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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1189-1196
Inhibitory Effect of the Transcription Factor Encoded by the
mi Mutant Allele in Cultured Mast Cells of Mice
By
Akihiko Ito,
Eiichi Morii,
Dae-Ki Kim,
Tatsuki R. Kataoka,
Tomoko Jippo,
Kazutaka Maeyama,
Hiroshi Nojima, and
Yukihiko Kitamura
From the Department of Pathology, Osaka University Medical School,
Suita, Osaka, Japan; the Department of Pharmacology, Ehime University
Medical School, Ehime, Japan; and the Department of Molecular Genetics,
Research Institute for Microbial Diseases, Osaka University, Suita,
Osaka, Japan.
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ABSTRACT |
The mi locus of mice encodes a transcription factor of the
basic-helix-loop-helix-leucine zipper protein family (MITF). The MITF
encoded by the mutant mi allele (mi-MITF) deletes 1 of
4 consecutive arginines in the basic domain. The mice of mi/mi
genotype express mi-MITF, whereas the mice of tg/tg
genotype have a transgene at the 5' flanking region of the
mi gene and do not express any MITF. To investigate the
function of mi-MITF in cultured mast cells (CMCs), we took two
approaches. First, mRNA obtained from mi/mi CMCs or
tg/tg CMCs was subtracted from complementary (c) DNA library of
normal (+/+) CMCs, and the (+/+-mi/mi) and
(+/+-tg/tg) subtraction libraries were obtained. When the
number of clones that hybridized more efficiently with +/+ CMC cDNA
probe than with mi/mi or tg/tg CMC cDNA probe was
compared using Southern analysis, the number was larger in the
(+/+-mi/mi) library than in the (+/+-tg/tg)
library. Second, we compared mRNA expression of six genes between
mi/mi and tg/tg CMCs by Northern analysis. The
transcription of three genes encoding mouse mast cell proteases was
impaired in both mi/mi and tg/tg CMCs. On the other
hand, the transcription of three genes encoding c-kit receptor,
tryptophan hydroxylase, and granzyme B was markedly reduced in
mi/mi CMCs, but the reduction was significantly smaller in
tg/tg CMCs. These results indicated the inhibitory effect of
mi-MITF on the transactivation of particular genes in CMCs.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE MUTANT MICE OF mi/mi genotype
were found by Hertwig1,2 among the offsprings of an
X-irradiated male mouse. The mi/mi mice show microphthalmia,
depletion of pigment in both hair and eyes, osteopetrosis, and decrease
in number of mast cells.3-7 The mutant gene was introduced
into C57BL/6 background and has been kept in the Jackson Laboratory
(Bar Harbor, ME). The mi locus was demonstrated to
encode a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip)
protein family of transcription factors (hereafter called
mi-transcription factor [MITF]).8,9 The MITF
encoded by the mutant mi allele deletes 1 of 4 consecutive
arginines in the basic domain (hereafter
mi-MITF).8,10,11 The mi-MITF is defective
in the DNA binding activity and the nuclear localization potential,12,13 and it does not transactivate target
genes.13-17
A VGA-9-tg/tg transgenic mouse possessing the
transgene-insertional mutation at the 5' flanking region of the
mi gene was produced by Hodgkinson et al8 and
Tachibana et al.18 The expression of MITF
transcripts was undetectable in various tissues of VGA-9-tg/tg
mice except the embryonal retina.8 We introduced the
tg transgene into the C57BL/6 background in the Osaka
University Medical School (Osaka, Japan). C57BL/6-tg/tg and
C57BL/6-mi/mi mice share several phenotypic features, such as
microphthalmia, white coat color, and the decrease of mast cells.
However, they are clearly distinguishable from each other at least in
one respect: the C57BL/6-tg/tg mice do not show osteopetrosis.
This fact indicates that the presence of mi-MITF is associated
with a more severe impairment than the absence of normal MITF (+-MITF).
Although the osteopetrosis of mi/mi mice is attributable to the
deficiency of osteoclasts,19,20 the effect of MITF has been
analyzed more intensely in mast cells than in osteoclasts. mRNA
expression of mi-MITF in mi/mi cultured mast cells
(CMCs) was comparable to that of +-MITF in +/+ CMCs,21 but
the expression of MITF was not detectable in tg/tg CMCs even
with reverse transcriptase modification of polymerase chain reaction
(RT-PCR).22 In the present study, we investigated the
inhibitory effect of mi-MITF on gene transactivation in CMCs.
Two approaches were taken. First, subtraction libraries of
complementary (c) DNAs were produced and analyzed. We have recently elaborated cDNA library from +/+ CMCs and mi/mi CMCs and
subtracted the latter from the former.21 The subtraction
process was so successful that we could isolate two genes encoding
granzyme (Gr) B and tryptophan hydroxylase (TPH) as novel MITF targets
from the resulting subtracted cDNA library of
(+/+-mi/mi).21 We applied the same subtraction
procedures here to tg/tg CMCs. By comparing two subtracted
libraries, we found that the (+/+-tg/tg) cDNA library contained
+/+ CMC-specific clones at an apparently lower frequency than the
(+/+-mi/mi) cDNA library. In addition to the Gr B and TPH
genes, we have isolated several genes whose expression was reduced
drastically in mi/mi CMCs.14-17 As the second
approach, mRNA expression levels of such genes were compared between
mi/mi and tg/tg CMCs by the Northern analysis. The
expression of mouse mast cell protease (MMCP)-4, MMCP-5, and MMCP-6 was
impaired in both mi/mi and tg/tg CMCs, but the
reduction of expression of three genes encoding Gr B, TPH, and
c-kit receptor was apparently smaller in tg/tg CMCs
than in mi/mi CMCs. The results obtained by the two experiments
consistently indicated that mi-MITF had an inhibitory effect on
transcription of particular genes that are expressed in +/+ CMCs.
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MATERIALS AND METHODS |
Mice and cells.
The original stock of C57BL/6J-mi/+ (mi/+) mice was
purchased from the Jackson Laboratory and was maintained in our
laboratory by consecutive backcrosses with our own inbred C57BL/6
colony (more than 12 generations at the time of the present
experiments). Female mi/+ mice were crossed with male
mi/+ mice, and the resulting mi/mi mice were selected
on the basis of their white coat color.3,4 The original
stock of VGA-9-tg/tg mice,8 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 our own inbred C57BL/6 colony (more than 12 generations at the time of the present experiments). Female and male
tg/+ mice were crossed together, and the resulting
tg/tg mice were selected by their white coat color.
Pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM)
was prepared according to the method described by Nakahata et
al.23 Mice of mi/mi or tg/tg genotype and
their normal (+/+) littermates were used to obtain CMCs at an age of 2 to 3 weeks. Mice were killed by decapitation after ether anesthesia and
the spleens were removed. Spleen cells derived from +/+, mi/mi,
and tg/tg 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 Biosupp Center,
Tokyo, Japan). Half of the medium was replaced every 7 days. Four weeks
after initiation of the culture, more than 95% of cells were
CMCs.24
The P-815 cell line was originally established from a mastocytoma of a
DBA/2 mouse by Dunn and Potter25 and was supplied by the
Japanese Cancer Research Bank (Tokyo, Japan). P-815 cells were cultured
in -MEM supplemented with 10% FCS.
Preparation of the subtracted cDNA library.
The detailed process for the preparation of the subtracted cDNA library
was described previously.21 To prepare single-stranded plasmid DNA, the plasmid DNA prepared from the +/+ CMC cDNA library was
introduced into E coli DH5F'IQ cells by
electroporation. After 1 hour of culture in rich medium (2× YT),
transformed cells were infected with R408 helper phages.
Single-stranded DNA was then purified from the supernatant of the
8-hour culture. To prepare biotinylated RNA drivers, total RNA was
extracted by the guanidine thiocyanate/CsTFA method from mi/mi
and tg/tg CMCs, and poly (A)+ RNA was purified and
labeled by photobiotin (Vector Laboratories, Burlingame, CA). One
microgram of single-stranded DNA prepared from the +/+ CMC cDNA library
was hybridized with 10 µg of biotinylated RNA at 42°C in 25 µL
hybridization buffer containing 40% formamide, 50 mmol/L HEPES (pH
7.5), 1 mmol/L EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.2 mol/L
NaCl, and 1 µg of oligo-poly(rA). After hybridization for 42 hours, the mixture was transferred to 400 µL of SB (50 mmol/L HEPES
[pH 7.5], 2 mmol/L EDTA, 500 mmol/L NaCl) and 10 µg of
streptoavidin was subsequently added. The mixture was incubated at room
temperature for 5 minutes and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The organic phase was back extracted with 100 µL
TE (10 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA). The aqueous phases
were pooled. Streptoavidin binding and phenol treatment were
repeated once more. The recovered single-stranded plasmid DNA was
subtracted with biotinylated RNA one more time. After repeating the
subtraction process, the recovered single-stranded DNA was converted to
double-stranded plasmid DNA by the BcaBEST DNA polymerase
(TaKaRa, Otsu, Japan) reaction at 65°C for 30 minutes. After phenol
extraction and ethanol precipitation, the DNA was dissolved in 20 µL
TE buffer and 3 µL aliquots were introduced into E coli
MC1061A cells by electroporation.
Screening of +/+ CMC-specific clones by Southern blot
analysis.
Four hundred cDNA clones were prepared from ampicillin-resistant
colonies randomly selected from the subtracted cDNA library by an
automated plasmid purification machine (PI-100: KURABO, Osaka, Japan).
After digestion with both Sma I and Not I to separate the cDNA insert, the plasmid DNA was electrophoresed in an agarose gel
and transferred to nylon membranes. For positive controls, the mouse
 -actin21 and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) HindIII-Apa I26 cDNA
fragments were also blotted. The cDNA probes were synthesized with
reverse transcriptase from poly (A)+ RNA prepared from +/+,
mi/mi, or tg/tg CMCs in a reaction mixture containing
[-32P]dCTP. Duplicate membranes were hybridized with
the reverse transcribed cDNA probes for 15 hours. The filters were
washed several times with a final stringency of 0.1× SSC
(1× SSC = 0.15 mol/L NaCl, 15 mmol/L sodium citrate, pH 7.2) and
0.1% SDS at 50°C and autoradiographed at  80°C with an
intensifying screen. The numbers of clones that hybridized with higher
efficiency to the +/+ cDNA probe compared with the mi/mi or
tg/tg cDNA probe were counted.
DNA sequencing.
The plasmid DNA was purified individually from the subtracted cDNA
libraries by PI-100 and directly subjected to DNA sequencing. Dideoxy-chain termination sequencing reactions were performed with T7
dye-labeled primers and thermal cycle sequencing kits purchased from
LI-COR (Lincoln, NE). The reaction products were analyzed by a
Model 4000L Automated DNA Sequencer (LI-COR).
Northern blot analysis.
Five micrograms of total RNA prepared from +/+, mi/mi, or
tg/tg CMCs was loaded per lane, fractionated on 1%
agarose-formaldehyde gels, and transferred to nylon membranes by
capillary action in 20× SSC. Baked membranes were
prehybridized for 3 hours at 42°C in a buffer containing 50%
formamide, 5× SSC, 5× Denhardt's solution, and 0.1% SDS.
The membranes were hybridized with the
[-32P]dCTP-labeled DNA probes at 42°C for 15 hours
in the same buffer. Preparation of the DNA probes was performed
according to the random hexamer labeling method. Preparation of MMCP-4,
MMCP-5, MMCP-6, c-kit, TPH, Gr B, MITF, and -actin cDNA
probes was described previously.21,27 After hybridization,
the membranes were washed to a final stringency of 0.1× SSC and
0.1% SDS at 50°C and autoradiographed at 80°C.
To characterize the subtracted cDNA libraries, 1 µg of the
Not I-digested plasmid DNA prepared from the +/+ CMC cDNA
library, the (+/+-mi/mi) subtracted library, or the
(+/+-tg/tg) subtracted library was used as a template for the
T7 RNA polymerase reaction (Stratagene, La Jolla, CA). Two micrograms
of synthesized RNA was loaded per lane, fractionated on 1%
agarose-formaldehyde gels, and transferred to nylon membranes by
capillary action in 20× SSC. The hybridization procedures were
the same as described above.
Construction of effector and reporter plasmids and the transient
cotransfection assay.
pEF-BOS expression vector28 was kindly provided by Dr S. Nagata (Osaka University). The expression plasmid containing +-MITF or
mi-MITF cDNA was constructed as described
previously.14,15 The luciferase gene subcloned into pSP72
(pSPLuc) was generously provided by Dr K. Nakajima (Osaka University
Medical School). To construct reporter plasmids, the promoter region of
the Gr B (nucleotides [nt] 910 to +42, +1 is the transcription
start site)29 gene was obtained with PCR and subcloned into
the upstream region of the luciferase gene in pSPLuc. Twenty micrograms
of a reporter, 5 µg of an effector, and 5 µg of an expression
vector containing the -galactosidase gene were added to cell
suspension of P-815 cells (1 × 107) in 0.4 mL of
-MEM. After incubation for 10 minutes on ice, electroporation was
performed by a single pulse (975 µF, 320 V) from Gene Pulser II
(Bio-Rad Laboratories, Hercules, CA). The cells were suspended in 5 mL
-MEM supplemented with 10% FCS and cultured in a 6-cm dish. The
expression vector containing the -galactosidase gene was used as an
internal control. The cells were harvested 24 hours after
electroporation and lysed with 0.1 mol/L potassium phosphate buffer (pH
7.4) containing 1% Triton X-100. Soluble extracts were then assayed
for luciferase activity with a luminometer LB96P (Berthold GmbH,
Wildbad, Germany) and for -galactosidase activity. The luciferase
activity was normalized using the -galactosidase activity, and the
total protein concentration was estimated according to the method
described by Yasumoto et al.30 The normalized value was
divided by the value obtained after cotransfection with the reporter
and pEF-BOS and was expressed as the relative luciferase activity.
Concentration of serotonin.
The concentration of serotonin was measured using high performance
liquid chromatography (HPLC) with electrochemical
detection.31 Briefly, CMCs were collected, washed with
phosphate-buffered saline (PBS), counted, and sonicated for 20 seconds
in a sonicator (Tomy, Tokyo, Japan) in 1 mL of ice-cold 3% perchloric
acid containing 5 mmol/L EDTA and 1 mmol/L sodium metabissulfite. The
homogenate was centrifuged at 10,000g for 15 minutes at 4°C
and the supernatant was applied directly to the HPLC column. The
concentration of serotonin per 1.0 × 106 cells was calculated.
Cytotoxicity assay.
Mast cell cytotoxicity was measured using a 51Cr release
assay according to the procedure described by Bissonnette and
Befus.32 As a positive control, spleen cells were freshly
prepared from 8-week-old +/+ mice. The target was YAC-1 cells, which
were obtained from the American Type Culture Collection (Bethesda, MD).
The cells were maintained in -MEM supplemented with 10% FCS. CMCs derived from +/+, mi/mi, and tg/tg mice, and
spleen cells of +/+ mice were washed, suspended in -MEM supplemented
with 10% FCS, and distributed at different concentrations (0.5, 1.0, and 2.5 × 106 cells) in triplicate into 96-well
microtiter plates with round bottoms. YAC-1 cells (5 × 106) were labeled with 100 µCi of [51Cr]
Na2CrO4 (Amersham, Arlington Heights, IL) for 2 hours, washed three times, and resuspended in -MEM supplemented with
10% FCS. Labeled YAC-1 cells (1.0 × 104) were mixed
with various numbers of CMCs in a total volume of 200 µL/well. Plates
were incubated at 37°C for 18 hours in a CO2 incubator
and spun at 150g for 10 minutes, and the radioactivity was
determined in 100 µL samples of cell-free supernatants. The radioactivity released in the well containing YAC-1 cells alone was
designated spontaneous release (SR). Total 51Cr release
(TR) was measured by adding 0.01% Triton X-100 to the well containing
YAC-1 cells alone. The percentage of specific 51Cr release
was calculated using the following formula: (cpm in the presence of
CMCs SR)/(TR SR) × 100.
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RESULTS |
Screening of two subtracted cDNA libraries by Southern analysis.
Using the same procedure that was used to establish the subtracted cDNA
library of (+/+-mi/mi),21 we elaborated the other subtracted library of (+/+-tg/tg) by the removal of clones
carrying inserts complementary to tg/tg CMC mRNAs from the +/+
CMC cDNA library. The (+/+-tg/tg) library was examined by the
library-Northern analysis.21 The sense RNAs of the cDNA
inserts were synthesized by the T7 RNA polymerase reaction using the
Not I-digested plasmid DNA of the +/+, (+/+-mi/mi), and
(+/+-tg/tg) cDNA libraries as templates. They were then fixed
to nylon membranes and probed with several cDNAs. MC-CPA and -actin
cDNAs were expressed equally in +/+, mi/mi, and tg/tg
CMCs, and hybridized bands were rarely detectable in the two subtracted
libraries (Fig 1). MITF expression was
comparable between +/+ and mi/mi CMCs but not detectable in tg/tg CMCs. When probed with MITF cDNA, a faint band was
detectable in the RNA obtained from the (+/+-tg/tg) library but
not in the RNA obtained from the (+/+-mi/mi) library. When
probed with the Gr B cDNA, the intensity of the hybridized band was
significantly stronger in the (+/+-mi/mi) library than in the
(+/+-tg/tg) library (Fig 1).
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| Fig 1.
Characterization of the subtracted cDNA library by
Northern blot analysis. In the left panel, 5 µg of total RNA prepared
from +/+, mi/mi, or tg/tg CMCs was loaded in each
lane and fixed onto nylon membranes by capillary action. In the right
panel, sense RNAs were synthesized by the T7 RNA polymerase reaction
using the Not I-digested plasmid DNA of the +/+ CMC cDNA
library, the (+/+-mi/mi) subtracted cDNA library, or the
(+/+-tg/tg) subtracted cDNA library as a template. Two
micrograms of synthesized RNA was loaded per lane and fixed onto nylon
membranes. Probes were prepared from the cDNAs for -actin, MC-CPA,
MITF, or Gr B using the random hexamer labeling method.
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The number of clones that are specifically expressed by +/+ CMCs was
compared between two subtracted libraries. Eighty cDNA clones were
isolated randomly from the (+/+-mi/mi) subtracted library, and
the cDNA insert of each clone was blotted on two membranes. For a
positive control, the mouse -actin and human GAPDH cDNAs were also
blotted. One membrane was hybridized with cDNA reverse-transcribed from
+/+ CMC poly (A)+ RNA as a probe, and another was
hybridized with cDNA reverse-transcribed from mi/mi CMC poly
(A)+ RNA (Fig 2, upper panel).
According to the same procedure, two blotted membranes were also made
using 80 clones from the (+/+-tg/tg) subtracted library and
probed with +/+ and tg/tg CMC cDNAs individually (Fig 2, lower
panel). After adjustment of the -actin-specific and GAPDH-specific
signals of each membrane to equal intensity, it may be expected that
the clones that hybridize more efficiently with the +/+ cDNA probe than
with the mi/mi or tg/tg cDNA probe carry cDNA inserts
transcribed specifically in +/+ CMCs. Five clones (denoted by
arrowheads with numbers in Fig 2, upper panel) corresponded to such
ones in the (+/+-mi/mi) library, whereas only one clone
(denoted by an arrowhead with a number in Fig 2, lower panel)
corresponded in the (+/+-tg/tg) library. By sequencing and a
computer-assisted homology search, all six clones turned out to be
reported genes. Clone no. 1 of Fig 2 encodes vimentin; clones no. 2 and
5 encode Gr B; clone no. 3 encodes E25 that is a marker for
chondro-osteogenic differentiation33; clone no. 4 encodes
MMCP-5; and clone no. 6 encodes MMCP-6. Although the expression of
vimentin and E25 in mast cells has not been studied intensively, the
mRNA expression was detectable in +/+ CMCs and significantly reduced in
mi/mi CMCs (data not shown).
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| Fig 2.
Screening of +/+ CMC-specific clones by Southern blot
analysis. After digestion with both Sma I and Not I to
separate the cDNA insert, plasmid DNAs of 80 clones randomly selected
from the subtracted cDNA library [(+/+-mi/mi) or
(+/+-tg/tg)] were electrophoresed in 1.0% agarose gel
(left panel, stained with ethidium bromide [EdBr]) and bound to nylon
membranes. Duplicate membranes were hybridized with
32P-labeled cDNAs synthesized from poly(A)+
RNA of +/+ (middle panel), mi/mi (right upper panel), or
tg/tg (right lower panel) CMCs. In some lanes, an equal
amount of the mouse -actin (A) and human GAPDH (G) cDNA fragments
and a  -BstEII size marker (M) were loaded as a control, and
we graphically equalized the intensity of their bands for two filters
to normalize the intensity of other sets of the bands. The clones that
hybridized to a greater degree with +/+ CMC cDNA than with
mi/mi or tg/tg CMC cDNA were identified by arrowheads
with numbers.
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The above-mentioned Southern experiment (experiment no. 1)
was repeated four more times (experiments no. 2, 3, 4, and 5). Consequently, we isolated 400 clones from the (+/+-mi/mi) or
(+/+-tg/tg) library, subjected them to Southern analysis, and
counted the number of clones yielding stronger signals with
+/+-probe than with mi/mi- or tg/tg-probe
(summarized in Table 1). In every experiment, the number of such clones was larger in the
(+/+-mi/mi) subtraction cDNA library than in the
(+/+-tg/tg) subtraction library. In total, the proportion of
such clones was greater in the (+/+-mi/mi) library than in
the (+/+-tg/tg) library. These results suggested that the
number of genes transcriptionally downregulated in mi/mi CMCs
is larger than the number of genes transcriptionally downregulated in
tg/tg CMCs.
Difference of transcriptional impairment between mi/mi
and tg/tg CMCs.
We have demonstrated that several genes indeed show a drastically
reduced expression in mi/mi CMCs.14-17,21
Therefore, we examined whether the transcription of these genes was
also impaired in tg/tg CMCs. We performed Northern analysis
with the six cDNA probes of the c-kit, TPH, Gr B, MMCP-4,
MMCP-5, and MMCP-6 genes. These genes were divided into two groups
according to their expression profile (Fig
3). The MMCP-4, MMCP-5, and MMCP-6 genes belonged to the first group.
They showed a transcriptional impairment in tg/tg CMCs as
severe as in mi/mi CMCs, although just a faint expression of
MMCP-4 and MMCP-5 genes was detected in tg/tg CMCs. The
c-kit, TPH, and Gr B genes belonged to the second group.
Although they showed a reduced expression in tg/tg CMCs, the
reduction was much smaller than the reduction observed in mi/mi
CMCs. By a densitometric analysis, the expression of the three genes
was reduced by 90% or more in mi/mi CMCs, whereas the
expression was reduced by only 50% in tg/tg CMCs (data not
shown). Therefore, the three genes were not included in the
(+/+-tg/tg) subtracted library. The presence of genes belonging
to the second group was consistent with the result obtained by
screening the subtracted libraries.
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| Fig 3.
Expression of the MMCP-4, MMCP-5, MMCP-6, Gr B, TPH, and
c-kit genes in +/+, mi/mi, and tg/tg CMCs.
Five micrograms of total RNA prepared from +/+, mi/mi,
or tg/tg CMCs was loaded in each lane and fixed onto nylon
membranes by capillary action. The membranes were hybridized with
specific DNA probes. An arrowhead indicates the specific signal for
c-kit. Reprobing with the -actin probe allowed verification
that an equal amount of mRNA was loaded per lane. With respect to the
expression profiles, the six genes were divided into two groups (groups
I and II).
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The mi-MITF appeared to function negatively in the
transcription of genes belonging to the second group. We examined the
effect of mi-MITF on the transactivation of the Gr B gene
promoter using the transient cotransfection assay. The
5'-flanking sequence of the Gr B gene (nt 910 to
+42)29 was cloned upstream of the luciferase gene. Three
CANNTG motifs were present in this region. The Gr B gene appeared to be
transactivated through these three CANNTG motifs.21 The
luciferase construct was cotransfected into P-815 cells with the
expression plasmid pEF-BOS containing no insert, +-MITF cDNA, or
mi-MITF cDNA. Neither +-MITF nor mi-MITF showed any
transactivation effect on the reporter without the Gr B promoter
sequence (Fig 4). When the Gr B
promoter-reporter construct was cotransfected with pEF-BOS containing
no insert, the luciferase activity increased threefold. The endogenous
MITF and other factors appeared to transactivate the luciferase
construct in P-815 cells. The cotransfection of pEF-BOS containing
+-MITF increased the luciferase activity remarkably. In contrast, that of pEF-BOS containing mi-MITF cDNA significantly reduced the
luciferase activity (Fig 4).
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| Fig 4.
The effect of coexpression of +-MITF or mi-MITF
cDNA on the luciferase activity under the control of the Gr B gene
promoter. Various forms of the reporter and effector constructs were
introduced into P-815 cells by electroporation. Three solid squares
represent CANNTG motifs between nt 910 and +42; ie, CAGATG (nt
563 to 558), CACGTG (nt 530 to 525), and CATTTG (nt 521
to 516) motifs. The bars represent the mean ± standard error (SE)
of the relative luciferase activities obtained by three independent
experiments: ( ) +-MITF; ( ) mi-MITF; ( ) pEF-BOS. In
some cases, the SE was too small to be shown by the bars. *P < .01 by t-test when compared with the luciferase activity,
in which pEF-BOS containing no insert was cotransfected.
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Serotonin concentrations.
Because mRNA expression levels of the TPH and Gr B genes were
significantly different between mi/mi and tg/tg CMCs,
we examined whether these two CMCs possess different phenotypes in
relation to the two genes. Because TPH is the rate-limiting
enzyme for serotonin synthesis,34 serotonin concentration
was compared between mi/mi and tg/tg CMCs. The
serotonin concentration was significantly lower in mi/mi CMCs
than in +/+ CMCs, as previously reported.21 The serotonin
concentration in tg/tg CMCs was in the middle of both, ie,
significantly lower than that of +/+ CMCs and
significantly higher than that of mi/mi CMCs
(Table 2). These values were well
correlated with the mRNA expression levels of the TPH gene.
Cytotoxicity to YAC-1 cells.
Gr B is essential for the induction of DNA fragmentation in target
cells caused by natural killer (NK) cells and cytotoxic T
lymphocytes.35-37 Because the expression of Gr B gene was
drastically reduced in mi/mi CMCs and mildly in
tg/tg CMCs, it seemed logical to assume that
mi/mi CMCs would show impaired cytotoxic activity but
that of tg/tg CMCs would be near the normal level. We examined this possibility using YAC-1 lymphoma cells as a target (Table 3). CMCs of +/+, mi/mi, or
tg/tg genotype were cultured together with
51Cr-labeled YAC-1 cells and 51Cr release from
YAC-1 cells was measured after 18 hours. At an effector/target (E/T)
ratio of 50, neither +/+, mi/mi, nor tg/tg CMCs
showed any cytotoxic activity to YAC-1 cells (Table 3). When the +/+
spleen cells were used as a positive control, they killed YAC-1 cells
at the E/T ratio. This was compatible with a previous
report36,37 indicating that our assays were reproducible. At an increased E/T ratio of 100 or 250, a significant difference in
cytotoxic activity between +/+ and mi/mi CMCs was observed; +/+
CMCs displayed approximately 20% cytotoxicity to YAC-1 cells, whereas
no cytotoxicity was observed with mi/mi CMCs. In contrast, tg/tg CMCs showed an apparent cytotoxicity comparable to that of +/+ CMCs.
| |
DISCUSSION |
We compared mi/mi and tg/tg CMCs to examine the effect
of mi-MITF on the transcription. The former possesses the
mutated MITF (mi-MITF) that is deficient in DNA-binding and
nuclear localization potential.12,13 The latter practically
lacks MITF.8,22 To study the difference of the gene
expression between these two types of CMCs, we evaluated subtracted
cDNA libraries: a subtraction of mi/mi CMC mRNA from +/+ CMC
cDNA library (+/+-mi/mi) and another subtraction of
tg/tg CMC mRNA from +/+ CMC cDNA library (+/+-tg/tg). As previously reported,21 we found a number of new genes
whose expression was downregulated in mi/mi CMCs when the
(+/+-mi/mi) library was screened. In contrast, the
(+/+-tg/tg) library that was newly established in the present
study contained a significantly smaller number of clones whose
expression might be downregulated in tg/tg CMCs. We have
isolated several genes whose expression severely decreases in
mi/mi CMCs.14-17,21 When we examined the expression
levels of such genes in tg/tg CMCs, we found that they were
divided into two groups. The first group showed a transcriptional impairment in tg/tg CMCs as severe as in mi/mi CMCs.
The MMCP-4, MMCP-5, and MMCP-6 genes are included in this group. The
presence of +-MITF appeared to be essential for the transactivation of the genes belonging to this group. The second group also showed a
reduced expression in tg/tg CMCs, but the magnitude of
reduction was much smaller when compared with that of mi/mi
CMCs. The c-kit, TPH, and Gr B genes belonged to the second
group. The result obtained by Southern analysis (Fig 2 and Table 1) and
that of Northern analysis (Fig 3) were consistent and indicated that
the number of genes whose expression was downregulated in tg/tg
CMCs is significantly smaller than the number of genes downregulated in
mi/mi CMCs. These results also suggested that mi-MITF
functioned negatively in the transactivation of genes belonging to the
second group.
We have reported that MITF is involved in transcriptional activation of
the c-kit,14 TPH,21 and Gr
B21 genes through the recognition of the CANNTG motif. The
transactivation effect of +-MITF on the c-kit, TPH,
and Gr B promoter-reporter construct was threefold or fourfold as large
as the control level.14,21 The effect was comparable with
the induction of the expression of those genes observed in +/+ CMCs
compared with the expression in tg/tg CMCs (Fig 3). On the
other hand, the expression level of the three genes in +/+ CMCs
appeared to be greater than 10-fold of the expression level observed in
mi/mi CMCs. The difference between mi/mi and
tg/tg CMCs may be explained by the involvement of other
transcription factor(s) in transactivation of the three genes. In fact,
Babichuk et al38,39 recently reported that AP-1 and PEBP2
binding sites are primarily responsible for the high level expression
of Gr B in lymphocytes. The two sites were located about 350 bases
downstream of three MITF binding sites that were reported by
us.21 Interaction of AP-1 or PEBP2 with mi-MITF may
occur in the cytoplasm, and the interaction with mi-MITF may
have an inhibitory effect on nuclear localization potential of AP-1 and
PEBP2.13 There is another explanation, because an appreciable amount of mi-MITF appeared to enter the
nucleus.13 In the nucleus, the mi-MITF might
prevent AP-1 or PEBP2 from binding their recognition sites.
We also examined the phenotypic differences in relation to the TPH and
Gr B genes. Serotonin concentrations of +/+, mi/mi, and
tg/tg CMCs were well correlated with the expression
levels of the TPH gene. The cytotoxic activity to YAC-1
cells showed a big difference between mi/mi and tg/tg
CMCs. CMCs of tg/tg genotype killed YAC-1 cells as effectively
as +/+ CMCs, whereas mi/mi CMCs were defective in the
cytotoxicity. The results were consistent with those obtained by the
Northern analysis that the Gr B gene expression reduced to
nondetectable levels in mi/mi CMCs and that the Gr B expression
level of tg/tg CMCs was approximately 50% that of +/+ CMCs.
The difference among these three types of CMCs with respect to their
cytotoxic activity suggests that Gr B is essential for cytotoxicity in
CMCs as well as in NK cells.
In conclusion, we examined the differences of transcriptional
impairment and the related phenotypes between mi/mi and
tg/tg CMCs. We found that mi/mi CMCs suffered from
transcriptional impairment more severely than tg/tg CMCs. Two
phenotypic features examined correlated with the mRNA expression. The
results reinforced our previous suggestion that mi-MITF
possessed an inhibitory effect on transactivation of particular target
genes.13 Further investigation by comparing mi/mi
and tg/tg CMCs will lead to the better understanding of the
transcriptional events mediated by MITF.
| |
ACKNOWLEDGMENT |
The authors thank Kirin Brewery Co Ltd (Tokyo, Japan) for supplying
with rmSCF.
| |
FOOTNOTES |
Submitted June 3, 1998; accepted October 7, 1998.
Supported by grants from the Ministry of Education, Science and
Culture, the Ministry of Health and Welfare, the Osaka Cancer Society,
the Naito Foundation, the Mochida Memorial Foundation, the Ryoichi
Naito Foundation, and Japan Foundation for Health Sciences.
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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Abstract
Introduction