From the CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, and Division of Oral Biology, Faculty of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
Received for publication, June 28, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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RANK ligand (RANKL) induces
activation of NF RANK1 ligand (RANKL) is
a member of the tumor necrosis factor superfamily that plays an
essential role in osteoclastogenesis, as well as the activation and
survival of mature osteoclasts. This factor is expressed on
osteoblasts, stromal cells, B-lymphoid lineage cells, and activated
T-cells as a transmembrane ligand and it also exists in a biologically
active soluble form (1-3). RANKL acts through its receptor RANK, which
is expressed on osteoclast precursors, mature osteoclasts, as well as
dendritic cells (4). Osteoprotegerin (OPG) is a soluble decoy receptor,
which binds RANKL and blocks its interaction with RANK (4).
Signaling through RANK involves the recruitment of cytosolic tumor
necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6, which in turn activate multiple signaling pathways (5-7). For example,
the association of RANK with TRAF2 induces activation of c-Jun
N-terminal kinase, which leads to phosphorylation of c-Jun and
activation of AP-1 (7-9). TRAF6 has been implicated in activation of
the nonreceptor tyrosine kinase c-Src and the transcription factor
NF NF Interaction of RANKL with RANK is crucial for osteoclast function,
however, there are gaps in our understanding of the signaling events
leading to activation of NF We tested the hypothesis that RANKL signaling in osteoclasts involves
elevation of [Ca2+]i, and examined the role of
cytosolic Ca2+ in cell survival and activation of NF Osteoclast Isolation and Culture--
Osteoclasts were isolated
from the long bones of neonatal Wistar rats or neonatal New Zealand
White rabbits as described previously (19). Briefly, long bones were
dissected free of soft tissue and cut with a scalpel to release bone
fragments into 2-3 ml of osteoclast culture medium that consisted of
Medium 199 buffered with 25 mM HEPES and
HCO Test Substances--
Soluble RANKL (murine recombinant 158-316)
and OPG (human recombinant 21-194 fused at the N terminus to the Fc
domain of human IgG1) were kindly provided by Amgen (Thousand Oaks, CA)
and RANKL (human recombinant 151-316 fused at the N terminus to a
linker peptide and a FLAG tag) was purchased from Alexis Corp. (San
Diego, CA). U73122 and U73343 were obtained from Calbiochem (La Jolla,
CA), dissolved in chloroform, aliquoted, evaporated under N2, and stored at Fluorescence Measurement of Cytosolic Free Ca2+
Concentration--
[Ca2+]i of single rat
osteoclasts and osteoclast precursors was monitored using
microfluorimetric techniques. Cells on glass coverslips were loaded
with 1.5 µM fura-2-AM (Molecular Probes) for 40 min at
room temperature in loading medium. Coverslips were then placed in a
chamber mounted on the stage of a Nikon Diaphot inverted phase-contrast
microscope, and superfused at room temperature with physiological
buffer containing (in mM): NaCl, 130; KCl, 5; glucose, 10;
MgCl2, 1; CaCl2, 1; HEPES, 20; adjusted to pH
7.4 with NaOH; 280-290 mOsmol/liter. The ratio of fluorescence
emission at 510 nm with alternate excitation wavelengths of 345 and 380 nm was measured using a Deltascan illumination system (Photon
Technology International, London, ON, Canada) as described previously
(21). RANKL was applied locally to cells by pressure ejection from a
micropipette. In some studies, cells were superfused with a
Ca2+-free physiological buffer, supplemented with 0.5 mM EGTA.
Electrophysiology--
The whole cell patch clamp configuration
was used to record membrane currents as described previously (22).
Electrode solution contained (in mM) KCl, 140; HEPES, 20;
MgCl2, 1; EGTA, 0.1; adjusted to pH 7.2 with KOH; 280-290
mOsmol/liter. Pipette resistance before seal formation was 3-5 M NF Statistical Analyses--
Data are presented as representative
traces, as percentages of total cells tested, or as mean ± S.E.
with sample size (n) indicating the number of osteoclasts
for Ca2+ fluorescence or electrophysiology studies, or the
number of separate cell preparations for immunofluorescence studies.
Differences were assessed by one-way analysis of variance for
correlated samples, followed by a Tukey or Bonferroni test and accepted
as statistically significant at p < 0.05. Sigmoid
curves were fit by nonlinear regression using Prism (GraphPad Software,
Inc., San Diego, CA). Error bars were omitted where they were smaller
than the symbol.
RANKL Induces Elevation of Cytosolic Free
Ca2+--
Rat osteoclasts were loaded with fura-2, and
Ca2+ was monitored by microspectrofluorimetry. Osteoclasts
had basal [Ca2+]i of 154 ± 4 nM
(n = 118, mean ± S.E.). Osteoclasts responded to
soluble RANKL with elevation of [Ca2+]i, which
typically peaked and then declined slowly, even in the continued
presence of RANKL. Upon washout of RANKL, [Ca2+]i
returned promptly to basal levels (Fig.
1A). Multiple [Ca2+]i transients could be elicited by
successive applications of RANKL (Fig. 1B), although the
subsequent responses were slightly decreased in amplitude. No responses
were observed when osteoclasts were stimulated with vehicle
(n = 25). Moreover, OPG blocked the ability of RANKL to
induce [Ca2+]i elevations in osteoclasts that
were responsive to multiple applications of RANKL alone
(n = 3).
The proportion of osteoclasts responding to RANKL with elevation of
[Ca2+]i was dependent on the concentration of
RANKL (Fig. 1C). [Ca2+]i elevations
were elicited by concentrations of RANKL as low as 10 pg/ml. The
maximum proportion of osteoclasts (~60%) responded to RANKL at
10-100 ng/ml, with half-maximal effects at ~0.1 ng/ml. When the
amplitudes of the RANKL-induced Ca2+ transients were
quantified, similar concentration dependence was observed (Fig.
1D). At concentrations of 10-100 ng/ml, RANKL elevated
[Ca2+]i to peaks of 220 ± 30 nM
above basal (based on 15 responsive osteoclasts of 24 tested). We also
assessed changes in Ca2+ upon application of RANKL to rat
osteoclast precursors. Even at concentrations of 1 µg/ml, RANKL
caused elevation of [Ca2+]i in only 3 of 27 osteoclast precursors tested, whereas 11 of 17 multinucleated
osteoclasts, tested in the same preparations, responded with elevation
of Ca2+. As a negative control, we tested the responses of
spindle-shaped stromal cells and found that none of the 12 cells tested
responded to RANKL. Thus, a proportion of osteoclast precursors
responded to RANKL with elevation of [Ca2+]i,
although the percentage of responsive precursors was significantly
lower than that of mature osteoclasts. All subsequent studies were
performed using multinucleated osteoclasts.
We next investigated the source of Ca2+ contributing to
RANKL-induced elevation of [Ca2+]i in
osteoclasts. RANKL elicited Ca2+ elevations of comparable
amplitude in Ca2+-containing and Ca2+-free
extracellular solutions, consistent with release of Ca2+
from intracellular stores (Fig. 2,
A and B, n = 5). Ca2+
release from stores often involves PLC-mediated production of inositol
1,4,5-trisphosphate. We have shown previously that the PLC inhibitor
U73122 blocks P2Y nucleotide receptor-mediated elevation of
[Ca2+]i in osteoclasts (21). Treatment of
osteoclasts with U73122 (1 µM for 10 min) abolished the
RANKL-induced rise of [Ca2+]i, whereas RANKL
still elicited [Ca2+]i elevations in the presence
of the control compound U73343 or vehicle (Fig. 2, C and
D, n = 5). Taken together, these data
indicate that RANKL signals through PLC leading to release of
Ca2+ from intracellular stores and transient elevation of
[Ca2+]i. Our findings are in contrast to previous
observations that RANKL caused sustained elevation of
[Ca2+]i in osteoclasts (23).
RANKL Activates Ca2+-dependent
K+ Current--
An independent approach was used to verify
the effect of RANKL on [Ca2+]i in osteoclasts.
Rat, rabbit, and human osteoclasts possess intermediate conductance
Ca2+-dependent K+ channels (24).
Because only a subpopulation of rat osteoclasts exhibits this current
(22), we used patch clamp techniques to monitor the effects of RANKL on
membrane currents of rabbit osteoclasts, which all demonstrate the
current (25). Cells were held at Role of Ca2+ in Osteoclast Survival--
It was shown
previously that RANKL prolongs osteoclast survival in vitro
(26). We investigated the role of Ca2+ in this process
using the intracellular Ca2+ chelator BAPTA. To establish
conditions for effective buffering of Ca2+ by BAPTA, we
used ATP, which activates P2Y nucleotide receptors on osteoclasts
leading to release of Ca2+ from intracellular stores and
reproducible elevation of [Ca2+]i (21).
Osteoclasts were stimulated with ATP (100 µM) to ensure
their responsiveness, then treated with different concentrations of
BAPTA-AM and rechallenged with ATP. We established that loading with 50 µM BAPTA-AM for 10 min at room temperature was optimal
for suppressing elevation of [Ca2+]i induced by
ATP (Fig. 4A,
n = 8). We then confirmed that, under these conditions,
BAPTA was effective in preventing RANKL-induced elevation of
[Ca2+]i (Fig. 4B).
To examine the role of [Ca2+]i in osteoclast
survival, cells were treated with BAPTA-AM or vehicle. The medium was changed and osteoclasts were incubated with RANKL (100 ng/ml) or
vehicle at 37 °C for 24 h. The number of osteoclasts per dish at 24 h was expressed as a percentage of the initial number of osteoclasts in the same dish. As expected, RANKL significantly increased the number of osteoclasts that survived 24 h (Fig.
4C, n = 4 independent experiments). BAPTA
suppressed this effect of RANKL, but did not affect osteoclast survival
in the absence of RANKL (Fig. 4C). Thus, elevation of
[Ca2+]i appears to be necessary for RANKL to
promote osteoclast survival.
Effect of PLC Inhibitor on RANKL-induced Nuclear Translocation of
NF
To examine a role of the PLC signaling pathway, we determined the
kinetics of RANKL-induced NF Effect of BAPTA on RANKL-induced Nuclear Translocation of
NF
Hydrolysis of BAPTA-AM results in release of small molecular weight
products because of the degradation of the acetoxymethyl ester
(AM) moieties. To determine whether these degradation products might affect NF Role of Calcineurin and Protein Kinase C in RANKL-induced Nuclear
Translocation of NF
PLC activation leads to the generation of 2 second messengers,
Ca2+ and diacylglycerol, both of which contribute to
activation of PKC. In other systems, PKC can activate I
The effects of cyclosporin A and bisindolylmaleimide I were additive at
7 min, however, the calcineurin inhibitor had no additional effect at
15 min (Fig. 7, data based on parallel samples from seven independent
experiments). Similarly, NF We demonstrate that RANKL induces transient elevation of
[Ca2+]i in osteoclasts because of activation of
PLC and release of Ca2+ from intracellular stores. The rise
of [Ca2+]i stimulates
Ca2+-dependent K+ current,
accelerates nuclear translocation of NF Half-maximal effects of RANKL on [Ca2+]i were
observed at ~0.1 ng/ml, with maximal actions at 10-100 ng/ml. These
findings are in keeping with the concentration dependence reported for the induction of osteoclastogenesis by RANKL (27, 28), suggesting that
elevation of [Ca2+]i is mediated by RANK.
Moreover, OPG prevented RANKL-induced elevation of
[Ca2+]i, ruling out possible nonspecific effects.
Repeated application of RANKL elicited multiple Ca2+
transients indicating lack of short term desensitization. In vivo, RANK signaling is thought to be mediated primarily by
interaction of osteoclasts and their precursors with cells expressing
RANKL (stromal cells, osteoblasts, and lymphocytes). Lack of
desensitization would allow osteoclasts to receive multiple signals
from neighboring cells, giving rise to temporal and spatial summation
of RANKL signals.
Our data demonstrate that RANK signaling in osteoclasts involves PLC.
PLC- As in other cell types, cytosolic Ca2+ likely plays
important roles in regulating a number of osteoclast functions. We have shown that RANKL activates the intermediate conductance
Ca2+-dependent K+ current
(IKCa) in osteoclasts. Our previous studies have shown that
elevation of [Ca2+]i results in activation of
this current (22), so our electrophysiological results provide
independent evidence to support RANKL-induced elevation of
[Ca2+]i. In vivo, the membrane
hyperpolarization resulting from activation of IKCa would
modulate the activity of electrogenic ion transport systems and
increase the driving force for influx of Ca2+, a vital
process in T cell signaling (34). Similarly, IKCa may play
an important role in osteoclast regulation, especially considering that
IKCa channels are selectively expressed in rat osteoclasts
having the morphological characteristics of actively resorbing cells
(22). Ca2+ also regulates cytoskeletal organization through
Ca2+-dependent actin-binding proteins, such as
gelsolin, that play critical roles in osteoclast motility (35).
Therefore, RANKL-induced alterations in [Ca2+]i
could have multiple downstream effects in osteoclasts. In this regard,
we found that elevation of [Ca2+]i is necessary
for RANKL to prolong osteoclast survival. Although previous reports
have suggested that elevation of [Ca2+]i in
osteoclasts leads to inhibition of resorption (36), more recent studies
have implicated Ca2+-dependent pathways in
promoting osteoclast formation (37). In osteoclasts, as in other
systems, the effects of [Ca2+]i elevation likely
depend upon the pattern, amplitude, and duration of the
Ca2+ signal as well as its interactions with other
signaling pathways.
NF RANK signaling involves a cascade of events, beginning with the
recruitment of TRAFs, leading to activation of NF Calcineurin is a serine/threonine phosphatase that is stimulated by
Ca2+. The immunosuppressant drugs cyclosporin A and FK506
bind distinct immunophilins and inhibit the activity of calcineurin
(38). We found that both of these inhibitors delayed the initial phase of NF Stimulation of PLC also leads to increased activation of PKC. To
investigate the involvement of PKC, we used bisindolylmaleimide I, a
selective inhibitor of the conventional and novel PKC isoforms, and
carried out control studies using the inactive analog,
bisindolylmaleimide V. Inhibition of PKC also caused delay in NF Because the concurrence of multiple transcription factors regulates
expression of genes, the kinetics of their activation is of critical
importance. In this regard, the temporal pattern of NFB, enhancing the formation, resorptive activity, and
survival of osteoclasts. Ca2+ transduces many
signaling events, however, it is not known whether the actions of RANKL
involve Ca2+ signaling. We investigated the effects of
RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp.
RANKL induced transient elevation of cytosolic free Ca2+
concentration ([Ca2+]i) to maxima 220 nM above basal, resulting in activation of
Ca2+-dependent K+ current. RANKL
elevated [Ca2+]i in Ca2+-containing
and Ca2+-free media, and responses were prevented by the
phospholipase C inhibitor U73122. Suppression of
[Ca2+]i elevation using the intracellular
Ca2+ chelator
1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NF
B was found predominantly in
the cytosol of untreated osteoclasts. RANKL induced transient translocation of NF
B to the nuclei, which was maximal at 15 min. U73122 or BAPTA delayed nuclear translocation of NF
B. Delays were
also observed upon inhibition of calcineurin or protein kinase C. We
conclude that RANKL acts through phospholipase C to release Ca2+ from intracellular stores, accelerating nuclear
translocation of NF
B and promoting osteoclast survival. Such
cross-talk between NF
B and Ca2+ signaling provides a
novel mechanism for the temporal regulation of gene expression in
osteoclasts and other cell types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (10, 11).
B transcription factors are dimers of the five mammalian NF
B
proteins: p65 (RelA), RelB, c-Rel, p50 (NF
B1), and p52 (NF
B2). NF
B regulates the expression of a large number of genes involved in
cell survival as well as in cellular responses to inflammation and
stress (12, 13). Typically, NF
B exists as a heterodimer of p50 and
p65 (12). NF
B is retained in the cytoplasm complexed with inhibitory
proteins I
Bs. RANK signaling involves activation of NF
B-inducing
kinase, leading to activation of I
B kinases (IKK)
and
, which
in turn phosphorylate serine residues on I
B, targeting it for
degradation in the proteasome (5, 11, 14). I
B degradation exposes
the NF
B nuclear localization sequence, permitting its nuclear
import. Within the nucleus, NF
B acts in concert with other
transcription factors to regulate gene expression, with termination of
the signal caused by binding of I
B (15). NF
B is essential for
osteoclastogenesis, as disruption of both p50 and p52 subunits of
NF
B leads to an osteopetrotic phenotype, because of impaired
osteoclast differentiation (16).
B in response to RANKL. Although interaction of RANK with TRAF6 is necessary and sufficient to activate
NF
B, dominant negative forms of TRAF molecules are unable to
completely block NF
B activation, suggesting that a TRAF-independent pathway is also involved (5, 11). Because Ca2+-sensitive
effectors such as calcineurin and protein kinase C (PKC) mediate NF
B
activation in T lymphocytes and monocytic cell lines (17, 18), we
considered the possible role of Ca2+ in the activation of
NF
B by RANKL in osteoclasts.
B.
Classical biochemical approaches for studying osteoclasts are limited
because of difficulty in isolating cells in sufficient number and
purity. Furthermore, osteoclasts are terminally differentiated, and
therefore do not proliferate in culture. We overcame these restrictions
by studying authentic osteoclasts using single-cell techniques:
microspectrofluorimetry and patch clamp to study changes in
[Ca2+]i and membrane currents, and
immunofluorescence to assess nuclear translocation of NF
B. We report
that RANKL stimulates phospholipase C (PLC) leading to release of
Ca2+ from intracellular stores, transient elevation of
[Ca2+]i, and activation of
Ca2+-dependent K+ current. The
effect of RANKL on osteoclast survival was found to be dependent on
elevation of [Ca2+]i. Moreover, nuclear
translocation of NF
B was slowed when elevation of Ca2+
was suppressed or when calcineurin or PKC were inhibited. Thus, phospholipase C and Ca2+ signaling are revealed to be
important regulators of NF
B activation and osteoclast survival.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. On the day of each
experiment, U73122 and U73343 were reconstituted in dimethyl sulfoxide
and added to the physiological buffer or medium bathing the cells.
BAPTA-acetoxymethyl ester (AM) and calcein blue-AM were obtained from
Molecular Probes (Eugene, OR) and stock solutions were prepared in
dimethyl sulfoxide. Osteoclasts were loaded with the BAPTA or calcein
blue by incubation in loading medium (HCO
.
Cells were superfused in physiological buffer at room temperature.
Currents were recorded with Axopatch-1D amplifier, filtered, and
digitized at 2-5 kHz using pClamp 6.0 (Axon Instruments, Union City, CA).
B Localization by Immunofluorescence--
Osteoclasts on
glass coverslips were incubated with or without RANKL in osteoclast
culture medium at 37 °C and at the indicated times fixed with 4%
paraformaldehyde (10 min), washed in PBS (3× 5 min); permeabilized
with 0.1% Triton X-100 in PBS (10 min), washed in PBS (3× 5 min); and
blocked with 1% normal goat serum in PBS (NGS) for 1-2 h at room
temperature. Monoclonal antibody to p65 (catalog number sc-8008, Santa
Cruz Biotechnology, Santa Cruz, CA) was diluted 1:100 in NGS and
applied overnight at 4 °C, followed by washing in PBS and incubation
for 2 h at room temperature with biotinylated goat anti-mouse IgG
(Vector Laboratories Inc., Burlingame, CA) diluted 1:200 in NGS. After
incubation (2 h, room temperature) with fluorescein-conjugated
streptavidin (Vector Laboratories Inc.) (1:100 in NGS), coverslips were
washed, mounted on slides with Vecta-Shield (Vector Laboratories Inc.), and examined using a Zeiss LSM 510 laser-scanning confocal microscope. We assessed localization of fluorescent label in all osteoclasts on
each coverslip (usually 40-70 cells/coverslip). Osteoclasts were rated
positive for nuclear localization if fluorescence intensity of one or
more nuclei exceeded that of the cytoplasm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RANKL elicits [Ca2+]i
elevations in rat osteoclasts. Single rat osteoclasts were loaded
with fura-2, bathed in physiological buffer, and
[Ca2+]i was monitored by microspectrofluorimetry.
A, RANKL (100 ng/ml) was applied to cells for 60 s, as
indicated by the bar below the Ca2+ trace.
B, illustrated is the response of one osteoclast to 2 successive stimulations with RANKL (10 ng/ml), where Ca2+
transients diminished in amplitude upon successive stimulation.
C, the percentage of osteoclasts responding to the single
application of RANKL increased with increasing RANKL concentration
(10 3 to 102 ng/ml, applied locally,
n = 12 osteoclasts for each concentration, except
10
3 ng/ml, where n = 6). Elevations in
[Ca2+]i >25 nM above basal were
considered to be responses. D, amplitudes of
Ca2+ transients were quantified as maximum elevations above
basal levels. The curve illustrates dependence of amplitude
on RANKL concentration. Data are mean ± S.E. of three to eight
responsive osteoclasts for concentrations of RANKL
10
2
ng/ml.
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Fig. 2.
RANKL causes release of Ca2+ from
intracellular stores. A, to examine the source of
Ca2+ contributing to the response, RANKL was applied
locally to rat osteoclasts in the presence or absence of extracellular
Ca2+. Illustrated are responses of one osteoclast to RANKL
(100 ng/ml, 60 s applications) superfused with physiological
buffer containing 1 mM Ca2+. Where indicated,
cells were superfused with nominally Ca2+-free buffer
containing 0.5 mM EGTA (0 Ca2+). Dotted
lines indicate when the recording was interrupted for 60 s to
allow buffer exchange. B, histogram illustrates the
amplitude of Ca2+ transients during applications of RANKL
(100 ng/ml) in the presence and absence of extracellular
Ca2+ (means ± S.E., n = 5 cells).
Withdrawal of Ca2+ did not significantly affect the
amplitude of the response. C, osteoclasts were stimulated
twice with RANKL (100 ng/ml) before application of test compounds to
ensure responsiveness to repeated stimulation with RANKL. Cells were
then treated for 10 min with vehicle (dimethyl sulfoxide, 0.1%),
U73122, or U73343 (1 µM in the bath). Traces
illustrate responses of separate osteoclasts challenged with RANKL (100 ng/ml) following treatment with the indicated test compound.
D, histogram illustrates the amplitude of RANKL-induced
Ca2+ transients following treatment with the indicated test
compound. Data are expressed as a percentage of response to RANKL in
the same cell prior to treatment. Data are means ± S.E. of 5 cells for each condition. U73122 significantly inhibited RANKL-induced
elevations of [Ca2+]i when compared with
responses elicited by RANKL in osteoclasts treated with vehicle or
U73343 (p < 0.05).
30 mV and voltage ramp commands were
applied every 2 s. RANKL (100 ng/ml) evoked outward current after
a delay of ~10 s (n = 6 out of 14 osteoclasts tested)
with inward current apparent at
100 mV (Fig.
3A). Current-voltage (I-V)
relationships were determined from the voltage ramp commands. Basal
current prior to application of RANKL was dominated by the inwardly
rectifying K+ current Kir2.1 that has been identified
previously in osteoclasts (Fig. 3B, Control).
Subtraction of the control current from that recorded at the peak of
the response to RANKL showed that the RANKL-induced current was linear
and reversed close to
70 mV, indicating K+-selective
current (Fig. 3C). A similar, linear K+ current
has been shown previously to closely follow elevations of
[Ca2+]i in osteoclasts (22). Thus, RANKL-induced
current likely represents activation of
Ca2+-dependent K+ channels because
of rise of [Ca2+]i. Hence, the voltage-clamp data
independently confirm that RANKL induces elevation of
[Ca2+]i in osteoclasts.
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Fig. 3.
RANKL activates
Ca2+-dependent K+
current in osteoclasts. Rabbit osteoclast was held under
voltage clamp at 30 mV, and voltage ramps from
100 to +100 mV in
340 ms were commanded every 2 s. A, RANKL (100 ng/ml),
applied locally for 20 s where indicated by the bar,
caused outward current at
30 mV, and inward current at
100 mV.
B, current-voltage (I-V) relationship for the same
osteoclast prior to stimulation (Control) and at the peak of
the response to RANKL. Control I-V relationship displayed inwardly
rectifying K+ current at negative potentials. RANKL
activated a large outward current. C, the RANKL-induced
current was determined by subtraction of control current from the peak
current during application of RANKL. The RANKL-induced current
exhibited a linear I-V relationship and reversed direction close to
70 mV, consistent with activation of the
Ca2+-dependent K+ current. Data are
representative of 6 responsive osteoclasts of 14 tested.
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Fig. 4.
Effect of intracellular Ca2+
chelator, BAPTA, on osteoclast survival. A, the ability
of BAPTA to suppress P2Y nucleotide receptor-induced elevation of
[Ca2+]i was demonstrated in rat osteoclasts.
Single osteoclasts were first stimulated with ATP (100 µM), then incubated with BAPTA-AM (50 µM)
or vehicle (0.05% dimethyl sulfoxide) for 10 min before rechallenging
with ATP. BAPTA abolished Ca2+ responses to ATP. Data are
representative of 5 osteoclasts treated with vehicle and 8 osteoclasts
loaded with BAPTA. B, parallel coverslips were
incubated with BAPTA-AM (50 µM) or vehicle for 10 min,
then the medium was changed and the osteoclasts were challenged with
RANKL (100 ng/ml). RANKL elicited Ca2+ responses in 4 of 9 osteoclasts treated with vehicle, whereas 0 of 9 BAPTA-loaded
osteoclasts responded to RANKL. C, to examine the role of
[Ca2+]i in osteoclast survival, rat osteoclasts
were treated with BAPTA-AM or vehicle as described above. The medium
was then changed and osteoclasts were incubated with RANKL (100 ng/ml)
or vehicle at 37 °C for 24 h. The number of osteoclasts per
dish at 24 h was expressed as a percentage of the initial number
of osteoclasts in the same dish. Osteoclast survival was significantly
greater in cultures treated with RANKL alone than under all other
conditions (p < 0.05). Initial cell numbers (100%)
were 182 ± 61, 169 ± 27, 125 ± 31, and 159 ± 39 osteoclasts/dish for samples treated with vehicle, RANKL alone, BAPTA
alone, or BAPTA and RANKL, respectively. Data are means ± S.E. from four independent experiments.
B--
NF
B is one of the major downstream effectors of RANK
signaling and activation of NF
B enhances cell survival in many cell types (13, 26). Therefore, we investigated the possible involvement of
the PLC/Ca2+ pathway in the nuclear translocation of NF
B
in osteoclasts. Activation of NF
B was assessed at the single-cell
level using immunofluorescence to monitor the spatial distribution of
the p65 subunit of NF
B. In the majority of untreated rat or rabbit osteoclasts, NF
B was located in the cytoplasm for the duration of
the experiment (Fig. 5A,
left). In a proportion of osteoclasts, RANKL induced
redistribution of NF
B to the nuclei, most often to all the nuclei
within a single osteoclast (Fig. 5A, right). Nuclear translocation of NF
B was rapid (within 7 min after addition of RANKL) and transient, reversing within 60 min (Fig. 5B).
Maximal translocation was evident by 15 min, with 52 ± 4% of
osteoclasts exhibiting nuclear localization of NF
B
(n = 12 independent experiments), compared with 4 ± 1% in untreated osteoclasts (n = 7 independent experiments).
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Fig. 5.
Effect of PLC inhibitor on RANKL-induced
nuclear translocation of NF B. Rat
osteoclasts were pretreated with test compounds prior to addition of
RANKL (100 ng/ml) or its vehicle at time 0. Samples were fixed at the
indicated times and localization of the p65 subunit of NF
B was
determined by immunofluorescence. A, confocal image on
left illustrates cytoplasmic localization of NF
B in
vehicle-treated osteoclast. Image on right illustrates
nuclear localization of NF
B in a different osteoclast treated with
RANKL (100 ng/ml) for 30 min. Calibration bar of 10 µm
applies to both panels. Micrographs are representative of results from
12 independent experiments. B, kinetics of RANKL-induced
nuclear translocation of NF
B. Cells were pretreated with the PLC
inhibitor U73122 (1 µM) or dimethyl sulfoxide (vehicle
for inhibitors, 0.1% final) for 1 h at 37 °C prior to addition
of RANKL (100 ng/ml) or its vehicle. The number of osteoclasts
exhibiting nuclear localization of NF
B was expressed as a percentage
of the total number of osteoclasts on the coverslip. RANKL caused rapid
translocation of NF
B to the nuclei. Treatment with U73122 delayed
nuclear translocation of NF
B. Data are representative of six
independent experiments. C, average nuclear translocation of
NF
B 15 min following addition of RANKL to cells pretreated with
U73122 or inactive control U73343 (1 µM). Data are
expressed as percentage of translocation observed 15 min following
addition of RANKL to parallel samples of control cells pretreated with
vehicle. Treatment with U73122 (n = 6), but not with
U73343 (n = 3), significantly reduced RANKL-induced
nuclear translocation of NF
B (p < 0.05). 100%
(control) value was 44 ± 6% of total number of
osteoclasts.
B translocation in osteoclasts treated
with the PLC inhibitor U73122, which prevents
[Ca2+]i elevations induced by RANKL. U73122
markedly delayed translocation of NF
B from the cytoplasm to the
nuclei in response to RANKL. In U73122-treated cells, maximum
translocation was delayed until 30 min following addition of RANKL
(versus 15 min in parallel samples treated with RANKL alone)
(Fig. 5B). U73122 did not significantly affect the maximum
proportion of osteoclasts exhibiting nuclear translocation of NF
B in
response to RANKL (100 ng/ml) (47 ± 6% in control
versus 44 ± 5% in U73122-treated, based on parallel
samples from six independent experiments). U73343, a closely related
analog of U73122, which does not inhibit PLC or block RANKL-induced
elevation of [Ca2+]i (Fig. 2, C and
D), had no significant effect on RANKL-induced translocation
of NF
B (Fig. 5C). Thus, we provide evidence that RANKL
signaling through PLC affects the kinetics of NF
B translocation.
B--
We next examined whether chelation of intracellular
Ca2+ using BAPTA affected the kinetics of RANKL-induced
translocation of NF
B. Using loading conditions established above, we
found that BAPTA delayed nuclear translocation of NF
B induced by
RANKL (Fig. 6, data based on parallel
samples from seven independent experiments). In BAPTA-loaded
osteoclasts, maximum translocation was observed 30-60 min following
treatment with RANKL (100 ng/ml) versus 15-30 min in cells
treated with RANKL alone. Loading of cells with BAPTA significantly
reduced the proportion of osteoclasts exhibiting nuclear localization
of NF
B at 15 min, whereas the proportion of cells exhibiting nuclear
localization at 60 min was significantly increased (as indicated by
asterisks in Fig. 6). Furthermore, BAPTA reduced the maximum
proportion of osteoclasts exhibiting nuclear translocation of NF
B in
response to treatment with RANKL (41 ± 6% for BAPTA-loaded
osteoclasts versus 57 ± 5% for control osteoclasts).
In the absence of RANKL, BAPTA did not significantly affect NF
B
distribution (Fig. 6).
View larger version (20K):
[in a new window]
Fig. 6.
Effect of BAPTA on the kinetics of
RANKL-induced nuclear translocation of NF B.
Rat osteoclasts were treated with BAPTA-AM (50 µM),
calcein blue-AM (50 µM, AM Control) or
dimethyl sulfoxide (0.05%, vehicle) for 10 min. The medium
was then changed and cells were incubated with or without RANKL (100 ng/ml) at 37 °C for the indicated times. The number of osteoclasts
exhibiting nuclear localization of NF
B was expressed as a percentage
of maximum translocation observed in each experiment. Loading with
BAPTA, but not calcein blue, significantly delayed RANKL-induced
nuclear translocation of NF
B. Data are means ± S.E. of seven
independent experiments, except for AM control where n = 3. 100% value was 59 ± 4% (of total number of osteoclasts).
*, indicates significant difference of BAPTA/RANKL compared with RANKL
alone, p < 0.05.
B translocation, we examined cells treated with calcein blue-AM (a compound that bears the same AM modification as
BAPTA-AM, but is ineffective as a Ca2+ chelator at
physiological [Ca2+]i). Loading cells with
calcein blue did not affect the kinetics or degree of RANKL-induced
NF
B translocation (Fig. 6, n = 3), arguing against
possible nonspecific effects of the AM degradation products. Taken
together, these data indicate that RANKL-induced elevation of
[Ca2+]i accelerates nuclear translocation of
NF
B.
B--
We examined the role of a
Ca2+-regulated effector known to contribute to activation
of NF
B in other systems, the
Ca2+-calmodulin-dependent phosphatase,
calcineurin. The calcineurin inhibitor, cyclosporin A (1 µM), applied 30 min before addition of RANKL, suppressed
the initial NF
B translocation (7 min), with no significant effect at
later times (15-30 min) (Fig. 7, data based on parallel samples from eight independent experiments). The
structurally distinct calcineurin inhibitor, FK506, had similar effects. FK506 (10 nM) significantly suppressed
RANKL-induced NF
B translocation at 7 min to 67 ± 7% of
control, but had no significant effect at 15 min (94 ± 7% of
control) (n = 8). These findings are consistent with
RANKL-induced elevation of [Ca2+]i causing
transient activation of calcineurin, which in turn accelerates
activation of NF
B.
View larger version (24K):
[in a new window]
Fig. 7.
Effect of inhibitors of calcineurin and
protein kinase C on RANKL-induced nuclear translocation of
NF B. Parallel samples of rat osteoclasts
were incubated with the indicated inhibitors or vehicle (0.05%
dimethyl sulfoxide) for 30 min at 37 °C before addition of RANKL
(100 ng/ml). Nuclear translocation of NF
B was assessed at 7 and 15 min following addition of RANKL. Data are expressed as percentages of
translocation observed in parallel samples of vehicle-treated control
cells at 7 and 15 min after addition of RANKL. Dashed line
indicates 100% level. Calcineurin inhibitor, cyclosporin A
(CsA, 1 µM, n = 8)
significantly reduced RANKL-induced nuclear translocation of NF
B at
7 min (p < 0.05), but had no effect at 15 min. The PKC
inhibitor bisindolylmaleimide I (Bis, 100 nM,
n = 7), significantly reduced RANKL-induced nuclear
translocation of NF
B at both 7 and 15 min (p < 0.05). The effects of cyclosporin A and bisindolylmaleimide I were
additive at 7 min, however, cyclosporin A had no additional effect at
15 min. *, indicates significant difference compared with control,
p < 0.05. #, indicates significant difference compared
with samples treated with only one inhibitor, p < 0.05. Data are means ± S.E., 100% (control) values were 32 ± 3 and 39 ± 3% of total number of osteoclasts for 7 and 15 min, respectively.
B kinases,
inducing translocation of NF
B (12). In osteoclasts, the PKC
inhibitor bisindolylmaleimide I (100 nM) applied 30 min
before addition of RANKL suppressed NF
B translocation at 7 and 15 min (Fig. 7, data based on parallel samples from seven independent
experiments), with no significant effect at 30 min. In contrast, the
control compound, bisindolylmaleimide V (100 nM) that does
not inhibit PKC, had no significant effect on RANKL-induced NF
B
translocation (107 ± 3 and 107 ± 4% of control at 7 and 15 min, respectively, n = 6). These findings are
consistent with a role for PKC, activated following stimulation of
phospholipase C, in mediating the effects of RANKL on NF
B.
B translocation at 7 min in samples
treated with FK506 together with bisindolylmaleimide I was 54 ± 6% of control, significantly less than in samples treated with
bisindolylmaleimide I or FK506 alone (77 ± 3 and 67 ± 7%, respectively, n = 7). Like cyclosporin A, FK506 had no
additional effect at 15 min (translocation in the presence of FK506 and
bisindolylmaleimide I was 78 ± 4% of control versus
71 ± 5% in osteoclasts treated with bisindolylmaleimide I alone,
n = 7). Taken together, these data indicate that
calcineurin and PKC are downstream effectors of RANK that accelerate
activation of NF
B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, and promotes osteoclast
survival, indicating key functional roles for PLC and Ca2+
in RANK signaling in osteoclasts.
is activated by growth factor receptor tyrosine kinases or
nonreceptor tyrosine kinases linked to cytokine receptors (29). In this
regard, RANK and TRAF6 interact with, and activate the nonreceptor
tyrosine kinase c-Src (10), which may serve to recruit PLC-
to the
RANK-signaling complex in osteoclasts, as was shown for endothelial
cells (30). Targeted disruption of c-src results in an
osteopetrotic phenotype because of compromised resorptive function of
osteoclasts (31). Interestingly, osteoclastogenesis is not impaired in
c-src knockout mice, indicating a different requirement for
c-Src signaling in osteoclasts and their precursors. In keeping with
these differences, we observed that RANKL-induced Ca2+
signaling is more prominent in mature osteoclasts than in precursors. Furthermore, TRAF6-deficient mice have been reported to display osteopetrosis because of nonfunctional osteoclasts (32), also suggesting critical differences between RANKL signaling in precursors and mature osteoclasts. However, osteoclastogenesis was found to be
impaired in another TRAF6 knockout model (33), leaving open the
question of the precise role for TRAF6-dependent pathways in RANKL-induced osteoclastogenesis and activation of resorption.
B is a key transcription factor that promotes cell survival in
many systems (13). In the present study, we have shown that
RANKL-induced Ca2+ signaling accelerates nuclear
translocation of NF
B. Several mechanisms may be considered.
Elevation of [Ca2+]i alone appears to be
insufficient to activate NF
B in osteoclasts. Nucleotides, such as
ATP (10-100 µM), which bind to the P2Y class of G
protein-coupled receptors causing even greater release Ca2+
from stores (21), do not activate NF
B in
osteoclasts.2 Similarly,
elevation of [Ca2+]i alone is insufficient to
activate NF
B in immune cells (18, 38). Therefore, Ca2+
appears to act in concert with other canonical signaling pathways to
accelerate activation of NF
B in osteoclasts.
B-inducing kinase
and the IKK complex (5, 11, 14). Enhanced activity of the enzyme
complex would increase phosphorylation of I
B, accelerating translocation of NF
B to the nucleus. Ca2+ may act to
increase activity of the IKK complex, and indeed, IKK
is reported to
be activated synergistically by two
Ca2+-dependent mediators of T cell receptor
signaling, calcineurin and PKC (18). We show in osteoclasts that RANK
signaling involves these same mediators.
B activation in osteoclasts, without affecting maximal
activation. Similar actions of distinct inhibitors support the
interpretation that their effects are mediated by calcineurin. The
actions of calcineurin inhibitors were restricted to early time points,
which might be explained by the fact that elevation of
[Ca2+]i in response to RANKL is transient, giving
rise to only brief activation of calcineurin. Our findings may have
relevance to previously observed inhibitory effects of cyclosporin A on osteoclastic resorption in vitro (39).
B
activation, again with no marked change in maximal activation. In
contrast to the calcineurin blockers, PKC inhibition resulted in a
greater delay in NF
B translocation, which could reflect the time
course for production of diacylglycerol. Therefore, it would appear
that early stages of NF
B activation involve the convergence of
several signaling pathways. Moreover, the effects of calcineurin and
PKC blockers were additive, and similar in amplitude to the effects of
BAPTA, all supporting a critical role for Ca2+ in
controlling the latency of NF
B activation.
B activation
has recently been shown to generate specificity in gene expression
(40). In concert with other transcription factors, NF
B controls cell
survival and the expression of genes encoding cytokines and adhesion
molecules (12, 13, 41). RANK also activates AP-1, which requires
cooperative interactions with other transcription factors and
coactivators, including NF
B, to achieve stimulus-specific regulation
of transcription (42, 43). In this regard, c-Jun and c-Fos have been
shown to interact directly with the p65 subunit of NF
B, enhancing
transactivation via both the
B and AP-1 response elements (44). In
addition, NF
B interacts with the cancer-amplified transcription
coactivator ASC-2 and other transcriptional regulators to control gene
expression (45). Therefore, the relative activities of NF
B and other
transcription factors at any point of time will determine the
composition of multipartner transcription complexes and consequently
gene expression. Thus, distinct responses will depend upon the kinetics
of activation of NF
B and other transcription factors stimulated by
RANKL or by other signaling molecules acting on the osteoclast. Whether the dependence of osteoclast survival on [Ca2+]i
elevation is because of changes in the kinetics of NF
B translocation
or because of the activation of other
Ca2+-dependent pathways is yet to be
determined. Nevertheless, the cross-talk between NF
B and
Ca2+ signaling demonstrated here provides a novel mechanism
for the temporal regulation of NF
B activity and gene expression in
osteoclasts and other cell types.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Lin Naemsch for performing preliminary electrophysiological and immunofluorescence studies on rabbit osteoclasts, and Caiqiong Liu for assistance in performing immunofluorescence labeling. We acknowledge Drs. John Hiscott (McGill University), David Litchfield (University of Western Ontario), and Michael Underhill (University of Western Ontario) for helpful comments on the manuscript. We thank Drs. William Boyle and Colin Dunstan (Amgen Inc., Thousand Oaks, CA) for providing soluble RANKL and OPG.
![]() |
FOOTNOTES |
---|
* This work was supported by The Arthritis Society, Canadian Arthritis Network, and Canadian Institutes of Health Research.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.
Present address: Dept. of Pharmacology, University of Cambridge,
Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
§ Present address: Dept. of Cell Physiology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.
¶ To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Faculty of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada. E-mail: stephen.sims@fmd.uwo.ca.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M206421200
2 S. J. Dixon and S. M. Sims, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RANK, receptor
activator of NFB;
AM, acetoxymethyl ester;
AP-1, activator protein
1;
[Ca2+]i, cytosolic free Ca2+
concentration;
NF
B, nuclear factor
B;
I
B, inhibitor of NF
B;
IKK, I
B kinase;
IKCa, intermediate conductance
Ca2+-dependent K+ current;
OPG, osteoprotegerin;
PLC, phospholipase C;
PKC, protein kinase C;
RANKL, receptor activator of NF
B ligand;
TRAF, tumor necrosis factor
receptor-associated factor;
PBS, phosphate-buffered saline;
BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Manabe, N.,
Kawaguchi, H.,
Chikuda, H.,
Miyaura, C.,
Inada, M.,
Nagai, R.,
Nabeshima, Y.,
Nakamura, K.,
Sinclair, A. M.,
Scheuermann, R. H.,
and Kuro-o, M.
(2001)
J. Immunol.
167,
2625-2631 |
2. | Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y. X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., and Boyle, W. J. (1998) Cell 93, 165-176[Medline] [Order article via Infotrieve] |
3. |
Yasuda, H.,
Shima, N.,
Nakagawa, N.,
Yamaguchi, K.,
Kinosaki, M.,
Mochizuki, S.,
Tomoyasu, A.,
Yano, K.,
Goto, M.,
Murakami, A.,
Tsuda, E.,
Morinaga, T.,
Higashio, K.,
Udagawa, N.,
Takahashi, N.,
and Suda, T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3597-3602 |
4. | Hofbauer, L. C. (1999) Eur. J. Endocrinol. 141, 195-210[Medline] [Order article via Infotrieve] |
5. |
Wong, B. R.,
Josien, R.,
Lee, S. Y.,
Vologodskaia, M.,
Steinman, R. M.,
and Choi, Y.
(1998)
J. Biol. Chem.
273,
28355-28359 |
6. |
Galibert, L.,
Tometsko, M. E.,
Anderson, D. M.,
Cosman, D.,
and Dougall, W. C.
(1998)
J. Biol. Chem.
273,
34120-34127 |
7. |
Darnay, B. G.,
Haridas, V.,
Ni, J.,
Moore, P. A.,
and Aggarwal, B. B.
(1998)
J. Biol. Chem.
273,
20551-20555 |
8. |
Lee, Z. H.,
Kwack, K.,
Kim, K. K.,
Lee, S. H.,
and Kim, H. H.
(2000)
Mol. Pharmacol.
58,
1536-1545 |
9. |
Zhang, Y. H.,
Heulsmann, A.,
Tondravi, M. M.,
Mukherjee, A.,
and Abu-Amer, Y.
(2001)
J. Biol. Chem.
276,
563-568 |
10. | Wong, B. R., Besser, D., Kim, N., Arron, J. R., Vologodskaia, M., Hanafusa, H., and Choi, Y. (1999) Mol Cell. 4, 1041-1049[Medline] [Order article via Infotrieve] |
11. |
Darnay, B. G.,
Ni, J.,
Moore, P. A.,
and Aggarwal, B. B.
(1999)
J. Biol. Chem.
274,
7724-7731 |
12. | Karin, M., and Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621-663[CrossRef][Medline] [Order article via Infotrieve] |
13. | Mak, T. W., and Yeh, W. C. (2002) Arthritis Res. 4, S243-S252[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Wei, S.,
Teitelbaum, S. L.,
Wang, M. W.,
and Ross, F. P.
(2001)
Endocrinology
142,
1290-1295 |
15. | Sun, S. C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259, 1912-1915[Medline] [Order article via Infotrieve] |
16. | Iotsova, V., Caamano, J., Loy, J., Yang, Y., Lewin, A., and Bravo, R. (1997) Nat. Med. 3, 1285-1289[Medline] [Order article via Infotrieve] |
17. | Steffan, N. M., Bren, G. D., Frantz, B., Tocci, M. J., O'Neill, E. A., and Paya, C. V. (1995) J. Immunol. 155, 4685-4691[Abstract] |
18. |
Trushin, S. A.,
Pennington, K. N.,
Algeciras-Schimnich, A.,
and Paya, C. V.
(1999)
J. Biol. Chem.
274,
22923-22931 |
19. |
Naemsch, L. N.,
Dixon, S. J.,
and Sims, S. M.
(2001)
J. Biol. Chem.
276,
39107-39114 |
20. | Tezuka, K., Sato, T., Kamioka, H., Nijweide, P. J., Tanaka, K., Matsuo, T., Ohta, M., Kurihara, N., Hakeda, Y., and Kumegawa, M. (1992) Biochem. Biophys. Res. Commun. 186, 911-917[Medline] [Order article via Infotrieve] |
21. |
Weidema, A. F.,
Dixon, S. J.,
and Sims, S. M.
(2001)
Am. J. Physiol.
280,
C1531-C1539 |
22. | Weidema, A. F., Barbera, J., Dixon, S. J., and Sims, S. M. (1997) J. Physiol. 503, 303-315[Abstract] |
23. | Myers, D. E., Collier, F. M., Minkin, C., Wang, H., Holloway, W. R., Malakellis, M., and Nicholson, G. C. (1999) FEBS Lett. 463, 295-300[CrossRef][Medline] [Order article via Infotrieve] |
24. | Komarova, S. V., Dixon, S. J., and Sims, S. M. (2001) Curr. Pharm. Des. 7, 637-654[Medline] [Order article via Infotrieve] |
25. |
Naemsch, L. N.,
Weidema, A. F.,
Sims, S. M.,
Underhill, T. M.,
and Dixon, S. J.
(1999)
J. Cell Sci.
112,
4425-4435 |
26. |
Lacey, D. L.,
Tan, H. L.,
Lu, J.,
Kaufman, S.,
Van, G.,
Qiu, W.,
Rattan, A.,
Scully, S.,
Fletcher, F.,
Juan, T.,
Kelley, M.,
Burgess, T. L.,
Boyle, W. J.,
and Polverino, A. J.
(2000)
Am. J. Pathol.
157,
435-448 |
27. | Kong, Y. Y., Yoshida, H., Sarosi, I., Tan, H. L., Timms, E., Capparelli, C., Morony, S., Oliveira-dos-Santos, A. J., Van, G., Itie, A., Khoo, W., Wakeham, A., Dunstan, C. R., Lacey, D. L., Mak, T. W., Boyle, W. J., and Penninger, J. M. (1999) Nature 397, 315-323[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Li, J.,
Sarosi, I.,
Yan, X. Q.,
Morony, S.,
Capparelli, C.,
Tan, H. L.,
McCabe, S.,
Elliott, R.,
Scully, S.,
Van, G.,
Kaufman, S.,
Juan, S. C.,
Sun, Y.,
Tarpley, J.,
Martin, L.,
Christensen, K.,
McCabe, J.,
Kostenuik, P.,
Hsu, H.,
Fletcher, F.,
Dunstan, C. R.,
Lacey, D. L.,
and Boyle, W. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1566-1571 |
29. |
Rebecchi, M. J.,
and Pentyala, S. N.
(2000)
Physiol. Rev.
80,
1291-1335 |
30. |
Kim, Y. M.,
Lee, Y. M.,
Kim, H. S.,
Kim, J. D.,
Choi, Y.,
Kim, K. W.,
Lee, S. Y.,
and Kwon, Y. G.
(2002)
J. Biol. Chem.
277,
6799-6805 |
31. | Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell. 64, 693-702[Medline] [Order article via Infotrieve] |
32. |
Lomaga, M. A.,
Yeh, W. C.,
Sarosi, I.,
Duncan, G. S.,
Furlonger, C.,
Ho, A.,
Morony, S.,
Capparelli, C.,
Van, G.,
Kaufman, S.,
van der Heiden, A.,
Itie, A.,
Wakeham, A.,
Khoo, W.,
Sasaki, T.,
Cao, Z.,
Penninger, J. M.,
Paige, C. J.,
Lacey, D. L.,
Dunstan, C. R.,
Boyle, W. J.,
Goeddel, D. V.,
and Mak, T. W.
(1999)
Genes Dev.
13,
1015-1024 |
33. |
Naito, A.,
Azuma, S.,
Tanaka, S.,
Miyazaki, T.,
Takaki, S.,
Takatsu, K.,
Nakao, K.,
Nakamura, K.,
Katsuki, M.,
Yamamoto, T.,
and Inoue, J.
(1999)
Genes Cells
4,
353-362 |
34. |
Fanger, C. M.,
Rauer, H.,
Neben, A. L.,
Miller, M. J.,
Wulff, H.,
Rosa, J. C.,
Ganellin, C. R.,
Chandy, K. G.,
and Cahalan, M. D.
(2001)
J. Biol. Chem.
276,
12249-12256 |
35. |
Chellaiah, M.,
Kizer, N.,
Silva, M.,
Alvarez, U.,
Kwiatkowski, D.,
and Hruska, K. A.
(2000)
J. Cell Biol.
148,
665-678 |
36. | Zaidi, M., Adebanjo, O. A., Moonga, B. S., Sun, L., and Huang, C. L. (1999) J. Bone Miner. Res. 14, 669-674[Medline] [Order article via Infotrieve] |
37. |
Ishida, N.,
Hayashi, K.,
Hoshijima, M.,
Ogawa, T.,
Koga, S.,
Miyatake, Y.,
Kumegawa, M.,
Kimura, T.,
and Takeya, T.
(2002)
J. Biol. Chem.
277,
41147-41156 |
38. |
Crabtree, G. R.
(2001)
J. Biol. Chem.
276,
2313-2316 |
39. | Chowdhury, M. H., Shen, V., and Dempster, D. W. (1991) Calcif. Tissue Int. 49, 275-279[Medline] [Order article via Infotrieve] |
40. |
Hoffmann, A.,
Levchenko, A.,
Scott, M. L.,
and Baltimore, D.
(2002)
Science
298,
1241-1245 |
41. |
Sha, W. C.
(1998)
J. Exp. Med.
187,
143-146 |
42. | Chinenov, Y., and Kerppola, T. K. (2001) Oncogene 20, 2438-2452[CrossRef][Medline] [Order article via Infotrieve] |
43. | Li, J. J., Cao, Y., Young, M. R., and Colburn, N. H. (2000) Mol. Carcinog. 29, 159-169[CrossRef][Medline] [Order article via Infotrieve] |
44. | Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993) EMBO J. 12, 3879-3891[Abstract] |
45. |
Lee, S. K.,
Na, S. Y.,
Jung, S. Y.,
Choi, J. E.,
Jhun, B. H.,
Cheong, J.,
Meltzer, P. S.,
Lee, Y. C.,
and Lee, J. W.
(2000)
Mol. Endocrinol.
14,
915-925 |