Endocrinology Vol. 138, No. 10 4384-4389
Copyright © 1997 by The Endocrine Society
Regulation of Osteoclastogenesis by Antisense Oligodeoxynucleotides Specific to Zinc Finger Nuclear Transcription Factors Egr-1 and WT1 in Rat Bone Marrow Culture System1
Toshio Kukita,
Akiko Kukita,
Hiroshi Harada and
Tadahiko Iijima
Second Department of Oral Anatomy, Faculty of Dentistry (T.K.,
T.I.), and the Department of Orthopedics, Faculty of Medicine (H.H.),
Kyushu University, 3–1-1 Maidashi, Fukuoka 812; and the Department of
Microbiology, Saga Medical School (A.K.), 5–1-1 Nabeshima, Saga 849,
Japan
Address all correspondence and requests for reprints to: Toshio Kukita, Ph.D., Second Department of Oral Anatomy, Faculty of Dentistry, Kyushu University, 3–1-1 Maidashi, Fukuoka 812, Japan.
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Abstract
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Differentiation of osteoclasts is defined by the transcription factors
expressed in response to bone microenvironments. In this work, we
examined the effects of an expressional blockage of Egr-1 and/or WT1 on
the differentiation of osteoclasts using specific antisense
oligodeoxynucleotides (ODN). In a culture system forming
preosteoclast-like cells (POC) from rat bone marrow cells depleted of
marrow stromal cells, POC formation was markedly stimulated by the
addition of Egr-1 antisense ODN compared to that in cultures in which
sense ODN was added, whereas Egr-1 antisense ODN inhibited the
formation of macrophage-like cells. The formation of multinucleated
osteoclast-like cells was also stimulated by the addition of Egr-1
antisense ODN in whole bone marrow cultures. In contrast, WT1 antisense
ODN did not affect POC formation induced by the treatment with Egr-1
antisense ODN; however, WT1 antisense ODN dramatically suppressed the
formation of osteoclast-like multinucleated cells induced by the
blockage of Egr-1 expression using Egr-1 antisense ODN. These data
suggest that Egr-1 acts as the suppressor, not as the inducer, in
osteoclastogenesis. The findings also suggested that WT1 could be
involved in the multinucleation step of osteoclastogenesis, at least
when Egr-1 expression was blocked.
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Introduction
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A TRANSCRIPTION factor designated the early
growth response gene-1 (Egr-1) with three zinc-finger motifs is one of
the EGR family that has zinc finger motifs at the C-terminal region of
the protein. It was originally discovered as a growth response gene in
cultured cells and in response to B cell maturation (1). However,
Nguyen et al. (2) demonstrated that the expression of Egr-1
is essential for the differentiation of macrophages; they reported a
series of findings revealing that Egr-1 has a critical role in the
differentiation of earlier progenitor cells to macrophages. Egr-1 binds
the G-rich consensus sequence (5'-GCGGGGGCG-3') of the control region
of the target genes followed by activation of the target genes (3, 4, 5).
WT1, also a member of the EGR family, has four zinc finger motifs, and
was discovered as a gene product of the tumor suppressor gene in
Wilms’ kidney tumors; it binds to the same consensus sequence as Egr-1
(6, 7, 8) and acts as the transcriptional repressor against target genes,
whereas Egr-1 acts as the transcriptional activator (9, 10, 11).
The cell lineage of the osteoclasts is considered to be very close to
that of the macrophages (12). It has been reported that mononuclear
phagocytes can be differentiated into osteoclasts when these cells are
cocultured with bone rudiments stripped off the osteum (13). The
formation of osteoclasts from alveolar macrophages has also been
reported (14). We recently found that a macrophage cell line, BDM-1,
has an ability to differentiate into osteoclast-like cells (15).
Further experiments using the osteopetrotic op/op mice
demonstrated that macrophage colony-stimulating factor (M-CSF) is an
essential cytokine for osteoclastogenesis (16, 17, 18, 19). Thus, it is widely
accepted that colony-forming unit macrophages are the most probable
candidates for the progenitor cells of the osteoclasts. As cells in the
osteoclast lineage are closely related to the monocyte/macrophages,
differentiation of osteoclasts may be regulated by transcription
factors common to those in macrophages.
In the present study, we studied the possible regulatory roles of the
zinc finger transcription factors Egr-1 and WT1 in osteoclastogenesis
by creating an expressional blockage of these transcription factors by
antisense oligodeoxynucleo-tides (ODNs) specific to each gene.
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Materials and Methods
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Materials
Male Sprague-Dawley rats (4–6 weeks old) were obtained from
SEAC Yoshitomi (Fukuoka, Japan). Cytochemical staining kits for
tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase
were purchased from Sigma Chemical Co. (St. Louis, MO). The ABC-AP kit
was obtained from Vector Laboratories (Burlingame, CA). Unmodified or
phosphorothioated ODNs of designed sequences were prepared and purified
with HPLC by Takara Biomedicals (Kyoto, Japan).
Bone marrow cultures
Rat bone marrow cells were obtained from 4- to 6-week-old rats
and cultured under the conditions described below, followed by staining
with the cytochemical staining kit for TRAP or with the
osteoclast-specific monoclonal antibody Kat1 (mAb Kat1) as described
previously (20). The presence of macrophages was also monitored by
staining for nonspecific esterase.
Stromal cell-deprived bone marrow cultures for forming
preosteoclasts.
Bone marrow cells were deprived of stromal cells
by use of a Sephadex G-10 column. Cells (4 x 105; in
100 µl 
MEM containing 15% FCS) were seeded into 96-well culture
plates, or 106 cells (in 500 µl MEM containing 15%
FCS) were seeded into 24-well culture plates in the presence of 10%
heat-treated conditioned medium with a molecular mass of more than 10
kDa (htROSCM) and 10-8 M
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] for 5 days, as described by
Kukita et al. (21), with or without ODNs.
Whole bone marrow cultures for forming osteoclast-like
multinucleated cells (MNCs) with high bone-resorbing activity.
Bone marrow cells were cultured in 24-well culture plates
(106 cells/well) in the presence of 10% htROSCM and
10-8 M 1,25-(OH)2D3
for 5 days, as described by Kukita et al. (22), in the
presence of various ODNs with a half-volume medium change on day 3 of
culture.
Design of antisense ODNs
Antisense ODN, sense ODN, and scrambled ODNs were designed and
prepared based on the DNA sequence database (GenBank). The optimum
sequence of 20 bases for generating specific ODNs was searched by
analyzing the 5'-upstream regions of the open reading frames of each
gene. In the case of Egr-1, the most specific regions for mouse Egr-1
(GenBank accession no. M20157) and rat Egr-1 (GenBank accession no.
M18416) were selected, and these regions exactly correspond to the
sequence of antisense ODN reported by Nguyen et al. (2).
Sequence 141–160 of mouse Egr-1 is identical to sequence 151–170 of
rat Egr-1, and this portion was selected for generating the Egr-1
antisense ODN. Scrambled sequences (Scr 1 and Scr
2) with the same base ratio as either the sense ODN or the
antisense ODN were confirmed to have no homology to any sequences
reported to date in the GenBank DNA database. For the WT1 antisense
ODN, two different portions of the sequence were selected after
analyzing the 5'-upstream region of the second open reading frame of
mouse WT1 (GenBank accession no. M55512) and the initial 300 bases of
this open reading frame. Sequence 441–460 of mouse WT1 was selected
because this region is highly specific to mouse WT1. Sequence 469–488
of mouse WT1 was also selected. This region is highly specific to mouse
WT1 and rat WT1. This sequence is identical to sequence 7–26 of rat
WT1 (GenBank accession no. X69716). These two regions were used for
generating the WT1 antisense ODNs. Scrambled sequences with the same
base ratio as that of the WT1 antisense ODNs were designed for each
portion. These scrambled sequences were confirmed to have no homology
to any sequences reported in the GenBank DNA database.
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Results
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Induction of preosteoclast-like cells and osteoclast-like MNCs by
treatment with Egr-1 antisense ODN
The 5'-upstream region of the Egr-1 open reading frame is highly
conserved between mouse and rat. The mouse 141–160 sequence is
completely conserved in rat 151–170, as shown in Fig. 1
. This mouse
sequence was used by Nguyen et al. (2) to generate antisense
ODNs that inhibit macrophage differentiation. Figure 2 shows the effect of Egr-1 antisense ODN
on the formation of POC from bone marrow cells deprived of stromal
cells and that of macrophage-like cells. Egr-1 antisense ODN
dramatically suppressed the formation of macrophage-like cells from
bone marrow cells (Figs. 2, lower panel, and
3); however, it markedly stimulated the
formation of POC (Figs. 2, upper panel, and 3). The
inhibition of macrophage formation by Egr-1 antisense ODN was also
confirmed by staining with a nonspecific esterase kit (data not
shown).
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Figure 1. Design of antisense ODNs. The outlines of the
messenger RNA structure are shown for mouse Egr-1 (gb:M20157), rat
Egr-1 (gb:M18416), mouse WT1 (gb:M55512), and rat WT1 (gb:X69716). The
sequences of all prepared ODNs are also shown. For the expressional
blockage of Egr-1, antisense ODN was prepared with the same sequence as
that reported by Nguyen et al. (2). They showed complete
inhibition of macrophage formation using this antisense ODN (141–160
of mouse Egr-1; AS Egr-1). This region corresponds to
sequence 151–170 of rat Egr-1. For control experiments, the sense ODN
(SEgr-1) with a sequence complementary to that of
antisense ODN and the scrambled ODN with the same base ratio as sense
ODN (Scr 1 Egr-1) or as antisense ODN (Scr
2 Egr-1) were used. For the expressional blockage of WT1, the
specific region of mouse WT1 was selected as described in
Materials and Methods. Two portions (441–460 and
469–488) were selected. The latter portion (mouse 469–488)
corresponds to sequence 7–26 of rat WT1. For control experiments, the
sense ODN (S WT1), with a sequence complementary to that
of antisense ODN, and the scrambled ODN (Scr WT1), with
the same base ratio as antisense ODN, were prepared. ORF, Open reading
frame.
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Figure 2. Egr-1 antisense ODN markedly stimulates the
formation of POC. Bone marrow cells deprived of stromal cells were
cultured for forming POC, as described in Materials and
Methods, in the presence of 4 µM Egr-1 sense OND
(S Egr-1) or antisense ODN (AS Egr-1)
followed by staining for TRAP. The numbers of TRAP-positive mononuclear
cells (POC; upper panel) and TRAP-negative adherent
cells (macrophages: lower panel) were counted from
quadruplicate cultures. Data represent the means (±SE) of
quadruplicate cultures. Data were analyzed by Student’s
t test. ***, P < 0.001; **,
P < 0.01 (compared with the sense control).
Results are representative data from three independent experiments.
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We examined whether antisense Egr-1 affects the formation of
osteoclast-like MNCs in whole bone marrow cultures induced with
1,25-(OH)2D3 and htROSCM, specific inducers
of osteoclast-like MNCs in rat bone marrow culture system (21, 22).
Egr-1 antisense ODN markedly stimulated the formation of TRAP-positive
MNCs compared to that in controls in which Egr-1 sense or scrambled ODN
was added to bone marrow cultures for forming osteoclast-like MNCs, as
shown in Fig. 4. We confirmed that
osteoclast-like MNCs induced by the presence of Egr-1 antisense ODN
have the ability to efficiently resorb dentin. We further confirmed
that these MNCs expressed the calcitonin receptor and the Kat1 antigen,
a specific cell surface marker for cells in the osteoclast lineage of
rats (data not shown). These data show that treatment of bone marrow
cultures with Egr-1 antisense ODN resulted in a marked increase in the
number of cells of osteoclast lineage. These data strongly suggest the
presence of another transcription factor that is required for
osteoclastogenesis when Egr-1 expression is blocked by Egr-1 antisense
ODN.
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Figure 4. Effect of Egr-1 antisense ODN on the formation of
osteoclast-like MNCs. Bone marrow cells were cultured in 24-well
culture plates for forming osteoclast-like MNCs, in the presence of 0.4
µM phos-phorothioated Egr-1 sense ODN
(S Egr-1), scrambled ODN (Scr 1, Scr 2
Egr-1), or antisense ODN (AS Egr-1) for 5 days followed
by staining for TRAP. Data are the means (±SE) of
quadruplicate cultures and were analyzed by Student’s t
test. **, P < 0.01 compared with the sense
control. Results are representative data from three independent
cultures.
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Regulation of osteoclast-like MNC formation by Egr-1 antisense ODN
and by WT1 antisense ODN
We next examined the possibility that WT1 is involved in the
stimulation of osteoclastogenesis induced by treatment with Egr-1
antisense ODN. Antisense ODNs that specifically bind WT1 messenger RNA
were designed, as shown in Fig. 1
. Two portions were selected for
generating antisense ODNs. One is the 5'-region upstream from the
initiation codon, and the other is the region including the initiation
codon of the second open reading frame of the mouse WT1 gene. Antisense
ODN, sense ODN, and scrambled ODN were prepared, and the effects of
these ODNs on the formation of osteoclast-like MNC were examined. As
shown in Fig. 5, WT1 antisense
ODN-(441–460) only slightly suppressed the formation of TRAP-positive
MNCs; however, WT1 antisense ODN-(469–488) did not affect their
formation. The stimulatory effect of osteoclast-like MNC formation
induced by Egr-1 antisense ODN was completely blocked by the addition
of WT1 antisense ODN, as shown in Fig. 6.
Both WT1 antisense ODNs (441–460 and 469–488) clearly suppressed the
formation of osteoclast-like MNCs induced with Egr-1 antisense ODN,
whereas neither WT1 sense nor WT1 scrambled ODN caused any significant
effect. Furthermore, this inhibitory effect of WT1 antisense ODN was
not accompanied by any suppressive effect on the formation of
TRAP-positive mononuclear cells, as shown in Fig. 7. We further confirmed this using
stromal cell-deprived bone marrow cells for forming POC. POC formation
induced by the addition of AS Egr-1 ODN was not suppressed
by the addition of AS WT1 ODN (Table 1). These data suggest that the POC
formation step is not under the control of WT1 and that the formation
of MNCs induced by the expressional blockage of Egr-1 was completely
suppressed by the expressional blockage of WT1 without altering the
formation of mononuclear precursors of the osteoclasts.
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Figure 5. Effect of antisense WT1 ODN on the formation of
osteoclast-like MNCs. Bone marrow cells were cultured in 24-well
culture plates for forming osteoclast-like MNCs in the presence of 0.4
µM phosphorothioated WT1 sense ODN (S
WT1), scrambled ODN (ScrWT1), or antisense WT1
(AS WT1) of two different portions of the WT1 gene
(441–460, left panel; 469–488, right
panel) for 5 days followed by staining for TRAP. Data are the
means (±SE) of quadruplicate cultures and were analyzed by
Student’s t test. *, P < 0.05
compared with the sense control. Results are representative data from
four independent experiments.
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Figure 6. Antisense WT1 suppresses the stimulatory effect of
antisense Egr-1 on the formation of osteoclast-like MNCs. Bone marrow
cells were cultured in 24-well culture plates for forming
osteoclast-like MNCs for 5 days in the presence of 0.4 µM
phosphorothioated sense Egr-1 (S Egr-1) or antisense
Egr-1 (AS Egr-1) with or without 0.4 µM
phosphorothioated sense WT1 (S WT1), scrambled WT1
(Scr WT1), or antisense WT1 (AS WT1) of
sequence 441–460 (left panel) or 469–488 (right
panel). Cultures were stained for TRAP, and the number of
TRAP-positive MNCs was counted. Data are the means (±SE)
of quadruplicate cultures and were analyzed by Student’s
t test. **, P < 0.02 compared with
the positive control using AS Egr-1.
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Figure 7. Antisense WT1 does not affect the formation of
preosteoclast-like cells induced by antisense Egr-1. Bone marrow cells
were cultured in 24-well culture plates for forming osteoclast-like
MNCs for 5 days in the presence of 0.4 µM
phosphorothioated sense Egr-1 (S Egr-1) or antisense
Egr-1 (AS Egr-1) with or without 0.4 µM
phosphorothioated sense WT1 (S WT1), scrambled WT1
(Scr WT1), or antisense WT1 (AS WT1).
Forty fields were randomly selected from quadruplicate cultures, and
the number of TRAP-positive mononuclear cells per unit area (2.60
mm2) was counted.
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Table 1. Absence of the inhibitory effect of antisense WT1
ODN on the formation of POC induced by antisense Egr-1 ODN
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Discussion
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It is quite true that osteoclasts and macrophages share many
characteristics; these two cell lineages originate in common progenitor
cells. We, therefore, examined whether osteoclast differentiation would
be affected when macrophage differentiation was blocked by suppressing
the expression of Egr-1, a zinc finger-type transcription factor able
to differentiate early progenitor cells into macrophages. Our findings
were contrary to our expectation that osteoclast differentiation would
also be inhibited by the expressional blockage of Egr-1. The formation
of mononuclear preosteoclast-like cells and multinucleated
osteoclast-like MNCs was markedly stimulated by the addition of Egr-1
antisense ODN compared to sense control as well as scrambled control,
as summarized in Fig. 8. Our data
strongly suggest that Egr-1 acts as a suppressor for osteoclastogenesis
and that there are other transcription factors required for
osteoclastogenesis, at least when Egr-1 expression is blocked. One
candidate for the transcription factor with the ability to antagonize
Egr-1 function is WT1, the product of the Wilms’ tumor suppressor
gene, which is a member of the EGR family with four C2H2-type zinc
finger motifs (6, 7, 8, 9, 10, 11). As we demonstrated in this study, WT1 antisense
ODN completely suppressed the formation of osteoclast-like MNCs induced
by an expressional blockage of Egr-1 as summarized in Fig. 8. Our data
suggest that WT1 has some role in the multinucleation step in
osteoclastogenesis when bone marrow cultures were treated with Egr-1
antisense ODN. WT1 is supposed to bind consensus sequence of Egr-1 in
place of Egr-1 itself when the expression of Egr-1 is blocked, thereby
this tumor suppressor gene product could express its unique biological
activity in osteoclastogenesis. This multinucleation step is one of the
most important steps in osteoclast differentiation; however, little is
known about the molecular mechanism of this step. The expression of key
molecules essential for the specific fusion process among osteoclast
precursors may be under the control of WT1. Interestingly, the
preosteoclast formation induced by Egr-1 antisense ODN in whole bone
marrow cultures was not affected by the expressional blockage of WT1,
suggesting that WT1 is not involved in regulation of the mononuclear
precursor formation. There may be some other strong inducer of
osteoclastogenesis in the formation of preosteoclasts from early
progenitors.
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Figure 8. Regulation of osteoclastogenesis by antisense ODN
specific to Egr-1 and WT1. Although antisense Egr-1 ODN
(AS Egr-1) inhibited macrophage formation, this ODN
markedly stimulated the formation of cells in the osteoclast lineage.
In contrast, antisense WT1 ODN (AS WT1) suppressed
osteoclastogenesis induced by a treatment with AS Egr-1
in the multinucleation step. Solid arrow, Stimulation;
broken arrow, inhibition.
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Grigoriadis et al. (23) reported that c-fos,
known as a transcription factor that binds the AP1 site after being
heteromerized with the partner protein of the Jun family, is
essential for osteoclastogenesis, as determined after detailed analysis
of c-fos gene-deficient mice generated by the homologous
recombination of the c-fos allele in embryonic stem cells
(24). Homozygous mutants of the c-fos allele have an
osteopetrotic phenotype due to a failure in normal osteoclast
formation. The lack of c-fos caused a lineage shift between
osteoclasts and macrophages resulting in the formation of increased
numbers of bone marrow macrophages, suggesting that c-fos is
essential for osteoclast differentiation, but not for macrophage
differentiation. In our culture system, the reason why the formation of
preosteoclasts induced by Egr-1 antisense ODN was not suppressed by WT1
antisense ODN may have been a high expression of c-fos in
this step. Other osteopetrotic mutant mice (mi/mi) have been
shown to have an abnormality in the helix-loop-helix-type transcription
factor (25). Mi/mi mice express an abnormality in mast cell
differentiation as well as osteoclast differentiation. Their study (25)
suggests an important role of the helix-loop-helix-type transcription
factor in osteoclastogenesis. As is shown in the differentiation of
other hematopoietic cells, there could be some hierarchy in the
expression of transcription factors in the differentiation of
osteoclasts. Further detailed analyses are required concerning the
time-course expression of transcription factors involved in
osteoclastogenesis in our in vitro culture system of
osteoclast differentiation.
M-CSF has been shown to be an essential cytokine for osteoclast
differentiation from a series of studies using osteopetrotic
(op/op) mice. Harrington et al. (26) reported
that the expression of M-CSF is induced by Egr-1 and suppressed by WT1.
They further demonstrated binding of SP1 and SP3 to the consensus
sequence of Egr-1 present at the upstream region of the M-CSF gene. To
give a simple interpretation, the expressional blockage of Egr-1 would
result in a reduced production of M-CSF, and that of WT1 would result
in an increased production of M-CSF. As the M-CSF is an essential
cytokine for osteoclastogenesis, a change in the level of M-CSF should
affect the formation of osteoclast-like MNCs. Additional studies are
required concerning the minute changes in the level of M-CSF in our
culture system.
In the present study, we obtained findings suggesting the role of WT1
in the multinucleation step in osteoclastogenesis. With respect to the
biological role of WT1, Kreidberg et al. (27) reported that
this zinc finger transcription factor has a crucial role in early
urogenital development. In their report, WT1-deficient mice die on
embryonic day 15 because of the abnormality of kidney development. As
these mutant animals die so early, the role of WT1 in skeletal tissue
remains unknown. The WT1 gene has 10 exons that give rise to several
protein isoforms through alternative splicing (28). In our present
study, WT1 antisense ODN was generated for the 5'-upstream region of
the initiation codon and for a region around the initiation codon of
the second open reading frame; thereby, the expression of every type of
splicing variant can be affected by treatment with this antisense ODN.
It will be interesting to determine which type of splicing variant is
used in osteoclastogenesis.
In this study, we successfully demonstrated the clear effect of
antisense ODNs on osteoclastogenesis in a rat bone marrow culture
system. These in vitro studies can be expanded to in
vivo studies. As the antisense ODN is not a stable reagent even as
a phosphorothioated ODN, this aspect provides an opportunity for
creating safer drugs that regulate bone metabolism by a specific
blocking of osteoclast differentiation but do not affect the
differentiation of macrophages, which play central roles in the
immunological defense of the body. In vivo studies examining
the direct regulation of osteoclastogenesis are underway in our
laboratories.
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Figure 3. Enhancement of POC formation by the addition of
Egr-1 antisense ODN. Bone marrow cells deprived of stromal cells were
cultured in the presence of Egr-1 sense ODN (A) or antisense ODN (B)
for 5 days, followed by staining for TRAP. Arrowheads,
TRAP-positive preosteoclast-like cells. Arrows,
TRAP-negative macrophage-like cells. Magnification, x125.
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Footnotes
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1 This work was supported in part by a Grant for Scientific Research
from the Japanese Ministry of Education, Science and Culture (Project
08672075). 
Received April 14, 1997.
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Abstract
Introduction
