Download Inhibitory effect of the transcription factor encoded by

From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
HEMATOPOIESIS
Inhibitory effect of the transcription factor encoded by the mutant mi
microphthalmia allele on transactivation of mouse mast cell protease 7 gene
Hideki Ogihara, Eiichi Morii, Dae-Ki Kim, Keisuke Oboki, and Yukihiko Kitamura
The transcription factor encoded by the mi
locus (MITF) is a transcription factor of the
basic-helix-loop-helix zipper protein family.
Mice of mi/mi genotype express a normal
amount of abnormal MITF, whereas mice of
tg/tg genotype do not express any MITFs
due to the transgene insertional mutation.
The effect of normal (ⴙ) and mutant (mi)
MITFs on the expression of mouse mast cell
protease (MMCP) 6 and 7 was examined.
Both MMCP-6 and MMCP-7 are tryptases,
and their coding regions with high homology are closely located on chromosome 17.
Both MMCP-6 and MMCP-7 genes are ex-
pressed in normal cultured mast cells (ⴙ/ⴙ
CMCs). Although the transcription of
MMCP-6 gene was severely suppressed in
both mi/mi and tg/tg CMCs, that of MMCP-7
gene was severely suppressed only in mi/mi
CMCs. The study identified the most significant segment for the transcription in the 5ⴕ
flanking region of MMCP-7 gene. Unexpectedly, no CANNTG motifs were found that are
recognized and bound by ⴙ-MITF in this
segment. Instead, there was an AP-1 binding motif, and binding of c-Jun to the AP-1
motif significantly enhanced the transcription of MMCP-7 gene. The complex forma-
tion of c-Jun with either ⴙ-MITF or mi-MITF
was demonstrated. The binding of ⴙ-MITF
to c-Jun enhanced the transactivation of
MMCP-7 gene, and that of mi-MITF suppressed the transactivation. Although the
former complex was located only in the
nucleus, the latter complex was predominantly found in the cytoplasm. The negative
effect of mi-MITF on the transcription of
MMCP-7 gene appeared to be executed
through the interaction with c-Jun. (Blood.
2001;97:645-651)
© 2001 by The American Society of Hematology
Introduction
We have been studying the involvement of the transcription
factor encoded by the mi locus (MITF) in the transcription of
genes expressed by mast cells. MITF is a member of the
basic-helix-loop-helix–leucine zipper (bHLH-Zip) protein of
transcription factors.1,2 The mutant mice of mi/mi genotype
were found by Hertwig among the offspring of an X-irradiated
male mouse.3,4 The mutant gene was introduced into C57BL/6
background and has been kept in the Jackson Laboratory (Bar
Harbor, ME). The MITF encoded by the mutant mi allele
(hereafter mi-MITF) deletes 1 of 4 consecutive arginines in the
basic domain.1,5,6 The mi-MITF is defective in the DNA-binding
activity7 and the nuclear localization potential.8
A VGA-9-tg/tg transgenic mouse possessing the transgeneinsertional mutation at the 5⬘ flanking region of the mi gene was
produced.1,9 The expression of MITF was undetectable in
various cells of VGA-9-tg/tg mice, including mast cells.1 We
introduced the tg transgene into C57BL/6 background and
compared messenger RNA (mRNA) expression between cultured mast cells (CMCs) derived from C57BL/6-mi/mi mice and
those of C57BL/6-tg/tg mice.10 The transcription of 3 genes
encoding mouse mast cell proteases (MMCPs), MMCP-4,
MMCP-5, and MMCP-6, was impaired in CMCs of both mice.
However, the transcription of the gene encoding the c-kit
receptor, tryptophan hydroxylase, or granzyme B was remarkably reduced in CMCs of C57BL/6-mi/mi mice, but the reduction was significantly smaller in CMCs of C57BL/6-tg/tg mice,
suggesting the inhibitory effect of mi-MITF on the transactivation of particular genes in CMCs.10
MMCPs are serine proteases and are useful for studying the
mechanism of gene expression in mast cells because of their
abundant expression. Nine MMCPs have been known.11-18
MMCP-1, 11 MMCP-2, 12 MMCP-4, 11,13 MMCP-5, 11,14 and
MMCP-918 are chymases, and the genes encoding each of them
reside on chromosome 14. Although the expression of both
MMCP-4 and MMCP-5 genes were impaired in mi/mi CMCs,
the mechanism of the impaired expression was different.19,20
MMCP-615 and MMCP-716 are tryptases, and the genes encoding each of them reside on the chromosome 17. The coding region
of the MMCP-6 gene is highly homologous with that of MMCP7.16 Both MMCP-6 and MMCP-7 mRNAs are present in mast cells
of the skin but not in mast cells of the intestinal mucosa.16
However, MMCP-6 and MMCP-7 showed some different biological characteristics. Mast cells in the peritoneal cavity express
MMCP-6 mRNA but not MMCP-7 mRNA.16 A considerable
amount of MMCP-6 mRNA is expressed in CMCs throughout the
culture period, but the amount of MMCP-7 mRNA decreases
markedly 3 weeks after starting the culture of CMCs.16
We attempted to examine the effect of MITF on the expression
of the MMCP-7 gene. However, the MMCP-7 gene is not
expressed in mast cells of the C57BL/6 strain due to a point
mutation at the first nucleotide of exon 2/intron 2 splice site.21,22
Because the MMCP-7 gene is normally expressed in the WB strain,
From the Department of Pathology, Osaka University Medical School, Suita,
Japan.
Medical School, Yamada-oka 2-2,
kitamura@patho.med.osaka-u.ac.jp.
Suita
565-0871,
Japan;
e-mail:
Submitted May 15, 2000; accepted October 5, 2000.
Supported by grants from the Ministry of Education, Science and Culture, the
Ministry of Health and Welfare, the Organization for Pharmaceutical Safety and
Research, and the Welfide Medicinal Research Foundation.
Reprints: Yukihiko Kitamura, Department of Pathology, Osaka University
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
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.
© 2001 by The American Society of Hematology
645
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
646
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
OGIHARA et al
we crossed WB-⫹/⫹ mice to C57BL/6-mi/⫹ or C57BL/6-tg/⫹
mice and finally obtained the mice possessing the WB strainderived MMCP-7 gene and double gene dose of either mi or tg
mutant allele. The expression of the WB strain-derived MMCP-7
gene remarkably decreased in CMCs of the mi/mi mice but
decreased only slightly in CMCs of the tg/tg mice. The regulation
of MMCP-7 gene expression appeared to be different from that of
MMCP-6 gene. We examined the mechanism and found that either
⫹-MITF or mi-MITF interacted with c-Jun and affected the
expression of the MMCP-7 gene through the c-Jun.
Materials and methods
Mice
The original stock of C57BL/6J-mi/⫹ mice was purchased from the
Jackson Laboratory (Bar Harbor, ME) and was maintained in our laboratory
by consecutive backcrosses with C57BL/6 mice of our own inbred colony.
The original stock of VGA-9-tg/tg mice,1 in which the mouse vasopressin–
Escherichia coli–galactosidase transgene was integrated at the 5⬘ flanking
region of the mi (MITF) gene, was kindly given by Dr H. Arnheiter
(National Institutes of Health, Bethesda, MD). The integrated transgene
was maintained by repeated backcrosses to C57BL/6 mice of our own
inbred C57BL/6 colony.
The mutant allele of MMCP-7 possessed by the C57BL/6 strain was
described as MMCP-7B6, and the normal allele of the WB strain as
MMCP-7WB. Mice of (MMCP-7WB/MMCP-7WB, mi/mi), (MMCP-7WB/
MMCP-7WB, tg/tg), and control (MMCP-7WB/MMCP-7WB, ⫹/⫹) genotype
were necessary in the present experiment. First, we crossed WB-(MMCP7WB/MMCP-7WB, ⫹/⫹) mice with C57BL/6-(MMCP-7B6/MMCP-7B6, mi/⫹)
mice or with C57BL/6-(MMCP-7B6/MMCP-7B6, tg/⫹) mice. Then we
selected (MMCP-7WB/MMCP-7B6, mi/⫹) mice by the presence of a white
belly spot3,4 and (MMCP-7WB/MMCP-7B6, tg/⫹) mice by the presence of a
tg transgene. Then we backcrossed these F1 hybrid mice to WB-(MMCP7WB/MMCP-7WB, ⫹/⫹) mice and selected (MMCP-7WB/MMCP-7WB, mi/⫹)
and (MMCP-7WB/MMCP-7WB, tg/⫹) mice by examining the MMCP-7
genotype.22 We crossed together mice of each genotype and finally selected
mice of (MMCP-7WB/MMCP-7WB, mi/mi) and of (MMCP-7WB/MMCP-7WB,
tg/tg) with white coat and mice of (MMCP-7WB/MMCP-7WB, ⫹/⫹) that had
completely black coat and no tg transgene. Because all mice were
homozygous at the MMCP-7WB allele, we describe them hereafter just as
mi/mi, tg/tg, and ⫹/⫹ mice.
Examination of genomic DNA
Tail tip was cut under ether anesthesia, and DNA was extracted as described
by Blin and Stafford.23 The presence or absence of tg transgene was
examined by polymerase chain reaction (PCR) with sense (5⬘-CATTAAATGTGAGCGAGTAACAACCCGTCG-3⬘) and antisense (5⬘-CTTTCGCCAGCTGGCGTAATAGCGAAGAGG-3⬘) primers.22
The region, including the exon 2/intron 2 splice site, of the MMCP-7
gene was amplified by PCR with the use of sense (5⬘-GCCCTGGCAGGTGAGCCTGC-3⬘) and antisense (5⬘-TTGTTGGGGTCAGCAACATC-3⬘)
primers.22 The correctly sized PCR products were purified by QIAquick
PCR Purification Kit (Qiagen, Santa Clarita, CA). The purified PCR
products were directly sequenced with standard dideoxy sequencing
techniques by Model 373A DNA sequencer (Applied Biosystems, Foster
City, CA).
Promoter region of MMCP-7 gene
The isolation of the promoter region of MMCP-7 gene was performed with
the Mouse Genome Walker Kit (Clonetech Laboratories, Palo Alto, CA)
according to the manufacturer’s instructions. The isolated promoter region
was cloned into Bluescript KS (⫺) plasmids (pBS; Stratagene, La
Jolla, CA).
Cells
Pokeweed mitogen-stimulated spleen cell condition medium (PWM-SCM)
was prepared according to the method described by Nakahata et al.24 Mice
of mi/mi, tg/tg, and ⫹/⫹ genotype were used at an age of 2 to 3 weeks to
obtain CMCs. Mice were killed by decapitation after ether anesthesia, and
the spleens were removed. Spleen cells of each genotype were cultured in
␣-minimal essential medium (␣-MEM; ICN Biomedicals, Costa Mesa, CA)
supplemented with 10% PWM-SCM and 10% fetal calf serum (FCS;
Nippon Biosupp Center, Tokyo, Japan). Half of the medium was replaced
every 7 days. Three weeks after initiation of the culture, more than 95% of
cells were CMCs.25 These CMCs were harvested and used for experiments.
FMA/3, mouse mastocytoma cell line, was maintained in ␣-MEM
supplemented with 10% FCS and antibiotics. MST,26 the stable heparinproducing cell line derived from the Furth murine masytoma, was also
maintained in ␣-MEM supplemented with 10% FCS and antibiotics. Jurkat,
human T-cell line, was maintained in RPMI 1640 supplemented with 10%
FCS and antibiotics. Monkey COS-7 cells were maintained in Dulbecco
modification of Eagle medium (DMEM) supplemented with 10% FCS and
antibiotics.
Construction of expression and reporter plasmids
The luciferase gene subcloned into pSP72 (pSPLuc) was kindly given by Dr
K. Nakajima (Osaka City University Medical School, Osaka, Japan).27 To
construct reporter plasmids, a DNA fragment containing a promoter region
and the first exon (noncoding region) of the MMCP-7 gene (⫺1892 to ⫹20)
was cloned into the upstream of luciferase gene in pSPLuc. The deletion of
the MMCP-7 promoter was done by using the appropriate restriction
enzyme or PCR. The mutation in the AP-1 binding consensus motif was
introduced by PCR with mismatch sense (5⬘-TGGAGTCTGAGTTGCCCATT-3⬘) and antisense (5⬘-AATGGGCAACTCAGACTCCA-3⬘) primers. Deleted or mutated products were verified by DNA sequencing.
pEF-BOS expression vector was kindly given by Dr S. Nagata (Osaka
University Medical School).28 pEF-BOS containing the whole coding
region of ⫹-MITF or mi-MITF had been constructed in our laboratory.29,30
The Myc-tagged ⫹- or mi-MITF constructs has been described.8 pBS
containing the whole coding region of c-Jun complementary DNA (cDNA)
(pBS-c-Jun) was constructed in our laboratory. c-Jun cDNA was subcloned
into pEF-BOS and pcDNA3-FLAG. pEF-BOS containing the dominant
negative c-Jun, TAM-67,31 was kindly given by Dr K. Shimozaki and Dr S.
Nagata (Osaka University Medical School).
Northern blot analysis
Total RNAs (20 ␮g) were isolated with the lithium chloride–urea method.32
Northern blot analyses were performed using the fragments of MMCP-7,
MMCP-6, MC-CPA, and GAPDH cDNAs as probes after being labeled
with [32P]-␣-dCTP (DuPont/NEN Research Products, Boston, MA; 10mCi/
mL) by random oligonucleotide priming. The probe of MMCP-7 cDNA was
a 247–base pair (bp) MMCP-7 gene specific fragment that corresponds to
the nucleotides 2071 to 2317 of the gene. The probes of MMCP-6,
MC-CPA, and GAPDH have been described.15,33 After hybridization at
45°C, blots were washed to a final stringency of 0.2 ⫻ SSC (1 ⫻ SSC is 150
mM NaCl, 15 mM trisodium citrate, pH 7.4) at 55°C and subjected to
autoradiography.
Luciferase assay
FMA/3 or Jurkat cells (5 ⫻ 106 cells in 300 ␮L of the culture medium in a
0.4-cm cuvette) were transfected with a luciferase-reporter plasmid (10 ␮g),
effector plasmids (each 1 ␮g), and an expression vector containing
␤-galactosidase gene (2 ␮g) by electroporation (Gene Pulser; Bio-Rad
Laboratories, Hercules, CA) at a setting of 975 ␮F/300V and at room
temperature. The expression vector containing ␤-galactosidase gene was
used to normalize the varying transfection efficiencies. The compositions of
transfected expression plasmids are described in figure legends. The total
amount of transfected DNA was always kept at 20 ␮g, using a backbone
plasmid, pEF-BOS. After 24 to 36 hours, cells were harvested, and
luciferase activity was assayed with a luminometer LB96P (Berthold,
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
GmbH, Wildbad, Germany). By determining the ␤-galactosidase activity
and the total protein concentration, the luciferase activity was normalized as
described previously.10
GENE EXPRESSION OF MOUSE MAST CELL PROTEASE 7
647
Results
Expression of MMCP-7
Overexpression of dominant negative c-Jun, TAM-67,
in MST cells
MST cells (2 ⫻ 107 cells) were transfected with 10 ␮g pEF-BOS containing
dominant negative c-Jun, TAM-67, or empty pEF-BOS with the electroporation, as mentioned above. After 4 days of culture, total RNAs (20 ␮g)
were extracted and subjected to Northern blot analysis.
Electrophoretic mobility shift assay
The production and purification of glutathione-S-transferase (GST) fusion
protein has been described.7 The wild-type oligonucleotide of the MMCP-7
promoter sequence (5⬘-AGCTGTCCCTGGAGTCTGAGTCACCCATTTAGCT-3⬘) was labeled with [32P]-␣-dCTP by filling 5⬘-overhangs and
was used as a probe of Electrophoretic mobility shift assay (EMSA). The
DNA binding reaction and electrophoresis were done as described previously.7 For the competition assay, unlabeled wild-type oligonucleotide or
mutated oligonucleotide (5⬘-AGCTGTCCCTGGAGTCTGAGTTGCCCATTTAGCT-3⬘) was added.
In vitro binding assays
The 35S-labeled c-Jun protein was synthesized, using the reticulocyte lysate
system (TNT system, Promega, Madison, WI). c-Jun cDNA in pBluescript
was transcribed with T7 RNA polymerase and translated in the presence of
35S-methionine. For the binding assays, the 35S-labeled c-Jun protein was
incubated for 1 hour at room temperature, with GST-⫹-MITF, GST-miMITF, 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. As controls, purified GST-⫹-MITF,
GST-mi-MITF, or GST was subjected to SDS-PAGE, and the gel was
stained with Coomassie brilliant blue.
Preparation of nuclear and cytoplasmic extracts
Nuclear and cytoplasmic extracts were prepared as described before.8
COS-7 cells were transfected with 5 ␮g each of the expression plasmids by
calcium phosphate method. Forty-eight hours after transfection, the cells
were collected and washed with PBS. Then the cells were resuspended in
100 ␮L buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 15 mM MgCl2, 0.1
mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride
[PMSF]). The cells were allowed to swell on ice for 3 minutes, and Nonidet
P-40 was added to a final concentration of 0.1%. After vigorous vortexing
for 10 seconds, the homogenate was centrifuged at 3000 rpm for 3 minutes.
The supernatant was used as cytoplasmic fraction. The pellet was resuspended in 100 ␮L buffer B (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, and 1 mM PMSF). The homogenate was
allowed to swell on ice for 30 minutes, subsequently diluted to 200 mM
NaCl in the same buffer lacking NaCl, and centrifuged at 15 000 rpm for 20
minutes at 4°C. The supernatant was used as the nuclear extract.
The expression of MMCP-7 gene is abnormal in the C57BL/6
strain because of the point mutation of an exon/intron splice site.22
However, the MMCP-7 gene is normally expressed in the WB
strain.21 We crossed WB-mice (MMCP-7WB/MMCP-7WB, ⫹/⫹)
with C57BL/6-mice (MMCP-7B6/MMCP-7B6, mi/⫹) or with C57BL/
6-mice (MMCP-7B6/MMCP-7B6, tg/⫹) and ultimately obtained
mice of (MMCP-7WB/MMCP-7WB, mi/mi), (MMCP-7WB/MMCP7WB, tg/tg), or (MMCP-7WB/MMCP-7WB, ⫹/⫹) genotype. Because
all mice used in the present experiment had MMCP-7WB/MMCP7WB genotype, we hereafter describe them just as mi/mi, tg/tg, or
⫹/⫹ mice.
CMCs were obtained using spleens of mi/mi, tg/tg, or ⫹/⫹
mice. Total RNA was extracted from CMCs of each genotype, and
the expression of MMCP-7 gene was examined by Northern blot
analysis. As controls, the expression of MMCP-6 and MC-CPA
genes was also examined. The expression of MMCP-6 gene was
severely impaired in both mi/mi and tg/tg CMCs; that of MC-CPA
gene was normal in both mi/mi and tg/tg CMCs. However, the
expression of MMCP-7 gene was different between mi/mi and tg/tg
CMCs. It was severely reduced in mi/mi CMCs but only slightly
reduced in tg/tg CMCs (Figure 1).
The promoter region of the MMCP-7 gene
McNeil et al16 reported the sequence of MMCP-7 gene 80 bp
upstream of the transcription initiation site. We cloned further 1811
bp upstream region to examine the regulation of transcription
(Figure 2). Although the coding regions of MMCP-7 and MMCP-6
genes showed a remarkable homology (73%),16 the promoter
regions of these genes showed less homology (51%) (Figure 2).
We constructed the reporter plasmid that contained the luciferase gene under the control of the MMCP-7 promoter starting
from nt ⫺1891 (⫹1 shows transcription initiation site, hereafter
referred to as pP7-1891). We also constructed the deleted reporter
plasmids containing the MMCP-7 promoter starting from nt
⫺1813, ⫺1117, ⫺672, ⫺542, ⫺368, ⫺342, ⫺280, ⫺235, ⫺185,
Immunoprecipitation
The nuclear or cytoplasmic extracts prepared as described above were
incubated with LIP buffer (10 mM HEPES, 250 mM NaCl, 0.1% Nonidet
P-40, 5 mM EDTA, and 1 mM PMSF) and Protein G Sepharose (Amersham
Pharmacia Biotech, 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-FLAG monoclonal antibody (mAb) (M2, Sigma, St. Louis, MO) or
anti-Myc mAb (9E10, Pharmingen, San Diego, CA) for 1 hour in LIP
buffer. Immunocomplexes were washed 4 times with LIP buffer, resuspended in loading buffer, boiled, and analyzed by Western blot with
anti-Myc or anti-FLAG mAb.
Figure 1. Expression of MMCP-7, MMCP-6, and MC-CPA mRNA in ⴙ/ⴙ, tg/tg,
and mi/mi CMCs. Twenty micrograms of total RNA prepared from ⫹/⫹, tg/tg, or
mi/mi CMCs was loaded in each lane. The membranes were hybridized with specific
DNA probes. Reprobing with GAPDH allowed verification that an equal amount of
mRNA was loaded per lane.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
648
OGIHARA et al
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Figure 3. Luciferase activity under the control of sequentially deleted MMCP-7
promoter. The luciferase gene under the control of intact or deleted MMCP-7
promoter was transfected to FMA/3 cells. The value of the luciferase activity was
divided by the value obtained with backbone vector pSPLuc and was shown as the
relative luciferase activity. The data represent the mean ⫾ standard error (SE) of 3
experiments. In some cases, the SE was too small to be shown by bars.
when c-Jun cDNA and pP7-185-AP-1-mut were cotransfected, the
transactivating effect of c-Jun was not detectable (Figure 4A).
A dominant negative c-Jun, TAM-67, was used to examine
whether endogenous c-Jun contributed to the promoter activity.
TAM-67 is a variant of c-Jun in which amino acids 3 to 122 are
deleted. TAM-67 may dimerize and bind DNA but lacks the
transactivating potential.31 When various amounts of TAM-67
cDNA were cotransfected with pP7-185 into FMA/3 cells, the
luciferase activity of pP7-185 decreased in a dose-dependent
manner (Figure 4A).
In the next experiment, TAM-67 was transfected into the MST
Figure 2. The nucleotide sequence of 5ⴕ flanking region of MMCP-7 gene. Each
CANNTG motif was boxed. The AP-1 binding consensus motif was underlined. A part
of the first exon is shown by capitals, and 5⬘ flanking region is shown by lower case.
The transcription initiation site was numbered as ⫹1.
or ⫺84. Each reporter plasmid was transfected to FMA/3 mastocytoma cells, which weakly expressed the MMCP-7 mRNA. When
the reporter plasmid deleted up to position ⫺235 (pP7-235) was
transfected, a small but significant luciferase activity was found
when compared to the basal activity. The reporter plasmid deleted
to position ⫺185 (pP7-185) showed a high luciferase activity, and
pP7-84 showed the basal luciferase activity. Thus, the promoter
region between nt ⫺185 and ⫺85 appeared important for the
expression of the MMCP-7 gene (Figure 3).
Role of AP-1 binding motif and c-Jun
Between nt ⫺185 and ⫺ 85, no CANNTG motif that is recognized
and bound by MITF was present. However, an AP-1 binding
consensus motif was found (Figure 2). The reporter plasmid
pP7-185 was mutated at the AP-1 binding motif (hereafter called
pP7-185-AP-1-mut) and transfected into FMA/3 cells. The mutation abolished the luciferase activity of pP7-185 (Figure 4A),
suggesting that the AP-1 binding motif was used for the transactivation of MMCP-7 gene.
c-Jun cDNA was cotransfected with pP7-185 to FMA/3 cells as
an effector molecule. The cotransfection increased the luciferase
activity of pP7-185 58-fold when compared to the basal level
(4.8-fold as compared with pP7-185 alone) (Figure 4A). However,
Figure 4. Involvement of c-Jun in transactivation of MMCP-7 promoter. (A) The
reporter plasmid containing the intact, mutated, or deleted AP-1 motif (pP7-185,
pP7-185-AP-1-mut, or pP7-84, respectively) was cotransfected with effector plasmids to FMA/3 cells. The value of luciferase activity was divided by that obtained with
the cotransfection of pP7-84 and pEF-BOS and was shown as relative luciferase
activity. (B) Reduced expression of MMCP-7 gene by the overexpression of dominant
negative form of c-Jun, TAM-67. Total RNA was extracted from MST cells (indicated
as Original), MST cells overexpressing TAM-67, or MST cells overexpressing empty
expression vector (indicated as Mock). The expression of MMCP-7 gene was
examined with Northern blot analysis. (C) Binding of c-Jun to the AP-1 motif in the
MMCP-7 promoter. The 5⬘-AGCTGTCCCTGGAGTCTGAGTCACCCATTTAGCT, designated as AP-1, was used as a probe (the AP-1 motif is underlined). The
oligonucleotide mutated in the AP-1 motif (TGAGTCA to TGAGTTG) was designated
as AP-1-mut (the mutated nucleotides are underlined). The excess amount of
nonlabeled AP-1 or AP-1-mut was added as a competitor.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
GENE EXPRESSION OF MOUSE MAST CELL PROTEASE 7
649
increased the luciferase activity of pP7-185 (2.1-fold and 3.5fold, respectively). The cotransfection of both c-Jun and
⫹-MITF cDNAs remarkably increased the luciferase activity of
pP7-185 (9.5-fold).
Physical interaction between c-Jun and MITF
Physical interaction between c-Jun and MITF was examined. The
coprecipitation of 35S-labeled c-Jun with GST-⫹-MITF or with
GST-mi-MITF was examined by SDS-PAGE. The complex of
c-Jun and GST-⫹-MITF and that of c-Jun and GST-mi-MITF were
detected, but the complex of c-Jun and GST was not (Figure 6A).
Inhibitory effect of mi-MITF on nuclear translocation of c-Jun
Figure 5. Functional cooperation between ⴙ-MITF and c-Jun in the promoter
activation of MMCP-7 gene. (A) The reporter plasmids were transfected to FMA/3
cells with the expression plasmid containing ⫹-MITF or mi-MITF cDNA or the
expression vector alone. (B) The reporter plasmids were transfected to Jurkat cells
with the effector plasmids in various combinations. Control vector was used to adjust
the total amount of DNA used in each transfection.
cells, because they express MMCP-7 mRNA at an easily detectable
level. After 4 days of culture, the expression of MMCP-7 gene was
examined by Northern blot analysis. The overexpression of TAM67, which dominantly inhibits the DNA binding of endogenous
c-Jun, significantly reduced the amount of MMCP-7 mRNA
(Figure 4B). This suggested that the endogenous c-Jun was
involved in the transactivation of MMCP-7 gene.
The binding of c-Jun with the MMCP-7 promoter was examined by
EMSA. GST–c-Jun fusion protein bound the oligonucleotide probe
containing the AP-1 binding motif (Figure 4C). The binding of c-Jun
was specifically abolished by the excess amount of the normal (5⬘AGCTGTCCCTGGAGTCTGAGTCACCCATTTAGCT-3⬘) nonlabeled oligonucleotide but not by the mutated (5⬘-AGCTGTCCCTGGAGTCTGAGTTGCCCATTTAGCT-3⬘) nonlabeled oligonucleotide.
Myc-tagged-⫹-MITF or Myc-tagged-mi-MITF cDNA was cotransfected with FLAG-tagged-c-Jun cDNA into COS-7 cells. Lysate of
transfected cells was divided into nuclear and cytoplasmic fractions. Each fraction was subjected to immunoprecipitation with
anti-Myc antibody and Western blot analysis with anti-Myc or
anti-FLAG antibody. When Myc-tagged–⫹-MITF and FLAGtagged–c-Jun were transfected, Myc-tagged–⫹-MITF and coprecipitated FLAG-tagged–c-Jun were detected only in the nuclear
fraction (Figure 6B). However, when Myc-tagged–mi-MITF and
FLAG-tagged–c-Jun were cotransfected, one third of Myc-tagged–
mi-MITF and FLAG-tagged–c-Jun were detected in the nuclear
fraction, and the remaining Myc-tagged–mi-MITF and FLAGtagged–c-Jun were in the cytoplasmic fraction (Figure 6B).
Discussion
Both MMCP-7 and MMCP-6 are tryptases,15,16 and genes encoding
each of them reside on chromosome 17.34,35 The coding region of
Functional cooperation between c-Jun and MITF
The expression of MMCP-7 gene was enhanced through cis
elements located between nt ⫺185 and ⫺85. An AP-1 binding
consensus motif but no CANNTG motif was present between nt
⫺185 and ⫺85. Therefore, c-Jun was considered to be involved in
the transactivation of MMCP-7 through the AP-1 binding sequence. Then we examined whether MITF affected the transactivation activity of c-Jun. When cDNA encoding ⫹-MITF was
cotransfected with pP-185 to FMA/3 cells, the luciferase activity
increased 38-fold when compared to the basal level (3.2-fold when
compared to pP7-185 alone) (Figure 5A). However, the promoter
activity of pP-185 was reduced by the cotransfection of mi-MITF in
a dose-dependent manner (Figure 5A).
To remove the possible involvement of the endogenous MITF,
we used the Jurkat human T-cell line that does not express MITF.
The reporter plasmid pP7-185 was not transactivated by the
endogenous factors in Jurkat cells (Figure 5B). The transfection of
either c-Jun cDNA alone or ⫹-MITF cDNA alone moderately
Figure 6. Physical interaction between MITF and c-Jun. (A) In vitro binding of
c-Jun to MITF. 35S-labeled c-Jun was subjected to coprecipitation with GST-⫹-MITF,
GST-mi-MITF, or GST-coated beads, and the protein complex was analyzed by
SDS-PAGE. Coomassie brilliant blue staining for each GST fusion protein was also
shown. (B) Coimmunoprecipitation of MITF and c-Jun. COS-7 cells were transfected
with FLAG-tagged–c-Jun and Myc-tagged–⫹MITF or Myc-tagged–mi-MITF. Nuclear
or cytoplasmic extract was subjected to immunoprecipitation with anti-Myc antibody.
Precipitates were separated by SDS-PAGE and immunoblotted with anti-Myc or
anti-FLAG antibody.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
650
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
OGIHARA et al
MMCP-7 gene is highly homologous with that of MMCP-6 gene,16
but the 5⬘ flanking region of both genes showed a remarkable
difference. Three MITF-binding consensus sequences are present
in the promoter region of MMCP-6 gene, all of which are
recognized and bound by ⫹-MITF.30 In fact, ⫹-MITF appeared to
play important roles for the transcription of MMCP-6 gene.30
However, we could not find any MITF-binding consensus sequences in the most significant segment (pP7-185) of the MMCP-7
promoter. Although the transcription of MMCP-6 gene was severely impaired in both mi/mi and tg/tg CMCs, that of MMCP-7
gene was severely impaired only in mi/mi CMCs. The abnormal
mi-MITF was expressed in mi/mi CMCs but practically no MITFs
were expressed in tg/tg CMCs. Therefore, the presence of mi-MITF
rather than the absence of ⫹-MITF appeared to have a negative
effect on the transcription of MMCP-7 gene.
We investigated the mechanism of the negative effect of
mi-MITF. In spite of the lack of the MITF-binding consensus
sequence, presence of ⫹-MITF enhanced the promoter activity of
pP7-185 and that of mi-MITF suppressed it. Both ⫹-MITF and
mi-MITF appeared to influence the promoter function through
binding to other transcription factors. There is an AP-1 binding
consensus motif in the pP-185 promoter. The mutation of the AP-1
binding motif abolished the promoter activity. The coexpression of
normal c-Jun enhanced the promoter activity, and that of dominant
negative mutant of c-Jun suppressed it. The binding of c-Jun to the
AP-1 binding motif activated the promoter, and binding of
⫹-MITF to the c-Jun further enhanced the promoter and that of
mi-MITF suppressed it. In fact, we demonstrated the physical
interaction of c-Jun with either ⫹-MITF or mi-MITF.
The complex of c-Jun and ⫹-MITF was detectable only in the
nuclear fraction, but that of c-Jun and mi-MITF was observed in
both nuclear and cytoplasmic fractions. A possible explanation of
suppressing potential of mi-MITF may be its deficient nuclear
localization potential. Not only mi-MITF by itself but also the
complex of mi-MITF and c-Jun appeared to have deficient nuclear
translocation potential. Hemesath et al5 showed a deficient DNAbinding ability of the heterodimer of mi-MITF and TEFB. Because
TFEB is another bHLH-Zip–type transcription factor, this mechanism may be understandable. However, because there was no
CANNTG consensus sequence that may be recognized and bound
by bHLH-Zip–type transcription factors in the pP-185 promoter, it
is difficult to explain the negative effect of mi-MITF by the
deficient DNA binding potential.
We produced a cDNA library from ⫹/⫹ CMCs.34 By subtracting mRNAs expressed by mi /mi CMCs or tg/tg CMCs, we obtained
(⫹/⫹-mi/mi) and (⫹/⫹-tg/tg) subtraction libraries.10,36 The number of clones that may hybridize cDNAs contained by the cDNA
library of ⫹/⫹ CMCs was significantly greater in the (⫹/⫹-mi/mi)
library than in the (⫹/⫹-tg/tg) library.10 This is consistent with the
present result. In addition to the loss of transactivation potential,
the presence of mi-MITF suppressed the transactivation potential
through other transcription factors. The transcription of c-kit,
granzyme B, and tryptophan hydroxylase genes was also suppressed by the presence of mi-MITF.10 Probably a similar suppressing mechanism may play a role, but counterpart transcription factors
that make a complex with mi-MITF may be different in each case.
Comparison of ⫹/⫹, mi/mi, and tg/tg CMCs may be a
convenient method to examine the influence of MITF on the
expression of genes that are expressed in CMCs. By screening the
(⫹/⫹-mi/mi) subtraction library, the maximum number of possible
target genes of MITF may be obtained. The following 2 types of
genes are included: (1) genes for which transcription ⫹-MITF is
essential and (2) genes of which transcription was suppressed by
mi-MITF. When the expression of type 2 genes was compared
between ⫹/⫹ CMCs and tg/tg CMCs, the expression in tg/tg
CMCs was slightly but significantly lower than that of ⫹/⫹ CMCs.
Even if such genes were not detected in the (⫹/⫹-tg/tg) subtraction
library, ⫹-MITF may enhance their expression. Taken together, the
mechanism of the transcription regulation by MITF was different
between MMCP-6 and MMCP-7 genes, and the comparison of
expression among ⫹/⫹, mi/mi, and tg / tg CMCs may be useful for
understanding the mechanism.
Acknowledgments
The authors thank Dr J. D. Esko of University of California, San
Diego for MST cells, Dr H. Arnheiter of National Institutes of
Health for tg/tg mice, Dr K. Shimozaki and Dr S. Nagata of Osaka
University for TAM-67 in pEF-BOS, Dr S. Nagata of Osaka
University for pEF-BOS, Dr I. Matsumura of Osaka University for
pcDNA3-FLAG, and T. Jippo and S. Adachi of Osaka University
and T. Sawado of Fred Hutchinson Cancer Research Center for
helpful discussion.
References
1. Hodgkinson CA, Moore KJ, Nakayama A, et al.
Mutations at the mouse microphthalmia locus are
associated with defects in a gene encoding a
novel basic-helix-loop-helix-zipper protein. Cell.
1993;74:395-404.
2. Hughes JJ, Lingrel JB, Krakowsky JM, Anderson
KP. A helix-loop-helix transcription factor-like
gene is located at the mi locus. J Biol Chem.
1993;268:20687-20690.
3. Hertwig P. Neue Mutationen und koppelungsgruppen bei der Hausmaus. Z Indukt Abstamm-u
VererbLehre. 1942;80:220-246.
4. Hertwig P. Sechs neue Mutationen bei der Hausmaus in ihrer bedeutung f ur allgemeine Vererbungsfragen. Z Menschl Vererbungs-u KonstL.
1942;26:1-21.
5. Hemesath TJ, Streingrimsson E, McGill G, et al.
Microphthalmia, a critical factor in melanocyte
development, defines a discrete transcription factor family. Gene Dev. 1994;8:2770-2780.
6. Steingrimsson E, Moore KJ, Lamoreux ML, et al.
Molecular basis of mouse microphthalmia (mi)
mutations helps explain their developmental and
phenotypic consequences. Nat Genet. 1994;8:
256-263.
7. Morii E, Takebayashi K, Motohashi H, Yamamoto
M, Nomura S, Kitamura Y. Loss of DNA binding
ability of the transcription factor encoded by the
mutant mi locus. Biochem Biophys Res Commun.
1994;205:1299-1304.
8. Takebayashi K, Chida K, Tsukamoto I, et al. Recessive phenotype displayed by a dominant
negative microphthalmia-associated transcription
factor mutant is a result of impaired nuclear localization potential. Mol Cell Biol. 1996;16:12031211.
9. Tachibana M, Hara Y, Vyas D, et al. Cochlear disorder associated with melanocyte anomaly in
mice with a transgenic insertional mutation. Mol
Cell Neurosci. 1992;3:433-445.
10. Ito A, Morii E, Kim DK, et al. Inhibitory effect of the
transcription factor encoded by the mi mutant allele in cultured mast cells of mice. Blood. 1999;
93:1189-1196.
11. Huang R, Blom T, Hellman L. Cloning and structural analysis of mMCP-1, mMCP-4, and
mMCP-5, three mast cell specific serine proteases. Eur J Immunol. 1991;21:1611-1621.
12. Serafin WE, Reynolds DS, Rogelj S, et al. Identification and molecular cloning of a novel mouse
mucosal mast cell serine protease. J Biol Chem.
1990;265:423-429.
13. Serafin WE, Sullivan TP, Conder GA, et al. Cloning of the cDNA and gene for mouse mast cell
protease 4: demonstration of its late transcription
in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the
mouse and rat. J Biol Chem. 1991;266:19341941.
14. McNeil HP, Austen KF, Somerville LL, Gurish MF,
Stevens RL. Molecular cloning of the mouse mast
cell protease-5 gene: a novel secretory granule
protease expressed early in the differentiation of
serosal mast cells. J Biol Chem. 1991;266:2031620322.
15. Reynolds DS, Gurley DS, Austen KF, Serafin WE.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 1 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 3
Cloning of the cDNA and gene of mouse mast cell
protease-6: transcription by progenitor mast cells
and mast cells of the connective tissue subclass.
J Biol Chem. 1991;266:3847-3853.
16. McNeil HP, Reynolds DS, Schiller V, et al. Isolation, characterization, and transcription of the
gene encoding mouse mast cell protease 7. Proc
Natl Acad Sci U S A. 1992;89:11174-11178.
17. Lutzelschwab C, Huang MR, Kullberg MC, Aveskogh M, Hellman L. Characterization of mouse
mast cell protease-8, the first member of a novel
subfamily of mouse mast cell serine proteases,
distinct from both the classical chymases and
tryptases. Eur J Immunol. 1998;28:1022-1033.
18. Hunt JE, Friend DS, Gurish MF, et al. Mouse
mast cell protease 9, a novel member of the chromosome 14 family of serine proteases that is selectively expressed in uterine mast cells. J Biol
Chem. 1997;272:29158-29166.
19. Jippo T, Lee YM, Katsu Y, et al. Deficient transcription of mouse mast cell protease 4 gene in
mutant mice of mi/mi genotype. Blood. 1999;93:
1942-1950.
20. Morii E, Jippo T, Tsujimura T, et al. Abnormal expression of mouse mast cell protease 5 gene in
cultured mast cells derived from mutant mi/mi
mice. Blood. 1997;90:3057-3066.
21. Ghildyal N, Friend DS, Freelund R, et al. Lack of
expression of the tryptase mouse mast cell protease 7 in mast cells of the C57BL/6J mouse.
J Immunol. 1994;153:2624-2630.
22. Hunt JE, Stevens RL, Austen KF, Zhang J, Xia Z,
GENE EXPRESSION OF MOUSE MAST CELL PROTEASE 7
Ghildyal N. Natural disruption of the mouse mast
cell protease 7 gene in the C57BL/6 mouse.
J Biol Chem. 1996;271:2851-2855.
651
cultured mast cell of mice. Blood. 1996;88:12251233.
23. Blin N, Stafford DW. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976;3:2303-2308.
30. Morii E, Tsujimura T, Jippo T, et al. Regulation of
mouse mast cell protease 6 gene expression by
transcription factor encoded by the mi locus.
Blood. 1996;88:2488-2494.
24. Nakahata T, Spicer SS, Cantey JR, Ogawa M.
Clonal assay of mouse mast cell colonies in
methylcellulose culture. Blood. 1982;60:352-362.
31. Brown PH, Chen TK, Birrer MJ. Mechanism of
action of a dominant-negative mutant of c-Jun.
Oncogene. 1994;9:791-799.
25. Nakano T, Sonoda T, Hayashi C, et al. Fate of
bone marrow-derived cultured mast cells after
intracutaneous, intraperitoneal, and intravenous
transfer into genetically mast cell-deficient W/W v
mice: evidence that cultured mast cells can give
rise to both connective tissue type and mucosal
mast cells. J Exp Med. 1985;162:1025-1043.
26. Montgomery RI, Lidholt K, Flay NW, et al. Stable
heparin-producing cell lines derived from the
Furth murine mastocytoma. Proc Natl Acad Sci
U S A. 1992;89:11327-11331.
27. Nakajima K, Kusafuka T, Takeda T, Fujitani Y, Nakae K, Hirano T. Identification of a novel interleukin-6 response element containing an Ets-binding
site and a CRE-like site in the junB promoter. Mol
Cell Biol. 1993;13:3027-3042.
28. Mizushima S, Nagata S. pEF-BOS, a powerful
mammalian expression vector. Nucleic Acids
Res. 1990;18:5322.
29. Tsujimura T, Morii E, Nozaki M, et al. Involvement
of transcription factor encoded by the mi locus in
the expression of c-kit receptor tyrosine kinase in
32. Auffray C, Rougenon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs
from total myeloma tumor RNA. Eur J Biochem.
1980;107:303-314.
33. Reynolds DS, Stevens RL, Gurley DS, Lane WS,
Austen KF, Serafin WE. Isolation and molecular
cloning of mast cell carboxypeptidase A: a novel
member of the carboxypeptidase gene family.
J Biol Chem. 1989;264:20094-20099.
34. Gurish MF, Nadeau JH, Johnson KR, et al. A
closely linked complex of mouse mast cellspecific chymase genes on chromosome 14.
J Biol Chem. 1993;268:11372-11379.
35. Gurish MF, Johnson KR, Webster MJ, Stevens
RL, Nadeau JH. Location of the mouse mast cell
protease 7 gene (Mcpt7) to chromosome 17.
Mamm Genome. 1994;5:656-657.
36. Ito A, Morii E, Maeyama E, et al. Systematic
method to obtain novel genes that are regulated
by mi transcription factor (MITF): impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells. Blood. 1998;91:
3210-3221.
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2001 97: 645-651
doi:10.1182/blood.V97.3.645
Inhibitory effect of the transcription factor encoded by the mutant mi
microphthalmia allele on transactivation of mouse mast cell protease 7
gene
Hideki Ogihara, Eiichi Morii, Dae-Ki Kim, Keisuke Oboki and Yukihiko Kitamura
Updated information and services can be found at:
http://www.bloodjournal.org/content/97/3/645.full.html
Articles on similar topics can be found in the following Blood collections
Gene Expression (1086 articles)
Hematopoiesis and Stem Cells (3444 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.