From the Department of Biochemistry, School of Dentistry, Showa
University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555 Japan and the
Department of Oral and Maxillofacial Surgery, Faculty of
Dentistry, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
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ABSTRACT |
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We previously reported that interleukin-1 (IL-1)
promoted the survival of murine osteoclast-like cells (OCLs) formed
in vitro and activated a transcription factor, NF-B, of
OCLs. The present study examined whether the activation of NF-
B is
directly involved in the survival of OCLs promoted by IL-1. The
expression of IL-1 type I receptor mRNA in OCLs was detected by the
polymerase chain reaction amplification of reverse-transcribed
mRNA. An electrophoretic mobility shift assay showed that IL-1
transiently activated NF-
B in the nuclei of the OCLs, and the
maximal activation occurred at 30 min. The degradation of I
B
coincided with the activation of NF-
B in the OCLs. The
immunocytochemical study revealed that p65, a subunit of NF-
B, was
translocated from the cytoplasm into almost all of the nuclei of
the OCLs within 30 min after IL-1 stimulation. The purified OCLs
spontaneously died via apoptosis, and IL-1 promoted the survival of
OCLs by preventing their apoptosis. The pretreatment of purified OCLs
with proteasome inhibitors suppressed the IL-1-induced activation of
NF-
B and prevented the survival of OCLs supported by IL-1. When OCLs
were pretreated with antisense oligodeoxynucleotides to p65 and p50 of
NF-
B, the expression of respective mRNAs by OCLs was
suppressed, and the IL-1-induced survival of OCLs was concomitantly
inhibited. These results indicate that IL-1 promotes the survival of
osteoclasts through the activation of NF-
B.
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INTRODUCTION |
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Osteoclasts are multinucleated giant cells responsible for bone
resorption (1-3). Osteoclastic bone resorption consists of several
important processes: the development of osteoclasts from hematopoietic
progenitor cells, the attachment of osteoclasts to the bone surface,
the formation of a ruffled border and clear zone, and the secretion of
acids and lysosomal enzymes into the space beneath the ruffled border
(2, 4). Osteoclasts are terminally differentiated cells with a limited
life span. Although osteoclasts are believed to die soon after they
play their role in bone (bone resorption), recent findings suggest that
the survival of osteoclasts is tightly regulated by several factors
(5-9). Two cytokines, macrophage colony stimulating factor
(M-CSF,1 also called CSF-1)
(5, 6) and interleukin 1 (IL-1) (6), and a calcium-regulating hormone,
calcitonin (7), stimulate the survival of osteoclasts. In contrast,
estrogen (8) and transforming growth factor- (TGF-
) (9)
induce the apoptosis of osteoclasts. However, it is not yet known how
the apoptosis and survival of osteoclasts is regulated by those
factors.
Nuclear factor B (NF-
B) is a ubiquitous transcription factor that
regulates the expression of many genes involved in immune and
inflammatory responses (10, 11). Conventional NF-
B is a heterodimer
that consists of p50 and p65 subunits. The amino acid sequences of both
subunits of NF-
B show a high homology to the Rel family, which
includes c-Rel, Rel B, and p52, and they are now categorized as the
NF-
B/Rel family (10, 11). The activity of NF-
B is strictly
regulated by an inhibitor, I
B
, that forms a complex with NF-
B
and keeps NF-
B in the cytoplasm (10, 11). When cells receive signals
that activate NF-
B, I
Bs are phosphorylated and degraded through a
ubiquitin/proteasome pathway. Multiple ubiquitin molecules attach to
the phosphorylated I
Bs, and then ubiquitinated I
Bs are degraded
by 26 S proteasome, an organella of intracellular protease complexes
(11-14). The degradation of I
Bs triggers the translocation of
NF-
B from the cytoplasm into the nucleus. Thus, proteasome is
believed to be a key enzyme which is involved in NF-
B activation
(11-14).
Recently several lines of evidence have reported that activation of
NF-B is involved in cell survival (15-19) besides in immune responses and inflammation. We have also reported that IL-1 stimulates the survival of osteoclasts (6) and activates NF-
B in osteoclasts (20), separately. Therefore, in this report, we examined whether the
effect of IL-1 on osteoclast survival is mediated by the activation of
NF-
B. IL-1 transiently induced an activation of NF-
B in
osteoclast-like cells (OCLs), which was concomitant with the
degradation of I
B
. The OCLs spontaneously died via apoptosis,
which was markedly blocked by the addition of IL-1. The pretreatment of
OCLs with either proteasome inhibitors or antisense
oligodeoxynucleotides to p65 and p50 prevented the IL-1-induced
survival of OCLs. These results indicate that IL-1 promotes the
survival of osteoclasts by preventing apoptosis through the activation
of NF-
B.
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EXPERIMENTAL PROCEDURES |
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Antibodies and Chemicals--
Anti-human p65 (sc-109) and
anti-human IB
rabbit polyclonal antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA) and New England BioLabs (Lake
Placid, NY), respectively. The peptide aldehydes
N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and
N-acetyl-leucinyl-leucinyl-methional (ALLM) were purchased from Wako Pure Chemical Co. (Osaka, Japan).
Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (ZLLLal),
carbobenzoxyl-leucinyl-leucinal-H (ZLLal) were purchased from Peptide
Institute Inc. (Osaka, Japan). Phenylmethylsulfonyl fluoride was
purchased from Sigma. Recombinant human interleukin 1
(IL-1
) and
murine IL-1 receptor antagonist (IL-1ra) were obtained from R&D Systems
(Minneapolis, MN).
Culture of Osteoclast-like Cells--
Osteoblasts obtained from
the calvaria of newborn mice and bone marrow cells obtained from the
tibiae of male mice were co-cultured in -minimal essential medium
(
-MEM) (Life Technologies, Inc., Grand Island, NY) containing 10%
fetal bovine serum (FBS), 1
,25-dihydroxyvitamin D3
(1
,25(OH)2 D3) (10
8
M) and prostaglandin E2 (PGE2)
(10
6 M) in 100-mm diameter dishes coated with
collagen gels (Nitta Gelatin Co., Osaka, Japan). OCLs were formed
within 6 days in culture and were removed from the dishes by treatment
with 0.2% collagenase (Wako Pure Chemical Co.). The purity of OCLs in
this fraction (crude OCL preparation) was about 5%. To further purify the OCLs, the crude OCL preparation was replated on culture dishes. After an 8-h culture, osteoblasts were removed with phosphate-buffered saline (PBS) containing 0.001% Pronase E (Calbiochem, La Jolla, CA)
and 0.02% EDTA for 10 min at 37 °C according to the method described previously (20).
Polymerase Chain Reaction Amplification of Reverse-transcribed mRNA-- Total RNA from purified OCLs, crude OCLs and primary osteoblasts in culture dishes (60 mm-diameter), was extracted using Trizol solution (Life Technologies, Inc.). Five % of the first-strand cDNA pool was submitted to polymerase chain reaction (PCR) amplification using gene-specific PCR primers (see Table I) by standard PCR protocols. The PCR program was as follows: 30 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min for IL-1 type I receptors (IL-1RI), calcitonin receptors (CTRs), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 40 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for osteocalcin; in a DNA thermal cycler (Program temperature control system, PC-700, Astec, Tokyo, Japan). The PCR products were separated by electrophoresis on 2% agarose gels and were visualized by ethidium bromide staining with ultraviolet light illumination.
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Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared according to the method described by Dignam et al.
(21). The sequence of the NF-B binding oligonucleotide used as a
radioactive DNA probe was 5'-AGCTTGGGGACTTTCCGAG-3'. The Oct 1-binding
oligonucleotide was 5'-TGTCGAATGCAAATCACTAGAA-3'. 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 dithiothreitol, 100 mg/ml bovine serum albumin), 3 µg
of poly(dI-dC), 1 × 105 cpm of
32P-labeled probe, and 8 µg of nuclear proteins. After
incubation for 15 min, the samples were electrophoresed on native 5%
acrylamide, 0.25 × Tris borate-EDTA gels. The gels were dried and
exposed to x-ray film.
Immunoblotting Analysis--
After purified OCL preparations
were cultured for various periods in the presence of IL-1 (10 ng/ml),
the cells were washed twice with ice-cold PBS and then lysed with an
SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue). The
cell lysates (20 µg of protein) were resolved by 10%
SDS-polyacrylamide gel electrophoresis and transferred onto PVDF
membranes (Millipore, Bedford, MA). After blocking with 5% skim milk
in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the
IB-
antibodies (1 µg/ml) were added in TBS-T containing 5%
bovine serum albumin and visualized by an enhanced chemiluminescence assay (ECL) using reagents from Amersham Pharmacia Biotech (UK) and by
exposure to x-ray film.
Immunofluorescence Microscopy-- For immunofluorescence analysis, OCLs were seeded onto sterile FBS-coated glass coverslips and purified by treatment with Pronase. After OCLs were purified, they were treated with or without IL-1 (10 ng/ml) for the indicated times and then fixed with 4% paraformaldehyde in PBS for 15 min, blocked with 5% skim milk in PBS for 15 min at room temperature, and incubated with 1 µg/ml polyclonal anti-p65 antibodies for 30 min at 37 °C. After extensive washes, the cells were incubated with FITC-conjugated anti-rabbit IgG (dilution 1:100) for 30 min at 37 °C. The cells were then washed and mounted in Immunon (Lipshaw, Pittsburgh, PA). The subcellular localization of FITC-labeled p65 was determined by fluorescence microscopy (Olympus BX-FLA, Osaka).
DNA Extraction and Electrophoretic Analysis-- DNA was prepared and analyzed by gel electrophoresis according to the method described by Bessho et al . (22). Briefly, purified OCLs were lysed by incubating them at 60 °C overnight in a digestion buffer containing 150 mM NaCl, 25 mM EDTA, 100 µg/ml proteinase K, and 0.2% SDS. The DNA was extracted twice with phenol/chloroform/isoamyl alchohol and once with chloroform and precipitated in ethanol with 150 mM CH3COONa, pH 5.2. The DNA was then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and treated with 20 µg/ml RNase A. The procedures for DNA extraction and precipitation were repeated. Two µg of DNA was separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining with ultraviolet light illumination.
Antisense Oligodeoxynucleotides and Cell Cultures--
Synthetic
phosphorothioate oligodeoxynucleotides (S-ODN) that include the ATG
initiation codon of the cDNA for mouse p50 or p65 were used for the
sense and antisense experiments (see
Table II). Crude OCLs were incubated in
the presence of 5 µM antisense or sense S-ODN/cationic
liposomes (DOTAP, Boehringer Mannheim, Mannheim, Germany) as the S-ODN
carrier in -MEM. They were then cultured for 2 h, 5% FBS was
added, and the OCLs were further cultured for 8 h. After OCLs were
purified, they were incubated in the presence of the antisense or sense
S-ODN/DOTAP in
-MEM. They were then cultured for 2 h, and 5%
FBS was added. After incubation for 6 h, OCLs were treated with
IL-1, and the incubation was continued for an additional 15 h. The
expression of p50 or p65 mRNA was detected by RT-PCR using
gene-specific PCR primers. The p50 primers (1)
5'-TCGGAGACTGGAGCCTGTGGTG-3' and (2) 5'-CCCTGCGTTGGATTTCGTGACT-3' (969-1551) define an amplicon of 604 base pairs. The p65 primers (1)
5'-GAAGAAGCGAGACCTGGAGCAA-3' and (2) 5'-GTTGATGGTGCTGAGGGATGCT-3' (423-1116) define an amplicon of 715 base pairs.
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Survival of OCLs-- The survival rate was measured as reported previously (6, 23). After OCLs were purified, some of the cultures were subjected to tartrate-resistant acid phosphatase (TRAP) staining. TRAP-positive multinucleated cells were counted as living OCLs. Other cultures were further incubated for indicated times in the presence or absence of IL-1. After incubation, the remaining OCLs were counted.
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RESULTS |
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Expression of IL-1 Type I Receptor mRNA by OCLs-- We first examined whether OCLs express IL-1RI mRNA. Total RNA isolated from the cell preparations of purified OCLs, crude OCLs, and primary osteoblasts was analyzed by RT-PCR using primers specific for IL-1RI, CTR (a marker of osteoclasts), and osteocalcin (a marker of osteoblasts) (Table I). CTR mRNA was detected in the purified OCL and crude OCL preparations (Fig. 1A, 1st middle panel, lanes 1-4), whereas osteocalcin mRNA was detected in the crude OCL and osteoblast preparations (Fig. 1A, 2nd middle panel, lanes 3-6). Osteocalcin mRNA was not detected in the purified OCL preparations (Fig. 1A, 2nd middle panel, lanes 1 and 2) even after PCR was performed at 40 cycles, suggesting that there was no contamination with osteoblasts in the purified OCL preparations. IL-1RI mRNA was detected in all preparations (Fig. 1A, upper panel, lanes 1-6), indicating that OCLs express IL-1RI mRNA. Expression of GAPDH mRNA was used as a control (Fig. 1A, bottom panel, lanes 1-6).
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Degradation of IB
Triggers the Activation of NF-
B in
OCLs--
A common feature of the regulation of NF-
B is their
sequestration in the cytoplasm as an inactive complex with I
B
.
The activity of NF-
B was then compared with the dynamics of I
B
in the purified OCLs after IL-1 stimulation. The activation of NF-
B
induced by IL-1 was first detected within 5 min, attained a maximal
level at 30 min, and declined thereafter. I
B
rapidly disappeared
upon IL-1 stimulation and reappeared after 30 min (Fig.
2A). The gene encoding
I
B
is one of the target genes of NF-
B (10, 11). When purified
OCLs were pretreated with actinomycin D (an inhibitor of mRNA
synthesis) and incubated with IL-1, the activation of NF-
B was first
detected within 5 min, and the activity was prolonged up to 2 h
(Fig. 2B). IL-1 also induced rapid disappearance of I
B
in the actinomycin D-pretreated OCLs. However, the reappearance of
I
B
was not observed in the OCLs during the incubation for 2 h (Fig. 2B), suggesting that the degradation of I
B
coincided with the activation of NF-
B in OCLs. Thus, IL-1 induces
the degradation of I
B
in OCLs, which triggers the activation of
NF-
B in OCLs.
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Translocation of NF-B into Multiple Nuclei of OCLs in Response
to IL-1--
The localization of NF-
B into the nuclei of OCLs
treated with IL-1 was also examined immunocytochemically using specific antibodies against p65 subunit. Before the IL-1 stimulation, p65 was
distributed throughout the cytoplasm (Fig.
3A) and especially around the
nuclei of OCLs, which were detected by staining with Hoechst 33342 (Fig. 3B). When purified OCLs were treated with IL-1 for 15 min (Fig. 3, C and D), p65 was translocated into
some nuclei in OCLs, but the remaining nuclei of OCLs showed no
accumulation of p65 at this time point. However, p65 was detected in
most of the nuclei of OCLs after IL-1 stimulation for 30 min (Fig. 3, E and F). The immunoreactivity of p65 completely
disappeared from the nuclei of the OCLs after stimulation for 90 min
(Fig. 3, G and H). When nonimmune immunoglobulins
were used as the first antibody, no specific immunolabeling was
detected in the OCLs (data not shown). Thus, the immunocytochemical
findings were well consistent with the findings by electrophoretic
mobility shift assay (EMSA) for NF-
B in OCLs treated with IL-1.
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Proteasome Inhibitors Block the Activation of NF-B and the
Survival of OCLs Promoted by IL-1--
Proteasome inhibitors were used
to explore whether the activation of NF-
B is directly involved in
the IL-1-induced survival of OCLs. The peptide aldehydes ALLN (calpain
inhibitor I) and ZLLLal inhibit the proteolytic activity of proteasome
(12). These inhibitors also suppress the protease activity of cathepsin B and calpain. Therefore, the structurally related compounds, ALLM
(calpain inhibitor II) and ZLLal, which inhibit cathepsin B and calpain
but not proteasome (12), were used as the controls. The pretreatment of
OCLs with ALLN or ZLLLal prior to IL-1 addition markedly decreased the
NF-
B activity induced by IL-1, whereas the pretreatment with
ALLM or ZLLal did not (Fig.
4A). Neither ALLN nor ZLLLal
affected the DNA-binding activity of another transcription factor,
Oct-1, of the OCLs (Fig. 4A).
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Antisense Oligodeoxynucleotides to NF-B Prevent the IL-1-induced
Survival of OCLs--
S-ODNs to p50 and p65 were also used to confirm
the notion that the activation of NF-
B is involved in the survival
of OCLs promoted by IL-1. When purified OCLs were treated with the
antisense S-ODNs to p50 or p65, expression of the respective mRNA
by OCLs was suppressed (Fig.
5A). Treatment of OCLs with
the sense S-ODNs showed no effect on the expression of p50 and p65
mRNA (Fig. 5A). The viability of OCLs treated with sense
S-ODNs to p50 and p65 went down slightly when compared with that
treated with IL-1 alone (p < 0.05, Fig.
5B). This indicates that the transfection of S-ODNs partially affects the viability of OCLs at a basal level. The survival
of OCLs was markedly reduced even in the presence of IL-1 by
pretreatment with antisense S-ODNs to p50 and p65, compared with sense
S-ODNs to p50 and p65 (Fig. 5, B and C).
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DISCUSSION |
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The present study clearly indicates that the activation of NF-B
is involved in the survival of OCLs promoted by IL-1. The OCLs formed
in our co-cultures expressed IL-1 type I receptors, and IL-1 directly
activated NF-
B of OCLs through the binding to the IL-1 type I
receptors. The degradation of I
B
appeared to trigger the
activation of NF-
B in the OCLs because the level of I
B
in OCLs
varied inversely with the activation of NF-
B. The immunocytochemical
study also showed that the nuclear translocation of p65 was well
correlated with the activation of NF-
B detected by the EMSA. Almost
all of the nuclei of OCLs accumulated NF-
B within 30 min in response
to IL-1 and discharged NF-
B after the stimulation for 90 min. This
suggests that all of the nuclei of OCLs are functionally active and
also that the function of multiple nuclei in osteoclasts is
harmoniously regulated by stimuli from outside of the cell. The
experiments using proteasome inhibitors and antisense S-ODNs to NF-
B
showed that the IL-1-promoted survival of OCLs was mediated by the
activation of NF-
B. These results indicate that activation of
NF-
B directly prevents the spontaneously occurring apoptosis of
OCLs.
Although IL-1 is a potent bone-resorbing factor in vivo (24) and in vitro (25), it is still not fully understood how IL-1 regulates osteoclast development and function. We previously reported that IL-1 stimulated OCL formation in murine bone marrow cultures by a mechanism involving prostaglandin E2 synthesis (26). Osteoblasts or marrow stromal cells have been proposed to play an essential role in OCL formation induced by osteotropic factors including IL-1 (3). Thomson et al. (27) showed that IL-1 stimulates the pit-forming activity of isolated rat osteoclasts through soluble factors secreted by osteoblasts. However, neither contaminating osteoblasts nor nonspecific esterase-positive macrophages were detected in our purified OCL preparation (20). Using an in situ hybridization technique, Xu et al. (28) showed the expression of mRNAs to IL-1 types I and II receptors by osteoclasts in normal bone tissues in mice and rats and inflammatory bone tissues in rats. Yu and Ferrier (29) also obtained evidence that osteoclasts are one of the target cells of IL-1. These findings together with the present report strongly suggest that IL-1 also acts on osteoclasts directly through their IL-1 receptors and regulates their functions without the help of other stromal cells.
M-CSF (5, 6, 23) and calcitonin (7) have been shown to prevent the
apoptosis of osteoclasts. We also reported that like IL-1, M-CSF
strongly supported the survival of OCLs (6, 23). It was shown that the
tyrosine phosphorylation of IB
represented a
proteolysis-independent mechanism of NF-
B activation that directly
coupled NF-
B to cellular tyrosine kinase (30). M-CSF, the receptors
of which possess tyrosine kinase in the cytoplasmic domain, activated
NF-
B of liver macrophages (Kupffer cells) of rats (31). However,
M-CSF did not activate NF-
B of OCLs, and antisense S-ODNs to NF-
B
failed to block the M-CSF-supported survival of OCLs in our culture
system (data not shown). Furthermore, the pretreatment of actinomycin D
suppressed the reappearance of I
B
protein in the purified OCLs
treated with IL-1. These results indicated that the IL-1-induced
NF-
B activation was mediated by a proteolysis-dependent
mechanism in OCLs. NF-
B of OCLs was not activated by the addition of
calcitonin (20). In our preliminary experiments, IL-1 as well as M-CSF
reduced the caspase activity in purified
OCLs.2 These results suggest
that signals other than the activation of NF-
B are involved in the
survival of OCLs promoted by M-CSF and calcitonin.
Among the several factors examined, tumor necrosis factor (TNF
,
10 ng/ml) also activated NF-
B of OCLs and prevented their spontaneous apoptosis (data not shown). It was recently reported that a
novel serine/threonine protein kinase, NF-
B-inducing kinase (NIK),
which binds to TRAF2 (TNF receptor associated factor 2) and activates
NF-
B, is a necessary component of an NF-
B-activating cascade
common to TNF
and IL-1 signalings (32). More recently, I
B kinase
was identified which binds to NIK (33, 34). These findings suggest that
OCLs also express functionally active TNF
receptors, and that
serine/threonine protein kinases such as NIK and I
B kinase are
commonly involved in the activation of NF-
B in OCLs treated with
IL-1 and TNF
.
On the contrary to the present report, it has been shown that the
activation of NF-B induces the apoptosis in certain cells (22, 35).
Abbadie et al. (35) reported that the overexpression of
c-Rel induced apoptosis of avian bone marrow cells. Bessho et
al. (22) also showed that the treatment of human leukemia cells
and thymocytes with a protease inhibitor, pyrrolidinedithiocarbamate (PDTC), which inhibited the activation of NF-
B, prevented their apoptosis. In addition, radiation or agents such as lipopolysaccharides and TNF
triggered the hydrolysis of membrane phospholipids to produce ceramide, which in turn activated NF-
B and induced apoptosis (11). It is not known at present if NF-
B is involved in promotion of
apoptosis in certain cells and if also responsible for inhibition of
apoptosis in other cells.
A novel function of NF-B in apoptosis was recently found in Rel A
(p65) knockout mice (15, 37). Beg et al. (36) showed that a
considerable apoptosis occurred in hepatocytes in Rel A-deficient embryos. They also reported that TNF
induced the apoptosis of fibroblasts and macrophages derived from Rel A knockout mice, and
transfection of Rel A into the cells rescued them from TNF
-induced apoptosis (15). Wang et al. (16) and Antwerp et
al. (17) independently demonstrated the anti-apoptotic role of
NF-
B using cell lines stably transfected with the dominant negative
I
B
. Liu et al. (18) also showed that three different
responses to TNF
were mediated by TNF
receptor complex: the
activation of Jun N-terminal kinase (JNK), the activation of NF-
B,
and the induction of apoptosis. The activation of NF-
B
protected the cells against TNF
-induced apoptosis. Ozaki
et al. (19) also reported that NF-
B inhibitors
pyrrolidinedithiocarbamate and choromethylketone stimulated apoptosis
of rabbit mature osteoclasts, which resulted in the inhibition of bone
resorption. These findings together with the present report indicate
that the NF-
B signaling cascade is closely involved in the
prevention of apoptosis of cells. Cell death therefore appears to be
tightly regulated through a balance between apoptosis-inducing and
apoptosis-preventing signals. The signaling cascade mediated by NF-
B
may be important in the survival of osteoclasts.
Osteoclasts reported to have a short half-life undergo apoptosis
within a few days (3, 5, 6, 23). Studying the control mechanism of
limited life span of osteoclasts may be important for understanding the
regulation of bone remodeling not only in vitro but also
in vivo. Treatment of OCLs with IL-1 for 1 h could support their survival, which was evaluated 20 h later (data not shown). This indicates that NF-B stimulates gene expression of long
term effective proteins on the survival of OCLs. Identification of such
proteins in the OCLs must be important in future studies. IL-1 has been
implicated in increased bone loss in pathological conditions such as
rheumatoid arthritis, lymphoma, and osteoporosis (37). The IL-1-induced
survival of osteoclasts may also play an important role in the bone
resorption stimulated by these pathological conditions. Further studies
are required to determine a more detailed mechanism of involvement of
NF-
B in the survival of osteoclasts and the gene expression of
proteins that exert anti-apoptotic effects promoted by IL-1.
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ACKNOWLEDGEMENTS |
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We thank Drs. Hiroshi Takeuchi (Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University), Nobuo Okahashi, and Tatsuji Nishihara (Department of Oral Science, The National Institute of Infectious Diseases) for helpful discussions and technical advice.
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FOOTNOTES |
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* This work was supported in part by Grants-in Aid (09771546 and 08557101) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 813-3784-8162; Fax: 813-3784-5555; E-mail: suda{at}dent.showa-u.ac.jp.
1
The abbreviations used are: M-CSF, macrophage
colony stimulating factor; TRAP, tartrate-resistant acid phosphatase;
NF-B, nuclear factor
B; IL, interleukin; ALLN, aldehydes
N-acetyl-leucinyl-leucinyl-norleucinal-H; ALLM,
N-acetyl-leucinyl-leucinyl-methional; ZLLLal,
carbobenzoxyl-leucinyl-leucinyl-leucinal-H; ZLLal,
carbobenzoxyl-leucinyl-leucinal-H; OCL, osteoclast-like cell;
-MEM,
-minimal essential medium; FBS, fetal bovine serum; RT-PCR,
reverse-transcribed polymerase chain reaction; PBS, phosphate-buffered saline; CTR, calcitonin receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate; S-ODN, synthetic phosphorothioate oligodeoxynucleotide; DOTAP,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts; EMSA, electrophoretic mobility shift assay; TNF
, tumor necrosis factor
; NIK, NF-
B-inducing kinase.
2 Okahashi, N., Koide, M., Jimi, E., Suda, T., and Nishihara, T., submitted for publication.
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REFERENCES |
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