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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1987-1997
Osteoclast-Derived Zinc Finger (OCZF) Protein With POZ Domain,
a Possible Transcriptional Repressor, Is Involved in Osteoclastogenesis
By
Akiko Kukita,
Toshio Kukita,
Mamoru Ouchida,
Hidefumi Maeda,
Hitomi Yatsuki, and
Osamu Kohashi
From the Department of Microbiology, Department of Biochemistry, Saga
Medical School, Saga, Japan; and the Second Department of Anatomy,
Department of Conservative Dentistry, Faculty of Dentistry, Kyushu
University, Fukuoka, Japan.
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ABSTRACT |
The differentiation of osteoclasts is regulated by transcription
factors expressed in cells of osteoclast lineage. We isolated here a
potential transcription factor from a cDNA library of an enriched
population of preosteoclasts and osteoclasts. The cDNA encodes a
protein with N-terminal POZ domain and C-terminal
Krüppel-like zinc fingers. We designate this protein as
osteoclast-derived zinc finger (OCZF). OCZF was found to be rat
homologue of mouse leukemia/lymphoma-related factor (LRF). Northern
blot and in situ hybridization analysis showed OCZF mRNA at a
high level in osteoclasts and kidney cells. OCZF had a nuclear
targeting sequence and was localized in the nucleus of transfected
cells. In addition, OCZF specifically bound to the guanine-rich
consensus sequences of Egr-1 and c-Krox. Transient transfection
assays indicate that OCZF can repress transcription activity like other
POZ domain proteins. Furthermore, antisense but not sense
phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA
suppressed the formation of osteoclast-like multinucleated cells (MNCs)
in bone marrow culture, whereas the same ODNs did not significantly
affect the formation of macrophage polykaryons and mononuclear
preosteoclast-like cells (POCs). These results suggest that OCZF is a
unique transcription factor that plays an important role in the late
stage of osteoclastogenesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
OSTEOCLASTS ARE DERIVED from
hematopoietic stem cells. They are multinucleated cells and are formed
by the fusion of mononuclear preosteoclasts. Their differentiation is
thought to be under the control of the bone microenvironment, which is
supplied by stromal cells or osteoblasts and local
factors.1 The detailed molecular mechanism underlying the
differentiation of osteoclasts is not yet understood. Extracellular
stimuli produced by the bone microenvironment induce an intracellular
signal of the precursor cells for osteoclasts. The signal leads to the
expression of target genes and differentiation toward osteoclasts. As
in many developmental systems, the cell fate decision and promotion of
differentiation are mediated by temporally expressed or activated
transcription factors.
PU.1- and Fos-deficient mice have an osteopetrosis phenotype,
suggesting that these transcription factors are involved in osteoclast
differentiation.2-4 Fos protein is a major component of the
AP-1 transcription factor complex. In c-fos mutant mice, the number of
macrophages is not decreased, but the development of osteoclast is
disordered. PU.1 mutation results in the elimination of the lineages
for both osteoclasts and macrophages. PU.1 is thought to regulate the
initial stages of myeloid differentiation, whereas Fos is thought to
promote the differentiation of bipotential precursors into osteoclasts,
indicating that these transcription factors are involved in the early
stage of osteoclastogenesis. Conversely, there is yet little
information regarding transcription factors expressed in the cells at
the late stage of osteoclastogenesis.
In hematopoiesis, a number of transcription factors are known to
participate in the proliferation and differentiation of each specified
cell lineage. The transcription factors are classified into different
types by specific motifs. The zinc finger domain, in particular, seems
to comprise a major class of DNA-binding motifs, because many proteins
possessing this domain have been described.5 Some zinc
finger transcription factors are important, because they play a key
role as a master switch at the branch point of differentiation in
hematopoiesis. For example, GATA-1, which contains 2 Cys4-type zinc fingers, has an essential role in
erythrocyte development.6,7 Erythroid
Krüppel-like factor (EKLF), with
Cys2His2-type zinc fingers, is an erythroid
cell-specific transcription factor regulated by GATA-1.8
Egr-1, another Krüppel-type zinc finger
transcription factor with 3 Cys2His2-type zinc
finger motifs, is expressed in macrophages and has been shown to play a
critical role in the differentiation of the macrophage
lineage.9,10 Such zinc finger transcription factors
expressed in osteoclasts may have an important role in osteoclastogenesis.
In this study, we characterized a zinc finger protein with POZ domain
that is preferentially expressed in osteoclasts. This protein, termed
osteoclast-derived zinc finger (OCZF), showed an ability to bind
specifically to a guanine-rich sequence and regulated transcriptional
activity, suggesting that OCZF is a transcription factor. The
functional role of OCZF in the differentiation of osteoclasts was
investigated using specific antisense phosphorothionate oligodeoxynucleotides (ODNs). Interestingly, the blockage of the expression of OCZF results in a significant reduction of the formation of osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures. Our results suggest that OCZF may be one of several important regulators involved in the late stage of osteoclastogenesis.
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MATERIALS AND METHODS |
Materials.
Male Sprague-Dawley (SD) rats were purchased from SEAC Yoshitomi
(Fukuoka, Japan). ROS17/2.8 and UMR106 cells (rat osteoblastic cells)
were generously provided by Dr L. Bonewald (University of Texas Health
Science Center, San Antonio, TX). L8 cells (rat myoblasts) and 293 (human kidney cells) cells were kindly provided by Drs S. Matsuhashi
and K. Miyake (Saga Medical School, Saga, Japan). NRK cells (rat kidney
cells) were obtained from the Riken Cell Bank (Tsukuba, Japan). 3Y1
(rat fibroblasts) and A11 (rat osteoblastic cells) cells were
generously provided by Drs G. Kimura and T. Inai (University of Kyushu,
Fukuoka, Japan), respectively. 293T cells (human kidney cells) were
provided by Drs T. Watanabe and H. Kishi (University of Kyushu). WRT-7
P2 (P2) cells (rat leukemia cells) were kindly provided by
Dr M. Kobayashi (University of Hokkaido, Sapporo, Japan).
1 ,25dihydroxyvitamin D3
[1,25(OH)2D3] was purchased from Biomol
Research Laboratories (Plymouth Meeting, PA).
Cell cultures.
A mouse hybridoma cell line for monoclonal antibody (MoAb) Kat6 and P2
cells were grown in Iscove's modified Dulbecco's medium (IMDM; GIBCO
BRL, Gaithersburg, MD) containing 10% fetal calf serum (FCS). The
differentiation of P2 cells into macrophages was induced by the
addition of 10 7 mol/L phorbol myristate acetate
(PMA; Sigma, St Louis, MO) for 3 days.11 The mouse
hybridoma cell line for the MoAb Kat1 was grown in the same medium in
the presence of 10 ng/mL human recombinant interleukin-6 (IL-6; R&D,
Minneapolis, MN).12 The other cell lines were grown in
Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) containing 10%
FCS. Rat primary osteoblasts were isolated by sequential digestion from
the calvariae of newborn rats according to the method of Takahashi et
al13 and cultured in minimum essential medium (MEM;
GIBCO BRL) containing 10% FCS.
For the formation of MNCs, bone marrow cells obtained from the tibiae
and femurs of rats were cultured in the presence of 108 mol/L 1,25(OH)2D3
and 10% heat-treated conditioned medium derived from
ROS17/2.8 cells (htROSCM), as described by Kukita et al.14 For the formation of preosteoclast-like cells (POCs), nonadherent bone
marrow cells from which adherent cells were eliminated using a Sephadex
G-10 column were cultured in the presence of 108
mol/L 1,25(OH)2D3 and 10% (vol/vol)
htROSCM, as described by Kukita et al.15 For the formation
of macrophage polykaryons, bone marrow cells were cultured in the
presence of 108 mol/L
1,25(OH)2D3 and 107 mol/L
PMA as described.16 These cells were cultured in MEM containing 15% FCS (Biowittaker, Walkersville, MA) for 4 or 5 days. At
the end of the culture, tartrate-resistant acid phosphatase (TRAP)
staining was performed with a commercial kit (Sigma). TRAP-positive mononuclear cells as POCs or TRAP-positive multinucleated cells (>3
nucleus) as MNCs were counted. TRAP-negative multinucleated cells were
counted as macrophage polykaryons.
Preparation of MoAb Kat6 and immunocytochemistry.
Immunization, hybridoma production, and screening were performed using
MNCs as the antigen.12 The immunocytochemistry of bone
marrow cells with MoAb Kat6 was performed as follows. Rat bone marrow
cells were cultured in the presence of 10% (vol/vol) htROSCM and
108 mol/L 1,25(OH)2D3.
After 5 days of culture, the cells were detached from the plastic
culture plates with 0.05% trypsin and 0.02% EDTA in
phosphate-buffered saline (PBS) and centrifuged onto glass slides with
the use of a cytospin. The cells were then fixed with 2%
paraformaldehyde (PFA) in PBS for 5 minutes, followed by treatment with
cold acetone for 2 minutes. The cells were stained by MoAb Kat 6 using
fluorescein isothiocyanate (FITC)-conjugated antimouse IgM (µ-chain
specific; Zymed, San Francisco, CA) as the second antibody.
Enrichment of osteoclasts and osteoclast-precursor cells by magnetic
cell separator system (MACS).
Rat bone marrow cells were cultured in the presence of 10% (vol/vol)
htROSCM and 108 mol/L
1,25(OH)2D3. After 4 days of culture, the
cells were detached from the plastic culture plates as described and
incubated with MoAb Kat1 (500 µg/mL), which recognizes the surface
antigen of osteoclasts, on ice for 20 minutes. After the cells were
rinsed in ice-cold PBS, they were reacted with biotinylated antimouse IgM (µ-chain specific) on ice for 10 minutes. The cells were then rinsed in ice-chilled PBS and reacted with FITC-conjugated
streptoavidin for 10 minutes, followed by the reaction with
biotinylated MACS microbeads for 5 minutes according to the
manufacturer's protocol. The cells were then resuspended in PBS
containing 2% bovine serum albumin (BSA) and applied to MACS (Miltenyi
Biotec, Sunnyvale, CA), and the positive fraction (Kat1-positive cells)
was collected.
Cloning of OCZF gene.
Poly(A)RNA was isolated from the Kat1-positive cell-enriched fraction
with the use of an mRNA isolation kit (Pharmacia Biotech, Uppsala,
Sweden), and cDNA synthesis was performed using a cDNA synthesis kit
(Stratagene, La Jolla, CA). Briefly, the synthesis of double-stranded
cDNA was primed with oligo dT linker-primer, and the cDNA was ligated
to EcoRI adapter and then digested by Xho I. Synthesized cDNA was size-fractionated by electrophoresis on a 4%
Nuisieve agarose gel (Takara, Tokyo, Japan). The cDNA larger than 1.0 kb was recovered and ligated to the EcoRI/Xho I site of
 Uni-ZAPII vector. A directional library was constructed with an in
vitro phage packaging kit using a packaging extract (Stratagene).
Screening of the library with MoAb Kat6 was performed by using an
ABC-AP kit (Vector, Burlingame, CA). The positive clone was
plaque-purified 3 times. The pBluscript plasmid containing the positive
cloned DNA insert was excised by coinfection with helperphage, and a
cDNA clone designated pK6-78 that encodes a partial sequence of the
OCZF gene was obtained. To obtain the full-length sequence of
OCZF, the insert of pK6-78 (0.9 kb, 933 to 1,872 bp of
pK6-78-4) was used as a probe to isolate overlapping clones from the
Kat1-positive cell library. Plaque hybridization was performed using a
32P-labeled insert of pK6-78 under high-stringency
conditions. The screening of the library with this probe led to the
isolation of several clones. The DNA sequence was determined using a
DNA sequencer (Model 370A; Applied Biosystems, Foster City, CA) and a
Taq DyeDeoxy cycle sequencing kit from Applied Biosystems. Sequencing of the full length of OCZF cDNA was performed using the longest clone, designated pK6-78-4.
In situ hybridization.
In situ hybridization was performed using sense and antisense RNA
probes corresponding to about 0.9 kb (insert of pK6-78) of OCZF
cDNA. The probes were labeled with digoxigenin with the use of an RNA
synthesis kit (Nippon Gene, Toyama, Japan). Four-micrometer sections of
rat mandible obtained from 6-day-old SD rats were prepared as described
previously, except that fixation was performed by perfusion of the
fixatives.17 Tissue sections were deparaffinized in xylene
and rehydrated in ethanol series. After being rinsed in PBS, sections
were treated with proteinase K (10 µg/mL; Sigma) for 10 minutes at
37°C, fixated in 4% PFA in PBS for 10 minutes, and then incubated
with 0.2 mol/L HCl for 10 minutes. The sections were treated in PBS
containing 0.2% glycine for 10 minutes 2 times, followed by rinsing in
PBS. The sections were acetylated with 0.25% acetic anhydride in 0.1 mmol/L triethanolamine (pH 8.0) for 10 minutes. The prehybridization
and hybridization were performed as previously described.17
Hybridized digoxigenin-labeled probes were detected by use of a DIG
Nucleic Acid Detection Kit (Boehringer Mannheim, Mannheim, Germany)
according to the manufacturer's instructions. After color reaction,
the sections were counterstained by 0.2% methylgreen.
Northern analysis of OCZF.
Poly(A)RNA was extracted from each of several tissues and cell lines
with the use of a mRNA isolation kit (Pharmacia Biotech). Rat liver,
testis, and brain poly(A)RNA were obtained from Clontech (Palo Alto,
CA). Poly(A)RNA was also extracted from bone marrow cells treated with
antisense, sense OCZF, and scramble ODNs after 4 days of culture. RNA
was denatured at 65°C for 5 minutes in 50% formamide,
electrophoresed through a 1.5% agarose gel containing 6.6%
formaldehyde, and transferred to nylon membranes (Genescreen; NEN
Research Products, Boston, MA). The insert of pK6-78 was labeled with
[-32P] cytosine triphosphate (CTP) using a random
primer labeling kit (Nippon Gene) and used as a probe. Hybridization
and washing were performed under stringent conditions at 60°C (the
preparation was finally washed twice at 60°C in 0.1× saline
sodium citrate plus 1% sodium dodecyl sulfate [SDS]).
DNA transfections.
For the expression of OCZF protein in cultured cells, full-length
OCZF cDNA was cloned into the EcoRI and Xho I
sites of the expression vector pME18S (provided by Dr K. Maruyama,
Tokyo University, Tokyo, Japan), which has the SR promoter. In some
experiments, OCZF cDNA (nt 159-2817) and human Egr-1 cDNA (nt
331-3132)18 were cloned into expression vector pCDL-FLAG,
which have the SR promoter (M. Ouchida, unpublished
results), restoring the open reading frame. Two types of
pCDL-FLAG vectors, pCDL-FLAGa and pCDL-FLAGc, which differ only in the
reading frame, were used for OCZF and Egr-1 cDNA, respectively. OCZF
and Egr-1 proteins produced from these plasmids have S-tag and FLAG-tag
epitope in its N-terminus and missed the first 10 and 21 amino acids,
respectively. For the isolation of nuclear extract, 293T cells were
plated in 10-mm dishes at a density of 4 × 105 cells
before transfection. For immunocytochemical study, 293T cells were
plated at a density of 2 × 104 cells on a glass
chamber (Nunc, Roskilde, Denmark) before transfection. DNA transfection
was performed by the calcium phosphate-DNA coprecipitation method as
described.19 The expression of FLAG-tagged protein was
confirmed by a Western analysis using anti-FLAG M2 MoAb (Sigma) as
described.20
Electrophoretic mobility shift assays (EMSA).
Complementary oligonucleotides containing the Egr-1-binding
sequence (underlined;
5'-ATCCCGGCGCGGGGGCGAGGGCGT-3' and
5'-ACGCCCTCGCCCCCGCGCCGGGAT-3') and the c-Krox-binding
sequence (5'-CCA CGTCCCTCCCCCCTCGGCTCCCTCCCCTA-3' and
5'-TAGG GGAGGGAGCCGAGGGGGGAGGGACGTGG-3')
were synthesized (Sawady Tec Co, Tokyo, Japan).21 The
oligonucleotides were annealed by heating to 65°C for 10 minutes
and then slowly cooled to room temperature. A consensus SP1
oligonucleotide (Promega, Madison, WI) was used as a competitor. Probes
were prepared by end-labeling oligonucleotides with
[ -32P] adenosine triphosphate (ATP) and T4
polynucleotide kinase. Nuclear extracts were prepared from the cell
lines 2 days after they were transfected with expression vector
containing cDNA for OCZF by the calcium-phosphate precipitation
method.22 The binding reaction was achieved by
preincubating nuclear extracts with 2 µg poly(dI-dC)/poly(dI-dC)
(Pharmacia Biotech) in a buffer containing 20 mmol/L HEPES, pH 7.9, 70 mmol/L KCl, 5 mmol/L MgCl2, 0.05% Nonidet P-40, 12%
glycerol, 1 mg/mL BSA, 0.5 mmol/L dithiothreitol, 100 µmol/L
ZnCl2, and 5 µmol/L p-Amidino phenylmethylsulphonyl fluoride (PMSF; Wakojunyaku, Osaka, Japan) at room temperature for 20 minutes as described.9 One nanogram of end-labeled probe (10,000cpm) was added to the reaction mixture containing the nuclear extract and incubated for 15 minutes at room temperature. For the
competition experiments, excess unlabeled DNA was incubated with the
reaction mixture for 15 minutes before the addition of the radiolabeled
probe. In supershift assays, 3 µg of anti-FLAG M2 MoAb (Sigma) was
incubated with the reaction mixture for 15 minutes before the addition
of the radiolabeled probe. The samples were analyzed by a 4%
polyacrylamide gel (PAGE) in 0.5× TBE (1× TBE consists of
0.089 mmol/L Tris-borate, 0.089 mmol/L boric acid, and 2 mmol/L EDTA)
buffer at 150 V at room temperature.
S-protein pulldown experiments.
Nuclear extracts were prepared from 293T cells 2 days after they were
transfected with pCDL-FLAG expression vectors (empty or encoding OCZF
or Egr-1 protein). To purify S-tagged protein, S-protein agarose beads
(Promega) were incubated with the nuclear extracts for 3 hours at
4°C and then washed 5 or 6 times in cold PBS. The bound proteins
were then used for DNA binding reaction. The binding reaction was
performed by incubating bound protein with 1 ng of end-labeled probe in
same buffer used in EMSA for 30 minutes at room temperature. To remove
unbound DNA, the bound DNA was then washed 3 times in the binding
buffer and then the oligomers retained by the beads were released by
the addition of deionizing water and boiling for 5 minutes. The
resulting DNA was analyzed by a 10% PAGE.
Luciferase assays.
The reporter plasmid, pOA-Egr-TK-Luciferase (M. Ouchida, unpublished
results), was constructed by inserting 4 tandem copies of
double-stranded oligonucleotides containing Egr-1 binding sequence in
upstream of the firefly luciferase gene of pOA-TK-Luciferase that
contained 250 bp 5' of the HSV thymidine kinase transcription initiation site. The internal control Renilla luciferase plasmid, p TK-RL, which has basal thymidine kinase promoter, was prepared from
pRLTK (Promega). The expression vectors, pME18S (either empty or
encoding OCZF protein), at 0.25 µg or 2.5 µg, were cotransfected with 1 µg of the reporter vector in 293 or 293T cells by calcium phosphate precipitation. The cells were harvested 48 hours
later in the Promega Lysis Buffer. The activity of firefly and Renilla luciferase was measured using a reagent kit (Promega) and normalized to
the Renilla luciferase activity of a cotransfected pTK-RL vector
(0.25 µg) to correct for variation in transfection efficiency.
Antisense ODN experiments.
The phosphorothioate ODNs (20 bases in length) ODN-1 and ODN-2,
complementary to the target sequence of OCZF mRNA, were
designed and purified by high-performance liquid chromatography (HPLC) by Greiner Japan Co (Tokyo, Japan) and Toa Synthesis Co (Tokyo, Japan),
respectively. Antisense OCZF ODN-1 located to a sequence starting at the ATG initiation codon of OCZF cDNA
(5'-CCGTCCACGCCGCCAGCCAT-3') and sense ODN-1
(5'-ATGCTGGCGGCGTGGGACGG-3') were used in the experiments,
the results of which are shown in Fig 8A (experiments no. 1 and 2).
Antisense OCZF ODN-2 targeted against a sequence located 16 bases upstream of the initial ATG codon
(5'-CATCTTCCGCGACACCTCT-3'), sense ODN-2
(5'-AGAGGTGTCGCGGAAGATG-3'), and scramble ODN-2
(5'-TGTCTCCTACC TGCCCAAC-3') as a control were used in the
experiments, the results of which are shown in Fig 8A (experiment no.
3), B, and C. Bone marrow cells or nonadherent bone marrow cells were
cultured in 3 types of conditions to form MNCs, POCs, and macrophage
polykaryons with ODNs at a final concentration of 1 µmol/L for 4 or 5 days at 37°C. Each culture was fed once every 3 days by replacing
half of the culture medium with fresh medium, hormone, ODNs, and
conditioned medium or PMA. Differentiation was assessed by staining the
cells with a TRAP kit (Sigma).
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RESULTS |
Reactivity of MoAb Kat6 to a nuclear antigen in MNCs in bone marrow
culture.
During the course of our search for the specific antigen expressed in
osteoclast-like cells in rat bone marrow culture by making a panel of
MoAbs,11 we found that MoAb designated Kat6 stained the
nuclei of osteoclast-like cells. Figure 1
demonstrates the positive staining of Kat6 in the nuclei of the
multinucleated cells derived from bone marrow cultures. Similar
positive staining with MoAb Kat6 was also seen in the mononuclear
cells, but not in primary osteoblasts (data not shown). Because little
information has been obtained regarding the nuclear proteins expressed
in osteoclasts, we attempted cDNA cloning of the gene for Kat6 antigen.
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| Fig 1.
Immunoreactivity of the MoAb Kat 6 to multinucleated
cells formed in bone marrow culture. Rat bone marrow cells were
cultured in the presence of htROSCM and 108 mol/L
1 ,25(OH)2D3 for 5 days. The detached cells
were centrifuged onto a glass slide by using a cytospin, fixed with 2%
PFA, and stained by MoAb Kat 6 using FITC-conjugated antimouse IgM as
the second antibody. Positive signals in the nucleus of the
multinucleated cells in the culture are indicated by arrows. (Original
magnification × 443.)
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Molecular cloning of OCZF cDNA.
We recently isolated MoAb Kat1, which recognizes the surface antigen of
osteoclasts.11 To perform the cDNA cloning efficiently, a
population of the cells including preosteoclasts and osteoclasts was
first enriched with this MoAb. A cDNA library (2.0 × 106 clones) in a phage vector ( ZAPII) was constructed
with mRNA from enriched cells. In the screening of cDNA library using
the MoAb Kat6, we obtained a candidate clone (pK6-78). The nucleotide sequence of pK6-78 and its predicted amino acid sequence from one open
reading frame (ORF) suggested that this protein encoded a partial
sequence of a novel zinc finger protein. First, to determine the
specificity of the expression of this cDNA, we performed an in situ
hybridization analysis of rat mandible tissue sections with RNA probes
prepared from pK6-78. As shown in
Fig 2A and B, positive signals
were detected with the antisense probe in the osteoclasts and some
mononuclear cells. In contrast, similar staining was absent with the
sense probe (Fig 2C). These results indicate that mRNA encoding a novel
zinc finger protein is expressed in osteoclasts. We therefore
designated this gene as an osteoclast-derived zinc finger gene,
OCZF. We then screened again approximately 3 × 105 plaques of the Kat1-positive library with the insert of
pK6-78 to obtain the full-length OCZF cDNA. The restriction
enzyme mapping of the inserts (0.9 to 2.8 kb) of the 4 positive clones
indicated that they overlapped. The longest clone (pK6-78-4) was
selected and characterized.
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| Fig 2.
Analysis of OCZF mRNA expression in osteoclasts
from rat mandible by in situ hybridization. Sections were hybridized
with a digoxygenin-labeled antisense (A and B) or sense (C) RNA probe.
(A and B) The intense signal of OCZF mRNA is detectable in the
osteoclasts with antisense RNA probe (the arrows). (C) No signal was
observed in control experiment in which sense RNA was used in
hybridization. (Original magnification × 239.)
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Structure of OCZF cDNA.
The DNA sequence analysis of this cDNA showed an ORF encoding a 569 amino acid polypeptide of 60,539 Daltons
(Fig 3). The putative ATG initial codon was
preceded by an in-frame termination codon located 49 bases upstream.
Because this terminal codon was confirmed by analyzing several
independent clones, we concluded that we have cloned cDNA encoding the
entire ORF. The predicted amino acid sequence contained 3 zinc fingers
of the Cys2-His2 type near the C-terminus. A
comparison of the OCZF sequence against Swiss Prot and
GenBank/EMBL databases using the BLAST algorithms23 showed
similarities with zinc finger proteins that are restricted to the
region of the zinc finger and N-terminus. A murine cDNA lymphocyte/leukemia-related factor (LRF) that has been isolated very
recently24 is 97% identical over their length, suggesting that OCZF is rat homologue of LRF. The high similarity between OCZF and
LRF was also found in N-terminus and zinc finger regions of the
proteins (Fig 3).
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| Fig 3.
Deduced amino acid sequence of rat OCZF protein and
comparison with that of mouse LRF. Dashes indicate shared identity
between the mouse and rat proteins. The N-terminal POZ-like domain is
underlined. The proline-rich sequence is double underlined. The domain
of 3 zinc fingers is underlined. The nuclear target sequence
corresponding to amino acids 480-496 is underlined with a dotted line.
Amino acids are numbered at the end of each line. OCZF nucleotide
sequence data are available from EMBL/GenBank/DDBJ under the accession
no. D88450.
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The comparison of the zinc finger sequence of OCZF showed features
representative of the Cys2His2
Krüppel-like family. The proteins that had the high
matches in the zinc finger region were human APM-1,25 mouse
c-Krox,26 and human hcKrox.27 The zinc finger
sequences of c-Krox and hcKrox were identical. To analyze the zinc
fingers in more detail, we aligned the zinc finger sequences of OCZF
with those of human APM-1, mouse c-Krox, Egr-1,28
and Krüppel,29 the segmentation gene in
Drosophila (Fig 4A). The homology
with APM-1, c-Krox, Egr-1, and Krüppel in the
zinc finger domains was 84%, 71%, 47%, and 47%, respectively. The
homology with other hematopoietic Krüppel-factors, such
as EKLF8 and lung Krüppel-like factor
(LKLF),30 was less than 30%. The N-terminal regions of
OCZF had homology with the sequence of the so-called pox virus and zinc
finger (POZ) domain seen in the N-terminal end of several proteins (Fig
4B). The highest homology was found in the N-terminal region of APM-1
and hcKrox protein. This region of OCZF did have homology with the POZ
domain of other zinc finger-transcription proteins, including the human
c-myc-interacting Zn finger protein-1 (Miz-1)31 and
promyelocytic leukemia zinc finger (PLZF; Fig 4B).32 The
N-terminal regions also had homology with the regions of
BCL-633 involved in chromosome translocation in
B-cell lymphoma, Drosophila kelch protein involved in nurse
cell-oocyte interaction,34 as well as viral proteins (for
example, VA55R) of the pox virus family (data not shown).35
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| Fig 4.
Comparison of amino acid sequence of OCZF to other zinc
finger proteins. (A) Homology of zinc finger domains of OCZF to those
of other Krüppel zinc finger proteins. The cysteines and
histidines of the zinc fingers are boxed. The homology with APM-1,
c-Krox, Egr-1, and Krüppel in the zinc finger
domains is shown on the right. The sequence of c-Krox is the same as
that of hcKrox. (B) Homology of the N-terminal region of OCZF to those
of other POZ domain proteins. The homology with APM-1, hcKrox, Miz-1,
and PLZF in these regions is shown on the right. Identical residues are
indicated with dashes.
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The N-terminal POZ-like region of OCZF was followed by a proline-rich
sequence that has been observed in the activator domains of other
transcription factors.8 In the C-terminus, the consensus sequence (KKDGCNGVPSRRGRKPR) for the nuclear targeting sequence was
found.36 This structural homology and characteristics
suggest that OCZF functions as a transcription factor that regulates differentiation.
Expression of OCZF mRNA.
To investigate the tissue specificity of the expression of
OCZF, we prepared poly(A)RNA from various rat tissues and cell lines and performed an RNA blot analysis. OCZF mRNA of 2 different sizes (5.3 and 3.3 kb) were detected. The length of the
smaller band (3.3 kb) was almost same as that of the OCZF ORF,
whereas the signal of larger bands (5.3 kb) was higher than that of the smaller band (3.3 kb). The highest level of OCZF mRNA was
detected in bone marrow cells, including MNCs that were stimulated with 1,25(OH)2D3 and htROSCM for 4 days and NRK
cells (kidney cells; Fig 5, lanes 6 and
12). OCZF mRNA was not seen in brain, unstimulated bone marrow
cells, L8 (myoblasts), or UMR (osteosarcoma cells) cells (Fig 5, lanes
1, 5, 9, and 11). POCs were induced from nonadherent bone marrow cells
(bone marrow cells depleted with stromal cells by using a Sephadex G-10
column) by treatment with 1,25(OH)2D3 and
htROSCM for 4 days. The OCZF mRNA level of the POCs (Fig 5, lane 15) was much lower than that of the bone marrow cells, including MNCs (Fig 5, lanes 6). Very low levels of the expression of
OCZF mRNA were seen in the testis, liver, spleen, primary
osteoblasts, 3Y1 (fibroblasts), A11 (osteoblastic cells), and P2
(leukemia cells) cells. P2 cells differentiated into macrophages in the presence of 107 mol/L PMA. The expression of
OCZF mRNA was not changed when P2 cells were differentiated
into macrophages in the presence of 107 mol/L PMA
(Fig 5, lanes 13 and 14).
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| Fig 5.
Expression of OCZF mRNA in various tissues and
cell lines. Poly(A)RNA was prepared, and approximately 1 µg of each
sample (indicated at the top of each lane) was separated on an
agarose/formaldehyde gel and transferred to a nitrocellulose filter.
The filter was hybridized at 60°C overnight with an OCZF
cDNA probe and then washed. The same filter was rehybridized with a
human  -actin probe as a control. Arrows indicate the bands of
OCZF and -actin mRNA.
|
|
Detection and localization of OCZF expressed in the cells with MoAb
Kat6.
To confirm that MoAb Kat6 recognizes OCZF expressed in the cells and to
examine the cellular localization of the protein, we transfected human
kidney cells (293T) with an expression vector containing cDNA for OCZF
and then stained with MoAb Kat6. As shown in Fig 6, OCZF
was detected in the nuclei of some of the cells. A mock transfection
(expression vector alone) was also performed and did not show any
nuclear staining in these cells (data not shown).
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| Fig 6.
Expression and detection of OCZF in human kidney cells.
OCZF cDNA was inserted into an expression vector and
transfected human kidney cells (293T) by the calcium phosphate-DNA
coprecipitation method. After 48 hours, the cells were fixed and
stained with MoAb Kat6 and FITC-labeled antimouse IgM. Dense staining
was seen in the nuclei of some cells. (Original magnification × 202.)
|
|
Detection of OCZF binding to Egr-1 and c-Krox consensus sequence.
To examine the DNA-binding activity of OCZF, we performed an EMSA. We
prepared nuclear extracts from 293T cells transfected with pCDL-FLAG
expression vectors (empty or encoding OCZF protein). The nuclear
extracts were then incubated with the probes. FLAG tagged OCZF did bind
the consensus sequences of Egr-1 and c-Krox. A binding complex was
detected in the EMSA using extract from 293T cells transfected with
expression vector pCDL-FLAGc containing OCZF cDNA, but not with
pCDL-FLAGc vector (Fig 7A, lanes 1, 2, 6, and 7). The formation of the complex was inhibited in the presence of a
100-fold molar excess of the unlabeled Egr-1 or c-Krox DNA but not in
the presence of SP-1 DNA, indicating that OCZF specifically recognize
these sequences (Fig 7A, lanes 3, 4, 8, and 9). Supershifting with FLAG
M2 MoAb confirmed that the shifted band contained OCZF protein (Fig 7A,
lanes 5 and 10).
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| Fig 7.
(A) EMSA of nuclear extracts from 293T cells transfected
with FLAG-tagged OCZF cDNA. c-Krox or Egr-1 DNA was
incubated with the nuclear extract from 293T cells transfected with
FLAG-OCZF cDNA (O; lanes 2 through 5 and 7 through 10) or vector cDNA (v; lanes 1 and 6), respectively. *The complex of OCZF and
DNA. Competition experiments were performed in the presence of 100-fold
molar excess of unlabeled c-Krox (lane 3), Egr-1 (lane 8), or
SP1 (lanes 4 and 9) DNA. Binding reactions were also performed after
preincubation of nuclear extracts with anti-FLAG MoAb (lanes 5 and 10).
Thirty micrograms of nuclear extracts was analyzed. (B) Analysis of DNA
binding activity with S-tagged Egr-1 and OCZF proteins. S-protein
pulldown experiments were performed as described in Materials and
Methods. The bound DNA was analyzed by a 10% PAGE. Samples purified on
S-protein beads were prepared from 293T cells transfected with
pCDL-FLAGa (v1; lane 1), pCDL-FLAGc (v2; lanes 3 and 5) or pCDL-FLAG
containing Egr-1 (lane 2) or OCZF (lanes 4 and 6). They were analyzed
for DNA binding activity using Egr-1 (lanes 1 through 4) or c-Krox
(lanes 5 and 6) consensus sequences as probes. Arrows indicate the
bound Egr-1 or c-Krox DNA. Lower bands indicate degraded products of
Egr-1 DNA. Autoradiography was performed for 6 days at  80°C for
OCZF protein or 3 days at room temperature for Egr-1 protein.
|
|
To further confirm that OCZF has the DNA binding activity, we performed
S-protein pulldown experiments using S-tagged OCZF protein produced
from pCDL-FLAGc vector containing OCZF cDNA. In the similar experiment,
S-tagged Eg-1 protein produced from pCD-FLAGa vector containing Egr-1
cDNA specifically bound its consensus sequence (Fig 7B, lane 2). We
found that S-tagged OCZF protein also could specifically bind Egr-1 and
c-Krox DNA (Fig 7B, lanes 4 and 6). In contrast, control samples
prepared from the extracts transfected with empty vectors, pCDL-FLAGa,
and pCDL-FLAGc did not show the band of bound DNA (Fig 7B, lanes 1, 3, and 5). This assay also indicated that the binding affinity of OCZF
with these DNA seems to be weaker than that of Egr-1, because the time of autragiography for OCZF was longer than that for Egr-1. Taken together, these results indicate that OCZF could bind specifically to
the consensus sequences of Egr-1 and c-Krox.
Transcriptional activity of OCZF.
The transcriptional function of OCZF was studied by DNA transfection
experiments with 293 and 293T cells using a reporter vector
pOA-Egr-TK-Luciferase containing 4 copies of the Egr-1 binding element
upstream of a basal TK promoter. When cotransfection of an expression
vector containing Egr-1 cDNA with this reporter plasmid, Egr-1
increased the luciferase activity in 293 cells (data not shown). In
293T cells, the cotransfection of expression vector containing OCZF
cDNA with a reporter vector pOA-Egr-TK-Luciferase induced a decrease in
luciferase activity. The cotransfection of empty expression vector had
no effect on the luciferase activity (Fig
8A). The transcriptional effect was further studied in 293 cells. The
cotransfection of expression vector containing OCZF cDNA with parental
reporter plasmid pOA-TK-Luciferase had no effect on the levels of
luciferase activity. But, cotransfection of expression vector with
pOA-Egr-TK-Luciferase decreased the luciferase activity (Fig 8B). These
results indicate that OCZF can act as a regulator of transcription
through Egr-1 binding elements.
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| Fig 8.
Transcriptional activity of OCZF. (A) 293T cells were
transiently transfected with 1 µg of reporter plasmid
pOAEgr-TKLuciferase, along with 0.25 or 2.5 µg of expression vector
containing OCZF cDNA (OCZF) or empty vector (pME18S). (B) 293 cells
were transiently transfected with 1 µg of reporter plasmid
pOATKLuciferase, or pOA-Egr-TKLuciferase with 0.25 µg expression
vector containing OCZF cDNA. Rennila luciferase expression vector,
p TK-RL (0.25 µg), was used as an internal control for
transfection. Bars represent the mean and SEM of 3 independent
transfections. *P < .05, **P< .01 compared with
control reporter vector.
|
|
Effect of blockage of OCZF mRNA on osteoclast differentiation.
To determine the functional role of OCZF in the osteoclast
differentiation, we added 2 types of OCZF sense and antisense
ODNs (ODN-1 and ODN-2) to bone marrow culture that form MNCs for
different periods. The addition of OCZF antisense ODN-1 for 1 to 5 days and 3 to 5 days partially inhibited the MNC formation
(Fig 9A; experiments no. 1 and 2). To
confirm the finding regarding the inhibitory effect of antisense OCZF
ODN-1, we added OCZF sense, scrambled, and antisense ODN-2,
which have sequences different from that of ODN-1, to 2 types of bone
marrow cultures that form POCs or MNCs. As shown in
Fig 9A (experiment no. 3), the treatment with OCZF antisense but not sense or scramble ODN-2 caused an inhibition of MNC formation. In contrast, the addition of antisense ODN-2 did not inhibit the formation of POCs in the stromal
cell-deprived bone marrow culture (Fig 9B). These data suggest that
OCZF is involved in the fusion process of mononuclear precursors in
osteoclast differentiation. We therefore added the same ODN-2 to
cultures that form macrophage polykaryons. In this culture system,
macrophage polykaryons were formed that were negative for
TRAP-staining. Interestingly, the antisense ODN-2 had no effect on the
formation of the macrophage polykaryons (Fig 9C).
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| Fig 9.
Effects of expressional blockage of OCZF cDNA
with antisense ODN on the formation of MNCs, POCs, and macrophage
polykaryons. (A) Inhibition of MNC formation with antisense ODN but not
sense or scrambled ODN in bone marrow culture. Bone marrow cells were
cultured with 108 mol/L
1,25(OH)2D3 and htROSCM for the formation of
MNCs. After 5 days of culture, TRAP-positive MNCs were counted. (B)
Effect of ODN-2 on the formation of POCs. Nonadherent bone marrow cells
were cultured in the presence of 108 mol/L
1,25(OH)2D3 and htROSCM for the formation of
POCs. After 5 days of culture, TRAP-positive mononuclear cells were
counted. (C) Effect of ODN-2 on the formation of macrophage
polykaryons. Bone marrow cells were cultured with 107
mol/L PMA and 108 mol/L
1,25(OH)2D3. After 4 days of culture,
TRAP-negative MNCs were counted. One micromole per liter of
OCZF antisense (A) or sense (S) ODN-1 ([A] experiments no. 1 and 2) or antisense (A), sense (S), or scrambled (Scr) ODN-2 ([A]
experiment no. 3, [B], and [C]) was added to the culture. Data are
the mean ± SEM of quadruplicate cultures. **P < .01 compared with control.
|
|
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| Fig 10.
Northern analysis of OCZF mRNA from antisense,
sense, and scrambled ODN-2-treated bone marrow cells. Bone marrow
cells were cultured with 108 mol/L
1,25(OH)2D3 and htROSCM for the formation of
MNCs in the presence of 1 µmol/L OCZF ODN-2 for 4 days. RNA
was isolated from the 4-day culture and approximately 1 µg of each
sample was analyzed. Northern analysis was performed as described in
Materials and Method. The same filter was rehybridized with a human
-actin probe as a control. Arrows indicate the 5.3- and 3.3-kb bands
of OCZF and -actin mRNA. Lanes correspond to RNA from scramble (lane
1), sense (lane 2), and antisense (lane 3) ODN-treated cells.
|
|
To confirm the antisense strategy, we next examined the effect of
antisense ODN-2 on OCZF mRNA levels. Poly(A)RNA was isolated from the
treated cells, and we performed a Northern analysis (Fig 10). The level
of OCZF mRNA of the cells treated with antisense ODN-2 was
significantly lower than those treated with sense or scrambled ODN-2.
This result confirmed that antisense ODN-2 successfully blocked the
expression of OCZF mRNA.
| |
DISCUSSION |
In the present study, we provide evidence that OCZF is a possible
transcription factor that regulates the fusion process in osteoclastogenesis. Osteoclasts are highly specialized multinucleated bone-resorbing cells originating from hematopoietic stem cells. The
process of osteoclast differentiation involves an early stage, the
proliferation and differentiation of osteoclast progenitors into
mononuclear preosteoclasts, and a late stage, the fusion of
preosteoclast progenitors into multinucleated osteoclasts, which are
under the control of the bone microenvironment. In the course of
osteoclast differentiation, the late-stage multinucleation step is a
critical step in bone resorption. Although data have accumulated
related to the involvement of cytokines in
osteoclastogenesis,37 the regulatory mechanism of this
important process is poorly understood. Interestingly, our analysis of
the functional role of OCZF with specific antisense ODN indicated that
OCZF is involved in the formation of MNCs but not in that of POCs. In
addition, OCZF antisense ODN did not inhibit the fusion of macrophages,
suggesting that the inhibition of fusion by this ODN was specific for
osteoclast differentiation. It was recently reported that surface
molecules such as E-cadherin and CD98 expressed on osteoclast precursor cells are involved in the fusion process in osteoclast
differentiation.38,39 The signal transduction of such
molecules may lead to the expression of OCZF.
To elucidate the molecular mechanism of osteoclast differentiation, it
is important to know the transcription factors specifically expressed
in osteoclasts. However, there is as yet little information about
nuclear transcription factors specifically expressed in mature
osteoclasts. Jimi et al40 found that NF B-like
transcription factor is expressed in osteoclast-like cells. In
addition, Iotsova et al41 recently reported that mice
lacking both of the transcription factors NF-B1 and NF-B2
developed osteopetrosis. However, NFB is a ubiquitous transcription
factor expressed in various cells. Inoue et al42 recently
reported that the expression of an osteoclast nuclear transcription
factor (NFOC-1) that bound to the human T-cell leukemia virus type
I-long terminal repeat enhancer element was found in osteoclast-like
cells but not in macrophages. The detailed sequence of NFOC-1 is not
known, but, interestingly, NFOC-1 seems not to be Fos, but had
reactivity to anti-JunD antibody.42 In this study, we
demonstrated by in situ hybridization that zinc finger protein OCZF
is preferentially expressed in mature osteoclasts. In addition, the
expression of OCZF mRNA is increased during the differentiation into
multinucleated osteoclast-like cells, but is not increased during
differentiation into macrophages.
OCZF encoded a protein with POZ domain and Krüppel-type 3 zinc fingers. OCZF was considered to be rat homologue of LRF that was
recently reported as a potential target for BCL-6 oncogene. High
conservation between rat OCZF and mouse LRF indicates that this factor
has a very important role. Because it is not known whether BCL-6 is
expressed in osteoclasts, further studies will be required to elucidate
the function of the interaction of BCL-6 and OCZF in
osteoclastogenesis. Five percent to 10% of Krüppel-type zinc finger proteins contain POZ domain in their N-terminus. This domain is known to form homomeric and heteromeric interactions with
other POZ domains43,44 and is required for transcriptional repression of several proteins, including hcKrox, BCL-6, and
PLZF.45-47 Punctuate nuclear staining patterns are commonly
seen in these zinc-finger POZ proteins.48,49 In this study,
we found that OCZF had the repressive activity of transcription like
other POZ domain proteins. Interestingly, OCZF by immunofluorescence
with Kat6 antibody showed the localization of this protein to discrete regions of the nucleus and did not show diffuse staining in osteoclasts (Fig 1).
The amino acid sequence of POZ domain of OCZF is very similar to those
of human APM-1 and hcKrox. APM-1 is recently isolated human protein
involved in human papillomavirus (HPV) integration region.24 APM-1 transcripts were detected in normal
cervical keratinocytes, but not in the cervical carcinoma cell lines.
Expression of APM-1 gene strongly reduced clonal cell growth,
suggesting that APM-1 may function as a tumor-suppressor gene. The
hcKrox gene was recently isolated as c-Krox homologue from a human
fibroblast cDNA library.26 hcKrox has an ability to repress
the transcription of extracellular matrix genes, including 1 type I
procollagen and fibronectin mRNA. The POZ domain of OCZF protein had
also homology with that of Miz-1 and PLZF, both of which were shown to
have potent suppressive effect on cell growth.30,50 It is interesting that POZ domain of OCZF had homology with APM-1, Miz-1, and
PLZF, which had cell growth inhibitory activity.
Recently, POZ domain proteins have been implicated in embryonic
development and in hematopoiesis.32,51 PLZF and BCL-6 have important roles in the terminal differentiation of the cells. Loss of
PLZF function blocks terminal differentiation of hematopoietic precursor cells.52 BCL-6 is expressed in many types of the
cells but is expressed at a high level in muscle cells. During the
differentiation of proliferating myoblasts into myotubes, BCL-6
expression is upregulated. BCL-6 was considered to be involved in
muscle terminal differentiation.53 In terminal
differentiation, the cells stop DNA synthesis. The terminal
differentiation step into osteoclasts, the fusion process is not
required for DNA synthesis.54,55 OCZF may be
involved in the regulation of cell growth or cell cycle, which is
possibly involved in the fusion process into osteoclasts.
The zinc finger region of OCZF had high homology with APM-1 and hcKrox.
However, the detailed binding DNA sequences for these factors have been
not shown. Because the zinc finger region of OCZF also had homology
with c-Krox and Egr-1, we have analyzed the ability of DNA binding of
OCZF to Egr-1 and c-Krox binding sequence. We have found that OCZF
bound these sequences specifically. In this study, we also found that
OCZF repressed the transcriptional activity through the Egr-1 binding
site. These data suggest that some genes that are expressed in
osteoclasts and have the Egr-1 binding sequence in the regulatory
region may be regulated by OCZF. However, OCZF may bind similarly to
another target sequence with high affinity, because the
binding affinity of OCZF for Egr-1 binding sequence seems to be lower
compared with that of Egr-1. To determine high-affinity binding
sequences and elucidate target genes of OCZF, further studies will be required.
The developmental process of hematopoietic cells is controlled by the
combination of several genes. The osteopetrotic phenotype of NF-B1
and NF-B2 double-knockout mouse model resembles that of
c-fos-deficient animals; however, the induction of c-fos expression was detected in this mutant mouse. Because these 2 transcription factors expressed in wide types of the cells, these
results suggest that some other common transcription factor expressed
specifically in the cells of osteoclast lineages may interact with
these factors to restrict their functions. In fact, some transcription
factors are known to self-associate to form dimmers44 or
interact with other transcription factors and regulate the
differentiation in a combined fashion.51,56 Thus, the
temporal expression of a set of several transcription factors is
thought to be required for the ongoing process of differentiation into
osteoclasts. The POZ domain of several transcription factors is a
region interacting with other proteins, suggesting that POZ domain
proteins have some role in the osteoclastogenesis. At present, the
molecular mechanism of the involvement of OCZF in the multinucleation
step in osteoclast differentiation is not entirely clear. Future
studies will clarify the possible functions of the OCZF protein in the regulation of differentiation and transcription in osteoclast-specific lineage cells.
| |
ACKNOWLEDGMENT |
The authors thank Dr T. Watanabe (Kyushu University Medical Institute
of Bioregulation) and Dr H. Kishi (Toyama University) for helpful
suggestions regarding cDNA cloning and Dr S. Matsuhashi (Saga Medical
School) for helpful suggestions regarding immunocytochemical study. We
also thank Dr M. Kobayashi (Hokkaido University) for donating the WRT-7
P2 cell line. We also thank Dr K. Hori (Vice-President, Saga Medical School) for encouragement and Dr Lin Xin Xu for assistance.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted May 24, 1999.
Supported in part by a Grant for Scientific Research from the Japanese
Ministry of Education, Science and Culture (Project No. 05671543).
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 Akiko Kukita, PhD, Department of
Microbiology, Saga Medical School, 5-1-1, Nabeshima, Saga 849-8501, Japan; e-mail: kukita{at}smsnet.saga-med.ac.jp.
| |
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