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Prepublished online as a Blood First Edition Paper on November 27, 2002; DOI 10.1182/blood-2002-07-2265.
HEMATOPOIESIS
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.
Microphthalmia transcription factor (MITF) is a
basic-helix-loop-helix-leucine zipper-type 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 B6+/+
CMCs that adhered to NIH/3T3 fibroblasts. From a cDNA library of B6+/+ 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 B6+/+ CMCs but not in CMCs derived from MITF mutants.
Immunocytochemical analysis showed that SgIGSF localized to the
cell-to-cell contact areas between B6+/+ 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) 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/N-sulfotransferase 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.
Mice
Cells
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 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 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-µL 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 (3µg/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 µg/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 µg/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 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' (nt1506 to
1472 of the SgIGSF promoter), and mE,
5'-GCTTTAATGTGTAACTCTTTAGATGGGTTGGCCGA-3'. EGMSA
was performed as described previously.7
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
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 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 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.
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 forms of SgIGSF that have received cell and tissue type-specific glycosylation.
Localization of SgIGSF CMCs of various genotypes were cultured in suspension in the presence of PWM-SCM. Cytospin preparations of the suspension-cultured CMCs were made and stained with anti-SgIGSF antibody. When aggregates of B6+/+ CMCs were observed, the SgIGSF-specific 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 +-MITF Spleen cells of B6tg/tg and B6mi/mi mice were cocultured with2 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, 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
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
anti-SgIGSF 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 +-MITF Single-cell suspension was prepared by pipetting aggregates of SgIGSF-transfected B6tg/tg and B6mi/mi CMCs. The resulting single-cell 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 +-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 (nt1501 to +19
[+1 is the transcription start site]) was cloned upstream of the
luciferase gene. This construct was cotransfected into MST mastocytoma
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).
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 adhesion of CMCs to NIH/3T3 fibroblasts was common among CMCs derived from all 3 MITF mutant mice. By screening the (+/+ 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 (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 E-cadherin 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
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 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.
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.
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.
Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology; the Osaka Cancer Society; the Sagawa Foundation for Promotion of Cancer Research; and the Naito Foundation.
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.
Reprints: Yukihiko Kitamura, Department of Pathology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: kitamura{at}patho.med.osaka-u.ac.jp.
1. Hodgkinson CA, Moore KJ, Nakayama A, et al. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell. 1993;74:395-404[CrossRef][Medline] [Order article via Infotrieve].
2.
Hughes JJ, Lingrel JB, Krakowsky JM, Anderson KP.
A helix-loop-helix transcription factor-like gene is located at the mi locus.
J Biol Chem.
1993;268:20687-20690
3.
Hemesath TJ, Streingrimsson E, McGill G, et al.
Microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family.
Genes Dev.
1994;8:2770-2780 4. Steingrimsson E, Moore KJ, Lamoreux ML, et al. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat Genet. 1994;8:256-263[CrossRef][Medline] [Order article via Infotrieve]. 5. Morii E, Takebayashi K, Motohashi H, et al. Loss of DNA binding ability of the transcription factor encoded by the mutant mi locus. Biochem Biophys Res Commun. 1994;205:1299-1304[CrossRef][Medline] [Order article via Infotrieve]. 6. Takebayashi K, Chida K, Tsukamoto I, et al. Recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nuclear localization potential. Mol Cell Biol. 1996;16:1203-1211[Abstract].
7.
Ito A, Morii E, Maeyama E, et al.
Systematic method to obtain novel genes that are regulated by mi transcription factor (MITF): impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells.
Blood.
1998;91:3210-3221
8.
Ito A, Morii E, Kim D-K, et al.
Inhibitory effect of the transcription factor encoded by the mi mutant allele in cultured mast cells of mice.
Blood.
1999;93:1189-1196 9. Tachibana M, Hara Y, Vyas D, et al. Cochlear disorder associated with melanocyte anomaly in mice with a transgenic insertional mutation. Mol Cell Neurosci. 1992;3:433-445. 10. Tsujimura T, Hashimoto K, Morii E, et al. Involvement of transcription factor encoded by the mouse mi locus (MITF) in apoptosis of cultured mast cells induced by removal of interleukin-3. Am J Pathol. 1997;151:1043-1051[Abstract]. 11. Silvers WK. The Coat Colors of Mice: A Model for Mammalian Gene Action and Interaction. New York, NY: Springer-Verlag; 1979.
12.
Steingrimsson E, Tessarollo L, Pathak B, et al.
Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development.
Proc Natl Acad Sci U S A.
2002;99:4477-4482
13.
Morii E, Ogihara H, Oboki K, et al.
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.
2001;97:3032-3039 14. Kasugai T, Oguri K, Jippo-Kanemoto T, et al. Deficient differentiation of mast cells in the skin of mi/mi mice: usefulness of in situ hybridization for evaluation of mast cell phenotype. Am J Pathol. 1993;143:1337-1347[Abstract].
15.
Ge Y, Jippo T, Lee Y-M, Adachi S, Kitamura Y.
Independent influence of strain difference and mi transcription factor on the expression of mouse mast cell chymases.
Am J Pathol.
2001;158:281-292
16.
Jippo T, Lee Y-M, Katsu Y, et al.
Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype.
Blood.
1999;93:1942-1950
17.
Morii E, Jippo T, Tsujimura T, et al.
Abnormal expression of mouse mast cell protease 5 gene in cultured mast cells derived from mutant mi/mi mice.
Blood.
1997;90:3057-3066
18.
Morii E, Tsujimura T, Jippo T, et al.
Regulation of mouse mast cell protease 6 gene expression by transcription factor encoded by the mi locus.
Blood.
1996;88:2488-2494
19.
Tsujimura T, Morii E, Nozaki M, et al.
Involvement of transcription factor encoded by the mi locus in the expression of c-kit receptor tyrosine kinase in cultured mast cells of mice.
Blood.
1996;88:1225-1233
20.
Ebi Y, Kanakura Y, Jippo-Kanemoto T, et al.
Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit.
Blood.
1992;80:1454-1462
21.
Fujita J, Nakayama H, Onoue H, et al.
Failure of W/Wv mouse-derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts.
Blood.
1988;72:463-468 22. Fujita J, Nakayama H, Onoue H, et al. Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype. J Cell Physiol. 1988;134:78-84[CrossRef][Medline] [Order article via Infotrieve].
23.
Kim D-K, Morii E, Ogihara H, et al.
Different effect of various mutant MITF encoded by mi, Mior, or Miwh allele on phenotype of murine mast cells.
Blood.
1999;93:4179-4186 24. Wakayama T, Ohashi K, Mizuno K, Iseki S. Cloning and characterization of a novel mouse immunoglobulin superfamily gene expressed in early spermatogenic cells. Mol Reprod Dev. 2001;60:158-164[CrossRef][Medline] [Order article via Infotrieve].
25.
Nakahata T, Spicer S, Cantey JR, Ogawa M.
Clonal assay of mouse mast cell colonies in methylcellulose culture.
Blood.
1982;60:352-362
26.
Montgomery RI, Lidholt K, Flay NW, Vertel B, Lindahl U, Esko JD.
Stable heparin-producing cell lines derived from the Furth murine mstocytoma.
Proc Natl Acad Sci U S A.
1992;89:11327-11331 27. Ito A, Kataoka TR, Watanabe M, et al. A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J. 2000;19:562-571[CrossRef][Medline] [Order article via Infotrieve].
28.
Morii E, Oboki K, Kataoka TR, Igarashi K, Kitamura Y.
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.
2002;277:8566-8571 29. Fujii T, Tamura K, Masai K, Tanaka H, Nishimune Y, Nojima H. Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermatogenesis. EMBO Rep. 2002;3:367-372[CrossRef][Medline] [Order article via Infotrieve]. 30. Oboki K, Morii E, Kataoka TR, Jippo T, Kitamura Y. Isoforms of mi transcription factor preferentially expressed in cultured mast cells of mice. Biochem Biophys Res Commun. 2002;290:1250-1254[CrossRef][Medline] [Order article via Infotrieve].
31.
Akagi T, Shishido T, Murata K, Hanafusa H.
v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation.
Proc Natl Acad Sci U S A.
2000;97:7290-7295
32.
Mizushima S, Nagata S.
pEF-BOS, a powerful mammalian expression vector.
Nucleic Acids Res.
1990;18:5322 33. Kataoka TR, Morii E, Oboki K, et al. Dual abnormal effects of mutant MITF encoded by Miwh allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation. Biochem Biophys Res Commun. 2002;297:111-115[CrossRef][Medline] [Order article via Infotrieve]. 34. Gomyo H, Arai Y, Tanigami A, et al. A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2. Genomics. 1999;62:139-146[CrossRef][Medline] [Order article via Infotrieve]. 35. Fukuhara H, Kuramochi M, Nobukuni T, et al. Isolation of the TSLL1 and TSLL2 genes, members of the tumor suppressor TSLC1 gene family encoding transmembrane proteins. Oncogene. 2001;20:5401-5407[CrossRef][Medline] [Order article via Infotrieve]. 36. Kuramochi M, Fukuhara H, Nobukuni T, et al. TSLC1 is a tumor suppressor gene in human non-small-cell lung cancer. Nat Genet. 2001;27:427-430[CrossRef][Medline] [Order article via Infotrieve]. 37. Pletcher MT, Nobukuni T, Fukuhara H, et al. Identification of tumor suppressor candidate genes by physical and sequence mapping of the TSLC1 region of human chromosome 11q23. Gene. 2001;273:181-189[CrossRef][Medline] [Order article via Infotrieve].
38.
Masuda M, Yageta M, Fukuhara H, Kuramochi M, Maruyama T, Nomoto A, Murakami Y.
The tumor suppressor protein TSLC1 is involved in cell-cell adhesion.
J Biol Chem.
2002;277:31014-31019 39. Takahasi K, Nakanishi H, Miyahara M, et al. Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. J Cell Biol. 1991;145:539-549.
40.
Miyahara M, Nakanishi H, Takahashi K, Satoh-Horikawa K, Tachibana K, Takai Y.
Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction.
J Biol Chem.
2000;275:613-618
41.
Guillemot J-C, Montcourrier P, Vivier E, Davoust J, Chavrier P.
Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in Fc
42.
Hayashi S, Kunisada T, Ogawa M, Yamaguchi K, Nishikawa S.
Exon skipping by mutation of an authentic splice site of c-kit gene in W/W mouse.
Nucleic Acids Res.
1991;19:1267-1271 43. Nocka K, Tan JC, Chiu E, et al. Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W. EMBO J. 1990;9:1805-1813[Medline] [Order article via Infotrieve]. 44. Adachi S, Ebi Y, Nishikawa S, et al. Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts. Blood. 1992;78:650-656.
© 2003 by The American Society of Hematology.
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 N. A. Meadows, S. M. Sharma, G. J. Faulkner, M. C. Ostrowski, D. A. Hume, and A. I. Cassady The Expression of Clcn7 and Ostm1 in Osteoclasts Is Coregulated by Microphthalmia Transcription Factor J. Biol. Chem., January 19, 2007; 282(3): 1891 - 1904. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
). 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-
),
B6tg/tg CMCs (
),
B6mi/mi CMCs transfected with SgIGSF cDNA
(mi/mi CMCSgIGSF; 
), and
B6tg/tg CMCs transfected with SgIGSF cDNA
(tg/tg CMCSgIGSF; 
). 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.
RI-activated rat basophilic leukemia (RBL-2H3) cells.
J Cell Sci.
1997;110:2215-2225