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INTRODUCTION |
Joint destruction because of matrix degradation and excessive bone
loss characterizes inflammatory bone diseases such as osteolysis, osteoarthritis, and rheumatoid arthritis (1-4). Accumulation of
inflammatory cells and their secreted products at the inflammation site
attracts osteoclasts and their precursor cells, leading to further
deterioration of the bone component (5-7). Tumor necrosis factor-
(TNF),1 interleukin-1 (IL-1),
and receptor activator of NF-
B ligand (RANKL, also known as
OPGL and ODF), are abundant in sites of inflammation and are known to
promote osteoclast recruitment, differentiation, and activation
(8-12). Osteoclast differentiation per se requires
activation of the RANK/RANKL pathway (13, 14). Recent evidence points
out that RANKL is also secreted by T helper 1 lymphocytes, the cells
responsible for secretion of the pro-inflammatory cytokines TNF and
IL-1 (12). More importantly, a direct role of T cell secreted RANKL in
promoting joint inflammation, bone, and cartilage destruction has been
established (12). Thus, RANKL and TNF may orchestrate bone and tissue
dissolution in inflammatory bone diseases.
In general, TNF receptor family members when activated recruit TNF
receptor-associated factor (TRAF) proteins to their cytoplasmic tail.
Acting as adaptor proteins, TRAFs bind to and activate several downstream tyrosine and serine/threonine kinases, including
c-Src, Akt/PKB, and MEKK-1. These in turn prompt activation,
primarily via phosphorylation events, of a signalsome-residing
molecules, such as (a) activation of I
B kinase
I
B
NF-
B pathway, (b) MEKK1
MEK
ERK,
and (c) MEKK1
JNK
c-Jun/AP-1. RANKL transmits its signal via a member of the TNF receptor family, RANK (15). The
complete repertoire of RANK intracellular signaling is unclear; however, similar to other TNF receptors, it recruits members of the
TRAF adapter proteins and activates AP-1 and NF-
B (16, 17).
Activation of these transcription factors entails phosphorylation of
inhibitory proteins followed by release and nuclear translocation of
the transcription factors. Thus, a considerable overlap exists in the
signal transduction pathways transmitted by RANKL and TNF receptors.
TNF (18, 19) recognizes two receptors: TNFr1 (also known as p55) and
TNFr2 (also known as p75) (20). We have previously shown that TNFr1
promotes osteoclastogenesis (21, 22), whereas TNFr2 is inhibitory.
Among the important events following TNF and RANKL occupancy of their
respective receptors, as already mentioned, are mobilization of NF-
B
and c-Jun/AP-1 (16, 17, 23). The pivotal role that NF-
B plays in
osteoclast recruitment is established by the fact that mice lacking
both the p50 and p52 NF-
B subunits develop a form of osteopetrosis
in which the animal is completely devoid of osteoclasts (24, 25).
Likewise, ablation of the c-fos component of AP-1
resulted in a similar osteopetrotic bone phenotype (26). Finally,
dominant-negative blockade of ERK signaling dampens osteoclastogenesis
(27). We and others have shown that inflammatory osteolysis reflects
TNF-induced recruitment of osteoclasts that resorb alveolar bone
leading to edentulism (21). Abundant RANKL in sites of bone erosion
further suggests that local differentiation and activation of
osteoclasts by pro-inflammatory cytokine is a likely occurring event.
Because TNF is also abundant in inflamed bone sites and plays a major role in progression of the disease, understanding the molecular mechanisms by which this inflammatory cytokine accelerates RANK-induced osteoclastogenesis will provide the basis for prevention of
inflammatory bone osteolysis.
In this study, we show that TNF closely regulates RANK/RANKL-induced
osteoclastogenesis. TNF markedly accelerates basal osteoclastogenesis induced by RANKL via its TNFr1. We also find that RANKL and TNF ultimately induce expression and recruitment of TRAF2, TRAF6, MEKK-1,
and c-Src. These events led to TNFr1-dependent activation of NF-
B, ERK, and c-Jun/AP-1, induction of RANK, and osteoclastogenesis.
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EXPERIMENTAL PROCEDURES |
Reagents--
All antibodies were purchased from Santa Cruz
(Santa Cruz, CA). Recombinant murine TNF and M-CSF were
purchased from R & D Systems Inc. (Minneapolis, MN). RANKL was
produced in Escherichia coli by fusion of the region from
lysine 158 to the C terminus of the mouse cDNA to thioredoxin and
will be described in detail elsewhere. ECL kit was obtained from
Pierce. All other chemicals were obtained from Sigma.
Animals--
C3H/HeN males were purchased from Harlan Industries
(Indianapolis, IN). Knockout mice for TNF receptors p55 and p75 and
their wild type controls were provided by Drs. Warner Lesslauer
(Hoffman-LaRoche) and Mark Moore (Genentech Inc., South San Francisco,
CA), respectively.
Cell Culture--
Bone marrow macrophages were isolated from
whole bone marrow of 4-6-week-old mice and incubated in tissue culture
plates, at 37 °C in 5% CO2, in the presence of 10 ng/ml
M-CSF (28). After 24 h in culture, the nonadherent cells were
collected and layered on a Ficoll-Hypaque gradient. Cells at the
gradient interface were collected and plated in
-minimum essential
medium, supplemented with 10% heat-inactivated fetal bovine serum, at
37 °C in 5% CO2 in the presence of 10 ng/ml M-CSF, and
plated according to each experimental conditions.
Osteoclast Generation--
Purified marrow macrophages were
cultured at 1 × 106 cells/ml in the presence of 10 ng/ml M-CSF and 20 ng/ml RANKL for 4 days. Cultures were supplemented
with M-CSF and RANKL on day 2 of culture.
Immunoblotting--
Total cell lysates were boiled in the
presence of 2× SDS-sample buffer (0.5 M Tris-HCl, pH 6.8, 10% (w/v) SDS, 10% glycerol, 0.05% (w/v) bromphenol blue, distilled
water) for 5 min and subjected to electrophoresis on 8-12%
SDS-polyacrylamide gel electrophoresis (29). Proteins were transferred
to nitrocellulose membranes using a semi-dry blotter (Bio-Rad) and
incubated in blocking solution (10% skim milk prepared in
phosphate-buffered saline containing 0.05% Tween 20) to reduce
nonspecific binding. Membranes were washed with phosphate-buffered
saline/Tween buffer and exposed to primary antibodies (1 h at room
temperature up to overnight at 4 °C), washed again four times, and
incubated with the respective secondary horseradish
peroxidase-conjugated antibodies (1 h, room temperature). Membranes
were washed extensively (5 × 15 min), and an ECL detection assay
was performed following the manufacturer's directions.
Electrophoretic Mobility Shift Assay--
Nuclear fractions were
prepared as described previously (30, 31). In brief, monolayers of bone
marrow macrophages grown in 100-mm2 tissue culture dish
were washed twice with ice-cold phosphate-buffered saline. Cells were
lifted from the dish by treating with 5 mM EDTA and 5 mM EGTA in phosphate-buffered saline. Cells were then resuspended in hypotonic lysis buffer A (10 mM HEPES, pH
7.8, 10 mM KCl, 1.5 mM MgCl, 0.5 mM
dithiothreitol 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 5 µg/ml leupeptin) and incubated on ice for 15 min, and
Nonidet P-40 was added to a final concentration of 0.64%. Nuclei were
pelleted, and the cytosolic fraction was carefully removed. The nuclei
were then resuspended in nuclear extraction buffer B (20 mM
HEPES, pH 7.8, 420 mM NaCl, 1.2 mM MgCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol,
0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml
pepstatin A, and 5 µg/ml leupeptin), vortexed for 30 s and
rotated for 30 min in 4 °C. The samples were then centrifuged, the
nuclear proteins in the supernatant were transferred to fresh tubes,
and protein content was measured using standard BCA kit (Pierce).
Nuclear extracts (10 µg) were incubated with an end-labeled double
stranded oligonucleotide probe containing the sequence 5'-AAA CAG GGG
GCT TTC CCT CCT C-3' (32) derived from the
B3 site of the TNF
promoter or with commercial cJun/AP-1 oligonucleotide (Santa Cruz). The
reaction was performed in a total of 20 µl of binding buffer (20 mM HEPES, pH 7.8, 100 mM NaCl, 0.5 mM dithiothreitol, 1 µg of poly(dI-dC), and 10%
glycerol) for 30 min at room temperature. Samples were then
fractionated on a 4% polyacrylamide gel and visualized by exposing
dried gel to film.
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RESULTS |
TNF Accelerates RANKL-induced Osteoclastogenesis by Bone Marrow
Macrophages via Its Type 1 Receptor--
We have shown that TNF
stimulates osteoclastogenesis in whole marrow cultures and in
co-culture of osteoclast precursors with stromal cells (21, 22). Basal
osteoclastogenesis in these two systems was induced by
1,25-dihydroxyvitamin D3. Given the presence of both
stromal and osteoclast precursors in those cultures, it is not clear
which cell type is targeted by TNF. To address this issue, a pure
population of osteoclast precursors, in the form of
monocytes/macrophages, was cultured in the presence of RANKL and M-CSF
for 4 days. Cells were then treated with carrier or TNF for 24 h,
after which cultures were fixed and stained for tartrate-resistant acid
phosphatase, a hallmark of osteoclasts (21). We found that TNF
dramatically enhances basal osteoclastogenesis by RANKL-induced bone
marrow macrophages (Fig. 1). A 4-5-fold increase in the number of multi-nucleated osteoclasts and a 4-12-fold increase in cell size were observed. The average number of osteoclasts in RANKL-treated wild type cultures (quadruplicate wells ± S.D., n = 3) was 134 ± 21 compared with 732 ± 115 in
RANKL + TNF-treated cells (p < 0.001).

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Fig. 1.
TNF accelerates RANKL-induced
osteoclastogenesis via its type 1 receptor. Osteoclast precursor
cells were isolated from the bone marrow of 4-6-week-old mice (wild
type, TNFr1 , and TNFr2 ) as described under "Experimental
Procedures." Pure marrow macrophages (>90%) were plated in 48-well
plates at 1 × 106 cells/ml using -minimum
essential medium supplemented with 10% heat-inactivated fetal calf
serum and 10 ng/ml M-CSF. Cultures were placed at 37 °C with 5%
CO2 incubator and were treated with 20 ng/ml soluble RANKL
for 3-4 days. TNF (10 ng/ml) was then added to one half of the
cultures for an additional 24 h. Developing osteoclasts were then
fixed and stained for tartrate-resistant acid phosphatase
(TRAP) activity following the manufacturer's directions.
Tartrate-resistant acid phosphatase-positive (purple) mono
and multi-nucleated large cells are osteoclasts and their committed
precursors. Results represent three independent experiments.
Images represent 20× magnification taken by light microscope.
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TNF transmits its biological signals via two receptors, TNFr1 and
TNFr2, and we have shown previously that TNFr1 is the pro-osteoclastic moiety (21, 22). Using cells derived from mice lacking individual TNF
receptor, we found that cells deleted of TNFr1 generate far less
osteoclasts in response to RANKL than their wild type counterparts (52 ± 14 versus 134 ± 21, respectively;
p < 0.005) (Fig. 1). More importantly, addition of TNF
to wild type cells while generating exuberant osteoclast response
overnight (top right panel), it failed to impact
osteoclastogenesis by TNFr1-null cells (middle right panel).
Number of osteoclasts in RANKL-treated TNFr1 conditions was 52 ± 14, which was statistically not different from the number of
osteoclasts from TNFr1 treated with RANKL and TNF (61 ± 21 osteoclasts). Furthermore, size and multi-nucleation of osteoclasts in
TNFr1 knockout cells were not affected by TNF treatment.
In contrast, osteoclastogenesis by cells lacking TNFr2 yet expressing
TNFr1 resumed in a similar manner seen in wild type cells (bottom
panels). Consistent with our previous observations (22), we found
an increased number of osteoclasts (189 ± 17) from TNFr2 mice in
basal conditions (+RANKL), which was dramatically increased with TNF
addition (847 ± 79, p < 0.001). Increased basal and TNF-induced osteoclastogenesis by TNFr2-null cells is consistent with the role of this receptor as an osteoclast suppressor (22) and
further enforces the requirement of TNFr1 for normal osteoclastogenesis.
TNFr1 Regulates Expression of TRAF2, TRAF6, c-Src, and MEKK-1 in
Osteoclasts--
We next turned to investigate the molecular
pathway(s) by which TNF accelerates RANKL-induced osteoclastogenesis.
Signaling of TNF and RANKL requires recruitment of TRAFs and activation of subsequent kinases. Thus, we examined the expression of these proteins in osteoclasts differentiated in vitro compared
with their precursors. We found that osteoclasts express elevated
levels of TRAF2, TRAF6, c-Src, and MEKK-1, whereas expression of these proteins by untreated precursor cells was negligible (Fig.
2A). Contrary to that, we
found that expression of these proteins in TNFr1-null osteoclasts is
reduced. These data are consistent with the pro-osteoclastic role of
TNFr1.

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Fig. 2.
A, higher expression of TRAF6, TRAF2,
c-Src, and MEKK1 in osteoclasts compared with their precursors is
TNFr1-dependent phenomenon. Macrophages (Mac)
from wild type or TNFr1 knockout mice were treated with M-CSF alone or
in combination with RANKL for 4 days to generate osteoclasts
(OC). Cells were then lysed in sample buffer, and expression
of TRAF6, TRAF2, c-Src, and MEKK1 was assessed in macrophages
versus osteoclasts using immunoblots. B,
osteoclastogenic agents induce the expression of TRAF6, TRAF2, and
MEKK1 by macrophages. Cells were cultured for 3 days with M-CSF after
which they were treated overnight with RANKL (20 ng/ml), TNF (10 ng/ml), lipopolysaccharide (10 ng/ml), IL-1 (20 ng/ml), and IL-1
(20 ng/ml). Cells were then lysed and subjected to immunoblots with
anti-TRAF6, TRAF2 and MEKK1 antibodies. Results represent three
independent experiments.
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Having established the expression levels of TRAF2, TRAF6, and MEKK1 in
osteoclasts, we examined expression of these proteins in precursor
cells in response to RANKL, TNF, and other pro-osteoclastic agents
following 24 h of treatment. We found that expression of TRAF2,
TRAF6, and MEKK1 is highly induced in wild type cells by RANKL, TNF,
and pro-osteoclastic agents, such as lipopolysaccharide and IL-1 (Fig.
2B). These observations point out that expression of these
proteins is likely essential for osteoclast differentiation evident by
their reduced expression in TNFr1-null osteoclasts, which were poorly
generated with RANKL (Fig. 2A). These findings further
support the possibility that TNFr1 is essential for TNF-enhanced activation of the RANK/RANKL pathway.
RANKL Activation of Erk and c-Jun Is Diminished in the Absence of
TNFr1--
Recruitment of TRAF2, TRAF6, and MEKK1 to the cytoplasmic
tail of TNF receptor family members facilitates binding and activation of multiple downstream kinases. These in turn, lead to activation of
(a) MEKK1
JNK
c-Jun/AP-1, (b) MEKK1
MEK
ERK, and (c) TRAFs
I
B kinases
I
B
NF-
B signaling pathways, all of which are essential for
osteoclast differentiation and survival (24, 26, 27). Thus, we asked
whether, similar to the impaired TRAFs and MEKK1 expression observed in
TNFr1-null cells, one or more of these downstream pathways are also impaired.
To this end we first examined expression and phosphorylation of MEK1
and ERK1/2 in RANKL-treated wild type and TNFr1 knockout cells.
Activation of MEK1 is manifested by its phosphorylation, at least in
part by MEKK1. MEK1 then phosphorylates ERK1/2, leading to their
dimerization and nuclear translocation. Reflecting reduced expression
of MEKK1 in RANKL-generated TNFr1-null osteoclasts, we found that MEK1
phosphorylation induced by RANKL is significantly reduced in TNFr1-null
compared with wild type cells (Fig.
3A). Hypophosphorylation of
the mitogen-activated protein kinase is specific because MEK1 protein
was equally expressed in all conditions. Similarly, we found that
ERK1/2 proteins are equally expressed in both cell types (Fig.
3B), and their expression is unaffected by the addition of
RANKL. Interestingly, however, and despite the equal expression of the
protein in both cell types, phosphorylation of the mitogen-activated
protein kinases resembled that observed with the upstream kinase MEK1.
Namely, although ERK1/2 undergo rapid phosphorylation within 10 min,
which is sustained up to 4 h after exposure to RANKL, little or no
phosphorylation was observed in RANKL-treated TNFr1-null cells.
Residual and transient phosphorylation of ERK1/2 in TNFr1 knockout
cells was detected only after 40 min of exposure to RANKL. This finding
correlates well with reduced expression of MEKK1 in TNFr1-null
osteoclasts (generated with RANKL) (Fig. 2A). Moreover,
given the established role of ERK activation as essential for
osteoclast activation and survival (27), these findings support the
notion that TNFr1 is required for normal osteoclastogenesis.

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Fig. 3.
RANKL induces phosphorylation of MEK1 as well
as ERK1 and 2 in a TNFr1-dependent manner. Osteoclast
precursors (macrophages) from wild type (WT) and TNFr1
knockout mice were treated with RANKL for the time points indicated.
Cell lysates were then subjected to western immunoblots using anti-MEK1
and phosphoMEK1 antibodies (A) and anti-ERK1/2 and
phosphoERK1/2 antibodies (B).
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MEKK-1, via JNK, also leads to c-Jun phosphorylation and nuclear
translocation. Using electrophoretic mobility shift assay, we provide
evidence that c-Jun is activated in osteoclast precursors induced by
RANKL (Fig. 4). More importantly, we
found that activation of c-Jun/AP-1 by RANKL is severely reduced in
TNFr1-null compared with wild type cells. Mirroring its
pro-osteoclastic effect (Fig. 1), TNF alone, although activating the
transcription factor in wild type cells as expected (Fig. 4,
lanes 6 and 7), fails to trigger such response in
TNFr1-null cells (Fig. 4, lanes 12-14). Lack of
transcription factor activation in TNFr1-null cells is in keeping with
its principal role in induction of RANKL-mediated osteoclastogenesis.

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Fig. 4.
Activation of cJun/AP-1 transcription factor
by RANKL and TNF is severely reduced in TNFr1 knockout cells. Wild
type and TNFr1- cells were treated with RANKL or TNF for the time
points indicated. Nuclear extracts were then prepared and subjected to
electrophoretic mobility shift assay using a cJun/AP-1 labeled
oligonucleotide.
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RANKL-induced Phosphorylation of I
B and Activation of NF-
B Is
TNFr1-dependent--
NF-
B is essential for osteoclast
formation. Activation of this transcription factor is a consequence of
I
B phosphorylation and dissociation of the I
B/NF-
B complex
followed by nuclear translocation of NF-
B and binding to DNA (23).
We have documented that expression of the adaptor proteins TRAF2 and
TRAF6 is impaired in RANKL-generated TNFr1 knockout osteoclasts (Fig.
2), a phenomenon consistent with reduced osteoclastogenesis by the same
cells (Fig. 1). Thus, we asked whether reduced osteoclastogenesis in
RANKL-treated TNFr1-null cells also reflect impaired NF-
B
activation, a process mediated through TRAF protein recruitment (23).
First, we document that RANKL induces rapid phosphorylation of I
B
manifested by the indicated slow migrating band (Fig.
5A, I
B-p). This
phosphorylation persists for 2 h and diminishes after 3 h.
Reflecting I
B phosphorylation, our data show that similar to TNF,
RANKL activates NF-
B in wild type cells, evidenced by increased DNA
binding activity with time (Fig. 5B, lanes 2-4).
In contrast and enforcing the role of TNFr1 in RANKL-induced
osteoclastogenesis, we found that I
B is hypophosphorylated (Fig.
5A) and that NF-
B nuclear translocation and DNA binding activity are severely reduced in cells lacking TNFr1 (Fig.
5B, lanes 8-14). Interestingly, in TNFr1-null
cells, activation of NF-
B was severely reduced in the presence of
RANKL and absent following TNF treatment. These data, once again,
highlight the essential role of TNFr1 as an activator of NF-
B, a
transcription factor critical for osteoclast formation.

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Fig. 5.
RANKL induces phosphorylation of
I B and activates NF- B
in a TNFr1-dependent manner. Wild type (WT)
and TNFr1 knockout cells were treated with RANKL as shown.
A, cells were then lysed and subjected to immunoblots with
an I B antibody that recognizes both native and phosphorylated form
of the protein. B, nuclear extracts were prepared and
electrophoretic mobility shift assay was performed using
32P-labeled NF- B oligonucleotide. Specificity of the
shifted band was documented previously (30) by competition with excess
unlabeled and mutated forms of this oligo.
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TNFr1 Regulates RANK--
Thus far, our data indicate that
signaling of osteoclastogenesis by TNFr1 and RANK is seemingly
overlapping, and activation of TNFr1-dependent events
accelerates RANKL-induced osteoclastogenesis. More specifically,
deletion of TNFr1 dampens RANKL-induced expression, phosphorylation,
and activation of key proteins required for normal osteoclastogenesis,
such as TRAFs, MEKK1, c-Src, I
B, NF-
B, AP-1/c-Jun, and ERK
proteins. Thus, it is reasonable to hypothesize that endogenous TNFr1
signaling regulates RANK expression/function. To address this issue, we
examined RANK expression by TNFr1-null cells. To this end, marrow
macrophages from wild type and TNFr1-null cells were cultured for 4 days in the absence or presence of RANKL, and levels of RANK expression
were assessed by immunoblots. We found that expression of RANK is
reduced in the absence of TNFr1 (Fig. 6,
lanes 1-4) and remain low in the presence of RANKL and/or TNF (Fig. 6, lanes 2 and 3). In contrast, levels
of RANK were elevated in wild type cells (Fig. 6, lane 5)
and were synergistically increased when treated with RANKL and TNF
(lane 8). These observations suggest that signals transmitted by the
TNFr1, likely to be NF-
B and/or AP-1-dependent genes,
regulate RANK expression.

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Fig. 6.
TNF, acting through its type 1 receptor,
induces RANK expression synergistically with RANKL. Wild type and
TNFr1 knockout cells were treated with vehicle, TNF, RANKL, or both
cytokines for 24 h. Cells were then lysed, and RANK expression was
measured by immunoblots of equal amounts of total cell proteins. Equal
protein loading was further confirmed by incubating nitrocellulose
membrane in Ponceau S solution.
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DISCUSSION |
Osteoclastogenesis is mediated via ligation of RANKL to its
transmembranal receptor, RANK (13, 14). Furthermore, osteoclast recruitment and activation is markedly induced in states of
inflammatory bone diseases (5-7). In this regard, we and others have
documented a primary role for TNF in stimulating osteoclastogenesis
in vitro and in vivo (21, 22, 33, 34). The recent
discovery of RANK signaling pathway in osteoclasts and their precursors
enables us to investigate the direct effect, if any, of pro- and
anti-inflammatory factors on RANK-induced osteoclastogenesis.
In this study, we established that TNF acts directly on osteoclast
precursors, thus providing a clear target to prevent TNF signaling in
states of bone inflammation. Similar to TNF, RANKL signaling involves
recruitment and activation of key proteins essential for
osteoclastogenesis, some of which include TRAFs, MEKK1, c-Src, I
B
kinases, ERKs, c-Jun, and NF-
B (16, 17). The obvious overlap between
TNF and RANKL stimulation of their respective pathways, their abundance
in sites of bone inflammation, and their documented pro-osteoclastic
role prompted us to investigate possible cooperative signaling between
TNF receptors and RANK with regard to events leading to
osteoclastogenesis. We found that levels of TRAF2, TRAF6, and MEKK1 are
increased in response to treatment of precursor cells with osteoclastic
agents, such as RANKL, TNF, IL-1, and lipopolysaccharide, and parallel
their high level of expression in differentiated osteoclasts.
Interestingly, and in support of its essential role in
osteoclastogenesis (35, 36), we found expression of TRAF6 is
significantly higher than TRAF2. In any case, expression of TRAF2 and
TRAF6 by RANKL or TNF-treated macrophages and high expression of these
factors in fully differentiated osteoclasts is in keeping with their
essential role for c-Jun and NF-
B activation, respectively (37, 38). Similar to our previous findings that TNF mobilizes and activates c-Src
(21, 30), we show in this report that expression of the tyrosine kinase
is elevated in RANKL-generated osteoclasts.
We also report that RANKL induces expression of the upstream
mitogen-activated protein kinase, MEKK-1. MEKK-1 normally binds to and
is activated by TRAF2 (37). In its activated form, MEKK-1 induces and
activates, among other functions, the JNK and MEK1, leading to
c-Jun/AP-1 and ERK activation, respectively (37). Based on these
observations, it is reasonable to propose that RANKL-activated pathways
converge at the level of TRAF adapter proteins. According to this
scenario and consistent with previous observations (36, 37),
TRAF6-c-Src complex leads to NF-
B activation, whereas TRAF2-MEKK1
primarily activates the ERK and c-Jun pathways. These pathways are not
entirely distinct because of cross-talk at different stages (39,
40).
By analogy to RANKL and using similar mechanisms, TNF is capable of
inducing NF-
B and c-Jun/AP-1 (20, 23). TNF exerts its effects via
two receptors, TNFr1 and TNFr2. Both receptors share functional
properties, but it is the TNFr1 that has been implicated as
pro-osteoclastic (21, 22). Although its direct RANKL-independent
osteoclastogenic potential remains disputed (41, 42), our data indicate
that TNF exerts robust osteoclastogenesis by RANKL-primed
pre-osteoclasts. Supporting this observation is our finding that
deletion of the TNFr1 attenuates endogenous and in vitro
RANKL-mediated osteoclastogenesis subsequent to reduced NF-
B, ERK,
and cJun/AP-1 activation. Reduced activation of NF-
B, ERKs, and
cJun/AP-1 in TNFr1 knockout cells is most likely secondary to
hypophosphorylation of upstream regulatory proteins, such as I
B,
MEK, and JNK, two of which are documented in this study. These impaired
signals appear to be TNFr1-dependent, because basal osteoclastogenesis resumes in TNFr1-expressing TNFr2-null cells in a
manner greater than that seen by their wild type counterparts.
The most challenging of our findings is the endogenous effect of TNF on
RANK-mediated osteoclastogenesis. Although equal amounts of total
protein were examined, expression levels of RANK were very little in
TNFr1-null cells compared with wild type controls. Interestingly,
administration of either TNF or RANKL has little or no impact on RANK
expression in both species. In contrast, combined addition of TNF and
RANKL, while eliciting no effect on TNFr1-null cells, synergistically
induce RANK expression within 24 h. Thus, regulation of RANK is
under the aegis of TNFr1 and requires preactivation with RANKL.
First, our findings point out that basal osteoclast formation by RANKL
is reduced in the absence of TNFr1. Thus, endogenous expression of this
receptor is essential to maintain higher level of osteoclastic pool.
Further treatment with TNF of cells derived from the TNFr1-null mice
fails to augment osteoclastogenesis. Second, examination of NF-
B and
AP-1 transcription factors in TNFr1-null nuclear extracts points to
reduced DNA binding of both factors compared with wild type-derived
cells. This finding mirrors reduced osteoclastogenesis by TNFr1-null
cells. These observations are in keeping with the essential role of
NF-
B and AP-1 transcription factors in osteoclast differentiation
and activation (16, 17, 26, 43). Third, the finding that TNFr1
transmits signals that up-regulate RANK expression and support
osteoclastogenesis favorably argues that RANK gene expression may be
regulated by NF-
B and/or AP-1 transcriptional machinery.
In conclusion, our findings suggest that overlapping signaling
mechanisms of RANKL and TNF result in exuberant osteoclastogenesis and
may mediate severe bone resorption. TNFr1 is required for both basal
RANK expression/signaling and for the cytokine-enhanced phase of osteoclastogenesis.