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(Received for publication, July 17, 1995; and in revised form, January 2, 1996) From the
We investigated the NF-
Osteoclasts play a crucial role in the resorption of bone
physiologically and pathologically. Extraordinary induction and
activation of osteoclasts results in excess bone resorption, which
overtakes the rate of bone formation, which in turn gives rise to the
pathological loss of bone such as in osteoporosis and periodontitis.
Thus, it is important to investigate the biochemical events occurring
in osteoclasts in order to understand the pathology of bone
destruction. It had been difficult to study osteoclasts because it
is very difficult to culture and amplify enough primary osteoclasts for
biochemical studies. Recently, a method where osteoclast-like cells
were induced from cultured bone marrow cells under a certain set of
conditions was developed(1, 2, 3) . The
resulting osteoclast-like cells have a similar phenotype to
osteoclasts, including their morphology as giant and multinucleated
cells (MNC), ( NF- In this study, we
investigated NF-
Mouse bone marrow cells were co-cultured with the primary
osteoblasts, which were derived from mouse calvaria, in the presence of
10
Figure 1:
Microscopic view of the enriched
osteoclast-like cell preparation. Mouse bone marrow cells were
co-cultured with mouse primary osteoblasts in the presence of
1
We treated these osteoclast-like cells with 10 ng/ml
IL-1
Figure 2:
A, NF-
PMA is a well
known potent NF- As for the origin of
NF-
Figure 3:
Comparison of the mobility of DNA binding
complexes in primary osteoblasts, crude OCL preparation, and purified
OCL preparation. Primary osteoblasts (POB, lanes 1 and 2), crude osteoclast-like cells (Crude OCL, lane 3), and purified OCL preparation (OCL, lanes
4 and 5) were treated for 30 min with (lanes
2-4) or without (lanes 1 and 4) IL-1 (10
ng/ml). The co-culture of primary osteoblasts and osteoclast-like cells
(the number of osteoblasts was 100 times higher than the number of
osteoclast-like cells) was also treated with IL-1 for 30 min. Nuclear
extracts were prepared from each cell population, and 10 µg of
protein from each nuclear extract was assayed for NF-
Active NF-
Figure 4:
Subunit composition of the NF-
This study indicates that IL-1 can induce an NF- There have been few studies on transcription factors in
osteoclasts. In situ hybridization showed the levels of
NF-IL-6 expression in osteoclasts of patients with osteoarthritic and
Paget's bone(22) . Roodman et al.(23) reported that IL-6 might be an autocrine/paracrine
factor that regulates osteoclastic bone resorption, and NF- Whether or not osteoclasts can
respond to IL-1 directly is still controversial (i.e. do
osteoclasts express IL-1 receptors or not?), even though IL-1 is the
most potent osteoclast-activating factor which promotes bone
resorption. We previously demonstrated that IL-1 can increase the
survival rate of osteoclast-like cells, as osteoclast-like cells could
adhere to plastic dishes for longer periods in the presence of
IL-1(31) . When measuring survival rates, osteoclast-like cells
must be cultured in the presence of IL-1 for 24 h. Culturing for 24 h
may be long enough for any contaminating cells, even if they are
present in very small numbers, to synthesize and release unknown
factor(s), which in turn could act on osteoclasts. In contrast, the
NF- The
osteoclast-like cells seem to be differentiated from mononuclear
phagocytes in bone marrow, in the presence of osteoblasts(32) .
Recently, Grigoriadis et al. reported that c-Fos is a key
regulator in osteoclast-macrophage lineage determination(27) .
Therefore, it will be interesting to see if differences in NF-
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4605-4608
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activates an NF-
B-like Factor in Osteoclast-like Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B transcription factor in
osteoclast-like cells. Osteoclast-like cells were differentiated from
mouse bone marrow cells in co-culture with mouse calvaria-derived
primary osteoblasts in the presence of 1
,25-dihydroxyvitamin
D
and prostaglandin E
in collagen gel-coated
dishes. We enriched osteoclast-like cells from the co-cultures by
Pronase treatment. When the enriched osteoclast-like cells were treated
with phorbol 12-myristate 13-acetate, interleukin-1 (IL-1), calcitonin,
or macrophage colony-stimulating factor, only IL-1 activated an
NF-B-like factor, which specifically bound to a B motif DNA
sequence, as detected by an electrophoretic mobility shift assay. IL-1
also activated NF-B induction in osteoblasts. However, the
NF-B-like factor induced by IL-1-stimulated osteoclast-like cells
is of smaller molecular size than the factor in osteoblasts, as shown
by an electrophoretic mobility shift assay. The NF-B activity of
osteoclast-like cells was recognized completely by antibodies against
the p50 subunit, and only partially by antibodies against the p65
subunit of NF-B. Antibodies against c-Rel, Rel B, and p52 did not
recognize the NF-B-like factor. These results suggest that IL-1
activates an NF-B-like factor in osteoclast-like cells, which
contains p50 and p65-related proteins.
)their expression of tartrate-resistant acid
phosphatase (TRAP) and calcitonin (CT) receptors, and their ability to
resorb dentine slices(1, 2, 3) . Therefore,
the use of these induced osteoclast-like cells enables scientists to
examine osteoclasts biochemically. Because of this, information about
the intracellular events occurring in osteoclasts has accumulated
rapidly, but little information on the intracellular signaling
molecules of osteoclasts, especially transcription factors, exists. B is a ubiquitous transcription factor which can activate a
number of genes including the IL-6, tumor necrosis factor-, major
histocompatibility complex class I, and adhesion molecule
genes(4) . The target genes of NF-B are involved in
inflammatory reactions where bone is degraded. Conventional NF-B
is a heterodimer composed of a p50 and a p65 subunit, both of which
show a high degree of amino acid sequence homology to the Rel family of
proteins, which includes c-Rel, Rel B, and
p52(5, 6, 7) . Latent NF-B, which
complexes with an inhibitor IB, resides in the
cytoplasm(8) . After stimulation, NF-B is released from
IB, and translocates into the nucleus to bind to a specific DNA
sequence. NF-B seems to be one of the central intracellular
signals for interleukin-1 (IL-1), which is one of the most potent
osteoclast activating factors(9) . Many genes activated by IL-1
are the target genes of
NF-B(4, 10, 11) .B activity in osteoclast-like cells treated with
IL-1. The NF-B-like factor of osteoclast-like cells appears to be
different from the NF-B in osteoblasts. Our results suggest that
osteoclasts possess an NF-B-like factor, which can be induced by
IL-1.
Culture of Osteoclast-like Cells
Bone marrow
cells were obtained from the tibiae and femora of 6-8-week-old
male ddY mice (Seiwa Experimental Animal, Fukuoka, Japan) as reported
previously (12) . Primary osteoblasts were prepared from the
calvaria of newborn ddY mice(13) . Bone marrow cells (5 
10
cells) were co-cultured with osteoblasts (1
10 cells) in -minimal essential medium (Life
Technologies, Inc.) containing 10% fetal bovine serum,
1,25-dihydroxyvitamin D (1,25(OH) D) (10M), and
prostaglandin E
(PGE) (10M) in 100 mm-diameter dishes coated with collagen gel
(Nitta Gelatin Co, Osaka, Japan) according to the method described by
Akatsu et al.(13) . Seven days after inoculation,
osteoclast-like cell formations had reached a maximum and all adherent
cells were detached from the dishes by treatment with 0.2% collagenase
(Wako Pure Chemical Co., Osaka, Japan). The cell suspension from each
dish was inoculated again into a 100-mm diameter dish. Ten hours later
the adherent cells were treated with phosphate-buffered saline (PBS)
containing 0.001% Pronase E (Sigma) and 0.02% EDTA for 10 min at
37° C to remove the osteoblasts and other bone marrow
cells(14) . More than 95% of the adherent cells remaining,
which were resistant to Pronase E, were TRAP-positive MNCs and
mononuclear cells(15) .
Preparation of Nuclear Extracts
Cell extracts were
prepared according to methods described by Dignam et al.(16) and Osborn et al.(17) . For samples
of enriched osteoclast-like cells, cells were scraped off each dish and
put in ice-cold PBS. The cells were washed in ice-cold PBS and then in
ice-cold hypotonic buffer (buffer A: 10 mM HEPES, pH 7.9, 1.5
mM MgCl, 10 mM KCl, 0.5 mM PMSF,
0.5 mM DTT), then lysed for 10 min on ice in buffer A
containing 0.1% Nonidet P-40. The lysates were centrifuged for 10 min
at 10,000 g. The pelleted nuclei were resuspended in a
high salt lysis buffer (buffer C: 20 mM HEPES, pH 7.9, 420
mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF, 0.5 mM DTT) and
incubated at 4° C for 15 min. Lysed nuclei were then centrifuged
for 10 min at 10000 g and the resulting supernatants,
which are referred to as nuclear extracts, were supplemented with five
volumes of storage buffer (buffer D: 20 mM HEPES, pH 7.9, 100
mM KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM PMSF, 0.5 mM DTT). One dish of osteoclast-like cells,
yielded 30 µg of nuclear extract.Electrophoretic Mobility Shift Assays (EMSA)
The
sequence of the NF-B binding oligonucleotide used as a radioactive
DNA probe was 5`-AGCTTGGGGACTTTCCGAG-3`. The mutated oligonucleotide
was 5`-AGCTTGCTCACTTTCCGAG-3`. The AP-1-binding oligonucleotide was
5`-CGCTTGATGACTCAGCCGGAA-3`. For use in the EMSA, the oligonucleotides
were labeled with [-
P]ATP by T4
polynucleotide kinase and purified by precipitation with ethanol as
described(10) . The DNA binding reaction was performed at room
temperature in a volume of 20 µl, which contained the binding
buffer (10 mM Tris/HCl, pH 7.5, 1 mM EDTA, 4%
glycerol, 100 mM NaCl, 5 mM DTT, 100 µg/ml bovine
serum albumin), 3 µg of poly(dI-dC), 1
10
cpm P-labeled probe, and 5-10 µg of nuclear
proteins. After 15 min of incubation, samples were electrophoresed on
native 5% acrylamide/0.25
TBE gels. Unlabeled oligonucleotides
were incubated with extracts for 10 min at room temperature prior to
the addition of the radiolabeled probe in cold competition experiments.
For the supershift assay, the nuclear extract samples were incubated
with 1 µl of either of the following polyclonal antibodies;
anti-p50, p65, c-Rel (residues 498-517), Rel B (residues
540-558), or p52 (residues 159-176) antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA) for 60 min at 4° C before the
binding buffer was added. Gels were dried and analyzed by an image
analyzer (BAS 2000, Fuji Film, Tokyo, Japan) after exposure to an IP
plate (Fuji Film) or x-ray film overnight.
M 1
,25(OH) D and 10M PGE
. Seven days
after inoculation, all cells including MNCs were detached from the
dishes by 0.2% collagenase treatment. The cell suspensions were
reinoculated into plastic dishes (Fig. 1A), and after
10 h adherent cells were treated with Pronase E to remove the weakly
adhered osteoblasts and other bone marrow cells. Most of the cells
still adhered to the dishes were TRAP-positive
MNCs(1, 2, 3) . Alkaline phosphatase-positive
cells (osteoblasts) and nonspecific esterase-positive cells
(mononuclear phagocytes) were very scarce in our purified osteoclast
preparation (Fig. 1B). The average number of nuclei in
the osteoclast-like cells used in this study was 8.0 ± 0.4
nuclei/cell (n = 100). The TRAP-negative cells
contaminating the purified osteoclast preparation were all mononuclear
cells.
,25(OH) D (10M) and PGE
(10M) for 7 days. Seven days after inoculation, all cells
including MNCs were detached from the dishes by treatment with 0.2%
collagenase. The cell suspension was inoculated again into plastic
dishes and cultured for 10 h (A). Then, osteoblasts and other
cells were removed by Pronase E treatment (B). The remaining
adherent MNCs were stained for TRAP and observed under the
microscope.
,10M CT, or 1 µg/ml PMA for
30 min, and prepared nuclear extracts from osteoclast-like cells from
each dish separately. NF-
B activity in the nuclear extracts was
measured by EMSA. Fig. 2A shows that IL-1, but not
PMA or CT, induced an NF-B-like factor in the nuclei of
osteoclast-like cells. This IL-1-induced NF-B activity appeared as
a single band in the EMSA. Excess amounts of the unlabeled wild type
B motif oligonucleotide, but not the mutated B motif
oligonucleotide, inhibited NF-B-like activity completely as shown
in the EMSA (Fig. 2B). This means that the
NF-B-like factor in osteoclast-like cells can recognize and bind
to the specific DNA sequence of the B motif.
B-like activity in the nucleus
of osteoclast-like cells after IL-1 stimulation. The enriched
osteoclast-like cells were treated with vehicle (lane 1), PMA
(1 µg/ml) (lane 2), IL-1 (10 ng/ml) (lane
3), or calcitonin (10M) (lane
4) for 30 min. NF-
B activity in the nuclear extracts was
measured by EMSA as described under ``Experimental
Procedures.'' B, specificity of NF-B-like factor
binding to the B sequence. The nuclear extracts (10 µg of
protein) from the osteoclast-like cells stimulated with IL-1 were
incubated without (lane 1) or with a 50-fold excess of
unlabeled wild type oligonucleotides (lane 2) or a 50-fold
excess of unlabeled mutant oligonucleotides (lane 3) at room
temperature for 10 min and then tested for their ability to bind to
radiolabeled wild type oligonucleotide probes in an EMSA. C,
effects of PMA and IL-1 on induction of AP-1 in osteoblasts and
osteoclast-like cells. POB (lanes 1-3) and purified
osteoclast-like cells (lanes 4-6) were treated for 30
min with either vehicle (lanes 1 and 4), PMA (lanes 2 and 5), or IL-1 (lanes 3 and 6). AP-1 activity in the nuclear extracts was measured by EMSA
as described under ``Experimental Procedures.'' The arrow shows the band with AP-1 activity from
osteoblasts.
B activator, as it activates protein kinase C to
phosphorylate IB, which in turn releases active NF-B (18) . Interestingly, in osteoclast-like cells PMA did not
activate the NF-B-like factor. AP-1 activity, another
transcription factor known to be activated by PMA, was also examined in
osteoclast-like cells. Both PMA and IL-1 greatly induced AP-1 activity
in primary osteoblasts (POB) but not in osteoclast-like cells (OCL) in
the experimental conditions used in this study (Fig. 2C). The same nuclear preparations of POB treated
with PMA and IL-1 and OCL treated with IL-1 contained an activated
NF-B-like factor (data not shown). Macrophage-colony stimulating
factor, which is essential for osteoclast formation in op/op mice(19) , also had no effect on NF-B induction in
osteoclast-like cells (data not shown).B-like activity, we were worried about the possibility of
contamination from other cell types, such as osteoblasts and bone
marrow cells, even though the purity of our osteoclast-like cell
preparation was more than 95%. In order to confirm whether this
IL-1-induced NF-B-like factor is derived from osteoclast-like
cells, we compared the mobility of the NF-B-like factor bands in
the enriched osteoclast-like cell population (purified OCL preparation)
with bands from osteoblasts alone, and mixed populations of osteoblasts
and osteoclast-like cells (crude OCL preparation) in an EMSA. IL-1
induced two NF-B bands, the upper band of which was weaker in
appearance, in the osteoblasts as well as the crude OCL preparation (Fig. 3). The NF-B-like band from the purified OCL
preparation, however, was the fastest migrator of any of the bands,
indicating that its molecular size in osteoclast-like cells is smaller
than in osteoblasts or in the crude OCL preparations. Thus the
NF-B-like factor band from the purified OCL preparation,
stimulated with IL-1, is from osteoclast-like cells and is not the
result of contamination.
B activity in
an EMSA. The arrow shows the NF-B-like activity band from
purified OCL. Arrowheads show the NF-B activity bands
from primary osteoblasts and crude OCL
preparation.
B consists of a p50 and p65
subunit as a heterodimer. In some cells, a p50-p50 homodimer is also
detected in the nucleus after
stimulation(6, 10, 20) . We examined whether
the NF-B-like factor, induced by IL-1 stimulation, in
osteoclast-like cells contained the p50 or p65 subunits of NF-B,
or other Rel family proteins in comparison with osteoblasts. As shown
in Fig. 4, anti-p50 antibodies bound and supershifted both the
upper and lower osteoblast NF-B-like factor bands in an EMSA,
while the anti-p65 antibodies supershifted only the upper band. This
indicates that the upper and lower band of NF-B-like factor
induced by IL-1 in osteoblasts are p65-p50 heterodimers and p50-p50
homodimers, respectively. On the other hand, anti-p50 antibodies, but
not anti-c-Rel, Rel B, or p52 antibodies, recognized the NF-B-like
factor of osteoclast-like cells in the EMSA (Fig. 4). The
NF-B-like factor band was partially reduced in the presence of
anti-p65 antibodies. It remains unknown whether the partial effect of
anti-p65 antibody is due to the weak affinity of the antibodies or to
the presence of a p65-related protein. It seems likely that the
NF-B-like factor in osteoclast-like cells is a heterodimer
composed of a p50 and a p65-related subunit.
B-like
factor of osteoclast-like cells. Nuclear extracts were prepared from
IL-1-stimulated primary osteoblasts (POB, lanes
1-3) and from IL-1-stimulated osteoclast-like cells (OCL, lanes 4-9). The nuclear extracts (5
µg of protein) were pretreated with vehicle (lanes 1 and 4), or 1 µl of one of the following polyclonal antibodies:
anti-p50 (lanes 2 and 5), anti-p65 (lanes 3 and 6), anti-c-Rel (lane 7), anti-Rel B (lane 8), or anti-p52 (lane 9) at 4° C for 60 min. EMSAs
were performed on these pretreated nuclear extracts. Arrowheads show the DNA binding complexes supershifted by antibodies. Arrows identify the two NF-B activity bands from primary
osteoblasts and the NF-B-like factor band from osteoclast-like
cells.
B-like
factor in the nuclei of osteoclast-like cells. It is unknown whether
this NF-B-like factor can actually activate the transcription of
certain genes, but it can bind to specific DNA sequences in the
nucleus. The molecular size of this NF-B-like factor is smaller
than the p50 homodimer in osteoblasts in view of its mobility in an
EMSA. From the supershift assay, we assume that this factor is a
heterodimer composed of a p50 and a p65 subunit, whose size could be
larger than a p50 homodimer. We speculate that the partner of the p50
subunit may be a p65-related protein, to which anti-p65 antibodies can
bind only weakly (21) . However, the function of this factor is
not clear.B and
NF-IL-6 were involved in the expression of IL-6 induced by inflammatory
cytokines, such as IL-1 and tumor necrosis
factor-(24, 25) . In addition, the 5`-flanking
region of the TRAP gene, whose expression is a useful marker for
osteoclasts, was reported to contain an AP-1 binding
sequence(26) . Furthermore, it is known that c-Fos, which is a
component of AP-1, is essential for the differentiation of osteoclast
precursors, probably from mononuclear phagocytes into
osteoclasts(27) . These findings indicate that AP-1 may be
present and functioning in osteoclasts, although we could not detect
AP-1 in osteoclast-like cells stimulated with PMA. Recently, Tezuka et al.(28) showed that the matrix metalloproteinase
(MMP)-9 gene, whose product can degrade some extracellular matrices,
was expressed in isolated rabbit osteoclasts(28) . NF-B
may be involved in the expression of the MMP-9 gene since the promoter
region of the MMP-9 gene has an NF-B binding
sequence(29, 30) .B-like factor in osteoclast-like cells was induced within 5 min
of the addition of IL-1 (data not shown). Even if other cells such as
osteoblasts were present in our enriched osteoclast-like cell cultures,
the number of the contaminating cells would be too small to exert any
influences on the osteoclast-like cells that rapidly. Moreover, PMA
activated AP-1 in osteoblasts, but not in osteoclast-like cells. Thus,
from our results, we conclude that IL-1 binds to putative IL-1
receptors on osteoclast-like cells, resulting in the induction of an
NF-B-like factor in the nucleus of these cells.B
expression between macrophages and osteoclasts depends on the stage of
differentiation of the osteoclasts. Further experiments need to be done
to determine this.
)B, nuclear factor B; IL, interleukin; PG, prostaglandin;
PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethanesulfonyl
fluoride; EMSA, electrophoretic mobility shift assay; OCL,
osteoclast-like cell(s); POB, primary osteoblast(s); DTT,
dithiothreitol; MMP, matrix metalloproteinase; 1,25(OH) D, 1,25-dihydroxyvitamin D.
We thank Dr. I. Kitajima (Department of Laboratory
Medicine, Faculty of Medicine, University of Kagoshima) and Dr. I.
Nakamura (Department of Orthopedic Surgery, Faculty of Medicine,
University of Tokyo) for their helpful discussions and technical
advice.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Sakuta, M. Tokuda, M. Tamura, E. Jimi, T. Ikebe, T. Koga, S. Nagaoka, and H. Takada Dual Regulatory Effects of Interferon-{alpha}, -{beta}, and -{gamma} on Interleukin-8 Gene Expression by Human Gingival Fibroblasts in Culture upon Stimulation with Lipopolysaccharide from Prevotella intermedia, Interleukin-1{alpha}, or Tumor Necrosis Factor-{alpha} Journal of Dental Research, August 1, 1998; 77(8): 1597 - 1605. [Abstract] [PDF]
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I. Nakamura, E. Jimi, L. T. Duong, T. Sasaki, N. Takahashi, G. A. Rodan, and T. Suda Tyrosine Phosphorylation of p130Cas Is Involved in Actin Organization in Osteoclasts J. Biol. Chem., May 1, 1998; 273(18): 11144 - 11149. [Abstract] [Full Text] [PDF]
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E. Jimi, I. Nakamura, T. Ikebe, S. Akiyama, N. Takahashi, and T. Suda Activation of NF-kappa B Is Involved in the Survival of Osteoclasts Promoted by Interleukin-1 J. Biol. Chem., April 10, 1998; 273(15): 8799 - 8805. [Abstract] [Full Text] [PDF]
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