HEMATOPOIESIS
SgIGSF: a new mast-cell adhesion molecule used for attachment to fibroblasts
and transcriptionally regulated by MITF
Akihiko Ito, Tomoko Jippo, Tomohiko Wakayama, Eiichi Morii, Yu-ichiro Koma, Hiroaki Onda, Hiroshi Nojima,
Shoichi Iseki, and Yukihiko Kitamura
Microphthalmia transcription factor (MITF)
is a basic-helix-loop-helix-leucine zippertype transcription factor. The mutant mi
and Miwh alleles encode MITFs with deletion and alteration of a single amino acid,
respectively, whereas the tg is a null
mutation. In coculture with NIH/3T3 fibroblasts, the numbers of cultured mast cells
(CMCs) derived from C57BL/6 (B6)mi/mi,
B6Miwh/Miwh, and B6tg/tg mice that adhered
to NIH/3T3 fibroblasts were one third as
large as the number of B6ⴙ/ⴙ CMCs that
adhered to NIH/3T3 fibroblasts. From a
cDNA library of B6ⴙ/ⴙ CMCs, we subtracted messenger RNAs expressed by
B6mi/mi CMCs and found a clone encoding
SgIGSF, a recently identified member of
the immunoglobulin superfamily. Northern and Western blot analyses revealed
that SgIGSF was expressed in B6ⴙ/ⴙ CMCs
but not in CMCs derived from MITF mutants. Immunocytochemical analysis
showed that SgIGSF localized to the cellto-cell contact areas between B6ⴙ/ⴙ CMCs
and NIH/3T3 fibroblasts. Transfection of
B6mi/mi and B6tg/tg CMCs with SgIGSF
cDNA normalized their adhesion to NIH/
3T3 fibroblasts. NIH/3T3 fibroblasts did
not express SgIGSF, indicating that SgIGSF
acts as a heterophilic adhesion molecule.
Transfection of B6tg/tg CMCs with normal
MITF cDNA elevated their SgIGSF expression to normal levels. These results indicated that SgIGSF mediated the adhesion of
CMCs to fibroblasts and that the transcription of SgIGSF was critically regulated by
MITF. (Blood. 2003;101:2601-2608)
© 2003 by The American Society of Hematology
Introduction
The mouse mi locus encodes a transcription factor belonging to the
basic-helix-loop-helix-leucine zipper family denoted hereafter as
the microphthalmia transcription factor (MITF).1,2 The mutant mi
allele produces an abnormal MITF protein that lacks 1 of the 4
consecutive arginines in the basic domain (denoted hereafter as
mi-MITF).1,3,4 The mi-MITF is defective in DNA binding, nuclear
translocation, and transactivation of target genes.5-8 Another mutant
allele is the tg allele, which is the MITF gene bearing a transgene
insertion mutation in its 5⬘ flanking region.1,9 Although the coding
region of the MITF gene in C57BL/6 (B6)tg/tg mice is normal,
significant amounts of MITF were not detectable in cultured mast
cells (CMCs) derived from the spleens of B6tg/tg mice.10
Both B6mi/mi and B6tg/tg mice show microphthalmia, lack of
melanocytes, and a decrease in skin mast cells.11 B6mi/mi mice show
osteopetrosis but B6tg/tg mice do not.12 Most B6mi/mi mice die upon
weaning due to the failure of tooth eruption caused by the
osteopetrosis, whereas most B6tg/tg mice survive to adulthood. Mast
cell numbers in skin tissues were comparable between B6mi/mi and
B6tg/tg mice.13 However, only B6mi/mi mice showed a decrease of
heparin content in skin mast cells.14
Gene expression profiles of CMCs were compared between
B6mi/mi and B6tg/tg mice. The transcription of mouse mast cell
protease 2 (mMCP-2), mMCP-4, mMCP-5, mMCP-6, and mMCP-9
genes decreased severely in both B6mi/mi CMCs and B6tg/tg
CMCs.15-18 The transcription of the genes encoding c-kit receptor
tyrosine kinase (KIT), granzyme B, tryptophan hydroxylase, and
N-deacetylase/N-sulfotransferase 2 was reduced severely in B6mi/mi
CMCs, but the reduction of transcription of these genes was not so
severe in B6tg/tg CMCs.8,13,19 This indicated that the mi-MITF
possessed an inhibitory effect on the transcription of KIT,19
granzyme B, tryptophan hydroxylase, and N-deacetylase/Nsulfotransferase 2 genes.8,13,19
We have shown that B6mi/mi CMCs have a variety of abnormal
phenotypes.5-8 One is that they adhere poorly to fibroblasts.20 A
considerable number of B6⫹/⫹ CMCs cultured on a monolayer of
fibroblasts adhere to the fibroblasts,20-22 but significantly fewer
B6mi/mi CMCs do this.20 In the present study, we examined the
number of B6tg/tg CMCs that adhered to NIH/3T3 fibroblasts. We
also assessed the adherence to fibroblasts of CMCs derived from
B6Miwh/Miwh mice, which have a single altered amino acid in the
basic domain of MITF.23 We found that B6tg/tg and B6Miwh/Miwh
CMCs were as poor as B6mi/mi CMCs in adhering to fibroblasts.
From a cDNA library of B6⫹/⫹ CMCs, we subtracted messenger
RNAs expressed by B6mi/mi CMCs. By screening the subtracted
cDNA library, we identified a new mast cell adhesion molecule,
SgIGSF (spermatogenic immunoglobulin superfamily),24 whose
transcription was critically regulated by normal MITF (⫹-MITF)
in CMCs. The deficient transcription of SgIGSF appeared to be a
cause of the defective adhesion of CMCs derived from MITF
mutant mice to NIH/3T3 fibroblasts.
From the Department of Pathology, Osaka University Medical School/Graduate
School of Frontier Bioscience, Suita, Osaka; the Department of Histology and
Embryology, Graduate School of Medical Science, Kanazawa University,
Kanazawa, Ishikawa; and the Department of Molecular Genetics, Institute for
Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Promotion of Cancer Research; and the Naito Foundation.
Submitted July 29, 2002; accepted November 19, 2002. Prepublished online as
Blood First Edition Paper, November 27, 2002; DOI 10.1182/blood-2002-07-2265.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by grants from the Ministry of Education, Culture, Sports, Science
and Technology; the Osaka Cancer Society; the Sagawa Foundation for
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
Reprints: Yukihiko Kitamura, Department of Pathology, Osaka University
Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail:
[email protected].
© 2003 by The American Society of Hematology
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Materials and methods
Mice
The original stock of B6mi/⫹ and B6Miwh/⫹ mice was purchased from the
Jackson Laboratory (Bar Harbor, ME). VGA-9tg/tg mice were kindly
provided by Dr H. Arnheiter (National Institutes of Health, Bethesda, MD).
All MITF mutant mice were maintained by consecutive back-crosses to our
own inbred B6 colony (more than 12 generations at the time of the present
experiment). Homozygous mice were produced by crosses between female
and male heterozygotes of each genotype and selected by their white coat
color. The WB⫹/⫹, WBW/W, WBB6F1⫹/⫹, and WBB6F1W/Wv mice were
purchased from Japan SLC (Hamamatsu, Japan).
Cells
Spleens from 2- to 3-week-old mice were first passed through a 23-gauge
needle and then cultured in ␣–minimal essential medium (␣-MEM; ICN
Biomedicals, Costa Mesa, CA) supplemented with 10% pokeweed mitogenstimulated spleen cell–conditioned medium (PWM-SCM) and 10% fetal
calf serum (FCS; Nippon Biosupply Center, Tokyo, Japan). PWM-SCM
was prepared as described previously.25 Half of the medium was replaced
every 7 days. Four weeks later, more than 95% of the cells were CMCs. To
examine growth kinetics of CMCs, 2.5 ⫻ 105 CMCs were suspended in 2
mL ␣-MEM supplemented with 10% PWM-SCM and 10% FCS and plated
onto 35-mm culture dishes. Various days after the plating, total cell
numbers were counted with a standard hemocytometer. MST mastocytoma
cells26 were kindly provided by Dr J. D. Esko (University of California, San
Diego, CA). ⌿2 helper virus–free packaging cells, NIH/3T3 mouse
fibroblastic cells, and Jurkat lymphoid cells were maintained as described
previously.19,27,28
Coculture of CMCs with fibroblasts and evaluation
of attachment
Coculture of CMCs with NIH/3T3 cells was performed as described
previously.21,22 Briefly, CMCs (1.0 ⫻ 105 cells per dish) were suspended in
2 mL ␣-MEM containing 10% FCS and added to a confluent culture of
NIH/3T3 cells in 35-mm culture dishes. In some experiments, PWM-SCM
was added to a concentration of 10%. After 3 hours of coculture, the dishes
were washed with warmed (37°C) ␣-MEM to remove nonadherent CMCs.
NIH/3T3 cells and adherent CMCs were harvested by trypsin treatment.
These cells were attached to microscope slides using the Cytospin 2
centrifuge (Shandon, Pittsburgh, PA), fixed with Carnoy solution, and
stained with alcian blue and nuclear fast red. The proportion of alcian
blue–positive mast cells to alcian blue–negative NIH/3T3 cells was
determined. Each experiment was done in triplicate and repeated 3 times
with similar results.
cDNA libraries and isolation of clones
A cDNA library of B6⫹/⫹ CMCs and the (⫹/⫹ ⫺ mi/mi) subtracted cDNA
library were constructed previously.7,29 Sequencing and isolation of clones
from the libraries were performed as described previously.7,27 The DNA
sequences were used to search the National Center for Biotechnology
Information database using the BLASTN algorithm.
Northern blotting and hybridization was performed using standard
methods. Relative signal intensity was calculated with the BAS 2000
system (Fuji Photo Film, Tokyo, Japan). cDNA inserts of clones from the
libraries and the ␤-actin cDNA fragment7 were labeled with ␣-32P dCTP by
the random labeling method.
Antibodies
A rabbit polyclonal antibody against SgIGSF was made in Kanazawa
University (by T.W. and S.I.). The method of preparation and the sensitivity
of the antibody are described in detail elsewhere (T.W. and S.I., manuscript
submitted, 2002). Briefly, rabbits were immunized against the synthetic
polypeptide containing 15 amino acids of the C-terminus of SgIGSF. Four
months later, the rabbit sera were purified with an affinity column
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
containing the synthetic polypeptide. The anti-MITF antibody has been
described previously.30 Other primary antibodies used were specific for KIT
(M-14; Santa Cruz Biotechnology, Santa Cruz, CA), E-cadherin (Clone 36;
Transduction Laboratories, Lexington, KY), N-cadherin (Clone 32; Transduction Laboratories), ICAM-1 (KAT-1; Seikagaku, Tokyo, Japan), integrin
␤3 (Transduction Laboratories), and ␣-tubulin (DM 1A; Sigma Chemical,
St Louis, MO). Secondary antibodies used were peroxidase-labeled antirabbit, antimouse, or antirat immunoglobulin G (IgG) antibodies (MBL,
Nagoya, Japan), and fluorescein isothiocyanate (FITC)–labeled antirabbit
IgG antibody (MBL).
Western blot analysis
CMCs and mouse tissues were lysed in a buffer containing 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. The resulting lysates were separated on 10% sodium dodecyl
sulfate (SDS)–polyacrylamide gels, transferred to Immobilon (Millipore,
Bedford, MA), and reacted with the primary antibodies indicated. After
washing, the blots were incubated with an appropriate peroxidase-labeled
secondary antibody and then reacted with Renaissance reagents (NEN,
Boston, MA) before exposure. After stripping, the blots were probed with
the anti–␣-tubulin antibody.
Enzymatic digestion of N-linked glycosylation
A 20-␮L volume of CMC pellet was denatured at 100°C for 10 minutes and
then one tenth of the sample was incubated at 37°C for 1 hour in the
presence or absence of Peptide:N-glycocidase F (PNGase F; 500 U)
according to kit instructions (New England Biolabs, Beverly, MA). The
samples were then separated on SDS-polyacrylamide gels and reacted with
the anti-SgIGSF antibody.
Transfection of CMCs with retroviral vector
The pCX4bsr vector, a modified pCXbsr vector,31 was kindly provided by
Dr T. Akagi (Osaka Bioscience Institute, Osaka, Japan). A clone containing
full-length SgIGSF cDNA was isolated from the B6⫹/⫹ CMC cDNA library.
The cDNA insert was excised by EcoRI digestion and inserted directionally
into the pCX4bsr retroviral vector via the EcoRI site. The resulting
pCX4bsr-SgIGSF vector or the empty pCX4bsr vector was then transfected
into the packaging cell line ⌿2, and blasticidin-resistant ⌿2 cell clones
were selected by culturing in ␣-MEM containing 10% FCS and blasticidin
(3␮g/mL; Invitrogen, Carlsbad, CA). To obtain infected CMCs, a subconfluent monolayer of the ⌿2 cell clones that produce high titers of retrovirus
containing either the SgIGSF cDNA or no insert was ␥-irradiated at a single
dose of 30 Gy. A freshly prepared spleen cell suspension was then added to
the monolayer and incubated for 5 days in ␣-MEM containing 10% FCS
and 10% PWM-SCM. Blasticidin-resistant CMCs were selected by continuing the culture in the presence of blasticidin (1.5 ␮g/mL) for 4 weeks.
Transfection of CMCs with a retrovirus vector containing the ⫹-MITF
cDNA was performed as described previously.19
NIH/3T3 cells were transfected with the pCX4bsr-SgIGSF vector by the
calcium phosphate coprecipitation method. Blasticidin-resistant NIH/3T3
cells were selected by continuing the culture in the presence of blasticidin
(3 ␮g/mL) for 4 weeks.
Immunocytochemistry
CMCs were washed with phosphate-buffered saline (PBS; pH 7.4), attached
to microscope slides by Cytospin 2 centrifugation (Shandon), and fixed with
methanol. For staining the coculture, an NIH/3T3 monolayer was established on a cover slip placed at the bottom of a culture dish and CMCs were
plated over this. After 3 hours’ coculture, the cover slips were washed with
PBS and fixed with methanol. Fixed samples were blocked with 2% bovine
serum albumin in PBS, incubated with the anti-SgIGSF antibody, and
stained with FITC-labeled antirabbit IgG antibody. For double staining with
phalloidin, the coculture samples were fixed with 3.7% paraformaldehyde
in PBS and permeabilized with 0.1% Triton X-100 in PBS. After staining
with the anti-SgIGSF antibody as described above, the samples were
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MITF-DEPENDENT MAST-CELL ADHESION MOLECULE
Table 1. Poor attachment of B6mi/mi, B6tg/tg, and B6 Miwh/Miwh CMCs
to NIH/3T3 fibroblasts
No. of adhering CMCs per NIH/3T3 cell*
CMC genotype
PWM-SCM⫺†
PWM-SCM⫹†
⫹/⫹
0.166 ⫾ 0.021
0.162 ⫾ 0.028
mi/mi
0.042 ⫾ 0.017‡
0.044 ⫾ 0.022‡
tg/tg
0.044 ⫾ 0.010‡
0.048 ⫾ 0.023‡
Miwh/Miwh
0.040 ⫾ 0.011‡
0.046 ⫾ 0.012‡
*Mean ⫾ SE of 3 dishes.
†The coculture of CMCs and NIH/3T3 cells was done with (⫹) or without (⫺)
PWM-SCM (10%).
‡P ⬍ .01 by Student t test when compared with the values of B6⫹/⫹ CMCs.
incubated with tetramethylrhodamine isothiocyanate (TRITC)–labeled phalloidin (1:5,000 dilution; Sigma). Cells were visualized using a confocal
laser scanning microscope (LSM510; Carl Zeiss, OberKochen, Germany).
Luciferase assay
The pEF-BOS vectors32 containing ⫹-MITF or mi-MITF cDNA, constructed previously,19 were used as effectors. The 5⬘ flanking sequence of
the SgIGSF gene was obtained from the database of the Celera Discovery
System (Celera, Rockville, MD). The genomic region of nucleotide (nt)
⫺1501 to ⫹19 of the SgIGSF gene was amplified by PCR and subcloned
into the upstream region of the luciferase gene in pSPLuc plasmid. A
reporter and an effector were electroporated together into MST mastocytoma and Jurkat lymphoid cells as described previously.13,27 The relative
luciferase activity was calculated as described previously.7
Electrophoretic gel mobility shift assay (EGMSA)
Glutathione S-transferase (GST) and GST–⫹-MITF were produced previously.5 Two oligonucleotides were synthesized: E, 5⬘-GCTTTAATGTGTAACTCATTTGATGGGTTGGCCGA-3⬘ (nt ⫺1506 to ⫺1472 of the SgIGSF
promoter), and mE, 5⬘-GCTTTAATGTGTAACTCTTTAGATGGGTTGGCCGA-3⬘. EGMSA was performed as described previously.7
2603
We performed Northern blot analysis on RNAs extracted from
B6⫹/⫹, B6mi/mi, and B6tg/tg CMCs, using clone 236 as a probe. Two
transcripts were detected near the positions of 28S and 18S in
B6⫹/⫹ CMCs: the expression of the longer transcript was much
stronger than that of the shorter one (Figure 1A). This result was
consistent with the result reported by Wakayama et al24 that the
SgIGSF gene has 2 transcripts of 4.5 and 2.1 kb in mouse testes. In
contrast to the case of B6⫹/⫹ CMCs, no hybridization signals were
detected in RNAs obtained from either B6mi/mi or B6tg/tg CMCs
(Figure 1A). We screened the original cDNA library of B6⫹/⫹
CMCs by using clone 236 as a probe and isolated 5 positive clones
carrying a cDNA insert of approximately 2.1 kb. Sequencing
revealed that the cDNA inserts of all clones were identical to
the reported full-length cDNA of the SgIGSF gene (accession
no. AB052293).
Protein expression of SgIGSF was examined by blotting the
lysates of CMCs from various genotypes with the anti-SgIGSF
antibody (Figure 1B). Two strong bands of approximately 110 and
38 kDa and a weak band of approximately 50 kDa were observed in
the B6⫹/⫹ CMC lysate. In the lysates of B6mi/mi, B6tg/tg, and
B6Miwh/Miwh CMCs, the bands of approximately 110 and 38 kDa
were not detectable but the band of approximately 50 kDa was
recognizable (Figure 1B).
Western blotting was also performed on the lysates of intact
NIH/3T3 cells and NIH/3T3 cells that had been transfected with
full-length SgIGSF cDNA. No band was observed in the lysate of
intact NIH/3T3 cells. In the transfected NIH/3T3 cells, 4 bands
were recognized, and the bands representing the longest and
shortest proteins were positioned at mobility sizes similar to those
of the 2 strong bands observed in B6⫹/⫹ CMCs (Figure 1B). Thus,
we considered that SgIGSF had 2 forms, of approximately 110 and
Results
Poor attachment of CMCs derived from MITF mutants
to NIH/3T3 fibroblasts
We examined numbers of B6⫹/⫹, B6mi/mi, B6tg/tg, and B6Miwh/Miwh
CMCs that adhered to NIH/3T3 cells 3 hours after the initiation
of the coculture. The number of adhering B6mi/mi, B6tg/tg, or
B6Miwh/Miwh CMCs was one third that of B6⫹/⫹ CMCs (Table 1). No
significant difference was observed among numbers of adhering
B6mi/mi, B6tg/tg, and B6Miwh/Miwh CMCs. As we reported previously,20
addition of PWM-SCM to the coculture did not affect the results
(Table 1).
Isolation of SgIGSF gene as a transcriptionally down-regulated
gene in B6mi/mi CMCs
We constructed a cDNA library from B6⫹/⫹ CMCs and subtracted
from it mRNAs expressed in B6mi/mi CMCs.7 The (⫹/⫹ ⫺ mi/mi)
subtracted cDNA library proved to be enriched with clones that
were transcriptionally down-regulated in B6mi/mi CMCs.7,8 From
the subtracted library, we attempted to isolate cDNA clones whose
gene product might explain the deficient adhesion of B6mi/mi and
B6tg/tg CMCs to NIH/3T3 cells. We sequenced approximately 600
clones from the library and found a clone (no. 236) that carried part
of the cDNA sequence encoding SgIGSF. The cDNA of SgIGSF
was recently cloned from mouse testes, and it has a putative
transmembrane domain and an extracellular domain consisting of 3
immunoglobulin-like loops.24
Figure 1. Expression of SgIGSF and intercellular adhesion molecules in CMCs
derived from MITF mutant mice. (A) Expression of SgIGSF mRNA in B6⫹/⫹, B6mi/mi,
and B6tg/tg CMCs. RNA (10 ␮g of total RNA) from CMCs of 3 genotypes was
electrophoresed and hybridized with the clone 236 probe. After stripping, the blot was
hybridized with the ␤-actin probe to indicate the amount of RNA loaded per lane.
(B) Expression of the SgIGSF protein and intercellular adhesion molecules in B6⫹/⫹,
B6mi/mi, B6tg/tg, and B6Miwh/Miwh CMCs. Lysates of the indicated cells were electrophoresed and blotted with antibodies against SgIGSF, E-cadherin, N-cadherin, ICAM-1,
and integrin ␤3. (C) Western blot analysis of NIH/3T3 cells and those transfected with
SgIGSF cDNA (NIH/3T3SgIGSF). The lysates of the indicated cells were electrophoresed and blotted with the SgIGSF antibody and then probed again with the
anti-␣-tubulin antibody to indicate the total amount of proteins loaded per lane. The
molecular weight scale is shown to the right of the blot.
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ITO et al
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forms of SgIGSF that have received cell and tissue type-specific
glycosylation.
Localization of SgIGSF
Figure 2. SgIGSF expression in CMCs derived from KIT mutant mice. Lysates
were prepared from CMCs derived from mice of the indicated genotypes and were
blotted with the anti-KIT and anti-SgIGSF antibodies. The molecular weight scale is
shown to the right of the blots. The blots were probed again with the anti–␣-tubulin
antibody to indicate the total amount of proteins loaded per lane.
38 kDa, in B6⫹/⫹ CMCs, but these forms were absent from B6mi/mi,
B6tg/tg, and B6-Miwh/Miwh CMCs. A weak band of approximately 50
kDa detected in all 4 types of CMCs remained uncharacterized.
Because there is no SgIGSF mRNA expression in B6mi/mi and B6tg/tg
CMCs, the approximately 50-kDa band appeared to arise from
cross-recognition of an unknown protein by the SgIGSF antibody
rather than its specific recognition of another form of SgIGSF.
We also examined the expression of several intercellular
adhesion molecules in CMCs with mutant MITFs. Expression
levels of E-cadherin, N-cadherin, ICAM-1, and integrin ␤3 in
B6mi/mi, B6tg/tg, and B6-Miwh/Miwh CMCs were comparable with
those of B6⫹/⫹ CMCs (Figure 1B).
CMCs were obtained from WB⫹/⫹, WBW/W, WBB6F1⫹/⫹, and
WBB6F1W/Wv mice, and the expression of KIT and SgIGSF was
examined using anti-KIT and anti-SgIGSF antibodies. KIT signals
were detected in the lysates of B6⫹/⫹, WB⫹/⫹, and WBB6F1⫹/⫹
CMCs, whereas no specific signals were found in the lysate of
WBW/W CMCs (Figure 2). In WBB6F1-W/Wv CMCs, KIT signals
were detectable at significantly reduced levels (Figure 2). Next, the
blot was reacted with the anti-SgIGSF antibody. WBB6F1W/Wv
CMCs expressed SgIGSF as abundantly as WBB6F1⫹/⫹ CMCs,
and WBW/W CMCs expressed a rather higher level of SgIGSF than
did WB⫹/⫹ CMCs (Figure 2). In the lysates of CMCs from WB and
WBB6F1 mice, the band of approximately 50 kDa was recognized
much more strongly by the SgIGSF antibody than in the lysates of
CMCs from B6 mice.
CMCs of various genotypes were cultured in suspension in the
presence of PWM-SCM. Cytospin preparations of the suspensioncultured CMCs were made and stained with anti-SgIGSF antibody.
When aggregates of B6⫹/⫹ CMCs were observed, the SgIGSFspecific fluorescence was detected in the area of cell-to-cell contact
(Figure 4A). The SgIGSF-specific fluorescence was not detectable
in B6⫹/⫹ CMCs that were isolated from each other. Even when
aggregates of B6tg/tg CMCs were observed in cytospin preparations,
no SgIGSF-specific fluorescence was detectable (Figure 4B). No
SgIGSF-specific fluorescence was observed in aggregated B6mi/mi
CMCs, either (data not shown). We also examined the localization
of SgIGSF in the coculture of CMCs and NIH/3T3 fibroblasts.
CMCs of various genotypes were plated onto the monolayer of
NIH/3T3 cells that had attached to a cover slip. After 3 hours’
coculture, the peripheral margin of B6⫹/⫹ CMCs adhering to
NIH/3T3 cells was clearly demarcated with the anti-SgIGSF
antibody (Figure 4C). The peripheral margin of neither B6tg/tg nor
B6mi/mi CMCs cocultured with NIH/3T3 cells was demarcated with
the anti-SgIGSF antibody (data not shown). A sectional view of the
coculture revealed that SgIGSF-specific fluorescence was concentrated on the lateral membrane of CMCs that faced the NIH/3T3
cells (Figure 4C). When the polymerized actin filaments in the
coculture of B6⫹/⫹ CMCs and NIH/3T3 cells were visualized with
phalloidin, densely stained actin filaments were detected in the
peripheral margins of adhering B6⫹/⫹ CMCs. These stains colocalized with SgIGSF (Figure 4D-F). This indicated the localization of
SgIGSF in lamellipodial structure. The immunocytochemical finding that NIH/3T3 cells were not stained with the anti-SgIGSF
antibody was consistent with the result of Western blot analysis
(Figures 1C and 4C).
Transfection of cDNAs encoding SgIGSF or ⴙ-MITF
Spleen cells of B6tg/tg and B6mi/mi mice were cocultured with ⌿2
packaging cells transformed with the retrovirus vector containing
the SgIGSF cDNA. As a control, B6tg/tg spleen cells were cocultured with the packaging cells transformed with either the retrovirus vector containing the ⫹-MITF cDNA or the empty vector. All 4
types of cocultures were maintained in the presence of PWM-SCM
and the selective drug. Four weeks after initiation of the coculture,
Tissue distribution of SgIGSF
We examined the expression of SgIGSF in various tissues of B6tg/tg
mice. Lysates of testes, spleens, lungs, and stomachs were obtained
from B6⫹/⫹ and B6tg/tg mice and were blotted with the anti-SgIGSF
antibody. In spite of the remarkable difference in SgIGSF expression between B6⫹/⫹ and B6tg/tg CMCs, the expression was comparable between testis, lung, spleen, and stomach tissues of B6⫹/⫹ and
B6tg/tg mice (Figure 3A). When the lysate of ⫹/⫹ CMCs was
treated with PNGase F, the mobility size of the approximately
110-kDa SgIGSF decreased to approximately 70 kDa, indicating
heavy N-glycosylation (Figure 3B). On the other hand, the
approximately 38-kDa form was not influenced by the PNGase F
treatment. Similar results were obtained when the lysate of B6⫹/⫹
testes were treated with PNGase F (Figure 3B). The variability of
the molecular weights of SgIGSF observed in CMCs, NIH/3T3
transfectants, and tissues seemed to reflect the presence of various
Figure 3. SgIGSF expression in various tissues of B6 tg/tg mice. (A) Expression of
the SgIGSF protein in testis, lung, spleen, and stomach tissues of B6⫹/⫹ and B6tg/tg
mice. Lysates were prepared from the indicated tissues and were blotted with the
anti-SgIGSF antibody. (B) N-linked glycosylation of SgIGSF proteins in B6⫹/⫹ CMCs
and testes. Lysates of B6⫹/⫹ CMCs and testes were incubated at 37°C for 1 hour in
the presence (⫹) or absence (⫺) of PNGase F and blotted with the anti-SgIGSF
antibody. The molecular weight scale is shown to the right of the blots. The blots were
probed again with the anti–␣-tubulin antibody to indicate the total amount of proteins
loaded per lane.
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Figure 5. Expression of the SgIGSF protein in B6tg/tg and B6mi/mi CMCs
transfected with SgIGSF cDNA. B6tg/tg CMCs were infected with the empty
retroviral vector (tg/tg CMCVector) or the vector containing either SgIGSF cDNA (tg/tg
⫹/⫺
CMCSgIGSF) or ⫹-MITF cDNA (tg/tg CMCMITF ). B6mi/mi CMCs were infected with the
vector containing the SgIGSF cDNA (mi/mi CMCSgIGSF). Lysates of the indicated cells
were electrophoresed and blotted with the anti-SgIGSF and anti-MITF antibodies.
After stripping, the blot was probed with the anti–␣-tubulin antibody to indicate the
total amount of proteins loaded per lane.
Figure 4. Immunolocalization of SgIGSF in CMCs. Immunocytochemical analysis
of B6⫹/⫹ (A) and B6tg/tg (B) CMCs. Cytospin preparations of CMCs were fixed with
methanol, reacted with the anti-SgIGSF antibody, and stained with FITC-labeled
secondary antibody. Bar, 10 ␮m. (C) Immunocytochemical analysis of the coculture
of B6⫹/⫹ CMCs and NIH/3T3 cells. CMCs were cocultured on the monolayer of
NIH/3T3 cells for 3 hours. The coculture was fixed with methanol, reacted with the
anti-SgIGSF antibody, and stained with FITC-labeled antibody. A representative set
of X-Y and Y-Z sections is shown. A red line indicates the plane of the Y-Z section.
(D-F) Colocalization of SgIGSF with polymerized actin filaments in the peripheral
margin of B6⫹/⫹ CMCs that have adhered to NIH/3T3 cells. After CMCs were
cocultured on the monolayer of NIH/3T3 cells for 3 hours, the coculture was fixed with
paraformaldehyde, reacted with the anti-SgIGSF antibody, and stained with FITClabeled secondary antibody (D). Subsequently, the culture was stained with TRITClabeled phalloidin (E) and the FITC and TRITC images were merged (F). *B6⫹/⫹ CMCs;
#NIH/3T3 cells. Bars, 10 ␮m.
more than 95% of the floating cells were CMCs in all 4 types of
coculture. Thus, coculture with the packaging cells did not
influence the purity of CMCs. Obtained CMCs were examined for
their expression levels of SgIGSF by Western blot. Prominent
expression of SgIGSF was detected in the lysates of B6tg/tg and
B6mi/mi CMCs transfected with SgIGSF cDNA (Figure 5). When the
band intensity was normalized with the immunoreactivity to the
antitubulin antibody, the expression levels of SgIGSF in B6tg/tg and
B6mi/mi CMCs transfected with SgIGSF cDNA were more than
10-fold higher than the level of B6⫹/⫹ CMCs. When the protein
samples were loaded equally per lane, the approximately 50-kDa
band was detected in the lysates of B6tg/tg and B6mi/mi CMCs
transfected with SgIGSF cDNA as strongly as in the lysates of
B6tg/tg and B6mi/mi CMCs (data not shown). The expression level of
SgIGSF in B6tg/tg CMCs transfected with ⫹-MITF cDNA was
comparable with that of B6⫹/⫹ CMCs (Figure 5). The blot was
probed again with the anti-MITF antibody. Although the expression
level of the endogenous ⫹-MITF in B6⫹/⫹ CMCs was below the
limit of detection, the expression of ⫹-MITF was detectable in
B6tg/tg CMCs transfected with ⫹-MITF cDNA (Figure 5). Neither
SgIGSF nor MITF expression was observed in the original B6tg/tg
CMCs or those transfected with the empty vector (Figure 5). B6tg/tg
CMCs transfected with SgIGSF cDNA started forming macroscopic aggregates beginning 4 weeks after culture of B6tg/tg spleen
cells was initiated (Figure 6A-B). Similar aggregates were formed
by B6mi/mi CMCs transfected with SgIGSF cDNA (data not shown).
On the other hand, B6tg/tg CMCs transfected with ⫹-MITF cDNA
did not form such aggregates (data not shown). The appearance of
B6tg/tg CMCs transfected with ⫹-MITF was similar to the appearance of B6⫹/⫹ CMCs.
In the culture of B6tg/tg and B6mi/mi CMCs transfected with
SgIGSF cDNA, both the number of aggregates and the number of
cells in each aggregate continually increased after the fourth week
of culture. In the fifth week of culture, the number of cells forming
aggregates reached more than half the number of total cells in each
culture, and some aggregates contained more than 100 cells. The
aggregated CMCs did not appear to grow as fast as intact B6tg/tg and
Figure 6. Phenotypes of B6tg/tg CMCs transfected with SgIGSF or ⴙ-MITF.
(A) Phase contrast image of B6tg/tg CMCs transfected with SgIGSF cDNA. Aggregates of the CMCs were floating in the medium. Bar, 50 ␮m. (B) Aggregates
composed of numerous alcian blue–positive cells. Cytospin samples of B6tg/tg CMCs
transfected with SgIGSF cDNA were stained with alcian blue and nuclear fast red.
Bar, 50 ␮m. (C,D) Immunocytochemical analysis of B6tg/tg CMCs transfected with
either SgIGSF cDNA (C) or ⫹-MITF cDNA (D). Cytospin preparations of either type of
cells were fixed with methanol, reacted with the anti-SgIGSF antibody, and stained
with FITC-labeled secondary antibody. Bar, 10 ␮m.
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ITO et al
Figure 7. Growth kinetics of SgIGSF-transfected CMCs. CMCs (2.5 ⫻ 105) were
suspended in 2 mL fresh medium containing PWM-SCM and plated onto 35-mm
culture dishes. On the indicated days after plating, total cell numbers were counted
with a standard hemocytometer. Four types of CMCs were examined: B6mi/mi CMCs
(f), B6tg/tg CMCs (F), B6mi/mi CMCs transfected with SgIGSF cDNA (mi/mi CMCSgIGSF;
䡺), and B6tg/tg CMCs transfected with SgIGSF cDNA (tg/tg CMCSgIGSF; E). Mean cell
numbers of triplicate samples were plotted; bars indicate SE. * P ⬍ .01 by Student t
test when compared with the values of intact B6mi/mi or B6tg/tg CMCs.
B6mi/mi CMCs. When intact B6tg/tg and B6mi/mi CMCs were plated in
fresh medium containing PWM-SCM, the total number of CMCs
increased nearly 3-fold after a week (Figure 7). In contrast, there
was only a 50% increase in the total cell number of B6tg/tg and
B6mi/mi CMCs transfected with SgIGSF after a week (Figure 7).
Cytospin preparations of B6tg/tg CMCs transfected with SgIGSF
cDNA or with ⫹-MITF cDNA were stained with anti-SgIGSF
antibody. The plasma membranes of aggregated SgIGSF-transfected B6tg/tg CMCs were strongly immunoreactive to the antiSgIGSF antibody (Figure 6C). The SgIGSF-specific fluorescence
was restricted to the cell-to-cell contact areas. Although B6tg/tg
CMCs transfected with ⫹-MITF cDNA did not form macroscopic
aggregates, microscopic aggregates of CMCs were detectable in
cytospin preparations. SgIGSF-specific fluorescence was detected
in the cell-to-cell contact areas of these aggregated cells, as with the
B6⫹/⫹ CMCs (compare Figures 6D and 4A). The SgIGSF-specific
fluorescence was significantly stronger in SgIGSF-transfected
B6tg/tg CMCs than in ⫹-MITF-transfected B6tg/tg CMCs. The result
of immunostaining of B6mi/mi CMCs transfected with SgIGSF
cDNA was similar to that of B6tg/tg CMCs transfected with SgIGSF
cDNA (data not shown).
Normalization of the adhesion of MITF mutant-derived CMCs
to NIH/3T3 cells by transfection with SgIGSF or ⴙ-MITF
Single-cell suspension was prepared by pipetting aggregates of
SgIGSF-transfected B6tg/tg and B6mi/mi CMCs. The resulting singlecell suspension of B6tg/tg and B6mi/mi CMCs transfected with
SgIGSF cDNA and the single-cell suspension of B6tg/tg CMCs
transfected with ⫹-MITF cDNA were added to the monolayer of
NIH/3T3 cells. After 3 hours’ coculture, we counted the number of
adhering CMCs per NIH/3T3 cell. Not only ⫹-MITF-transfected
B6tg/tg CMCs but also SgIGSF-transfected B6tg/tg and B6mi/mi CMCs
adhered to NIH/3T3 cells as frequently as B6⫹/⫹ CMCs (Table 2).
Transcriptional activation of the SgIGSF gene by ⴙ-MITF
We examined the effect of ⫹-MITF and mi-MITF on the transcription of the SgIGSF gene, using the transient cotransfection assay.
The 5⬘ flanking sequence of the SgIGSF gene (nt ⫺1501 to ⫹19
[⫹1 is the transcription start site]) was cloned upstream of the
luciferase gene. This construct was cotransfected into MST masto-
cytoma cells with an empty pEF-BOS plasmid or with the vectors
expressing either ⫹-MITF or mi-MITF. Cotransfection with the
⫹-MITF cDNA increased the luciferase activity 3-fold as strongly
as cotransfection with the empty vector, but cotransfection with
mi-MITF cDNA did not increase the luciferase activity (Figure 8A,
upper panel). Previously we reported that ⫹-MITF directly transactivated a number of genes by binding to CANNTG motifs.7,17,18 The
region between nt ⫺1501 and ⫹19 of the SgIGSF gene contained 2
CANNTG motifs: CATTTG (nt ⫺1490 to ⫺1485) and CACTTG
(nt ⫺682 to ⫺677). We mutated the CATTTG (nt ⫺1490 to
⫺1485) motif to CTTTAG and found that the mutation abolished
the transactivation effect of ⫹-MITF (Figure 8A, upper panel).
Next we deleted the CATTTG (nt ⫺1490 to ⫺1485) motif from the
SgIGSF promoter. The resulting shorter luciferase construct, which
contained only the CACTTG (nt ⫺682 to ⫺677) motif, was not
transactivated by cotransfection with the ⫹-MITF cDNA (Figure
8A, upper panel). We performed similar luciferase assays using
Jurkat lymphoid cells instead of MST cells. Neither ⫹-MITF nor
mi-MITF produced any transactivation effects on the 3 luciferase
constructs (Figure 8A, lower panel).We examined in vitro binding
of ⫹-MITF to the CATTTG motif by EGMSA. Two oligonucleotides containing the CATTTG (nt ⫺1490 to ⫺1485; oligonucleotide E) and mutated (CTTTAG; oligonucleotide mE) motifs were
synthesized. When oligonucleotide E was incubated with the
GST–⫹-MITF fusion protein, a slowly migrating band was
detected (Figure 8B, lane 2). The band appeared to be due to the
specific binding of oligonucleotide E to ⫹-MITF but not to GST,
because incubation of this oligonucleotide with the GST protein
alone did not yield any bands (Figure 8B, lane 1). To examine
whether the binding between oligonucleotide E and GST–⫹-MITF
was dependent on the CATTTG motif, the binding reaction was
performed in the presence of an excess amount of unlabeled
competitors. The binding of labeled oligonucleotide E to GST–⫹MITF was completely canceled out by adding an excess amount of
unlabeled oligonucleotide E, but not by adding the same amount of
unlabeled oligonucleotide mE (Figure 8B, lanes 3 and 4).
Discussion
In the present study, we examined cell-to-cell adhesion phenotypes
of CMCs derived from 3 types of MITF mutant mice, B6tg/tg,
B6mi/mi, and B6Miwh/Miwh. The mi and Miwh mutant alleles encode
MITFs with deletion (mi-MITF)1,3,4 and alteration (Miwh-MITF),23
respectively, of a single amino acid at the basic domain,4 whereas
tg is a null mutation.1,9,10 The mi-MITF has a significant inhibitory
effect on transcription of various genes.8,13,19 The Miwh-MITF has a
decreased but detectable transcription activity on some genes and
an inhibitory effect on other genes.23,33 Although these mutant
alleles have different structural and functional abnormalities, poor
Table 2. Normalization of attachment of B6tg/tg and B6mi/mi CMCs to NIH/3T3
fibroblasts by transfection with SgIGSF or ⴞ MITF cDNA
CMCs
⫹/⫹ CMCs
No. of adhering CMCs per NIH/3T3 cell*
0.163 ⫾ 0.006
tg/tg CMCs
0.053 ⫾ 0.006†
tg/tg CMCs transfected with SgIGSF
0.189 ⫾ 0.026
tg/tg CMCs transfected with ⫹-MITF
0.169 ⫾ 0.017
tg/tg CMCs transfected with vector
0.057 ⫾ 0.005†
mi/mi CMCs
0.045 ⫾ 0.005†
mi/mi CMCs transfected with SgIGSF
0.156 ⫾ 0.029
*Mean ⫾ SE of 3 dishes.
†P ⬍ .01 by Student t test when compared with the values of B6⫹/⫹ CMCs.
BLOOD, 1 APRIL 2003 䡠 VOLUME 101, NUMBER 7
Figure 8. Luciferase assay and EGMSA. (A) Transactivation of the SgIGSF
promoter by ⫹-MITF. The promoter sequences of the SgIGSF from ⫺1501 to ⫹19
were inserted upstream of the luciferase (LUC) gene of the pSPLuc vector. In one
construct, the CATTTG motif in the promoter was mutated to CTTTAG (indicated by
X). The deletion construct lacking the CATTTG motif was also made. These reporter
constructs were cotransfected into MST (upper panel) or Jurkat (lower panel) cells
with the empty pEF-BOS vector or the vector containing either ⫹-MITF or mi-MITF
cDNA. Mean values of the luciferase activity obtained with triplicate samples were
plotted; bars indicate SE. In most cases, the SE values were too small to be shown by
bars. (B) In vitro binding between the CATTTG motif and ⫹-MITF. Oligonucleotide E
was labeled with ␣-32P dCTP and used as a probe. The unlabeled oligonucleotides E
and mE were used as competitors. The probe was incubated with either GST or
GST–⫹-MITF protein in the absence (⫺) or presence of an excess amount of either
competitor. An arrow indicates a protein-DNA complex.
adhesion of CMCs to NIH/3T3 fibroblasts was common among
CMCs derived from all 3 MITF mutant mice.
By screening the (⫹/⫹ ⫺ mi/mi) subtracted cDNA library, we
identified SgIGSF as a transcriptionally down-regulated gene in
B6mi/mi CMCs. SgIGSF is a member of the immunoglobulin
superfamily that was recently cloned from the cDNA library of
adult mouse testes. Wakayama et al24 showed that SgIGSF was
preferentially expressed by spermatogenic cells and suggested that
SgIGSF may participate in the interaction of spermatogenic cells
with Sertoli cells. Expression of SgIGSF was readily detectable in
B6⫹/⫹ CMCs, but not in CMCs derived from 3 types of MITF
mutant mice examined.
A human homolog of SgIGSF is known as IGSF4 (immunoglobulin superfamily number 4)34: there is 98% identity between
the 2 molecules at amino acid levels. Both SgIGSF and IGSF4 have
an extracellular domain with significant homology to neural cell
adhesion molecule 1 (NCAM-1) and NCAM-2.24 A putative motif
sequence that connects to actin cytoskeleton was present in the
intracellular domain of SgIGSF and IGSF4.35 Recently, IGSF4 was
found to be identical to tumor suppressor in lung cancer 1
MITF-DEPENDENT MAST-CELL ADHESION MOLECULE
2607
(TSLC1).36,37 TSLC1 was originally cloned from the genomic
region that frequently exhibits loss of heterozygosity in human
lung cancers and possesses tumor-suppressor activity. A recent
study by Masuda et al38 showed that TSLC1/IGSF4 localized to the
plasma membrane in cell-to-cell contact areas and that cells overexpressing TSLC1/IGSF4 more frequently formed aggregates. SgIGSF as
well as TSLC1/IGSF4 appeared to mediate intercellular adhesion.
Immunofluorescence analysis of cytospin preparations showed
that the subcellular localization of SgIGSF was restricted to
cell-to-cell contact areas among B6⫹/⫹ CMCs. This localization
pattern was common not only to TSLC1/IGSF438 but also to
well-characterized intercellular adhesion molecules, such as Ecadherin and nectins.39,40 In B6⫹/⫹ CMCs adhering to NIH/3T3
fibroblasts, SgIGSF was located primarily in the lamellipodial
structure, where cytoskeletal components and regulators, such as
vinculin and Rac-1, are known to accumulate in mast cells.41 These
results suggested that SgIGSF may mediate adhesion either among
CMCs or between CMCs and NIH/3T3 fibroblasts.
Consistent with the results of immunofluorescence, overexpression of SgIGSF in B6tg/tg and B6mi/mi CMCs resulted in the
formation of macroscopic aggregates in suspension culture, although intact B6tg/tg and B6mi/mi CMCs did not form such aggregates. SgIGSF appeared to function as a homophilic adhesion
molecule. However, this homophilic interaction may take place
only when SgIGSF is overexpressed. In fact, B6⫹/⫹ CMCs did not
form such macroscopic aggregates. B6tg/tg and B6mi/mi CMCs
overexpressing SgIGSF adhered to NIH/3T3 fibroblasts as well as
B6⫹/⫹ CMCs, although original B6tg/tg and B6mi/mi CMCs did not.
This indicates that SgIGSF is necessary for adhesion of CMCs to
NIH/3T3 fibroblasts. Because NIH/3T3 fibroblasts did not express
SgIGSF, SgIGSF appeared to serve as a heterophilic adhesion
molecule in the interaction between CMCs and NIH/3T3 fibroblasts. The counterpart of SgIGSF that is expressed by NIH/3T3
fibroblasts remains to be identified. Probably the heterophilic
interaction may be more physiological than the homophilic interaction, because the overexpression of SgIGSF was not necessary for
the heterophilic interaction.
Expression levels of the SgIGSF protein were not reduced in
CMCs derived from WBW/W and WBB6F1W/Wv mice. The W allele
encodes the mutant KIT without the extracellular domain,42 and the
Wv allele encodes the mutant KIT with an intact extracellular
domain and a mutated tyrosine kinase domain.43 In the coculture
with NIH/3T3 fibroblasts, WBB6F1W/Wv CMCs normally adhered to
NIH/3T3 fibroblasts, whereas WBW/W CMCs did not.44 Because
WBW/W CMCs expressed SgIGSF, their deficient adhesion to
NIH/3T3 fibroblasts was attributable to the deficient expression of
the extracellular domain of KIT. On the other hand, B6mi/mi, B6tg/tg,
and B6Miwh/Miwh CMCs did not adhere to NIH/3T3 fibroblasts.
Although B6mi/mi and B6tg/tg CMCs showed deficient expression of
KIT,8 B6Miwh/Miwh CMCs did show normal expression levels of
KIT.23 Therefore, the deficient adhesion of CMCs of MITF mutants
was attributable to the deficient expression of SgIGSF. Both KIT
and SgIGSF appeared necessary for adhesion of CMCs to NIH/3T3
fibroblasts.
Defective expression of SgIGSF in CMCs derived from all
MITF mutant mice suggested that the presence of ⫹-MITF was
essential for the expression of SgIGSF in mast cells. However,
⫹-MITF did not appear necessary for the expression of SgIGSF in
cells other than mast cells. In fact, the expression of SgIGSF was
comparable between testes, spleens, lungs, and stomachs of B6⫹/⫹
and B6tg/tg mice. Probably, other transcription factors may compensate for ⫹-MITF in these tissues. The transactivation effect of
⫹-MITF on the SgIGSF gene promoter was detected in MST
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ITO et al
mastocytoma cells but not in Jurkat lymphoid cells. In addition,
transfection with the ⫹-MITF cDNA normalized the expression of
SgIGSF in B6tg/tg CMCs. The transactivation of ⫹-MITF was
mediated through CATTTG motif (nt ⫺1490 to ⫺1485) in the
promoter region of the SgIGSF gene, at least in mast cells.
In summary, we identified a new mast cell adhesion molecule,
SgIGSF. Transcription of the SgIGSF gene was critically regulated
by ⫹-MITF in mast cells. SgIGSF appeared to mediate not only the
aggregation of CMCs through its homophilic interaction, but also
the adhesion of CMCs to NIH/3T3 fibroblasts through its heterophilic interaction.
Acknowledgments
We thank J. D. Esko for MST cells, H. Arnheiter for VGA-9tg/tg mice,
S. Nagata for pEF-BOS, and T. Akagi for pCX4bsr. We also thank
M. Kohara, T. Sawamura, and K. Hashimoto for technical assistance.
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