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From the Departments of Pharmacology/Pathology,*
Cell
Biology,
Protein
Chemistry,
Mammalian Cell Molecular
Biology,§
and Mammalian
Genomics,¶
Amgen, Inc., Thousand Oaks, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and increased the expression
of bcl-2 and bcl-XL mRNA, but had no effect on
JNK activation. In contrast, OPGL enhanced both NF-B
and JNK kinase activation and increased the expression of
c-src, but not bcl-2 and bcl-XL mRNA. These data
suggest that aspects of both OPGL’s and CSF-1’s signaling/survival
pathways are required for optimal osteoclast survival. In mice,
a single dose of OPG, the OPGL decoy receptor, led to a
>90% loss of osteoclasts because of apoptosis within 48 hours of
exposure without impacting osteoclast precursor cells.
Therefore, OPGL is essential, but not
sufficient, for osteoclast survival and endogenous CSF-1 levels
are insufficient to maintain osteoclast viability in the absence of
OPGL.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the mouse, and likely in humans as well, two factors seem to be critical for osteoclast differentiation and activation.3,4 The monocyte lineage colony stimulating factor, CSF-1, was discovered to be one of these factors based on molecular analysis of the op/op osteopetrotic mouse. These mice, which are severely osteoclast-deficient, have a stop codon mutation in the CSF-1 coding region that abrogates the production of active protein.5 The second factor is osteoprotegerin ligand (OPGL),3,6 a novel tumor necrosis factor (TNF) family member that has also been described as TRANCE, RANKL, and ODF.6-8 This long sought after factor complements CSF-1 to stimulate the formation of osteoclasts from hematopoietic precursors in the absence of other cytokines or colony factors.3,6 Both OPGL and CSF-1 are made by stromal cells and osteoblasts and their production is regulated by factors known to modulate bone resorption activity.6 Osteoblasts also secrete a soluble decoy receptor for OPGL, OPG,9,10 which can down-regulate the effects of OPGL by sequestering it from the OPGL cell-surface receptor.11,12
OPGL can also stimulate the activities of mature osteoclasts.13,14 Using preformed osteoclasts from neonatal rat long bones, we have shown that OPGL stimulates bone resorption in a pit-forming assay and rapidly induces the formation of actin ring structures, which are required for bone resorption to occur. Finally, injections of OPGL in normal mice lead to hypercalcemia too rapidly for osteoclast formation to occur indicating that OPGL targets pre-existing osteoclasts in bone.
The above studies have clearly shown that OPGL plays a key role in the
differentiation of osteoclasts from hematopoietic precursors as well as
the activation of mature osteoclasts. What is not clear is the
importance and potential mechanisms of OPGL in the determination of
osteoclast survival once the mature cell has formed. Recent reports
using neonatal rat osteoclasts14
or purified
cultured-murine osteoclasts15
suggest that OPGL/ODF/TRANCE
alone is sufficient to maintain osteoclast viability in
vitro with NF-B activation playing some role in this effect. In
this study, we have examined the effects of OPGL and its withdrawal on
the survival of osteoclasts both in vitro and in
vivo. In vitro, removal of both OPGL and CSF-1 from
bone marrow cultures where CSF-1 + OPGL had driven osteoclast
differentiation quickly leads to a significant reduction in osteoclast
numbers because of apoptosis. Withdrawal of either CSF-1 or OPGL alone
also leads to osteoclast apoptosis with CSF-1 removal leading to a more
rapid disappearance of osteoclasts indicating that neither CSF-1 nor
OPGL alone are sufficient for optimal osteoclast survival. The
apoptotic mechanism seems to involve at least two distinct pathways,
one of which is caspase 3-mediated. In vivo, blockade of
either administered or endogenous OPGL using a single, high dose of
recombinant OPG delivered intravenously leads to the disappearance of
virtually all of the osteoclasts along both the endosteal and
periosteal surfaces throughout a 48-hour period. These findings
indicate that OPGL is critical for osteoclast survival in
vivo in normal animals and that its depletion leads to programmed
cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoclast forming cultures were established as
described3
using bone marrow from C3H/HeN mice (Charles
River Laboratories, Wilmington, MA) as a source of osteoclast
progenitors. The media used to drive osteoclast differentiation was
-minimal essential medium (MEM) supplemented with 10%
heat-inactivated fetal calf serum (termed basal media) that was
supplemented with murine recombinant CSF-1 (30 ng/ml, R&D Systems,
Minneapolis, MN) and murine recombinant OPGL (158 to 316, 100
ng/ml).3
When osteoclasts appeared, the media was removed
and the plates gently rinsed. The media was then replaced with basal
media, or basal media supplemented with CSF-1, OPGL, or CSF-1/OPGL. In
some experiments, the caspase 3 or nonspecific caspase inhibitors,
DEVD-FMK or zVAD-FMK (100 µmol/L; Enzyme Systems Products, Dublin,
CA), were also added as indicated. At various times, the supernatants
were collected and the adherent layer stained cytochemically for
tartrate resistant acid phosphatase (TRAP) activity as
described.11
Supernatant cytospins were prepared using a
Shandon Cytospin 3 cytocentrifuge (Shandon Scientific, Ltd., Runcorn,
UK) set at 200 rpm for 3 minutes and then stained with Wright’s Giemsa
or TRAP stains. In the osteoclast-forming assay using osteoclast
progenitors from OPG or saline-treated mice, various concentrations of
OPGL were used together with CSF-1 (30 ng/ml) and a solution assay that
quantitates TRAP culture activity was used to assess osteoclast
differentiation.11
Ultrastructural Analysis
Osteoclast cultures were established on polyethylene terephthalate track-etched membranes (0.4 µm; Becton Dickinson, Franklin Lakes, NJ) and manipulated as above. At various times, the supernatants were collected and the detached cells gently pelleted (5 minutes at 500 relative centrifugal force) and resuspended in 3% glutaraldehyde in cold 0.1 mol/L sodium cacodylate buffer at pH 7.4. After 24 hours fixation at 4°C, the cells were rinsed in buffer, postfixed for 1 hour with 1% aqueous osmium tetroxide, rinsed in water, and transferred to 2-ml microcentrifuge tubes. The adherent cells were fixed in situ for 24 hours, rinsed, then postfixed with 1% aqueous osmium tetroxide. The cells were gently dislodged using a rubber policeman and then pelleted in 2-ml microcentrifuge tubes. The pellets were dehydrated in ethanol and embedded in an epoxy resin. Light microscopic examination of all embedded materials was conducted on toluidine blue-stained, 1-µm sections. Ultra-thin sections were collected on 200-mesh copper grids and contrast-enhanced with uranyl acetate and lead citrate before examination on a Philips CM120 transmission electron microscope.
Molecular Analyses
The following sequences were generated by reverse transcription-polymerase chain reaction from mouse thymus (bcl-2 and bcl-XL) or mouse osteoclasts (c-src)3 and cloned into the transcription vector pGEM-T (Promega, Madison, WI): bcl-2 (GenBank: M16506, bases 1846 to 2264), bcl-XL (GenBank: L35049, bases 527 to 735), and c-src (GenBank: M17031, bases 259 to 431). A 105-bp murine cyclophilin probe (Ambion, Austin, TX) was used as an internal control. After cloning, the vectors were linearized, and radiolabeled-antisense transcript was synthesized using SP6 or T7 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) and [32P]rUTP (800 Ci/mol; Amersham, Arlington Heights, IL). The probes were purified on a 6% polyacrylamide/7 mol/L urea gel. The caspase probes were synthesized from the mAPO-1 template (Pharmingen, San Diego, CA). The RNase protection assay was performed using the RPA II kit (Ambion, Inc., Austin, TX) and 15 µg (for bcl-2 and bcl-XL), 10 µg (for caspases), or 5 µg (for c-src) of total RNA from each sample. Quantitation was performed with a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The integrated volume of the probe band and internal control band was calculated and the ratio of the two was averaged.
Caspase Assays
Cytosolic extracts of cells were prepared from osteoclast cultures as described.16 For caspase activity assays, 20 to 40 µg of cell lysates were diluted 10-fold in assay buffer (50 mmol/L HEPES, pH 7.5, 10% sucrose, 100 mmol/L NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate) containing 50 µmol/L substrate and incubated at room temperature for up to 1 hour. The substrates tested included, YVAD-AFC (caspase 1), DEVD-AFC (caspase 3), LETD-AFC (caspase 8), and IETD-AFC (caspase 9) and were from Enzyme Systems Products (Dublin, CA). The release of AFC was measured in a Cytofluor II, fluorescence multiwell plate reader (PerSeptive Biosystems, Framingham, MA) using an excitation filter of 400/30 nm and an emission filter of 508/20 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were performed as described previously.16 The mouse anti-caspase 3 antibody was obtained from Transduction Laboratories (Catalog no. C76920, Lexington, KY). Densitometry on autoradiograms was performed using a Fluorchem 8000 and AlphaEase Software (AlphaInnotech Corp., San Leandro, CA).
NF-B and JNK Kinase Assays
Osteoclasts cultures were rinsed and then treated for 30 minutes
with temperature (37°C) and CO2-equilibrated
basal media alone, or basal media supplemented with CSF-1 (30 ng/ml),
OPGL (100 ng/ml), or CSF-1 + OPGL. For NF-B activation, gel
retardation assays were performed using the Gel Shift Assay Kit
(Stratagene, La Jolla, CA) using the manufacturer’s recommended
conditions to detect NF-B activity. For endogenous JNK kinase
activity, culture lysates were prepared and assayed as previously
described.17,18
The GST-cjun (1 to 143) substrate was
purified with a commercially available purification module (Pharmacia
Biotech, Piscataway, NJ) using the manufacturer’s recommended
conditions.
In Vivo Studies
C3H/HeN male mice, 6 to 7 weeks old, were injected subcutaneously with either saline or OPGL (1 mg/kg/day) for 7 days. Three hours after the last treatment, each group was further subdivided into two groups. These subgroups were administered a single dose of either saline or OPG (10 mg/kg) intravenously. The recombinant OPG used was a truncated human molecule lacking the c-terminal heparin binding domain that was fused at the amino terminus to a human Fc sequence.11 At time = 0 (time of OPG treatment) and at various times up to 192 hours, groups of animals were sacrificed and the femurs and tibiae were collected.
Histology/Immunohistochemistry
The femurs and tibiae were processed into paraffin as described.11 Bone sections were stained with hematoxylin and eosin and were also subjected to immunohistochemistry using cathepsin K antibodies.19 For immunohistochemistry, sections (4 µm) were blocked with CAS Block (Zymed Laboratories, San Francisco, CA) and incubated with affinity-purified anti-cathepsin K antibodies (10 µg/ml). A biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame CA) was used as the secondary antibody. The tertiary reagent was an alkaline phosphatase conjugated avidin-biotin complex (Vector Laboratories) and Vector Red (Vector Laboratories) was the substrate used to develop the staining reactions. All sections were lightly counterstained with hematoxylin.
Histomorphometry
Osteoclast numbers were assessed in the distal 5 mm of the femur from the midshaft to the metaphyseal side of the growth plate on cathepsin K-immunostained sections using image analysis (MetaMorph; Universal Imaging Co., West Chester, PA) and were expressed in terms of osteoclasts/total tissue area. Apoptotic osteoclasts were identified in cathepsin K-stained sections and were expressed as apoptotic osteoclasts/total tissue area. The total tissue area examined in the femur sections averaged 4.64 ± 0.504 mm2 (X ± SD, n = 200).
Statistical Analysis
The in vivo data were analyzed by analysis of variance using a computer-based statistical program (Statview; Abacus Concepts Inc., Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for
example) after which the media was replaced as indicated. In the
presence of media alone, osteoclasts rapidly disappeared from the
cultures with observable declines seen by 6 hours (Figure 1)
with <5%
remaining at 24 hours. When CSF-1 alone was re-added, osteoclasts still
disappeared from the cultures, but the rate was slower than that
observed in the media-alone group with significant differences present
only by the 24-hour time point where the osteoclast number was ~30%
of the starting number. Surprisingly, re-addition of OPGL did not
prevent the disappearance of osteoclasts, because their rate of
disappearance was intermediate between the CSF-1-alone and medium-alone
conditions. However, osteoclast numbers were maintained when both OPGL
and CSF-1 were re-added.
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. This suggests that the
osteoclasts seen after 24 hours in the CSF-1 + OPGL condition were
there primarily as a result of improved survival. Electron micrographs
(Figure 2)
provide ultrastructural evidence that osteoclast numbers
decline because of apoptosis. Apoptotic osteoclasts were present in all
conditions suggesting that once the cell death program had been
initiated, the path(s) of self-destruction could not be altered.
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in total RNA from in
vitro-derived osteoclasts. Using culture lysates and various
caspase substrates including YVAD-AFC, DEVD-AFC, LETD-AFC, or IETD-AFC,
which are somewhat selective for caspases 1, 3, 8, or 9, respectively,
we find that culture lysates contain enzymatic activity selective for
DEVD-AFC (Figure 3B)
. In all circumstances except for the combination
of CSF-1 + OPGL, elevations of caspase 3-like activity were seen by 24
hours with a modest delay for caspase 3-like activity elevations in the
CSF-1 alone condition compared to media-alone and OPGL-alone
conditions. Western blots of the osteoclast lysates (Figure 3C)
demonstrate the presence of procaspase 3 in culture lysates that
disappears over time, particularly in the OPGL- and medium-alone
conditions. This observation is consistent with activation of
procaspase 3 leading to the generation of active caspase 3, which can
be difficult to detect.21
DEVD-FMK and zVAD-FMK were used
to block osteoclast apoptosis in vitro and neither of these
inhibitors blocked apoptosis when used alone (Figure 3D)
. However, both
inhibitors complemented OPGL, but not CSF-1, in improving osteoclast
survival. These data suggest that lack of CSF-1 causes caspase 3
activation leading to apoptosis. On the other hand, the loss of OPGL
activates a pathway involving a novel caspase or noncaspase enzyme
which concomitantly activates caspase 3 either directly or indirectly
(Figure 3B)
and is insensitive to specific (DEVD-FMK) and nonspecific
(zVAD-FMK) caspase inhibitors (Figure 3D)
.
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B and/or JNK kinase in other
cell types raising the possibility that these pathways may contribute
to osteoclast survival. As shown in Figure 4A
, CSF-1 or OPGL produce a modest
increase in NF-B activity in mature osteoclasts after a 30-minute
exposure. The combination of OPGL and CSF-1 synergized to produce
higher levels of NF-B binding activity. OPGL, but not CSF-1, also
stimulates JNK kinase activity in these cultures as shown in Figure 4B
.
We have not seen any evidence for an interaction between OPGL and CSF-1
on JNK kinase activation. Hence, in cultures enriched for mature
osteoclasts, OPGL alone stimulates both NF-B and JNK kinase whereas
CSF-1 seems to impact only NF-B activity.
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, osteoclast cultures exposed to CSF-1,
whether alone or combined with OPGL, express increased amounts of
bcl-XL mRNA. While bcl-2 mRNA levels were only
~10% of bcl-XL, CSF-1 treatment again led to
increases in bcl-2 mRNA expression similar in magnitude to
bcl-XL mRNA expression. OPGL did not alter the
expression of either bcl-2 or bcl-XL mRNA, nor
did it seem to enhance the effect of CSF-1 on the expression of these
anti-apoptotic proteins. However, OPGL, but not CSF-1, re-addition did
serve to maintain c-src mRNA expression at elevated levels in the
presence or absence of CSF-1.
To study the role of OPGL in osteoclast survival in vivo, a
recombinant form of OPG, the secreted high-affinity OPGL receptor, was
used to sequester OPGL, either endogenously expressed or administered,
in mice. Groups of mice were treated for 7 days with daily injections
of either saline or OPGL (1 mg/kg) subcutaneously.3
Three
hours after the last injection of OPGL or saline, a single large dose
of recombinant OPG (10 mg/kg) or saline was administered. After a week
of OPGL injections, just before OPG injection, osteoclast numbers were
increased approximately twofold compared to control (Figure 5A
; Figure 6, A and B
). After cessation of OPGL
treatments, osteoclast numbers declined slightly throughout the next 48
hours, but not to control levels (Figure 5A
; Figure 6D
). In contrast,
OPGL-treated animals given OPG exhibited a marked, rapid decline in
osteoclasts such that by 48 hours essentially none remained (Figure 5A
;
Figure 6F
). There was some lag between OPG administration and
osteoclast disappearance as the greatest osteoclast decline was seen
between 12 and 24 hours after OPG treatment (Figure 5A)
. Similar
changes were seen in saline-treated animals given OPG (Figure 5A
;
Figure 6, A and E
), but the osteoclast starting number was obviously
less. Saline-treated animals challenged with saline had stable
osteoclast numbers throughout the 48-hour period (Figure 5A
; Figure 6C
).
|
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). In the OPG-treated, OPGL group
apoptotic osteoclasts were evident at 6 hours, maximizing at hour 24,
and declining thereafter (Figure 5B)
. In the OPG-treated, saline group
maximal osteoclast apoptosis was also seen at 24 hours. Apoptotic
osteoclasts were also seen in the OPGL-treated group given saline. In
this group, apoptotic osteoclasts become detectable at hour 24 and
remain elevated at hour 48. These observations strongly indicate that
the removal of OPGL via OPG led to osteoclast apoptosis in
vivo.
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, osteoclast counts in OPG-treated
animals nadir to nearly 0 at 48 hours and remain very low up to 96
hours after OPG treatment. After 8 days, the OPG-treated groups have
slightly increased numbers of osteoclasts relative to controls
suggesting that a modest rebound has occurred. In OPGL-treated animals
that had received saline instead of OPG, osteoclast numbers remained
elevated with only a trend toward reduction at the 8-day time point.
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. These are the
same areas that exhibit osteoclast activity in normal animals (Figure 8B)
and additionally, exhibit the most evident OPGL mRNA expression by
in situ hybridization.3
To exclude the possibility that OPG exposure in vivo had
deleterious effects on osteoclast precursor cells, bone marrow
collected from animals 48 hours after OPG treatment (10 mg/kg, i.v.)
was used to generate osteoclasts in vitro. As shown in
Figure 8D
, the osteoclast-forming potential of bone marrow cells was
not impacted by OPG exposure in vivo. We also examined bone
marrow cells for OPGL-FITC binding 24 hours after exposure to CSF-1 (30
ng/ml) and OPGL (100 ng/ml) and found that the percentage of OPGL-FITC+
cells were virtually identical between the saline- (5.43%) and OPG
(5.04%)-treated groups. Together these findings indicate that mature
osteoclasts are more sensitive to loss of OPGL than their precursor
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6 days after OPG injection to occur. This delay in
osteoclast recovery likely reflects the pharmacokinetics of this form
of OPG in mice (mean residence time ~21 hours; S. Martin, unpublished
data) as well as the necessity for osteoclasts to regenerate de
novo from precursor cells. Lastly, this in vivo OPG
effect likely does not reflect blockade of endogenous TRAIL, another
TNF family member to which OPG appears to bind, because TRAIL does not
seem to have direct effects on osteoclast formation or osteoclast
activity in vitro or in vivo.24,25 Our data differ from Fuller et al14 and Jimi et al15 who demonstrate that in vitro either OPGL/TRANCE/ODF or CSF-1 (M-CSF) alone are sufficient to ensure osteoclast survival. Additionally, we have found that osteoclasts derived from neonatal rat bones cultured on bone slices can survive 24 hours in the absence of OPGL.13 Methodological differences leading to the presence of contaminating cells and the effects of bone matrix constituents on cell survival may in part explain these differing results. The relevance of our observations showing that the combination of CSF-1 + OPGL together determine osteoclast survival are underscored by the in vivo experiments that demonstrated that the removal of OPGL through OPG administration led to osteoclast apoptosis. One would predict that administration of sufficient levels of a CSF-1-blocking reagent would also lead to osteoclast apoptosis in vivo.
Osteoclast cultures contained mRNA for caspases 1, 2, 3, 6, 7, 8, and
11 based on an RNase protection assay (Figure 3)
. We have detected
caspase 3-like activity in osteoclast cultures and caspase inhibition
with either DEVD-FMK or zVAD-FMK had significant anti-apoptotic effects
only when combined with OPGL. This suggests that loss of CSF-1 leads to
activation of caspase 3, an observation which confirms and extends some
of the findings of Okahashi et al.26
Unlike these
investigators, however, we did not find that the use of DEVD-FMK alone
improved osteoclast survival in our system. Because these inhibitors
did not complement CSF-1 alone, it is probable that a distinct
apoptosis pathway may be operative when OPGL is withdrawn from
osteoclasts. Whether this pathway involves a caspase (potentially
novel) or perhaps a novel apoptosis-inducing, noncaspase protein such
as AIF27
that we have not detected nor searched for in our
experiments is unknown.
OPGL signaling in osteoclast precursors and osteoclasts seems to be
mediated by RANK and involves TRAFs 2, 5, and 6.1,17
While
not identifying a specific pathway responsible for apoptosis
prevention, we have characterized the effects of OPGL and CSF-1 on
select signaling pathways in osteoclast cultures. OPGL stimulates both
NF-B and JNK kinase activation just as it seems to do in dendritic
cells.8
In contrast, CSF-1 alone only stimulates NF-B
activity with no effects on JNK kinase. Based on earlier studies,
NF-B activity seems to be essential for osteoclast formation and may
play an important role in osteoclast survival.28
In fact,
interleukin 1 has been reported to enhance osteoclast survival in
vitro through NF-B activation.29
While the
magnitude of NF-B activation may be important for osteoclast
survival, from our data it is clear that activation of NF-B alone
per se is insufficient to promote osteoclast survival.
CSF-1, but not OPGL, enhances the expression of mRNA for both bcl-2 and
bcl-XL, two proteins that are
anti-apoptotic.30
In contrast to its effects on
osteoclasts, OPGL/TRANCE does seem to increase
bcl-XL expression in murine dendritic
cells.31
The stimulatory effect of CSF-1 on bcl-2 and
bcl-XL may explain the modest effects that CSF-1
has on osteoclast survival (Figures 1 to 3)
. On the other hand, OPGL,
but not CSF-1, maintained c-src mRNA at elevated levels. Obviously
elevated c-src alone was not sufficient to prevent osteoclast
apoptosis. From these data one must conclude that the mechanism able to
most effectively prevent osteoclast apoptosis relies on the combined
signaling pathways of OPGL and CSF-1. Additionally, as some of the
genes induced by OPGL on osteoclasts include proteins with signaling
pathways of their own (c-src, ß3 integrin for instance), it is
possible that these signaling pathways may also synergize with those
driven by CSF-1. Identification of pivotal intersects in these pathways
may yield known or novel molecules that would present logical targets
for innovative therapeutic strategies.
In a broader context, these data provide additional insights into how the osteoblast may modulate both the beginning and end of the resorption component of bone remodeling. At resorption initiation, osteoblast presentation of OPGL and CSF-1, perhaps attended by down-regulated OPG production, promotes recruitment, and activation of osteoclasts and their precursors. As the stimulus for resorption abates, osteoblasts reduce the supply of available OPGL through OPGL down-regulation, increased OPG production, or perhaps some combination of the two. The loss of OPGL immediately blocks further osteoclast differentiation from precursor cells and provides a potential death signal for previously formed osteoclasts. These events likely are anatomically quite specific as both OPGL and OPG are local factors because of osteoblast membrane expression and matrix affinity, respectively. Lastly, the capacity for a single, pharmacologically administered dose of OPG to cause rapid, massive osteoclast apoptosis provides both a potent mechanism for some of its in vivo activity and a strong rationale for its use as an anti-resorptive therapeutic.
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Footnotes |
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Accepted for publication April 20, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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saline group: *, P < 0.05, **,
P < 0.01; different from OPGL 
