transcript were used as the source of mRNA. The
5⬘-RACE product of the NDST-2 cDNA started from an adenine,
which was located 1013 bp upstream of the 5⬘-end reported by
Orellana and coworkers (Figure 3A).37 The 5⬘-UTR of the NDST-2
gene was composed of 1525 bp (512 bp for the previously reported
part ⫹ 1013 bp for the isolated part). Then, we isolated the DNA
fragment containing the promoter region. The isolated fragment
contained the above-mentioned 5⬘-RACE product (1013 bp) and
the remaining upstream sequence (1300 bp) (Figure 3B, the
transcription initiation site was indicated as ⫹1).
To determine the motif(s) that mediate the transactivation, we
Figure 4. Luciferase assay in MST cells that are spontaneously expressing the
NDST-2 gene. (A) Luciferase activity under the control of promoter region and
5⬘-UTR of the NDST-2 gene in MST cells. (B) A part of 5⬘-UTR of the NDST-2 gene
between ⫹649 and ⫹668. Two GGAA motifs are underlined. (C) Luciferase activity of
the reporter plasmid containing the intact GGAA motifs or the motifs mutated at either
5⬘-GGAA or 3⬘-GGAA sequence. The data represent the mean ⫾ SE of 3 experiments. In some cases, the SE was too small to be shown by bars. *P ⬍ .01 by t test
when compared with the luciferase activity obtained by the transfection of the reporter
plasmid containing ⫺196 to ⫹668 region.
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3036
MORII et al
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
Figure 7. The effect of coexpression of dominant negative GABP cDNA on the
luciferase activity. The reporter plasmids were transfected to MST cells with the
expression plasmid containing the dominant negative GABP cDNA (5 or 10 ␮g). The
transfected DNA was always kept in equal amount using the backbone expression
vector in each transfection assay. The data represent the mean ⫾ SE of 3
experiments. In some cases, the SE was too small to be shown by bars. P ⬍ .01 by t
test when compared with the luciferase activity obtained by the cotransfection of the
reporter plasmid with the expression vector alone.
Figure 5. EGMSA using nuclear extract of MST cells. Oligo 1 containing the intact
GGAA motifs was used as a probe. As a competitor, nonlabeled oligo 1, oligo 2
mutated at the 5⬘-GGAA motif, or oligo 3 mutated at the 3⬘-GGAA motif was added.
Arrows indicate the 2 specific DNA/protein complexes and the free probe. The
super-shift assay was performed using the anti-GABP␣, GABP␤, or Ets-1 Ab. The
arrowhead indicates the super-shifted band.
MST cells specifically bound the 5⬘-GGAA motif but not the
3⬘-GGAA motif. To characterize the protein that specifically bound
the 5⬘-GGAA motif, we performed super-shift assay with the Ab
against the GABP␣, GABP␤, or Ets-1. The addition of the Ab
against GABP␣ or GABP␤ showed the super-shifted band, but the
addition of the Ab against Ets-1 did not (Figure 5).
To examine whether the endogenous GABP increased the
amount of NDST-2 transcript through the 5⬘-GGAA motif, we used
a dominant negative form of GABP. The dominant negative form of
GABP is a mutant of ␣ subunit that lacks the DNA binding ability.47
We overexpressed the dominant negative GABP in MST cells.
After 6 days of culture, the expression of the NDST-2 gene was
examined by Northern blotting. The overexpression of dominant
negative GABP significantly reduced the amount of NDST-2
Figure 6. Reduced expression of NDST-2 transcript by the overexpression of a
dominant negative GABP cDNA. Total RNA was extracted from original MST cells
(indicated as original), MST cells overexpressing empty expression vector alone
(indicated as vector), or MST cells overexpressing the dominant negative GABP (indicated as dn-GABP). The expression of the NDST-2 gene was examined with
Northern blot.
mRNA (Figure 6). Next, we cotransfected the dominant negative
GABP to MST cells with the reporter plasmid. When various
amounts of dominant negative GABP were cotransfected, the
luciferase activity of the reporter plasmid containing the intact
5⬘-GGAA motif deceased in a dose-dependent manner (Figure 7).
We examined the effect of MITF on the expression of NDST-2
transcript by GABP. We cotransfected the reporter plasmid to MST
cells with the effector plasmid containing ⫹-MITF or mi-MITF
cDNA. The coexpression of ⫹-MITF did not affect the luciferase
activity, but the coexpression of mi-MITF reduced the luciferase
activity in a dose-dependent manner (Figure 8).
The physical interaction between MITF and GABP was examined by an in vitro binding experiment. The 35S-labeled ⫹-MITF or
35S-labeled mi-MITF was subjected to coprecipitation with GST,
GST-GABP␣, or GST-GABP␤ fusion protein that was immobilized on glutathione-agarose beads. Protein complexes were analyzed by SDS-PAGE. The complex of ⫹-MITF and GST-GABP␣
and that of ⫹-MITF and GST-GABP␤ were detected, but that of
⫹-MITF and GST was not (Figure 9). We also examined the
complex formation of mi-MITF. The complex of mi-MITF and
GST-GABP␣ and that of mi-MITF and GST-GABP␤ were detected, but that of mi-MITF and GST was not (Figure 9).
We carried out coimmunoprecipitation studies to confirm the
interaction between MITF and GABP. The Myc-tagged ⫹-MITF or
Figure 8. The effect of coexpression of ⴙ-MITF or mi-MITF cDNA on the
luciferase activity. The reporter plasmids were transfected to MST cells with the
expression plasmid containing ⫹-MITF or mi-MITF cDNA (5 or 10 ␮g). The
transfected DNA was always kept in equal amount using the backbone expression
vector in each transfection assay. The data represent the mean ⫾ SE of 3
experiments. In some cases, the SE was too small to be shown by bars. P ⬍ .01 by t
test when compared with the luciferase activity obtained by the cotransfection of the
reporter plasmid with the expression vector alone.
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BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
INHIBITION OF HEPARIN SYNTHESIS BY MUTANT MITF
3037
Figure 9. In vitro binding of MITF to GABP. 35S-labeled ⫹-MITF or 35S-labeled
mi-MITF was subjected to coprecipitation with GST-GABP␣, GST-GABP␤, or GST,
which had been attached to beads, and the protein complex was analyzed
by SDS-PAGE.
Myc-tagged mi-MITF was coexpressed with GABP in COS-7 cells,
and their whole cell lysate was analyzed. The immnoprecipitated
product with anti-GABP␣ Ab or with anti-GABP␤ Ab was
immunoblotted by anti-Myc Ab. The immunoprecipitated product
with anti-GABP␣ Ab contained either ⫹-MITF or mi-MITF
(Figure 10A). The immunoprecipitated product with anti-GABP␤
Ab also contained either ⫹-MITF or mi-MITF (Figure 10B).
The effect of MITF on nuclear translocation of GABP was
examined by immunocytochemistry. First, we examined the subcellular localization of GABP␣ or GABP␤ without any MITFs. When
expressed alone, GABP␣ diffused both in the nucleus and cytoplasm, whereas GABP␤ was localized in the nucleus (Figure 11A).
GABP␣ became localized in the nucleus when coexpressed with
GABP␤ (Figure 11A, left). Next, we coexpressed ⫹-MITF with
GABP␣ or GABP␤. By coexpressing ⫹-MITF, GABP␣ was
localized in the nucleus (Figure 11B, left). GABP␤ was also
localized in the nucleus when coexpressed with ⫹-MITF (Figure
11B, right). Then, we coexpressed mi-MITF with GABP␣ or
GABP␤. GABP␣ diffused in both nucleus and cytoplasm when
coexpressed with mi-MITF (Figure 11C, left). GABP␤ also diffused in both nucleus and cytoplasm when coexpressed with
mi-MITF (Figure 11C, right). When expressed alone, the ⫹-MITF
Figure 11. Subcellular localization of GABP␣, GABP␤, ⴙ-MITF, or mi-MITF
examined by immunocytochemistry. (A) GABP␣ or GABP␤ was expressed alone,
or GABP␣ and GABP␤ were coexpressed. Cells were stained with either anti-GABP␣
Ab or anti-GABP␤ Ab. (B) GABP␣ and ⫹-MITF, or GABP␤ and ⫹-MITF were
coexpressed. Cells were stained with the mixture of anti-GABP␣ Ab and anti-MITF
Ab, or the mixture of anti-GABP␤ Ab and anti-MITF Ab. (C) GABP␣ and mi-MITF, or
GABP␤ and mi-MITF were coexpressed. Cells were stained with the mixture of
anti-GABP␣ Ab and anti-MITF Ab, or the mixture of anti-GABP␤ Ab and anti-MITF Ab
(original magnification ⫻ 2000).
was localized in the nucleus, whereas the mi-MITF diffused both
in the nucleus and cytoplasm, as reported previously (data
not shown).22
Discussion
Figure 10. Coimmunoprecipitation of MITF and GABP. Myc-tagged ⫹-MITF or
Myc-tagged mi-MITF was cotransfected to COS-7 cells with GABP␣ (A) or GABP␤
(B). The whole cell extract was subjected to immunoprecipitation with anti- GABP␣ or
GABP␤ Ab. Precipitates were separated by SDS-PAGE and immunoblotted with
anti-Myc Ab.
NDST-2 catalyzes the initial sulfation step of heparin synthesis.37,48
Recently, 2 groups reported the phenotype of the mice whose
NDST-2 genes were disrupted (NDST-2⫺/⫺).33,34 In NDST-2⫺/⫺
mice, skin mast cells show depletion of heparin and CMCs do not
form normal granules. We previously reported that the synthesis of
heparin is abnormal in the skin mast cells of mi/mi mice.9 In the
present study, we compared the expression of the NDST-2 gene
among mast cells of ⫹/⫹, mi/mi, and tg/tg genotypes. The NDST-2
mRNA was detected by in situ hybridization in the skin mast cells
of ⫹/⫹ and tg/tg mice, but not in the skin mast cells of mi/mi mice.
The amount of NDST-2 mRNA in CMCs of mi/mi genotype
decreased significantly when compared to the value of ⫹/⫹ or tg/tg
genotype. This indicated that the mi-MITF possessed the inhibitory
effect on the expression of NDST-2 transcript.
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3038
BLOOD, 15 MAY 2001 䡠 VOLUME 97, NUMBER 10
MORII et al
In mast cells of NDST-2⫺/⫺ mice, the unsulfated heparin
precursors are found instead of heparin.33,34 We previously reported
that mast cells of mi/mi mice not only reduced the content of
heparin but contained unidentified glycosaminoglycans.9 The unidentified glycosaminoglycans in mi/mi skin mast cells might be
unsulfated heparin precursors, which were found in mast cells of
NDST-2⫺/⫺ mice.
To examine the inhibitory effect of mi-MITF, we cloned the
promoter region and 5⬘-UTR of the NDST-2 gene and found that the
5⬘-UTR between ⫹649 and ⫹668 was important for the transactivation of the NDST-2 gene. Unexpectedly, no CANNTG motifs,
which were recognized and bound by ⫹-MITF, were present
between ⫹649 and ⫹668. Instead, a GGAA motif, which was
recognized and bound by GABP, was observed. The expression of
dominant negative GABP reduced the luciferase activity of the
reporter plasmid containing the GGAA motif, indicating that
GABP increased the amount of NDST-2 transcript through the
GGAA motif. The mi-MITF reduced the luciferase activity of the
reporter plasmid containing the GGAA motif in a dose-dependent
manner. This suggested that the mi-MITF possessed the inhibitory
effect on the ability of GABP to express NDST-2 transcript.
The GGAA motif was located in the 5⬘-UTR of the NDST-2 gene.
The protein that binds to the motif in 5⬘-UTR regulates the expression of
genes in the following 2 ways.49-51 One is that the protein binds to the
transcribed RNA and stabilizes it, in which the protein regulates the
expression of genes in a post-transcription level.49,50 Another is that the
protein binds to the DNA and regulates the expression in a transcription
level, as in the case of usual transcription factors.51-55 Further studies
including nuclear run-on analysis will clarify whether the GABP
regulated the expression of NDST-2 transcript as an RNA-binding factor
or a DNA-binding factor.
GABP is a heterodimer and is composed of GABP␣ and
GABP␤.45,46 GABP␣ itself had no nuclear localization ability, but
was localized in the nucleus when coexpressed with GABP␤, as
reported previously.47 GABP␣ was also localized in the nucleus
when coexpressed with ⫹-MITF. The ⫹-MITF and GABP␤
showed the same effect on the nuclear localization of GABP␣.
Because ⫹/⫹ CMCs expressed both ⫹-MITF and GABP␤,
GABP␣ may enter into the nucleus of ⫹/⫹ CMCs.
When coexpressed with mi-MITF, either GABP␣ or GABP␤
was not localized in the nucleus. This was reminiscent of the
previous finding that the mi-MITF disturbed the nuclear accumulation of other transcription factors, that is, Pu.1 and c-Fos.56 The
mi-MITF may inhibit the ability of GABP to express NDST-2
transcript by disturbing the nuclear localization.
The ⫹-MITF did not affect the luciferase activity of the reporter
plasmid under the control of the 5⬘-UTR of the NDST-2 gene containing
the GGAA motif. The ⫹-MITF might not be necessary for the
expression of NDST-2 transcript, because the NDST-2 gene was
normally expressed in tg/tg mast cells that lacked any MITFs. Besides
the NDST-2 transcript, the expression of c-kit, Gr B, and TPH transcripts
were negatively affected by the presence of mi-MITF.25 The level of
expression of c-kit, Gr B, and TPH transcripts did decrease in tg/tg
CMCs25 but that of NDST-2 transcript did not decrease in tg/tg CMCs.
In other words, the ⫹-MITF may enhance the expression of c-kit, Gr B,
and TPH transcripts but not the expression of NDST-2 transcript.
NDST-2 is the first gene whose expression was affected negatively by
mi-MITF but whose expression was not affected positively by ⫹-MITF.
In vitro binding assay and immunoprecipitation assay showed
that GABP and MITF physically interacted with each other. The
physical interactions between the ets family transcription factors
and the bHLH-Zip family transcription factors have been reported.57 The present result may be another example of such
interactions. GABP␤ contains a Zip-like motif.47 de la Brousse and
coworkers58 reported that the Zip-like motif of GABP␤ can be
replaced by a Zip domain. Because Zip domains are known to
mediate the protein-protein interactions,59 GABP␤ and MITF
might interact with each other through the Zip-like domain of
GABP␤ and the Zip domain of MITF. Further biochemical studies
are necessary to reveal the detailed mechanism of the interaction.
Heparin plays an important role for the storage of mediators in
mast cell granules, because amounts of various mediators, such as
MMCP-4, MMCP-5, and MMCP-6, decrease in NDST-2⫺/⫺ mast
cells.33,34 We have demonstrated that MITF regulates the transcription of MMCP-4, MMCP-5, and MMCP-6 genes.11-13 Here we
showed that MITF was involved in the expression of NDST-2
transcript. MITF appeared to regulate both transcription and
storage of MMCP-4, MMCP-5, and MMCP-6.
Heparin is synthesized with multiple steps. Besides NDST-2,
various enzymes, such as copolymerase EXT-1 and EXT-2, are
involved in heparin synthesis in mast cells.60 Further studies will
reveal the importance of such enzymes.
Taken together, the mi-MITF possessed the inhibitory effect on
the expression of NDST-2 transcript by disturbing the nuclear
localization of GABP. The decreased heparin content in mi/mi skin
mast cells may be attributable to the deficient expression of the
NDST-2 transcript.
Acknowledgments
The authors thank Dr H. Arnheiter of National Institutes of Health
for VGA9-tg/tg mice, Dr K. Nakajima of Osaka City University for
pSP-Luc, Dr S. Nagata of Osaka University for pEF-BOS, and Dr I.
Matsumura of Osaka University for CS2⫹MT.
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2001 97: 3032-3039
doi:10.1182/blood.V97.10.3032
Inhibitory effect of the mi transcription factor encoded by the mutant mi allele
on GA binding protein−mediated transcript expression in mouse mast cells
Eiichi Morii, Hideki Ogihara, Keisuke Oboki, Chika Sawa, Takahiko Sakuma, Shintaro Nomura, Jeffrey D.
Esko, Hiroshi Handa and Yukihiko Kitamura
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