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B Ligand Activates Nuclear Factor-B in Osteoclast Precursors1
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: F. Patrick Ross, Ph.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital North Mail Stop #90–30-669, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail: rossf{at}medicine.wustl.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand [RANK ligand
(RANK-L)] stimulates mature osteoclasts to resorb bone, a process
associated with NF-B activation. RANK-L also prompts macrophages to
develop the osteoclast phenotype. Although NF-B is essential for
osteoclast differentiation, it is not known whether RANK-L activates
this transcription complex in osteoclast precursors. We report that
RANK-L rapidly induces NF-B activation in both authentic osteoclast
precursors, namely bone marrow macrophages, and RAW 264.7 cells, a
murine macrophage line also capable of RANK-L-mediated
osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA
binding moiety contains p50/p65, the most common NF-B complex.
Subcellular translocation of p50 and p65 subunits is confirmed by
Western blots and immunofluorescence analysis. RANK-L activates NF-B
in both bone marrow macrophages and RAW 264.7 cells by serine
phosphorylation of IB
within 5 min, resulting in rapid IB
degradation and resynthesis. Attesting to function, RANK-L treatment of
RAW 264.7 cells transiently transfected with a plasmid containing
NF-B consensus elements linked to luciferase greatly enhances
reporter activity. Our data suggest that activation of the NF-B
pathway is an integral component of RANK-L-induced osteoclast
differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B) develop osteopetrosis because
of failed osteoclastogenesis (7, 8).
Recent developments have made it possible to generate both human
and murine osteoclasts by treating purified myeloid precursors
with the cytokines, macrophage colony-stimulating factor (M-CSF) and
receptor activator of NF-B ligand (RANK-L) (9, 10, 11).
Although M-CSF is a well characterized cytokine that supports the
survival and proliferation of cells in the macrophage lineage
(12), the identity and function of RANK-L were determined
only recently (9, 10, 13, 14). RANK-L is a member of the
tumor necrosis factor (TNF) cytokine superfamily and functions as an
osteoclast-specific protein that exists in both soluble and
membrane-bound forms (9, 10). RANK, the surface receptor
for RANK-L, initiates osteoclastogenic signal transduction after
ligation with RANK-L. The proximal RANK-derived signals include binding
of TRAF family members such as TRAF2, TRAF3, TRAF5, and TRAF6,
which, in turn, initiates a cascade of kinases. Two critical distal
events in RANK signaling are activation of the NF-B complex and the
transcription factor, activator protein-1 (15).
RANK-L activates NF-B in mature osteoclasts (16, 17).
It is not known, however, whether this transcription complex is
activated by RANK-L in osteoclast precursors. For example, it has been
previously reported that NF-B activation is not detected in
RANK-L-treated RAW 264.7 cells, a murine myelomonocytic cell line that
differentiates into osteoclasts under the influence of the cytokine
(18). Given that RAW 264.7 cells represent an immortalized
line, uncertainty exists as to whether RANK-L-induced NF-B is a
component of the initial stage of osteoclastogenesis.
We found that RANK-L, via serine 32/36 phosphorylation and degradation
of IB, readily activates the most common NF-B dimer, p50/p65,
in both bone marrow macrophages (BMMs) and RAW 264.7 cells.
Furthermore, RANK-L promotes the expression of an NF-B-dependent
reporter plasmid transiently transfected into RAW cells. Thus,
RANK-L-stimulated osteoclastogenesis is associated with NF-B
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and polyclonal
anti-p50NF-B antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal
antiphospho-IB was obtained from New England Biolabs, Inc. (Beverly, MA). Polyclonal anti-p65NF-B was purchased
from Upstate Biotechnology, Inc. (Lake Placid, NY).
Anti-p50NF-B for electrophoretic mobility shift assay (EMSA) was
obtained from Geneka (Montréal, Canada). The bicinchoninic acid
kit for protein determination and enhanced chemiluminescence kits were
obtained from Pierce Chemical Co. (Rockford, IL).
Recombinant murine M-CSF was purchased from R\|[amp ]\|D Systems, Inc. (Minneapolis, MN). Murine RANK-L was expressed in our
laboratory as described previously (19). All other
chemicals were obtained from Sigma (St. Louis, MO).
Cell culture
BMMs were isolated from whole bone marrow of 4- to 6-week-old
C3H/Hen males (Harlan Industries, Indianapolis, IN) and incubated in
tissue culture dishes at 37 C in 5% CO2 in the
presence of recombinant mouse M-CSF (5 ng/ml). After 24 h in
culture, the nonadherent cells were collected and layered on a
Ficoll-Hypaque gradient, and the cells at the gradient interface were
collected. The cells were replated at 65,000/cm2
in MEM, supplemented with 10% heat-inactivated FBS at 37 C in 5%
CO2 in the presence of M-CSF (5 ng/ml). RAW 264.7
cells, obtained from American Type Culture Collection
(Manassas, VA), were grown in DMEM supplemented with10%
heat-inactivated FBS.
Immunoblotting
Untreated or RANK-L-stimulated monolayers of BMMs or RAW cells
were washed twice with ice-cold PBS. Cells were lifted from the dish
after treatment with 5 mM EDTA and 5 mM EGTA in
PBS. Cells were then resuspended in hypotonic lysis buffer A [10
mM HEPES (pH 7.8), 1.5 mM
MgCl2, 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 6.4%. Nuclei were pelleted, and both these
and cytosolic fractions were carefully transferred to fresh tubes.
Nuclei were washed and then resuspended in nuclear extraction buffer B
[20 mM HEPES (pH 7.8), 420 mM NaCl, 1.2
mM MgCl2, 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] and rotated for 30 min at 4 C. The samples were
then centrifuged, and nuclear proteins in the supernatant were
transferred to fresh tubes.
Nuclear or cytosolic extracts were boiled in the presence of SDS sample buffer [0.5 M Tris-HCl (pH 6.8), 10% (wt/vol) SDS, 10% glycerol, and 0.05% (wt/vol) bromophenol blue] for 5 min and subjected to electrophoresis on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad Laboratories, Inc., Richmond, CA) and incubated in blocking solution (5% nonfat dry milk or 3% BSA prepared in PBS containing 0.1% Tween-20) for 1 h to reduce nonspecific binding. Membranes were then exposed to primary antibodies (1 h at room temperature or overnight at 4 C), washed four times, and incubated with secondary goat antimouse or rabbit IgG horseradish peroxidase-conjugated antibody for 1 h. Membranes were washed extensively, and an enhanced chemiluminescence detection assay was performed following the manufacturer’s directions.
Immunofluorescence analysis
BMMs grown on chamber slides were treated with or without RANK-L
(100 ng/ml) for the indicated times, fixed with methanol, permeabilized
with 0.5% Triton X-100 for 30 min, and then blocked with 20% normal
serum in PBS for 20 min at room temperature. Polyclonal antibodies to
NF-B p50 and p65 were applied overnight at 4 C, followed by a 45-min
incubation at room temperature with fluorescein-conjugated goat
antirabbit IgG. The cells were washed and mounted with an antifade kit
(Molecular Probes, Inc., Eugene, OR). Immunofluorescence
analysis was performed with a Nikon E-800 microscope
(Nikon, Melville, NY) and a Microradiance confocal system
(Bio-Rad Laboratories, Inc.). Data were collected and
analyzed with LaserSharp NT-2000 software.
EMSA
Nuclear extracts (1.5–3 µg) were incubated with an
end-labeled double stranded oligonucleotide probe containing the
sequence 5'-AAACAGGGGGCTTTCCCTCCTC-3' derived from the B3 site of
the TNF promoter (20) (GenBank accession no. U68415, bases
481–502) for BMMs and 5'-AGTTGAGGGGACTTTCCCAGCC-3' (Santa Cruz Biotechnology, Inc.) for RAW 264.7 cells. The reaction was
performed in a total of 20 µl binding buffer [20 mM
HEPES (pH 7.8), 100 mM NaCl, 0.5 mM
dithiothreitol, 1 µl of poly(dI-dC), and 10% glycerol] for 20 min
at room temperature. For supershift assay, the nuclear extract was
incubated with specific antibodies for an additional 30 min. The
samples were fractionated on a 4–20% TBE gel (Novex, San
Diego, CA) and visualized by exposing dried gel to film.
Transfection and reporter gene assays
RAW 264.7 cells were transiently transfected with a plasmid
containing a luciferase reporter gene driven by two repeats of human
immunodeficiency virus type 1 B enhancer (provided by Dr. David V.
Goeddel, Tularik, Inc, South San Francisco, CA) and a plasmid
containing cytomegalovirus 
-galactosidase using SuperFect
transfection reagent (QIAGEN, Valencia, CA). Transfected
cells were lysed in reporter lysis buffer (Promega Corp.,
Madison, WI) and mixed with luciferase assay reagent (Promega Corp.), and luciferase activity was measured in a luminometer
(MGM Instruments, Hamden, CT). -Galactosidase activity was
determined by mixing the same amount of cell lysate and assay 2 x
buffer (Promega Corp.) and reading the absorbance at 420
nm after 30-min incubation at 37 C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in osteoclast precursorsB is an event required for osteoclast
differentiation in vivo (7, 8). To determine
whether NF-B is activated by RANK-L in osteoclast precursors, in the
form of BMMs, as well as in RAW 264.7 cells, a line capable of forming
osteoclasts in response to RANK-L (18), cells were treated
with the cytokine, and cytosolic extracts were subjected to Western
blotting. We found that RANK-L treatment of both BMMs and RAW 264.7
cells causes rapid serine phosphorylation of IB at 5 min, an
event followed by degradation and resynthesis of the protein (Fig. 1
). Consistent with the known capacity of
NF-B to transactivate the IB gene (21) cytosolic
levels of the inhibitory protein gradually increase.
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B involves phosphorylation of
IB serine residues 32/36, resulting in ubiquitination and rapid
proteosomal degradation of the phosphorylated inhibitory protein
(22, 23, 24, 25). We previously reported that activation of
NF-B in BMMs by another osteoclastogenic cytokine, TNF, is
mediated by c-Src-dependent tyrosine phosphorylation of IB
without degradation of the protein (26). TNF and RANK-L
are both members of the TNF superfamily, raising the possibility that
each cytokine prompts tyrosine phosphorylation of IB. Tyrosine
phosphorylation of IB is not detectable within 1 h of RANK-L
treatment (data not shown), suggesting that NF-B activation in BMMs
induced by RANK-L involves the classical pathway involving IB
serine phosphorylation and degradation.
RANK-L induces nuclear translocation of p50/p65
Treatment of both osteoclast precursors and RAW 264.7 cells with
RANK-L leads to degradation of IB, the protein that retains
NF-B members in the cytosol. Thus, the disappearance of IB is
likely to be followed by nuclear translocation of NF-B, which
comprises dimers of specific subunits. To confirm subcellular
translocation of NF-B and to identify the subunits contained in the
NF-B complex in response to RANK-L, Western blots were performed
using nuclear extracts from both BMMs and RAW 264.7 cells. As shown in
Fig 2, RANK-L promoted rapid
appearance of p50 and p65 in nucleus in both cell types. In parallel
studies, intact cells treated under the same experimental conditions
were processed for immunofluorescence and analyzed using anti-p50 and
anti-p65 antibodies. Both p50 and p65 were predominantly localized in
cytoplasm in naive cells, whereas treatment with RANK-L led to their
nuclear accumulation (Fig. 3).
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B activation
and 3 demonstrate that RANK-L
treatment of osteoclast precursors leads to translocation of the
p50/p65 complex, they do not provide direct evidence that the event is
followed by DNA binding and activation of transcription. To examine
these questions, both BMMs and RAW 264.7 cells were treated with
RANK-L, and nuclear extracts were subjected to EMSA.
As shown in Fig. 4A, a complex
recognizing the B3 sequence of the TNF promoter (TNF-B3), appears
after 5 min of RANK-L treatment of BMMs and persists for at least 30
min. The specificity of the EMSA probe was established by competition
with excess unlabeled TNF-B3 and a consensus binding site for
NF-B (Santa Cruz Biotechnology, Inc.). In contrast,
mutated forms of the consensus oligonucleotides fail to compete with
TNF-B3 (Fig. 4B). Use of the same probe to identify nuclear-residing
NF-B in RANK-L-treated RAW cells yielded positive, but less
dramatic, results (data not shown). On the other hand, when an NF-B
consensus oligonucleotide (Santa Cruz Biotechnology, Inc.)
was used, specific RANK-L-stimulated NF-B nuclear translocation was
also clearly evident in RAW cells (Fig. 4, C and D).
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B
complex, we performed EMSA, using TNF-B3 as a probe for BMMs and an
NF-B consensus oligonucleotide (Santa Cruz Biotechnology, Inc.) for RAW cells in the absence or presence of anti-p50 and
p65 antibodies. Addition of anti-p50, anti-p65, or both antibodies led
to supershift of the specific DNA-bound protein in both cell types,
suggesting that the RANK-L-induced DNA binding moiety consists largely
of p50/p65, the most common NF-B heterodimer (Fig. 5).
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B-responsive reporter in RAW 264.7
cellsB typically results in activation
of genes containing one or more consensus binding sequences to which
the heterodimeric complex can bind. To determine whether RANK-L induced
nuclear translocation of the p50/p65 is functionally important, we
transiently transfected RAW 264.7 cells with an NF-B-responsive
reporter construct. RANK-L stimulated luciferase expression by
NF-B-dependent reporter plasmid containing cells in a dose-dependent
manner (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differentiation along the macrophage/osteoclast lineage requires the
transcription factors, c-Fos, which activates the c-Jun N-terminal
kinase (JNK) signaling pathway (4, 5), and NF-B
(7, 8). Although RANK-L is central to the osteoclastogenic
process, whether it does so via NF-B and/or JNK is controversial.
Thus, Wong et al. found that RANK-L treatment of murine
osteoclasts fails to activate JNK (17). In contrast, Jimi
and his colleagues demonstrated a time- and dose-dependent increase in
JNK activation in osteoclasts following exposure to RANK-L
(16). Although their studies do not address
osteoclastogenesis, Hsu et al. reported that RANK-L
stimulated JNK, but not NF-B, in RAW 264.7 cells (18).
This latter study stands in contrast to ours, which establishes
RANK-L-induced NF-B activation in this osteoclastogenic line as well
as authentic osteoclast precursors. We also found that RANK-L-induced
NF-B nuclear translocation is RAW 264.7 clone dependent (data not
shown). We posit, therefore, that the inconsistency between Hsu’s data
and ours reflex their use of a nonresponsive clone.
The NF-B family has been implicated in osteoclast formation,
function, and survival (22). Thus, mice lacking p50 have
fewer osteoclasts, whereas neither mature osteoclasts nor
tartrate-resistant acid phosphatase-positive precursors are present in
p50/p52 null mice (7). On the other hand, a recent report
suggests that NF-B is involved in activation, but not survival, of
osteoclasts (27), diverging from the posture that
interleukin-1 (IL-1) promotes osteoclast survival (28)
through activation of an NF-B complex containing p50 and p65
(29).
NF-B is activated by numerous agonists, including inflammatory
cytokines such as TNF, phorbol myristyl acetate, DNA-damaging
agents, double stranded RNA, and viruses, including human
immunodeficiency virus type 1 (22, 23, 24, 25). We now
report that RANK-L activates NF-B during the process of osteoclast
differentiation. Treatment of primary osteoclast precursors, in the
form of BMMs, and RAW 264.7 cells results in rapid nuclear
translocation of p50 and p65, the two most common subunits of the
NF-B family, where they associate with a consensus DNA-binding
sequence. The capacity of RANK-L to transactivate an NF-B reporter
construct in RAW cells establishes that the nuclear translocated
transcription complex is functional. Importantly, we found that M-CSF
fails to activate NF-B in BMMs (data not shown), confirming the
specificity and importance of RANK-L in this process.
We have previously shown that TNF activates NF-B in BMMs by c-Src
tyrosine phosphorylation of IB (26). To determine
whether such is the case for RANK-L, we examined the phosphorylation
status of IB in BMMs and RAW 264.7 cells after exposure to the
cytokine. We found that the inhibitory protein is rapidly
phosphorylated at serine 32 by RANK-L, degraded, and then
resynthesized. In contrast to TNF, tyrosine phosphorylation of
IB does not occur in BMMs in response to RANK-L.
The fact that NF-B activation is required for osteoclast formation
and function may be central to the pathogenesis of postmenopausal
osteoporosis and rheumatoid arthritis, in which osteoclastogenesis is
accelerated. In addition to RANK-L, increased generation and/or
activity of osteoclasts can be mediated by IL-1 and TNF
(30, 31, 32), which are abundant in rheumatoid synovium and
are potent activators of NF-B. Interleukin-1 targets stromal cells
and/or osteoblasts, leading to the release of RANK-L (33).
In addition, the cytokine acts directly on preosteoclasts generated in
a stroma coculture system (and hence differentiated with RANK-L) to
stimulate their fusion and survival (34). Additionally,
IL-1 enhances the survival and activation of mature osteoclasts
(28). The fusogenic, survival, and activation functions of
IL-1 are accompanied by activation of NF-B (28, 34, 35). In contrast, there are no reports that IL-1 can itself
replace RANK-L in the differentiation of osteoclast precursors.
Although earlier studies suggested that TNF, acting directly on
precursors, could act independently of RANK-L to induce osteoclast
formation (36, 37), recent efforts reveal that cells need
to be primed by preexposure to RANK-L before TNF alone can be
osteoclastogenic (38). In summary, we provide the first
data that RANK-L activates the NF-B signaling pathway in murine
osteoclast precursors. As neither IL-1 nor TNF, both of which
activate the same NF-B signals as does RANK-L, can replace the
latter molecule in the osteoclastogenic process, we conclude that
NF-B activation is necessary, but not sufficient, for
osteoclastogenesis. However, the fact that RANK-L and these
inflammatory, bone-resorptive cytokines prompt NF-B activation
postures this transcription complex as a potential antiosteoclastogenic
therapeutic target.
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Footnotes |
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Received August 11, 2000.
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