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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1942-1950
Deficient Transcription of Mouse Mast Cell Protease 4 Gene in
Mutant Mice of mi/mi Genotype
By
Tomoko Jippo,
Young-Mi Lee,
Yee Katsu,
Kumiko Tsujino,
Eiichi Morii,
Dae-Ki Kim,
Hyung-Min Kim, and
Yukihiko Kitamura
From the Department of Pathology, Osaka University Medical School,
Suita, Japan; and the Department of Oriental Pharmacy, College of
Pharmacy, Wonkwang University, Chonbuk, Korea.
 |
ABSTRACT |
The mi locus encodes a member of the
basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of
transcription factors (hereafter called MITF). We reported that
expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes
were deficient in cultured mast cells (CMC) derived from mutant mice of
mi/mi genotype. Despite the reduced expression of both MMCP-5
and MMCP-6, their regulation mechanisms were different. Because MMCP-5
is a chymase and MMCP-6 a tryptase, there was a possibility that the
difference in regulation mechanisms was associated with their different
characteristics as proteases. We compared the regulation mechanisms of
another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The
expression of the MMCP-4 gene was also deficient in mi/mi CMC.
The overexpression of the normal (+) MITF but not of mi-MITF
normalized the poor expression of the MMCP-4 gene in mi/mi CMC,
indicating the involvement of +-MITF in transactivation of the MMCP-4
gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4
expression was more similar to MMCP-6 than to MMCP-5. We also showed
the deficient expression of granzyme B and cathepsin G genes in
mi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and
MMCP-5 are located on chromosome 14. Because all these genes showed
deficient expression in mi/mi CMC, there is a possibility that
MITF might regulate the expression of these genes through a locus
control region.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE mi LOCUS OF MICE encodes a
member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein
family of transcription factors (mi-transcription factor,
MITF).1,2 The MITF encoded by the mutant mi locus
deletes one of four consecutive arginines in the basic domain
(hereafter called mi-MITF).1,3,4 The mi/mi
mice show microphthalmia, depletion of pigment in both hair and eyes,
osteopetrosis, and decrease in the number of mast cells.5-9 In addition to the decrease in number, the phenotype of mast cells was
abnormal in mi/mi mice.9-13 We have shown that
normal (+) MITF (hereafter called +-MITF) is involved in the expression
of the mouse mast cell protease 6 (MMCP-6),14
c-kit,15 p75 nerve growth factor (NGF)
receptor,16 and MMCP-5 genes.17 We have also
shown that the deficient transactivation ability of the mi-MITF is caused by two different mechanisms: the loss of DNA binding and the
impaired nuclear localization.18,19
Mast cells of mice contain various proteases. The complementary (c) DNA
and genes that encode mast cell carboxypeptidase A (MC-CPA) and six of
the seven mouse mast cell-specific serine proteases have been cloned
and sequenced.20-27 The MC-CPA is an exopeptidase that
prefers C-terminal aliphatic amino acids. The MMCP-1, MMCP-2, MMCP-4,
and MMCP-5 are predicted to be chymases from the deduced amino acid
sequences, whereas MMCP-6 and MMCP-7 to be tryptases. The MMCP-1,
MMCP-2, MMCP-4, and MMCP-5 genes reside on the chromosome 14 and link
with a gene complex encoding blood cell proteases, such as the
cathepsin G and the granzymes specific to cytotoxic T
lymphocytes.28-31 On the other hand, the MC-CPA gene
resides on the chromosome 3, and the MMCP-6 and MMCP-7 genes reside on
the chromosome 17.28 Despite the significant reduction in
expression of both MMCP-5 and MMCP-6 in mi/mi cultured mast
cells (CMC), their regulation mechanisms appeared to be
different.17 Although +-MITF directly bound CANNTG motifs
in the promoter region of the MMCP-6 gene and transactivated it, the
binding of +-MITF to the CANNTG motif in the promoter region of the
MMCP-5 gene was not detectable. The +-MITF appeared to regulate the
transactivation of the MMCP-5 gene indirectly.17 Moreover,
addition of stem cell factor (SCF) to the medium normalized the
expression of MMCP-5 but not of the MMCP-6 gene in mi/mi
CMC.17
In the present study, we investigated whether this difference of the
regulation mechanisms between MMCP-5 and MMCP-6 were associated with
the difference between chymase and tryptase. For this purpose, we
examined the regulation of MMCP-4, whose expression was also poor in
mi/mi CMC. Although MMCP-4 is chymase as MMCP-5, the regulation
mechanisms of MMCP-4 expression was more similar to MMCP-6 than to
MMCP-5.
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MATERIALS AND METHODS |
Mice.
The original stock of C57BL/6-mi/+ (mi/+) mice was
purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained
in our laboratory by consecutive backcrosses to our own inbred C57BL/6 colony (more than 18 generations at the time of the present
experiment). Female and male mi/+ mice were crossed together,
and the resulting mi/mi mice were selected by their white coat
color.5,6 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 (MITF) gene, were kindly given by H. Arnheiter (National Institutes of Health, Bethesda, MD). The integrated transgene
was maintained by repeated backcrosses to our own inbred C57BL/6 colony
(more than eight generations at the time of the present experiment).
Female and male tg/+ mice were crossed together, and the
resulting tg/tg mice were selected by their white coat color.1
Cells.
Pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM)
was prepared according to the method described by Nakahata et
al.32 Mice of mi/mi or tg/tg genotype and
control C57BL/6-+/+ (+/+) mice were used at 2 to 3 weeks of age to
obtain CMC. Mice were killed by decapitation after ether anesthesia and
spleens were removed. Spleen cells derived from mi/mi, tg/tg,
or +/+ mice 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 7 days. More than
95% of cells were CMC 4-weeks after initiation of the culture.33,
34 The helper virus-free packaging cell line ( 2) was
maintained in Dulbecco's modified Eagle's medium (DMEM; ICN
Biomedicals) supplemented with 10% FCS.35 The NIH/3T3
fibroblast cell line was generously provided by Dr S.A. Aaronson
(National Cancer Institute, Bethesda, MD) and maintained in DMEM
supplemented with 10% FCS. The IC-2 cell line was provided by Dr I. Yahara (Tokyo Metropolitan Institute of Medical Science, Tokyo,
Japan)36 and maintained in -MEM supplemented with 10%
PWM-SCM and 10% FCS. When various amounts of recombinant mouse SCF
(rmSCF, a generous gift of Kirin Brewery Co Ltd, Tokyo, Japan) were
added, mi/mi and +/+ CMC were cultured in -MEM containing
10% PWM-SCM and 10% FCS.
In situ hybridization.
Skin pieces were removed from the back of 20-day-old mice and smoothed
onto a piece of the filter paper to keep them flat. The skin pieces
were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB;
pH 7.4), and embedded in paraffin. CMC were collected, washed with
phosphate-buffered saline (PBS), and immobilized by
agarose.37 The pieces of agarose containing CMC were fixed with 4% paraformaldehyde in 0.1 mol/L PB overnight, and then embedded in paraffin. To obtain the MMCP-4, MMCP-5, and MMCP-6 probes, total RNA
was extracted from CMC of C57BL/6-+/+ mouse origin by lithium
chloride-urea method.38 Single-stranded cDNA was generated from the RNA. A polymerase chain reaction (PCR) product was then amplified from the cDNA with specific primers for each
protease.13,39 The products were subcloned into the
EcoRV site of Bluescript KS (-) plasmid (pBS; Stratagene, La
Jolla, CA) that contains T3 and T7 promoters to generate probes. The
technique of in situ hybridization was described previously.37,
40 The number of MMCP-4 messenger RNA (mRNA)-positive
cells and that of alcian blue-positive cells were counted in the
serial sections, and the proportion of MMCP-4 mRNA-positive cells to alcian blue-positive cells was calculated.
Construction of retrovirus vector and its infection.
Plasmid pBS containing the whole coding region of +-MITF or
mi-MITF (pBS-+-MITF and pBS-mi-MITF, respectively) had
been constructed in our laboratory.18,19 A retroviral
vector, pM5Gneo,41 a derivative of myeloproliferative
sarcoma virus vector, was a kind gift from Dr W. Ostertag
(Universität Hamburg, Hamburg, Germany). The purified
SmaI-HincII fragment from pBS-+-MITF or
pBS-mi-MITF was introduced into the blunted EcoRI site
of pM5Gneo. The resulting pM5Gneo-+-MITF and pM5Gneo-mi-MITF
were transfected into the packaging cell line (2)35 by
the calcium phosphate precipitation method,42 and
neomycin-resistant 2 cell clones were selected by culturing in DMEM
supplemented with 10% FCS and G418 (0.8 mg/mL; GIBCO-BRL, Grand
Island, NY). For gene transfer, spleen cells obtained from mi/mi mice were incubated on irradiated (30 Gy) subconfluent
monolayer of virus-producing 2 cells for 72 hours in -MEM
supplemented with 10% PWM-SCM and 10% FCS. Neomycin-resistant CMC
were obtained by continuing the culture in -MEM containing 10%
PWM-SCM, 10% FCS, and G418 (0.8 mg/mL) for 4 weeks. In our previous
study, Northern blotting analysis showed the apparent expression of the introduced +-MITF or mi-MITF cDNA in mi/mi CMC, whereas
the mRNA expression of the endogenous mi gene was hardly
detectable in either +/+ CMC or mi/mi CMC.14, 15
Cathepsin G cDNA probe.
Sense (5'-CATTGCTTGGGAAGCTCCATA-3', nucleotide [nt] 328 to 348) and antisense (5'-ACCAGAATCACCCCTGAAGG-3', nt 725 to 744) oligonucleotide primers were synthesized by conventional means. Nucleotide numbers were based on the report of Aveskogh et
al43 Total RNA (5 µg) obtained from +/+ CMC was used as a
template, and the single-strand cDNA was synthesized with a random
primer by avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany). The cDNA was amplified by
PCR using sense and antisense primers. The products were subcloned into
the plasmid pBS for further analysis. Nucleotide sequence was
determined by Model 373A DNA sequencer (Applied Biosystems, Foster
City, CA) using Taq dye deoxyterminater cycles sequencing kit
(Applied Biosystems).44
Northern blot analysis.
Total RNA from CMC or IC-2 cells was prepared by the lithium
chloride-urea method.38 Northern blot analysis was
performed using MMCP-4,11,13 mouse
-actin,45 and mouse cathepsin G cDNA labeled with
-[32P]-dCTP (DuPont/NEN Research Products, Boston, MA)
by random oligonucleotide priming as probes. After hybridization
at 42°C, blots were washed to a final stringency of 0.2 × standard saline citrate (SSC; 1 × SSC is 150 mmol/L NaCl and 15 mmol/L trisodium citrate, pH 7.4) at 50°C and subjected to autoradiography.
Structure of MMCP-4 transcript.
Five micrograms of total RNA obtained from +/+ or mi/mi CMC
were reverse transcribed in 20 µL of the reaction mixture containing 20 U of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim GmbH Biochemica) and random hexamer. One microliter of each
reaction product was amplified in 25 µL of PCR mixture containing 0.125 U of Taq DNA polymerase (Takara Shuzou, Kyoto, Japan) and 12.5 pmol each of sense (5'-AGAATCTCTCTCCAAGCTGTGACCG-3',
nt 1 to 25) and antisense
(5'-GGAGGTTAGGTCTTTACTGAGGTGCA-3', nt 974 to
999)22 primers for the MMCP-4 gene by 30 cycles of
denaturation at 94°C (30 seconds), annealing at 57°C (30 seconds), and synthesis at 72°C (1 minute). Inserts were
subcloned into the plasmid pBS for further analysis. Nucleotide
sequence was determined as described previously.44
Nuclear run-on assays.
CMC of C57BL/6-+/+ or -mi/mi mice were cultured in -MEM
supplemented with 10% PWM-SCM and 10% FCS. CMC (2 × 107) were washed with ice-cold PBS and lysed in 5 mL of
ice-cold lysis buffer (10 mmol/L NaCl, 3 mmol/L MgCl2,
0.5% Nonidet P-40, and 10 mmol/L Tris-HCl, pH 7.4). After
centrifugation of the lysates at 800g for 5 minutes at 4°C,
the nuclei recovered in each bottom fraction were resuspended in 200 µL of storage buffer (40% glycerol, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 50 mmol/L Tris-HCl, pH 7.5).46 Each
nuclear run-on reaction was initiated in 400 µL of reaction buffer
containing 30 mmol/L Tris-HCl, pH 8.0, 20% glycerol, 5 mmol/L
MgCl2, 150 mmol/L KCl, 1 mmol/L dithiothreitol, 10 units of
RNase inhibitor, 0.5 mmol/L adenosine triphosphate (ATP), 0.5 mmol/L
cytidine triphosphate (CTP), 0.5 mmol/L guanosine triphosphate (GTP),
and 100 µCi of -[32P]-UTP (Amersham, Arlington
Heights, IL). After a 30-minute incubation at 30°C, the reaction
mixture was digested with DNase I (25 µg/mL, RNase-free) for 5 minutes and the radiolabeled transcripts were purified. Each slot blot,
containing 5 µg of pBS DNA and gene-specific probes for MMCP-4,
MMCP-5, MMCP-6, and -actin11,13,17,45 immobilized onto
separate regions of the nitrocellulose membrane, was placed in a small
vial containing 1 to 2 mL of hybridization buffer and 1 to 5 × 106 cpm/mL 32P-labeled RNA. After a 36- to
48-hour incubation at 65°C, the slot blot was treated with RNase A
for 20 minutes at 37°C, washed three times (20 minutes each) with
2 × SSC at 65°C, and subjected to autoradiography.
Construction of effector and reporter plasmids.
pEF-BOS expression vector47 was kindly provided by Dr S. Nagata (Osaka University Medical School, Osaka, Japan). The
SmaI-HincII fragment of pBS-+-MITF or
pBS-mi-MITF was introduced into the blunted XbaI site
of pEF-BOS. The resulting pEF-+-MITF and pEF-mi-MITF expression
vectors were used as effectors. The luciferase gene subcloned into
pSP72 (pSPLuc)48 was generously provided by Dr K. Nakajima
(Osaka University Medical School, Osaka, Japan). To construct reporter
plasmids, a DNA fragment containing a promoter region and the first
exon (noncoding region) of the MMCP-4 gene (nt-1556 to +35, +1 is the
transcription initiation site)22 obtained from genomic DNA
of C57BL/6-+/+ mouse was cloned into the upstream region of the
luciferase gene in pSPLuc. The deletion of the MMCP-4 promoter was
produced by using the appropriate restriction enzyme. The mutations
were introduced by PCR with mismatched primers. Deleted or mutated
products were verified by sequencing.
Transient assay.
NIH/3T3 cells (5 × 105) were plated in a 10 cm dish 1 day before the procedure. Cotransfection with 10 µg of a reporter,
500 ng of an effector, and 5 µg of an expression vector containing the -galactosidase gene was performed by the calcium phosphate precipitation method.42 The expression vector containing
the -galactosidase gene was used as an internal control. Because IC-2 cells expressed effector gene by themselves,16 the
reporter and the expression vector containing the -galactosidase
gene were added to cell suspension (1 × 107) in 0.7 mL PBS, mixed gently, and incubated on ice for 10 minutes. For gene
transfer, cells were electroporated by a single pulse (975 microfarads
at 350 V) from a Gene Pulser II (Bio-Rad Laboratories, Richmond, CA).
After incubation on ice for 10 minutes, the cells were suspended in 10 mL complete culture medium. NIH/3T3 cells were harvested 48-hours after
transfection; IC-2 cells were harvested 8-hours after transfection.
Cells were lysed with 0.1 mol/L potassium phosphate buffer (pH 7.4)
containing 1% Triton X-100 (Sigma). Extracts were then used to
assay luciferase activity with luminometer model LB96P (Berthold,
Wildbad, Germany) and -galactosidase activity. Luciferase activity
was normalized by -galactosidase activity and total protein
concentration according to the method described by Yasumoto et
al.49 The normalized value was divided by the value
obtained with the cotransfection of the reporter and pEF-BOS, and was
expressed as relative luciferase activity.
Electrophoretic gel mobility shift assay (EGMSA).
The production and purification of glutathione-S-transferase
(GST)-+-MITF and GST-mi-MITF fusion proteins were described
previously.18,19 Oligonucleotides were labeled with
-[32P]-dCTP by filling 5'-overhangs, and were
used as probes for EGMSA. DNA binding assays were performed in a
20-µL reaction mixture containing 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 75 mmol/L KCl, 1 mmol/L dithiothreitol, 4% Ficoll type
400, 50 ng poly (dI-dC), 25 ng of the labeled DNA probe, and 3.5 µg
of GST-+-MITF or GST-mi-MITF fusion protein. After 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 hydroxymethyl aminomethane-borate,
ethylenediaminetetraacetic acid (TBE) buffer (1 × TBE is 90 mmol/L Tris-HCl, 64.6 mmol/L boric acid, and 2.5 mmol/L EDTA, pH 8.3).
The polyacrylamide gels were dried on Whatman 3MM chromatography paper
(Whatman, Maidstone, UK) and subjected to autoradiography. Competitive
DNA binding assays were performed as described above, except that the
unlabeled competitive DNA was added to the reaction mixture before
addition of GST-+-MITF fusion protein.
| |
RESULTS |
We examined the expression of the MMCP-4 gene in skin mast cells of
+/+, mi/mi, and tg/tg mice by in situ hybridization.
The criterion of the positive expression was arbitrarily determined, and we assessed that approximately 20% of the skin mast cells expressed the MMCP-4 mRNA in +/+ mice (Fig
1A and B). Although approximately 2% of the skin mast cells showed the
positive signals of the comparable degree in mi/mi and
tg/tg mice (Fig 1C to F), most skin mast cells in these mutant
mice did not show such significant signals. Then we calculated the
proportion of MMCP-4 mRNA-positive mast cells by counting 180 to 210 alcian blue-positive mast cells in each mice
(Table 1). The proportion was significantly
greater in +/+ mice than in mi/mi and tg/tg mice.
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| Fig 1.
Mast cells expressing MMCP-4 mRNA in the skin of +/+,
mi/mi, and tg/tg mice. Serial sections from the skin of
a +/+ mouse (A and B), a mi/mi mouse (C and D), and a
tg/tg mouse (E and F). A, C, and E, stained with alcian blue
and nuclear fast red; B, D, and F, in situ hybridization with MMCP-4
probe. In each set of serial section, arrows show identical cells that
expressed MMCP-4 gene and arrowheads show identical cells that did not
express MMCP-4 gene. Original magnification ×400.
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Table 1.
Proportion of MMCP-4 mRNA Expressing Mast Cells in the
Skin of +/+, mi/mi, and tg/tg Mice Shown by In Situ
Hybridization
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The expression of the MMCP-4 gene was also examined in +/+,
mi/mi, and tg/tg CMC by Northern blot. The mRNA
expression of the MMCP-4 gene was easily detectable in +/+ CMC but
hardly in mi/mi and tg/tg CMC
(Fig 2). To examine the involvement of
+-MITF in the expression of the MMCP-4 gene, we used mi/mi CMC,
to which cDNA encoding +-MITF or mi-MITF had been introduced.
Overexpression of either +-MITF or mi-MITF in mi/mi CMC
had been confirmed in previous studies.14,15 The poor mRNA
expression of the MMCP-4 gene was normalized in mi/mi CMCs
overexpressing +-MITF but not in mi/mi CMC overexpressing of
mi-MITF (Fig 2).
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| Fig 2.
Reduced expression of MMCP-4 mRNA in mi/mi and
tg/tg CMC and normalization of the MMCP-4 expression in
mi/mi CMC by the introduction of the +-MITF cDNA but not of
mi-MITF cDNA. The blot was hybridized with
32P-labeled cDNA probe of MMCP-4 or of -actin.
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The poor expression of the MMCP-7 mRNA in the C57BL/6 strain is due to
a splice-site mutation causing instability of its
transcript.50 Both +/+ and mi/mi mice used in the
present experiment were of C57BL/6 background. First the nucleotide
sequence of MMCP-4 cDNA was compared between +/+ and mi/mi CMC.
No difference was detectable (data not shown). Then we compared the
sequence to that of the MMCP-4 cDNA obtained from the Kirsten sarcoma
virus-immortalized mast cells (KiSV-MC1) derived from a DBA/2
mouse22 and also to that of the cDNA obtained from the
mastocytoma cell line derived from a (Leaden × A1) F1
(hereafter LAF1) mouse.25 Eight nucleotides were different between C57BL/6 and DBA/2 strains, but 5 of 8 nucleotide changes did not result in the alternation of amino acids
(Fig 3). The remaining three changes caused
the amino acid changes: an ATG codon at nt 217 to 219 changed to a TTG
codon, leading to the change from Met to Leu at codon 41; an ACA codon
at nt 514 to 516 changed to an ATA codon, leading to the change from Thr to Ile at codon 140; a GAG codon at nt 772 to 774 changed to an AAG
codon, leading to the change from Glu to Lys at codon 226. Complete
cDNA sequence of the mastocytoma cell line from LAF1 mouse
has not been reported. The reported sequence started from codon
18, nt 53. Between nt 53 and 780, two nucleotide alternations leading to changes of an amino acid were observed between C57BL/6 and
LAF1 mice: a TAG codon at nt 775 to 777 changed to an AAG codon, abolishing the stop codon observed in C57BL/6 mice (Fig 3). As a
result, MMCP-4 of LAF1 mice appeared to contain one more amino acid (Lys) than that of C57BL/6 mice. These results suggested that the poor expression of the MMCP-4 mRNA in mast cells of
C57BL/6-mi/mi mice was not attributable to the specific gene
structure of the C57BL/6 strain.
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| Fig 3.
Comparison of the structure of MMCP-4 transcript among
KiSV-MC1 cells derived from a DBA/2 mouse, CMC derived from
C57BL/6-+/+ mice and the mastocytoma cells derived from a
LAF1 mouse. Numbering of the nucleotides begins at the
transcription initiation site of the MMCP-4 transcript of KiSV-MC1
cells. Dashes indicate identical nucleotides. Dots indicate deletion of
nucleotides in the sequence.
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Because amounts of mRNA of mast cell-specific chymases were reported
to be regulated by a posttranscriptional mechanism,51 we
examined whether such a mechanism was responsible to the decreased expression of MMCP-4 mRNA in mi/mi CMC. The nuclear run-on
assay showed that MMCP-4, MMCP-5, and MMCP-6 genes were transcribed in
+/+ CMC but not in mi/mi CMC (Fig
4). In contrast, the transcription of the -actin gene in +/+
CMC was comparable to that of mi/mi CMC. Because the MMCP-4,
MMCP-5, MMCP-6, and -actin probes were inserted in pBS, the
pBS-derived DNA was used as a negative control. Hybridization of
radiolabeled transcripts to pBS-derived DNA was not detected in the
nuclear RNA preparation.
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| Fig 4.
Nuclear run-on analysis of +/+ and mi/mi CMC.
32P-labeled nuclear transcripts obtained from +/+ CMC
(left lane) and mi/mi CMC (right lane) were examined for their
ability to hybridize to DNA probes for -actin, MMCP-4, MMCP-5, and
MMCP-6. pBS-derived DNA was used as a negative control.
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The transcription of MMCP-4 gene was poor in both mi/mi skin
mast cells and mi/mi CMC. Skin mast cells of mice were
considered to be supported by SCF52 and CMC by
T-cell-derived growth factors.53 We compared the effect of
SCF on the expression of MMCP-4, MMCP-5, and MMCP-6 between +/+ and
mi/mi CMC. The poor expression of MMCP-5 in mi/mi CMC
was normalized by the addition of SCF but the poor expression of MMCP-4
and MMCP-6 remained unchanged (Table 2). The expression of MMCP-4, MMCP-5, and MMCP-6 in +/+ CMC was not influenced by the addition of SCF, either (Table 2).
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Table 2.
Effect of rmSCF on Proportions of CMCs Expressing
MMCP-4, MMCP-5, or MMCP-6 mRNA in +/+ and mi/mi CMCs Shown
by In Situ Hybridization
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Granzyme B is located at the 5' most end of the granzyme gene
cluster, which includes (from 5' to 3') genes encoding
granzymes B, C, F, G, D, E, cathepsin G, and mast cell chymase (MMCP-1, MMCP-2, MMCP-4, and MMCP-5).28 Pham et al54
showed that the knock out of granzyme B gene affected the expression of
granzymes C, D, and F genes but not the expression of cathepsin G gene. The knock out of cathepsin G did not affect the expression of the
upstream granzymes. Pham et al54 suggested that there was probably a locus control region (LCR) for the granzyme genes and another LCR for cathepsin G and the downstream chymase genes. Because
the expression of granzyme B, MMCP-4, and MMCP-5 genes are deficient in
mi/mi CMC,17, 55 we investigated the involvement of
the LCR in the expression of these genes. The MITF might have an effect
on LCR. Because cathepsin G gene is located between the granzyme gene
cluster and the genes encoding mast cell chymases, we compared the
expression of cathepsin G gene between mi/mi and +/+ CMC. In
fact, the mRNA expression of the cathepsin G gene was significantly
lower in mi/mi CMC than that in +/+ CMC
(Fig 5). This suggested a possibility that
MITF may regulate the expression of granzyme B, cathepsin G, MMCP-4,
and MMCP-5 through a single LCR or LCRs.
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| Fig 5.
Expression of cathepsin G mRNA in +/+ and mi/mi
CMCs. The blot was hybridized with 32P-labeled cDNA
probe of cathepsin G or of -actin.
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Although MITF may have an effect on LCR that might regulate the
expression of the genes encoding granzyme B, cathepsin G, MMCP-4, and
MMCP-5, we could not exclude another possibility that MITF could
activate these genes in their different and appropriate elements. We
have reported the direct and functional binding of +-MITF with
5'-upstream region of granzyme B.55 Thus, we
attempted to study the interaction of MITF with 5'-upstream
region of MMCP-4. We cloned 1556 bases of the 5'-upstream region
of the MMCP-4 gene (Fig 6). The reporter
plasmid that contained the luciferase gene under the control of the
MMCP-4 promoter starting from nt-1556 was constructed. We also
constructed the deleted reporter plasmids containing the MMCP-4
promoter starting from nt-791, -313 to examine the elements that
mediate the transactivation. The expression plasmid containing +-or
mi-MITF cDNA was cotransfected into NIH/3T3 fibroblasts with
the luciferase construct under the control of the MMCP-4 promoter.
Coexpression of +-MITF but not mi-MITF significantly increased
the luciferase activity when we used the reporter plasmids containing
the MMCP-4 promoter starting from both nt-1556 and -791 (Fig 7). Coexpression of +-MITF did not
increase the luciferase activity when we used the reporter plasmid
containing the MMCP-4 promoter starting from nt-313. Between nt-791 and
-313, there were two CANNTG motifs, CATGTG and CAGATG.
Mutation of the CATGTG motif (nt-383 to -378) to
C TG G abolished the transactivation ability of
the +-MITF when we used the reporter plasmids containing the MMCP-4
promoter starting from nt-791 (Fig 7). However, mutation of the CAGATG
motif (nt-366 to -361) to CGAG did not
abolish the transactivation of the +-MITF.
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| Fig 6.
The nucleotide sequence of 5' flanking region of
the MMCP-4 gene. The CANNTG motif was boxed. A part of first exon was
shown by capitals, and the 5' flanking region is shown by lower
case. The transcription initiation site was numbered as +1.
|
|
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Effect of coexpression of cDNA encoding +-MITF or
mi-MITF on luciferase activity. The luciferase gene under
control of the normal, deleted, or mutated MMCP-4 promoter was
cotransfected to NIH/3T3 fibroblasts. Bars indicate the standard error
of these assays.
|
|
Because NIH/3T3 cells did not express +-MITF, we examined whether
endogenous MITF can drive expression of the MMCP-4 reporter construct
by using the IC-2 mast cell line that expressed +-MITF.16 IC-2 cells also expressed the MMCP-4 mRNA
(Fig 8A). We introduced the luciferase gene
under the control of the MMCP-4 promoter into IC-2 cells. Luciferase
activity was enhanced when we used the reporter plasmids containing the
MMCP-4 promoter starting from both nt-1556 and -791 (Fig 8B). In
contrast, the luciferase activity did not increase when we used the
reporter plasmid containing the MMCP-4 promoter starting from nt-313.
Transcription activity was abolished when the CATGTG motif (nt-383 to
-378) was mutated, but not when the CAGATG motif (nt-366 to -361) was
mutated. When we compared the reporter gene activity between IC-2 and
NIH/3T3 cells, no significant difference was detectable in spite of the expression of +-MITF in IC-2 cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 8.
(A) Expression of MMCP-4 mRNA in IC-2 cells. The blot was
hybridized with 32P-labeled cDNA probe of MMCP-4 or of
-actin. (B) Luciferase reporter gene promoter assay in IC-2 cells
that expressed +-MITF. The luciferase gene under control of the
normal, deleted, or mutated MMCP-4 promoter was introduced into the
IC-2 mast cell line with electroporation. Bars indicate the standard
error of three assays.
|
|
To examine whether the +-MITF protein practically bound the CATGTG
motif (nt-383 to -378) and/or the CAGATG motif (nt-366 to
-361), EGMSA was performed with each oligonucleotide probe containing
the CATGTG or CAGATG motif. The
5'-GTCCTCTCCTC CTCATA-3' oligonucleotide
(probe 1: the hexametric motif is shown by the underline) and the
5'-CACTA TCTGTGTTTGTG-3' oligonucleotide (probe 2) were synthesized and labeled. EGMSA was performed using GST-+-MITF or GST-mi-MITF fusion protein. No band was detected in the sample that contained no proteins (data not shown). A retarded band was observed in the sample containing the probe 1 and GST-+-MITF but not in the sample containing the probe 1 and GST-mi-MITF
(Fig 9A). In contrast to the probe 1, the
probe 2 was bound by neither GST-+-MITF nor GST-mi-MITF (Fig
9A). To examine the specificity of the binding of the GST-+-MITF to
probe 1, we added unlabeled oligonucleotide that had the identical
sequence with probe 1 (oligo 1, Fig 9B) to the reaction mixture
containing the GST-+-MITF and probe 1. The binding of the GST-+-MITF to
probe 1 was completely inhibited (Fig 9B). We then performed
competition binding with the oligonucleotide 3, in which the hexametric
motif of the probe 1 was mutated from CATGTG to
CTGG (oligo 3, Fig 9B). The addition of the
unlabeled oligo 3 did not affect the binding of GST-+-MITF to the probe
1 (Fig 9B).
View larger version (20K):
[in this window]
[in a new window]
| Fig 9.
Binding of +-MITF to the CATGTG motif in 5'
flanking region of the MMCP-4 gene. (A) EGMSA using GST-+-MITF and
GST-mi-MITF fusion proteins. The
5'-GTCCTCTCCTCCTCATA oligonucleotide containing
a CATGTG motif (probe 1, nt -394 to -372; numbers refer to the sequence
shown in Fig 5) and the
5'-CACTATCTGTGTTTGTG-3' oligonucleotide
containing a CAGATG motif (probe 2, nt -371 to -349) were used (CANNTG
motifs are boxed). The DNA-protein complex is indicated by an
arrowhead. (B) Competitive DNA binding assay with GST-+-MITF. Two
competitors were synthesized. The oligo 1 was identical to the probe 1;
oligo 3 had the mutation at the CTGG motif
(to CTGG). DNA-protein complexes are
indicated by an arrowhead.
|
|
| |
DISCUSSION |
The amount of MMCP-4 mRNA was significantly greater in +/+ CMC than in
mi/mi and tg/tg CMC. In situ hybridization also
suggested the higher content of MMCP-4 mRNA in the skin mast cells of
+/+ mice when compared with that in the skin mast cells of
mi/mi and tg/tg mice. Although the poor expression of
the MMCP-7 gene in C57BL/6 strain is due to a splice-site mutation
causing instability of its transcript,50 the nucleotide
sequence of MMCP-4 cDNA obtained from CMC of C57BL/6-mi/mi mice
was identical to that of CMC of C57BL/6-+/+ mice. Thus, the poor
content of MMCP-4 mRNA in mi/mi CMC was not attributable to the
abnormal structure of the MMCP-4 transcript. Because the nuclear run-on
assay showed the lack of the significant amount of the MMCP-4
transcript in mi/mi CMC, the primary reason why the MMCP-4 gene
was not expressed in mi/mi mast cells was a decreased rate of
the transcription. Moreover, the overexpression of +-MITF but not of
mi-MITF in mi/mi CMC normalized the transcription of
the MMCP-4 gene. These results showed the involvement of
+-MITF in the transcription of the MMCP-4 gene.
We have reported the poor expression of MMCP-517 and
granzyme B55 in mi/mi CMC. In the present study, we
also showed the poor expression of MMCP-4 and cathepsin G in mi/mi
CMC. The expression of genes encoding all MMCP-5, granzyme B,
MMCP-4, and cathepsin G appeared to be affected by MITF. Genes encoding
granzyme B, cathepsin G, and mast cell chymases are located on
chromosome 14 (from 5' to 3').28 These results
suggest that MITF may affect some global regulatory element responsible
for organizing the domains in which these genes lie, or some LCR
element that may interact with multiple local regulatory sequences. In
fact, Pham et al54 suggested that there is probably a LCR
for the granzymes and another LCR for cathepsin G and the downstream
chymases. MITF might be involved in the regulatory mechanism of the
single LCR or LCRs that may regulate the expression of these proteases.
Although the single LCR or LCRs appeared to regulate the expression of
genes that encode mast cell-specific or hematopoietic cell-specific
proteases, we could not exclude the other possibility that MITF could
activate all of these genes in their different and appropriate
compartments. GST-+-MITF fusion protein but not GST-mi-MITF
fusion protein bound the CATGTG motif in the
5'-upstream region of the MMCP-4 gene. When the luciferase
construct containing the MMCP-4 promoter with the CATGTG motif was
transfected into NIH/3T3 fibroblasts, the coexpression of +-MITF but
not of mi-MITF significantly increased the luciferase activity,
suggesting that the +-MITF transactivated the luciferase gene through
the direct binding with the CATGTG motif. We have also reported the
direct and functional binding of MITF with the proximal transcriptional element of granzyme B.55 The direct binding of MITF with
the 5'-upstream region of MMCP-5 gene was not
detectable,17 and the interaction of MITF with the
5'-upstream region of cathepsin G gene has not been examined. We
are now planning to investigate the interaction of MITF with 5'
flanking regions of all mast cell-specific chymases located on
chromosome 14.
We previously reported that the expression of the MMCP-6 gene was also
deficient in both skin mast cells and CMCs of mi/mi mice.10,11,14 Overexpression of +-MITF in mi/mi CMC
normalized the transcription of the MMCP-6 gene.14
The +-MITF protein bound the CANNTG motifs in the
5'-upstream region of the MMCP-6 gene appeared to transactivate
it. Although MMCP-4 is a chymase and the MMCP-6 is a tryptase, their
transactivation mechanism by +-MITF appeared to be similar. Both MMCP-6
and MMCP-7 are tryptase, and genes encoding MMCP-6 and MMCP-7 are
located on chromosome 17. If the expression of MMCP-7 is also deficient
in mi/mi CMC, there is a possibility that a LCR in chromosome
17 might regulate the expression of both MMCP-6 and MMCP-7.
The expression of MMCP-5 was also deficient in mi/mi
CMC.17 In contrast with MMCP-4 and MMCP-6, the expression
of MMCP-5 was not deficient in the skin mast cells of mi/mi
mice. This was attributable to SCF-dependent expression of MMCP-5 mRNA
in mi/mi mice. In the culture condition without SCF, the
overexpression of the +-MITF but not of mi-MITF normalized the
poor expression of the MMCP-5 gene in mi/mi CMC, indicating the
involvement of +-MITF in transactivation of the MMCP-5
gene.17 Although +-MITF directly bound CANNTG motifs in the
promoter regions of the MMCP-4 and MMCP-6 genes, +-MITF did not bind to
the CAGTTG motif in the promoter region of the MMCP-5 gene. The +-MITF
appeared to transactivate the MMCP-5 gene indirectly. The expression of
MMCP-5 gene might be regulated only by the LCR(s) on chromosome 14, whereas the expression of MMCP-4 might be regulated by both the LCR(s)
and the 5'-upstream region of the MMCP-4 gene. Although both
MMCP-4 and MMCP-5 are chymases, their transactivation mechanisms by
+-MITF appeared to be different.
| |
ACKNOWLEDGMENT |
The authors thank Dr H. Arnheiter of NIH for VGA9-tg/tg mice,
Dr S. Nagata of Osaka University for pEF-Bos, Dr K. Nakajima of Osaka
University for pSPLuc, Kirin Brewery Company Ltd for rmSCF, Dr H. Hamada of Osaka University for technical advice on the nuclear run-on
assay, and Dr M. Yamamoto of Tsukuba University for valuable discussion.
| |
FOOTNOTES |
Submitted August 22, 1997; accepted November 9, 1998.
Supported by Grants from the Ministry of Education, Science and
Culture, the Ministry of Health and Welfare, the Ryoichi Naito Foundation for Medical Research, the Joint Research Project under the
Japan-Korea Basic Scientific Promotion Program, and the Organization for Pharmaceutical Safety and Research. Y-M.L. is a postdoctoral fellow
supported by the Japan Society for the Promotion of Science.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yukihiko Kitamura, MD, Department of
Pathology, Osaka University Medical School, Yamada-oka 2-2, Suita
565-0871, Japan.
| |
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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]
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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]
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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]
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A. Ito, T. Jippo, T. Wakayama, E. Morii, Y.-i. Koma, H. Onda, H. Nojima, S. Iseki, and Y. Kitamura
SgIGSF: a new mast-cell adhesion molecule used for attachment to fibroblasts and transcriptionally regulated by MITF
Blood,
April 1, 2003;
101(7):
2601 - 2608.
[Abstract]
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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]
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C. M. Takemoto, Y.-J. Yoon, and D. E. Fisher
The Identification and Functional Characterization of a Novel Mast Cell Isoform of the Microphthalmia-associated Transcription Factor
J. Biol. Chem.,
August 9, 2002;
277(33):
30244 - 30252.
[Abstract]
[Full Text]
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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]
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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]
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E. Morii, H. Ogihara, K. Oboki, C. Sawa, T. Sakuma, S. Nomura, J. D. Esko, H. Handa, and Y. Kitamura
Inhibitory effect of the mi transcription factor encoded by the mutant mi allele on GA binding protein-mediated transcript expression in mouse mast cells
Blood,
May 15, 2001;
97(10):
3032 - 3039.
[Abstract]
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E. Morii, H. Ogihara, D.-K. Kim, A. Ito, K. Oboki, Y.-M. Lee, T. Jippo, S. Nomura, K. Maeyama, M. L. Lamoreux, et al.
Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mice/mice mutant mice of which MITF lacks the zipper domain
Blood,
April 1, 2001;
97(7):
2038 - 2044.
[Abstract]
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H. Ogihara, E. Morii, D.-K. Kim, K. Oboki, and Y. Kitamura
Inhibitory effect of the transcription factor encoded by the mutant mi microphthalmia allele on transactivation of mouse mast cell protease 7 gene
Blood,
February 1, 2001;
97(3):
645 - 651.
[Abstract]
[Full Text]
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Y. Ge, T. Jippo, Y.-M. Lee, S. Adachi, and Y. Kitamura
Independent Influence of Strain Difference and mi Transcription Factor on the Expression of Mouse Mast Cell Chymases
Am. J. Pathol.,
January 1, 2001;
158(1):
281 - 292.
[Abstract]
[Full Text]
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S. Adachi, E. Morii, D.-k. Kim, H. Ogihara, T. Jippo, A. Ito, Y.-M. Lee, and Y. Kitamura
Involvement of mi-Transcription Factor in Expression of {alpha}-Melanocyte-Stimulating Hormone Receptor in Cultured Mast Cells of Mice
J. Immunol.,
January 15, 2000;
164(2):
855 - 860.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION