From The H. Hubert Humphrey Center for Experimental Medicine and
Cancer Research, The Hebrew University Faculty of Medicine, P. O. Box
12272, Jerusalem 91120, Israel
Received for publication, December 9, 2002, and in revised form, February 27, 2003
Regulation of osteoclastogenesis by
lipopolysaccharide (LPS) is mediated via its interactions with
toll-like receptor 4 (TLR4) on both osteoclast- and osteoblast-lineage
cells. We have recently demonstrated that CpG oligodeoxynucleotides
(CpG ODNs), known to mimic bacterial DNA, modulate osteoclastogenesis
via interactions with osteoclast precursors. In the present study we
characterize the interactions of CpG ODNs with osteoblasts, in
comparison with LPS. We find that, similar to LPS, CpG ODNs modulate
osteoclastogenesis in bone marrow cell/osteoblast co-cultures, although
in a somewhat different pattern. Osteoblasts express receptors for both
LPS and CpG ODN (TLR4 and TLR9, respectively). The osteoblastic TLR9 transmits signals into the cell as demonstrated by NF
B activation as
well as by extracellular-regulated kinase (ERK) and p38
phosphorylation. Similar to LPS, CpG ODN increases in osteoblasts the
expression of tumor necrosis factor (TNF)-
and macrophage-colony
stimulating factor (M-CSF). The two TLR ligands do not affect
osteoprotegerin expression in osteoblasts. CpG ODN does not
significantly affect receptor activator of NF
B ligand (RANKL)
expression, in contrast to LPS, which induces the expression of this
molecule. In the co-cultures CpG ODN induces RANKL expression in
osteoblasts as a result of the more efficient TNF-
induction. CpG
ODN activity (modulation of osteoclastogenesis, gene expression, ERK
and p38 phosphorylation, and nuclear translocation of NF
B) is
specific, because the control oligodeoxynucleotide, not containing CpG, is inactive. Furthermore, these effects (unlike the LPS effects) are
inhibited by chloroquine, suggesting a requirement for endosomal maturation/acidification, the classic CpG ODN mode of action. We
conclude that CpG ODN, upon TLR9 ligation, induces osteoblasts osteoclastogenic activity.
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INTRODUCTION |
Osteoblasts, the bone-forming cells, play a central role
in modulating the differentiation and activity of the bone-resorbing cells, the osteoclasts (1-6). Molecules produced by osteoblasts, in
particular receptor activator of NF
B
(RANK)1 ligand (RANKL) and
tumor necrosis factor (TNF)-
, are potent inducers of osteoclasts,
and therefore modulation of the expression of these proteins in
osteoblasts should have an impact on resorption (7-10). It is well
documented that the bacteria-derived lipopolysaccharide (LPS) exerts
its resorption stimulatory activity, at least in part, via modulation
of osteoblasts (11, 12).
In addition to LPS, a variety of bacterial products such as teichoic
acid and other cell wall components have been shown to stimulate
osteoclastic bone resorption (11). These pathogen-derived molecules are
known to induce innate immunity via toll-like receptors (TLRs)
(13-18). Recent advances demonstrate that bacterial DNA is also a
pathogen-derived molecule activating the innate immune system (19-22).
This activity of bacterial DNA depends on its content of unmethylated
CpG dinucleotides in particular base contexts ("CpG motif")
(20-25). Vertebrate DNA contains a lower than expected amount
of CpG dinucleotides, and these are highly methylated, which prevents
their immune stimulatory effects. Oligodeoxynucleotides containing CpG
motifs, CpG oligodeoxynucleotides (CpG ODNs), mimic the activity of
bacterial DNA. These, together with the ability of CpG ODN to activate
NF
B (26-28), a critical transcription factor in osteoclast
differentiation (29, 30), prompted us to examine modulation of
osteoclastogenesis by the oligodeoxynucleotides. We demonstrated that
CpG ODNs exert dual effect on osteoclastogenesis: they inhibit
RANKL-induced osteoclastogenesis of osteoclast precursors not exposed
to RANKL prior to CpG ODN addition. On the other hand, the
oligodeoxynucleotides are potent osteoclastogenic agents to osteoclast
precursors pretreated with RANKL (31).
The present study was designed to examine if osteoblasts are targets to
CpG ODNs, and thus these cells mediate, in part, the modulation of
osteoclastogenesis by the ODN. We find that CpG ODN interacts with
osteoblastic TLR9 and elicits intracellular events leading to the
increased expression of molecules regulating osteoclastogenesis.
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EXPERIMENTAL PROCEDURES |
Mice--
Newborn BALB/c mice and 7- to 9-week-old male
BALB/c mice were obtained from Harlan Laboratories Ltd. (Jerusalem, Israel).
Reagents--
Nuclease-resistant phosphorothioate
oligodeoxynucleotides were purchased from BTG (Rehovot, Israel) and had
undetectable endotoxins according to a limulus amoebocyte lysate assay
(BioWhittaker, Walkersville, MD). The sequences of the
oligodeoxynucleotides used are: 5'-TCCATGACGTTCCTGACGTT-3' (ODN 1826)
and 5'-TCCAGGACTTCTCTCAGGTT-3' (ODN 1982) (27, 32). Purified
Escherichia coli 055:B5 LPS, chloroquine, dexamethasone, and
mouse monoclonal anti-
-actin antibody were purchased from Sigma (St.
Louis, MO). 1,25-Dihydoxyvitamin D3
(1,25(OH)2D3) was purchased from BIOMOL
(Plymouth Meeting, PA). Osteoprotegerin/Fc (OPG/Fc) (chimera containing
OPG amino acids 1-398 residues and Fc) and goat polyclonal
anti-mouse TNF-
neutralizing antibody were purchased from R&D
systems Inc. (Minneapolis, MN). Rabbit polyclonal anti-NF
B
antibodies and rabbit polyclonal anti-mouse M-CSF receptor antibody
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat
anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories
(West Grove, PA). Mouse monoclonal anti-phospho extracellular-regulated
kinase (ERK), and anti-phospho p38 antibodies and rabbit polyclonal
anti-ERK antibody were purchased from Cell Signaling Technology Inc.
(Beverly, MA). IL-1 receptor antagonist (IL-1ra) was a gift from Dr. C. Dinarello (University of Colorado, Denver, CO). Gliotoxin
was purchased from Calbiochem (San Diego, CA). Media and sera were purchased from Biological Industries (Beth Haemek, Israel). All chemicals and reagents were of analytical grade.
Calvarial Osteoblastic Cells--
Primary calvaria-derived
osteoblasts were isolated using the collagen-gel culture as described
previously (33-35).
Bone Marrow Macrophages--
Primary BMMs were isolated as
described (36) and incubated for 3 days prior to the experiment.
In Vitro Osteoclast Formation Assay--
Osteoclasts were
generated using the mouse BMM/osteoblast co-culture system in the
presence of 1,25(OH)2D3 (10 nM) and
dexamethasone (100 nM), as described previously (37).
Tartrate-resistant acid phosphatase (TRAP)-positive cells containing
more than two nuclei were counted on day 7, following removal of the
osteoblasts by collagenase.
Methylene Blue Staining--
The relative cell number was
estimated by the methylene blue staining assay using a Dynatech plate
reader (Vienna, VA) (38).
Western Blot Analysis--
Western analysis was performed as
described previously (37). Bands were quantified by densitometry.
Reverse Transcriptase-PCR Analysis--
Total RNA was prepared
using TRI REAGENT (Sigma) according to the manufacturer's
instructions. First-strand cDNA was synthesized from 1 µg of
total RNA (1 h, 42 °C) using Moloney murine leukemia virus reverse
transcriptase and oligo-dT (Promega, Madison, WI) and was subjected to
PCR amplification with Taq DNA polymerase (Roche Applied
Science, Mannheim, Germany) using specific PCR primers: TLR9 (sense,
5'-CTACAACAGCCAGCCCTTTA-3'; antisense, 5'-GCTGAGGTTGACCTCTTTCA-3'); TLR4 (sense, 5'-CAGCTTCAATGGTGCCATCA-3'; antisense,
5'-CTGCAATCAAGAGTGCTGAG-3'); L19 (sense, 5'-CTGAAGGTGAAGGGGAATGTG-3';
antisense, 5'-GGATAAAGTCTTGATGATCTC-3). PCR cycling conditions were as
follows: 94 °C and either 54 °C (TLRs) or 58 °C (L19) (30 s
each) and 72 °C (60 s) (35 cycles for TLR9, 30 cycles for TLR4, and
28 cycles for L19).
Northern Blot Analysis--
Osteoblasts or BMM/osteoblast
co-cultures were grown in
-MEM with 10% fetal calf serum (39).
Total cellular RNA was extracted using TRI reagent, fractionated by
electrophoresis on 1.2% agarose formaldehyde gels (10 µg/lane), and
transferred to nylon membranes (Hybond-N, Amersham Biosciences, Little
Chalfont, UK). 32P-Labeled mouse TNF-
, macrophage colony
stimulating factor (M-CSF), M-CSF receptor, IL-1
, RANKL, OPG, rat
alkaline phosphatase, or mouse ribosomal protein L32 cDNA probes
were used for hybridization. The hybridized membranes were then
subjected to autoradiography, and the density of each of the mRNA
bands was quantified.
Electrophoretic Mobility Shift Assay (EMSA)--
Osteoblasts
were cultured in
-MEM containing 10% fetal calf serum for 6 days
and then treated as described in the Fig. legends. The NF
B
binding site oligodeoxynucleotide 5'-GATCAAACAGGGGGCTTTCCCTCCTC-3' derived from the
B3 site of the TNF promoter was labeled with [
-32P]CTP and Klenow polymerase (40). Nuclear extracts
(5 µg) were incubated with the labeled probe in 20 µl of reaction
buffer (10 mM Tris, pH 7.9, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 µg of
poly(dI-dC), and 4% glycerol) for 20 min at room temperature. Where effects of antibodies were examined, 1 µg of the corresponding antibody was added to nuclear extracts 20 min before addition of DNA
probe. Samples were then fractionated on a 7% polyacrylamide gel and
visualized by exposing dried gel to film.
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RESULTS |
CpG ODN Modulates Osteoclastogenesis in BMM/Osteoblast
Co-cultures--
We have previously shown that CpG ODNs and LPS (31,
37) exert dual effect on osteoclast differentiation. They inhibit RANKL-induced osteoclastogenesis in BMMs not exposed previously to
RANKL but strongly stimulate osteoclastogenesis in RANKL-primed BMMs.
These studies were performed in the absence of osteoblasts/stromal cells essential for in vivo osteoclastogenesis. In the
present study we used BMM/osteoblast co-cultures to mimic the in
vivo interactions (41).
The inclusion of ODN 1826 from the beginning of the co-cultures
resulted in blocking (almost 100%) the osteoclastogenesis (Fig.
1A); when ODN 1826 was added
24 h after the beginning of the culture ~50% inhibition was
observed. The addition of the ODN at later time points (days 5 and 6)
caused a marked enhancement of the osteoclastogenesis. The effect was
specific, because the control oligodeoxynucleotide, not containing the
CpG motif (ODN 1982), was inactive. In contrast, LPS stimulated
osteoclastogenesis even when it was present from the beginning of the
co-culture (Fig. 1B), unlike the LPS effect in BMMs in the
absence of osteoblasts (37). The inclusion of either ODN 1826 or LPS
resulted in increased cellular contents of the monolayer (Fig. 1,
C and D). ODN 1826 exerted its inhibitory (Fig.
2A) and stimulatory (Fig.
2B) effects on osteoclastogenesis already at 20 nM.

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Fig. 1.
Modulation of osteoclastogenesis by
oligodeoxynucleotides and LPS in BMM/osteoblast co-cultures. BMMs
were cultured for 7 days with primary osteoblasts in the presence of
1,25(OH)2D3 (10 nM) and
dexamethasone (100 nM). At the indicated time, ODN 1826 or
ODN 1982 (100 nM) (A) or LPS (20 ng/ml)
(B) were added. TRAP-positive cells containing more than two
nuclei were counted. C and D,
methylene blue staining of the experiments presented in A
and B, respectively.
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Fig. 2.
Modulation of osteoclastogenesis by
oligodeoxynucleotides in BMM/osteoblast co-cultures: dose
response. BMMs were cultured as in Fig. 1. Oligodeoxynucleotides
at different doses were added for the whole 7 days (A) or
for the last 24 h (B). TRAP-positive cells containing
more than two nuclei were counted.
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In Fig. 3A we see that
osteoprotegerin (OPG) inhibited the stimulation of osteoclastogenesis
caused by LPS when presents throughout the experiment (7 days). OPG
also inhibited the basal activity (in the absence of LPS). When LPS or
ODN 1826 (Fig. 3B) were added for the last 24 h of the
7-day experiment, OPG inhibited the activity of both. Chloroquine
inhibited the activity of ODN 1826, confirming the classic mode of the
ODN action, involving endosomal maturation and/or acidification. As
expected, chloroquine did not affect the activity of LPS.

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Fig. 3.
Modulation of ODN 1826- and LPS-induced
osteoclastogenesis by OPG and chloroquine. Cells were cultured as
in Fig. 1. A, ODN 1826 (100 nM) or LPS (20 ng/ml) were added to the culture for 7 days in the presence or absence
of OPG (100 ng/ml). B, ODN 1826 (100 nM) or LPS
(20 ng/ml) were added to the culture for the last 24 h in the
presence or absence of OPG (100 ng/ml) or chloroquine (2.5 µg/ml).
TRAP-positive cells containing more than two nuclei were counted.
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M-CSF and RANKL interactions with their respective receptors on
osteoclast precursors are essential for the differentiation of these
cells. Therefore, we examined the hypothesis that the inhibitory effect
of CpG ODN observed in the co-cultures is caused by reduction in the
levels of these receptors. To this end, co-cultures were incubated in
the presence or absence of ODN 1826 for 4-6 days. The
osteoblasts were removed by collagenase (see Fig. 10A below), and RNA and protein were prepared from the remaining adherent cells. Northern analysis revealed a significant reduction (~67%) in
M-CSF receptor mRNA abundance (Fig.
4A). A slight non-significant reduction was observed in the abundance of RANK transcript levels (not
shown). Western analysis (Fig. 4B) showed that M-CSF
receptor protein level was also markedly reduced (~72%) in ODN
1826-treated cells. These findings are in agreement with our studies on
osteoclasts precursors in the absence of osteoblasts (31).

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Fig. 4.
Modulation M-CSF receptor expression by ODN
1826. BMM/osteoblast co-cultures were grown with or without
ODN 1826 (100 nM) for 4 (A) or 6 days
(A and B). Monolayers were then treated with
collagenase. The collagenase-resistant adherent cells were analyzed
(see Fig. 10). Northern (A) and western (B)
analyses were performed.
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CpG ODN Induces NF
B Activation and ERK and p38 Phosphorylation
in Osteoblasts--
Because effects of LPS and CpG ODNs are mediated
via TLR4 and TLR9, respectively, we examined the expression of these
receptors in osteoblasts using reverse transcriptase-PCR (Fig.
5). TLR4 is present in comparable levels
in osteoclast precursors and in osteoblasts. In contrast, TLR9
expression is lower in osteoblasts as compared with osteoclast
precursors. The lack of expression in the absence of reverse
transcriptase serves as a control.

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Fig. 5.
TLR4 and TLR9 expression in BMMs and
osteoblasts. Total RNA was prepared from BMMs and osteoblasts.
Templates for PCR were synthesized with (+RT) or without
( RT) reverse transcriptase.
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LPS and CpG ODNs are known to induce activation of NF
B in the target
cells. In Fig. 6A we
demonstrate (using EMSA) that the two modulators induce NF
B
activation in osteoblasts in a time-dependent manner.
Controls (NE, absence of nuclear extract; cold,
excess of unlabeled nucleotides) are shown. In Fig. 6B we
see that anti-p65 and anti-p50 antibodies caused a supershift and a
block-shift, respectively, in osteoblasts treated with TLR ligands,
indicating that these members of the NF
B family are mobilized into
the nucleus. On the other hand, no effects were observed with anti-p52,
anti-Rel B, or anti-c-rel antibodies. In Fig. 6C it is shown
that NF
B nuclear association induced by either LPS or ODN 1826 is
not affected by OPG. Chloroquine inhibits the effect of ODN 1826 but
not the LPS effect. The control oligodeoxynucleotide, ODN 1982, did not induce NF
B nuclear association.

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Fig. 6.
CpG oligodeoxynucleotide activates
NF B in osteoblasts. A,
osteoblasts were treated with ODN 1826 (100 nM) or LPS (20 ng/ml) for the indicated time. Nuclear extracts were prepared and
subjected to EMSA. B, osteoblasts were treated with ODN 1826 (100 nM) or LPS (20 ng/ml) for 90 min. Effect of the
indicated anti-NF B antibodies was performed as described under
"Experimental Procedures." C, osteoblasts were treated
with ODN 1826 (100 nM), ODN 1982 (100 nM), or
LPS (20 ng/ml) for 90 min in the presence or absence of OPG (100 ng/ml)
or chloroquine (2.5 µg/ml). Nuclear extracts were prepared and
subjected to EMSA.
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The signaling pathways mediating the cellular effects of CpG ODN were
studied (42, 43). To demonstrate modulation of TLR9 signaling pathway
in osteoblasts by CpG ODN we chose to measure ERK (p42/44) and p38
phosphorylation. In Fig. 7 we show that
indeed p38 and, to a lesser extent, ERK are phosphorylated in response to ODN 1826 but not in response to ODN 1982. Consistent with the low
TLR9 expression, as compared with BMMs, ERK and p38 phosphorylation occurs to a smaller degree in osteoblasts (Fig. 7, compare A
to B).

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Fig. 7.
CpG ODN induces ERK and p38 phosphorylation
in osteoblasts. Monolayers of BMMs (grown for 3 days)
(A) or osteoblasts (grown for 6 days) (B) were
washed extensively with PBS and incubated for the indicated time with
ODN 1826 or ODN 1982 (100 nM each) in -MEM. Total cell
lysate was subjected to Western blot analysis.
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CpG ODN Modulates Osteoblast Gene Expression--
We next studied
how the TLR ligands affect osteoblastic genes known to participate in
regulation of osteoclastogenesis. Both ODN 1826 and LPS did not affect
significantly the expression of the osteoclastogenesis inhibitor OPG
(Fig. 8A). LPS caused a marked increase in expression of RANKL, TNF-
, and M-CSF in osteoblasts. The
increase in TNF-
and M-CSF expression by ODN 1826 was moderate, and
no significant effect was exerted by the ODN on RANKL expression (about
50% increase in transcript abundance, as compared with 1500% increase
with LPS). The CpG ODN induction of TNF-
, but not the LPS induction,
was blocked completely by chloroquine (Fig. 8B). We wondered
if the fact that RANKL induction is observed with LPS, but not with ODN
1826, could be responsible to the differential effect of the two
modulators on TNF-
expression. The failure of OPG to modulate the
effect of either LPS or ODN 1826 (Fig. 8B) rules out this
possibility.

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Fig. 8.
Modulation of osteoclastogenesis-related
genes expression in osteoblasts. Osteoblasts were grown for
6 days. A, monolayers were washed and incubated with LPS,
ODN 1826, or ODN 1982. B, alternatively, osteoblasts were
treated with LPS (20 ng/ml) or ODN 1826 (100 nM) for 4 h, in the presence or absence of OPG (100 ng/ml) or chloroquine (2.5 µg/ml). RNA was then prepared and examined using Northern blot
analysis for transcript abundance of RANKL, TNF- , M-CSF, IL-1, and
OPG (A) or TNF- (B). L32 was used as a
loading control.
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The inability of ODN 1826 to induce RANKL expression in osteoblasts
does not correlate with the ability of OPG to inhibit the
osteoclastogenic effect of the oligodeoxynucleotide in BMM/osteoblast co-cultures (Fig. 3). We examined, therefore, the effect of ODN 1826 on
gene expression in the co-culture and found that the ODN is much more
efficient in increasing the expression of IL-1
and TNF-
in
BMM/osteoblast co-cultures than in osteoblasts in the absence of BMMs
(Fig. 9A). Furthermore, in the
co-cultures a significant increase in RANKL expression by ODN 1826 is
also observed. In the co-culture, similarly to what we observed with
osteoblasts, chloroquine selectively inhibited the effect of ODN 1826, whereas OPG did not change the effects of both CpG ODN and LPS (Fig.
9B). To examine which of the cells in the co-culture produce
TNF-
and RANKL, we treated the BMM/osteoblast co-cultures at the end of the experiment with collagenase (Fig. 10) and analyzed separately the cells that were removed by the collagenase and the cells that remained adherent. In Fig.
10A we see that the cells
removed by collagenase are enriched for alkaline phosphatase (an
osteoblastic marker), whereas the remaining adherent cells are enriched
for the M-CSF receptor (a marker for macrophages and osteoclast-lineage cells). Consistent with results presented in Fig. 9, we see that ODN
1826 increases TNF-
more efficiently in the co-culture than in
osteoblasts alone (Fig. 10B). Furthermore, an increase in
RANKL expression in response to the ODN is observed in the co-culture, but very little in osteoblasts. The collagenase treatment revealed that
most of the TNF-
expression is derived from the osteoclast precursors-enriched fraction, whereas most of the RANKL expression is
derived from the osteoblast-enriched population.

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Fig. 9.
Modulation of osteoclastogenesis-related
genes expression in BMM/osteoblast co-cultures. A,
osteoblasts or BMMs/osteoblasts were cultured for 6 days. Cells were
then washed and incubated with ODN 1826 (100 nM) to the
indicated time. B, BMMs/osteoblasts were cultured for 6 days. Cells were then washed and treated with LPS (20 ng/ml) or ODN
1826 (100 nM) for 4 h, in the presence or absence of
OPG (100 ng/ml) or chloroquine (2.5 µg/ml). RNA was then prepared and
examined for transcript abundance of RANKL, TNF- , M-CSF, and IL-1
(A) or RANKL and TNF- (B). L32 was as a
loading control.
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Fig. 10.
Modulation of osteoclastogenesis-related
genes expression in BMM/osteoblast co-cultures: analyses of collagenase
adherence resistant and sensitive subpopulations. A,
osteoblasts and BMMs/osteoblasts were cultured for 6 days. Some of the
BMMs/osteoblasts were treated with collagenase. The adherent and
non-adherent cell populations were collected separately prior to RNA
isolation. Alkaline phosphatase and M-CSF receptor mRNA levels were
examined by Northern analyses in osteoblasts, in BMMs/osteoblasts
co-cultures, and in the two sub-populations. B,
BMMs/osteoblasts were cultured for 6 days. Cells were then washed and
treated with LPS (20 ng/ml) or ODN 1826 (100 nM) for 4 h. At the end of the experiment, cells were treated with collagenase as
in A. Abundance of TNF- and RANKL transcripts was
measured in osteoblasts, in BMMs/osteoblasts, and in the two
subpopulations.
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Next we attempted to understand the mechanism of RANKL modulation by
the ODN. We found that TNF-
and IL-1
increase the expression of
RANKL in osteoblasts (not shown), confirming previous studies (44, 45).
CpG ODNs increase the expression of these two cytokines in
osteoclast precursors and, to a lesser extent, in osteoblasts; therefore, we hypothesize that (a) the ODN increases
osteoblastic RANKL expression indirectly via TNF-
and/or IL-1
and
(b) because the effect on the cytokines levels in
co-cultures is stronger than in osteoblasts alone, the efficiency of
ODN in inducing RANKL is more pronounced in the former conditions. To
test these hypotheses we have examined the ability of
anti-TNF-
neutralizing antibody and of IL-1ra to inhibit the
induction of RANKL by CpG ODN in the co-culture. Using Northern
analysis we show in Fig. 11A
that anti-TNF-
neutralizing antibody blocks the induction of RANKL by ODN 1826, whereas control IgG was not effective. IL-1ra did not
affect RANKL induction by ODN 1826 in the absence or presence of
anti-TNF-
-neutralizing antibody. The inhibition of RANKL induction by ODN 1826 results in inhibition of the ODN ability to stimulate osteoclastogenesis (Fig. 11B).

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Fig. 11.
Anti-TNF- antibody
inhibits ODN 1826-induced RANKL expression and osteoclast
differentiation in BMM/osteoblast co-cultures. A,
BMM/osteoblast co-cultures were grown for 6 days. Cells were then
washed and treated with ODN 1826 (100 nM) for 4 h, in
the presence or absence of anti-TNF- , IL-1ra, anti-TNF- plus
IL-1ra or IgG (20 µg/ml each). Transcript abundance of RANKL was
measured using Northern analysis. L32 was used as a loading control.
B, BMM/osteoblast co-cultures were grown for 6 days. Then
ODN 1826 was added for 24 h in the presence or absence of either
anti-TNF- (20 µg/ml) or IgG (20 µg/ml). Osteoclast formation was
measured.
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Importance of NF
B in CpG ODN Induction of
Osteoclastogenesis--
Finally we have tested if NF
B activation in
osteoblasts plays a role the osteoclastogenic activity of these cells.
To this end we have examined the effects of gliotoxin, a known
inhibitor of NF
B activation (46), on ODN 1826 induction of NF
B
activation, TNF-
, and RANKL expression and osteoclastogenesis. In
Fig. 12 we see that gliotoxin inhibits
ODN 1826 stimulatory activities on osteoblasts: NF
B activation
(A), TNF-
(B and C) and RANKL (C) expression, as well as osteoclastogenesis
(D).

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Fig. 12.
Modulation of ODN 1826 activities by
gliotoxin. A, osteoblasts were cultured for 6 days and
then treated with ODN 1826 (100 nM) for 90 min, with or
without gliotoxin (1 µg/ml, added 30 min before the ODN). Nuclear
extracts were prepared and subjected to EMSA. Osteoblasts
(B) or BMM/osteoblast co-cultures (C) were grown
for 6 days and then treated with ODN 1826 (100 nM) for
4 h with or without gliotoxin. RNA was then prepared and examined
using Northern blot analysis for transcript abundance of TNF-
(B and C) or RANKL (C). L32 was used
as a loading control. D, BMM/osteoblast co-cultures were
grown for 6 days in the presence of 1,25(OH)2D3
(10 nM) and dexamethasone (100 nM). Then ODN
1826 was added for 24 h with or without of gliotoxin. Osteoclast
formation was measured.
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DISCUSSION |
Normal bone resorption is maintained by systemic hormones and
local growth factors and cytokines that regulate the differentiation and activation of osteoclasts (1, 8, 47-50). Accelerated osteoclastogenesis is the cause of pathological bone loss induced by
bacterial products in a variety of diseases, including periodontitis, osteomyelitis, bacterial arthritis, and infected metal implants (11,
51-54). Most data on involvement of bacterial products in osteoclastogenesis and resorption were obtained with LPS. For example,
it has been identified as an important factor in the pathogenesis of
periodontitis, characterized by gingival inflammation and alveolar bone
resorption (55). LPS stimulates osteoclastic bone resorption in
vivo (56, 57) and in vitro in organ culture (58, 59)
and promotes osteoclast differentiation in whole bone marrow cell
culture (60). LPS induces RANKL expression in osteoblasts (12) and
stimulates these cells to secrete IL-1, prostaglandin E2,
and TNF-
, each of which seems to be involved in LPS-mediated bone
resorption (11). We showed that CpG ODNs, known to be responsible for
the ability of bacterial DNA to elicit innate immunity (19-25),
modulate osteoclastogenesis via interactions of the ODN with osteoclast
lineage cells (37). These findings were confirmed recently (61).
LPS and CpG ODNs exert their activities via interactions with TLR4 and
TLR9 on the target cells, respectively (16, 18, 62, 63). It is
recognized that modulation of osteoclastogenesis by LPS occurs
directly, via interactions with TLR4 on osteoclast lineage cells, as
well as indirectly via interactions with TLR4 on osteoblasts (12,
64).
We examined in the present study the hypothesis that CpG ODNs, in
addition to their interactions with osteoclast lineage cells, are also
capable of modulating the osteoclastogenic activity of osteoblasts. We
demonstrate here that osteoblasts express TLR9, the receptors for CpG
ODNs. Furthermore, the interaction of the oligonucleotides with
osteoblasts elicits phosphorylation of ERK and p38, activation of
NF
B, and modulation of genes participating in the regulation of
osteoclastogenesis. Gliotoxin blocks the activation of NF
B by
ODN 1826, the induction of TNF-
and RANKL, and osteoclast
differentiation. In fact, also the basal osteoclastogenesis (in the
absence of ODN 1826) is inhibited, consistent with the role of NF
B,
TNF-
, and RANKL in basal osteoclastogenesis. Methylene blue uptake
was not affected (not shown), ruling out the possibility that the
reduction in osteoclast number is caused by toxicity of the gliotoxin.
Modulation of TNF-
, M-CSF, and IL-1 expression in osteoblasts by CpG
ODN was less efficient than the activity exerted by LPS. Moreover, CpG
ODN did not markedly affect RANKL expression in osteoblasts, in
contrast to LPS. It is of note that TLR9 abundance in osteoblasts is
significantly lower than in osteoclast precursors, whereas TLR4 levels
are comparable in the two cell lineages. This might be the reason that
in osteoblasts the effects mediated by TLR4 are stronger than those
mediated by TLR9. All effects of CpG ODN were stronger in the
co-culture than in osteoblasts alone. Moreover, in the co-culture CpG
ODN was able to up-regulate RANKL mRNA abundance. Using collagenase
treatment of the co-cultures, we analyzed separately the osteoblasts
and the osteoclast lineage cells and showed that the CpG ODN-induced
increase in RANKL expression is in the osteoblasts. Using anti-TNF-
antibody and IL-1ra, we found that CpG ODN induces RANKL via TNF-
(but not IL-1
) that was shown previously to increase RANKL (44, 45).
In osteoblasts alone, the increase in RANKL by the ODN is moderate, due
to the relatively low levels of TNF-
. Consistent with this, when a
higher density of osteoblasts is examined the level of RANKL induction by the ODN is more pronounced (not shown). Our results indicate that
CpG ODN-induced RANKL expression is mediated via autocrine and
paracrine mechanisms, due to TNF-
produced by osteoblasts and
osteoclast lineage cells, respectively. Under the experimental conditions that we use, the paracrine mechanism is dominant.
Our findings show that CpG ODN interacts with osteoblasts and modulates
their osteoclastogenic activity. The expression of TLR9 by osteoblasts,
and the ability of chloroquine to inhibit the ODN effects indicate the
involvement of TLR9 in mediating the CpG ODN interaction with
osteoblasts. The comparison with the effects of LPS shows that ligation
of either TLR4 or TLR9 has an impact on osteoclastogenesis via
interactions with both osteoclast precursors and osteoblasts. It is of
note, however, that there are differences between the activities of the
TLR4 ligand, LPS, and of the TLR9 ligand, CpG ODN. Thus, the mechanisms by which TLR ligands lead to osteolysis in diseases such as
periodontitis and rheumatoid arthritis could include interactions of
the bacteria-derived products (LPS and CpG ODN representing bacterial
DNA) with osteoblasts.
CpG ODNs interactions with bone cells result in "pro" and
"anti" osteoclastogenic signals (increase in TNF-
and RANKL
expression and reduction in osteoclastic M-CSF receptors,
respectively). In vivo studies are underway to examine if
the net result of the ODNs administration will be increased or
decreased osteoclastogenesis.
Clinical implications of CpG ODN-mediated effects are directed toward
modulation of immune functions. Several therapeutic concepts have been
developed, including the use of CpG ODN as adjuvant for vaccine therapy
in infectious disease and cancer (65). It is important to know whether
CpG ODN therapy has an impact on bone resorption. It is possible that
this therapy is associated with increased bone loss, due to increased
circulating RANKL levels. This is highly relevant for CpG ODN-mediated
immunotherapy in general. Furthermore, a better understanding of the
interaction of CpG ODN with bone cells, osteoclasts and osteoblasts,
might also potentially lead to new therapies to treat bone disease.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M212473200
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