Granulocyte Macrophage-Colony Stimulating Factor Reciprocally Regulates
v-Associated Integrins on Murine Osteoclast Precursors
Masaru Inoue,
Noriyuki Namba,
Jean Chappel,
Steven L. Teitelbaum and
F. Patrick Ross
Department of Pathology Washington University School of
Medicine St. Louis, Missouri 63110
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ABSTRACT
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The integrins
vß5 and
vß3 are expressed
reciprocally during murine osteoclastogenesis in vitro.
Specifically, immature osteoclast precursors, in the form of bone
marrow macrophages, contain exclusively
vß5, surface
expression of which declines with commitment to the osteoclast
phenotype, while levels of
vß3 increase
concomitantly. The distinct functional significance of
vß5 is underscored
by the integrins capacity, unlike
vß3, to mediate
both attachment and spreading on ligand, of marrow macrophages,
suggesting
vß5
negotiates initial recognition, by osteoclast precursors, of bone
matrix. Northern analysis demonstrates changes in the two ß-subunits,
and not
v, are responsible for these
alterations. Treatment of early precursors with granulocyte-macrophage
colony stimulating factor (GM-CSF) leads to alterations in
ß3 and ß5 mRNA and
vß5 and
vß3,
paralleling those occurring during osteoclastogenesis. Nuclear run-on
and message stability studies demonstrate that while GM-CSF treatment
of precursors alters ß5 transcriptionally,
the changes in ß3 arise from prolonged mRNA
t1/2. Similar to GM-CSF treatment, the rate
of ß5 transcription falls during authentic
osteoclastogenesis. In contrast to cytokine-induced
vß3, however, that
attending osteoclastogenesis reflects accelerated transcription of the
ß3-subunit. Thus, while GM-CSF may participate in
modulation of
vß5
during osteoclast differentiation, signals other than those derived
from the cytokine must regulate expression of
vß3.
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INTRODUCTION
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The osteoclast is a multinucleated cell whose ability to degrade
bone requires matrix attachment. Commitment to the osteoclast
phenotype, by mononuclear precursors, involves substrate recognition,
an event mediated largely by integrins. Integrins are a family of
membrane-spanning, cell surface receptors consisting of noncovalently
bound
- and ß-subunits which mediate cell-matrix binding. After
attachment, integrins also transmit extracellular signals, thereby
regulating intracellular events, including cytoskeletal reorganization,
migration, proliferation, differentiation, and coagulation (1, 2, 3).
The
v-, ß1-, or ß2-subunits
are the promiscuous components of integrin families, each combining
with multiple partners, thereby generating heterodimers that effect
cell anchoring (1). While mature osteoclasts express ß1-
and
v-integrins (4, 5), it is the heterodimer
vß3 that plays a major functional role in
these cells. Thus, a series of in vitro and in
vivo experiments involving antibodies (5, 6), disintegrins (7, 8),
peptides containing the integrin recognition motif Arg-Gly-Asp,
RGD (9, 10, 11), or a small peptidomimetic (12) indicate that
vß3-blockade inhibits osteoclastic bone
resorption. In fact, the capacity of an RGD peptide mimetic capable of
recognizing
vß3 to arrest bone loss in the
oophorectomized rat (12) raises the possibility that osteoclast
integrin blockade may prevent postmenopausal osteoporosis.
Osteoclasts are polykaryons derived from macrophages, the latter
expressing the two closely related integrins,
vß3 and
vß5
(13, 14). Given the homology of their protein sequences, it is not
surprising that
vß3 and
vß5 share a number of matrix ligands (1),
including the RGD-containing proteins, vitronectin, osteopontin, and
bone sialoprotein, all present in bone (15). Since osteoclasts derive
from cells known to express the two
v-integrins, we
examined expression of
vß3 and
vß5 in a murine model of
osteoclastogenesis. We find
vß5 to be the
sole
v-integrin on immature precursor cells and that
this heterodimer is replaced by
vß3 during
osteoclast differentiation. Furthermore, the two integrins serve
separate functions in osteoclast precursors attached to the
RGD-containing protein vitronectin. While
vß5 mediates both attachment and
spreading,
vß3 regulates only attachment.
Finally, the changes in
v-integrin expression during
osteoclastogenesis are mimicked by treatment of precursors with the
hematopoietic cytokine granulocyte macrophage-colony stimulating factor
(GM-CSF). While the molecular mechanism by which GM-CSF diminishes
ß5 mirrors that occurring during osteoclastogenesis, the
pathways by which the cytokine enhances ß3 mRNA differs
from that occurring in osteoclastogenic culture, an indication that
molecules other than GM-CSF regulate
vß3
on bone-resorptive cells.
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RESULTS
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mRNA Levels of the ß3- and
ß5-Integrin Subunits Vary Reciprocally during
Osteoclastogenesis
We examined expression of
v-integrins during murine
osteoclastogenesis, using an in vitro system wherein
osteoclast precursors, supported by the mouse stromal line ST-2,
differentiate, with time, into functional osteoclasts. Most
importantly, the macrophage-derived population is easily separated from
stromal cells, during culture (16), thereby permitting analysis of
integrin expression on osteoclast precursors as they differentiate.
Northern analysis reveals that early osteoclast precursors express
almost exclusively ß5 mRNA, with ß3 message
virtually undetectable (Fig. 1
). With
increasing time of coculture [and with a concomitant rise in the
number of osteoclasts formed (16)], levels of ß3 mRNA
rise, while those of ß5 decline.

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Figure 1. Levels of mRNA for the Integrin ß3
and ß5 Vary Reciprocally during Murine Osteoclastogenesis
Adherent bone marrow macrophages (osteoclast precursors) were cultured
for varying periods of time with the stromal line ST-2 in the presence
of 1,25-(OH)2D3 and dexamethasone. Stromal
cells were totally removed by collagenase digestion, and total RNA was
extracted from the remaining adherent cells (osteoclasts and their
precursors), electrophoresed, and transferred to nylon membranes.
Northern analysis was performed with a murine ß5 CDNA.
The same membranes were stripped and reprobed with ß3
cDNA and an oligomer specific for 18S ribosomal RNA, to show equal
loading.
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GM-CSF Reciprocally Regulates Expression of
ß3- and ß5-Integrin
Subunit mRNAs
To identify molecules that may play a role in regulating
ß3 and/or ß5 expression, osteoclast
precursors were treated with a range of hematopoietic cytokines.
GM-CSF decreases steady-state ß5 mRNA (Fig. 2
). Several cytokines, such as
interleukin-3 (IL-3) or IL-6, are not as effective, while others fail
to alter ß5 mRNA levels.

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Figure 2. Effects of Different Cytokines on ß5
mRNA Levels in Murine Osteoclast Precursors
Cells were stimulated by cytokines for 16 h, and total RNA was
analyzed by Northern blot using a murine ß5 cDNA probe.
Cells were treated with vehicle (C), IL-1ß (5 ng/ml), IL-3 (300
U/ml), IL-4 (50 U/ml), IL-6 (1000 U/ml), IL-8 (100 ng/ml), IL-11 (10
ng/ml), GM-CSF (10 ng/ml), ciliary neurotrophic factor (CNTF, 10 ng/ml)
or oncostatin M (OSM, 20 ng/ml).
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To characterize further the role of GM-CSF in regulating expression of
v-integrins, we examined the time course for changes in
both ß3 and ß5 mRNA after treatment with a
single concentration (10 ng/ml) of the cytokine. As shown in Fig. 3A
, ß5 mRNA begins to
decrease between 4 and 8 h and reaches its nadir after 48 h.
In contrast to ß5, ß3 mRNA is detectably
increased after 48 h of GM-CSF treatment and maximizes at 96
h.

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Figure 3. GM-CSF Reciprocally Alters ß3 and
ß5 mRNA Levels in Osteoclast Precursors in a Time- and
Dose-Dependent Manner
A, Adherent bone marrow macrophages were stimulated by a single
addition of 10 ng/ml GM-CSF. Total RNA was isolated at the indicated
times and analyzed by Northern blot. B, Adherent bone marrow
macrophages were stimulated with the indicated doses of GM-CSF. After
96 h, total RNA was isolated and analyzed by Northern blot. The
membranes were stripped and rehybridized with v,
ß3, and 18S probes.
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Osteoclast precursors were treated with varying concentrations of
GM-CSF and, after 96 h, analyzed for alterations of
ß5 and ß3 mRNA. Levels of both subunits
respond in a dose-dependent manner, with the effective minimum GM-CSF
concentration required for change in both integrin subunits mRNAs being
the same, namely, 0.3 ng/ml. In contrast to ß3 and
ß5,
v is not substantially regulated by
GM-CSF (Fig. 3B
).
GM-CSF-Induced Changes in ß5 and
ß3 mRNA Are Paralleled by Surface Expression
of the Integrins
vß3 and
vß5
Having documented the reciprocal effect of GM-CSF on
ß5 and ß3 mRNA levels, we asked whether
these events are paralleled by surface expression of
vß5 and
vß3.
Cells were surface labeled with 125I, lysed, and
immunoprecipitated with antibodies that recognize ß5 or
ß3 specifically. Reflecting the levels of mRNA, control
cells express abundant
vß5 and little
vß3 (Fig. 4
). GM-CSF induces surface expression of
vß3 while reducing that of
vß5. While the size of the upper band,
about 150 kDa, is consistent with that for
v, the two
associated ß-chains differ significantly, with ß3 being
smaller than ß5, which displays the expected size of
about 95 kDa.

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Figure 4. GM-CSF Reciprocally Regulates Surface Expression of
the Integrins vß3 and
vß5 on Cultured Bone Marrow Macrophages
Cells treated for 4 days with or without GM-CSF were surface
radioiodinated and lysates immunoprecipitated with a monoclonal
antibody to ß3 or a polyclonal rabbit antibody to
ß5. The immune complexes were analyzed by SDS-PAGE.
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The Integrin
vß5 Is Functional
in Osteoclast Precursors
Regardless of the mechanism of expression, we find
vß5 to be the dominant
v-integrin on osteoclast precursors. However, in
contrast to the resorptive importance of
vß3, the functional role, if any, of
vß5 in development of the osteoclast
phenotype is unknown. We examined this issue by performing both
attachment and spreading assays, using marrow macrophages whose
predominant integrin is either
vß3 or
vß5. To obtain these respective
populations, immature osteoclast precursors were cultured, nonadherent
(Teflon beakers), for 3 days and treated with either vehicle (yielding
cells expressing
vß5) or 10 ng/ml GM-CSF
(in which case
vß3 is the dominant
v-integrin; see Fig. 4
). As determined by Northern
analysis, mRNA levels of ß3 and ß5 are
similar under conditions of adherence or nonadherence (data not shown).
Cells bearing one or other
v-integrin were allowed to
adhere for 45 min to plates coated with either vitronectin or type 1
collagen. Cells expressing either
vß3 or
vß5 attached equally well to vitronectin,
but neither cell type exhibited significant collagen binding (Fig. 5
). These results indicate that integrins
are capable of mediating attachment to the RGD-containing matrix
protein vitronectin, but only those cells expressing
vß5 (i.e. untreated with
GM-CSF) spread, an event inhibited by a peptidomimetic recognizing both
v-integrins (12) (Fig. 6
).

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Figure 5. Macrophages Expressing Either
vß5 or vß3
Attach to Vitronectin, but not Collagen
Macrophages were isolated and cultured in Teflon beakers with or
without GM-CSF. Cells were added to plates coated with vitronectin or
collagen I. After 45 min, plates were washed, stained with methylene
blue and the cell-associated dye was solubilized. The absorbance of the
wells was read at 650 nm.
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GM-CSF Regulates ß5 and
ß3 mRNA Levels by Different Mechanisms
To determine the molecular mechanism by which GM-CSF alters
ß-integrin subunit mRNA, we performed both nuclear run-on and message
stabilization studies. Nuclei from control or GM-CSF-treated cells were
isolated and used to measure ß3- and
ß5-gene transcription in vitro. The cytokine
decreases the rate of ß5 transcription, but has no effect
on that of ß3 (Fig. 7
).
Given the failure of GM-CSF to impact ß3 transcription in
the face of increased steady-state ß3 mRNA, we measured
the cytokines impact on ß3 message stability, using
actinomycin D as a specific inhibitor of RNA polymerase ll. As seen in
Fig. 8
, GM-CSF increases ß3
mRNA t1/2 from 6.5 to 38 h.

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Figure 7. GM-CSF Alters the Rate of Transcription of the
ß5, but not ß3, Gene
Nuclei isolated from control or GM-CSF-treated cells were used for
in vitro transcription in the presence of
[32P]UTP. Transcripts containing equal amounts of
trichloroacetic acid-precipitable counts were hybridized to excess
ß3, ß5, GAPDH and empty vector cDNAs
immobilized on a nylon membrane.
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Figure 8. GM-CSF Treatment Leads to Increased Stability of
ß3 mRNA
Cells treated with or without GM-CSF for 4 days. At this time
actinomycin D (5 µg/ml final concentration) was added to inhibit
transcription of mRNA. After 0, 4, 8, and 12 h, total RNA was
extracted and analyzed by Northern blot. The intensity of the
ß3 specific band was normalized to the level of the 18S
ribosomal RNA band. Results are expressed as the percentage of the
normalized ß3 mRNA values obtained before the addition of
inhibitor. The t1/2 of ß3 in untreated cells
is 6.5 h, while that in cells treated with GM-CSF is considerably
longer (assuming linear extrapolation of the experimental data, the
value would be 38 h).
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Transcription of the ß5 and
ß3 Genes Is Altered during
Osteoclastogenesis
To delineate the mechanisms by which steady-state levels of
ß3 and ß5 mRNAs alter during in
vitro osteoclastogenesis, we once again turned to nuclear run-on
and mRNA stability studies. In this instance, we compared generated
osteoclasts, purified by collagenase treatment, to a homogenous
population of bone marrow macrophages maintained in the absence of
GM-CSF and ST2 stromal cells, the latter essential for osteoclast
differentiation. Similar to GM-CSF-treated cells (Fig. 8
) the rate of
ß5 transcription in cells undergoing osteoclast
differentiation, normalized to that of the housekeeping gene,
GAPDH, is 5.4-fold less than that occurring in untreated
macrophages. In contrast to the stabilization of ß3-mRNA
induced by the cytokine, no such alteration occurs with
osteoclastogenesis, in which ß3-message is enhanced by a
2-fold increase in transcription (data not shown).
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DISCUSSION
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Reflecting development of experimental models in which
osteoclasts may be isolated or generated in culture, a number of
insights have been gained into the mechanisms by which these cells
resorb bone. Among those molecules identified as crucial to the
resorptive process is the
vß3-integrin. In
osteoclasts, the heterodimer mediates attachment to bone matrix (5, 18), generation of matrix-derived intracellular signals (19, 20, 21, 22), and
cytoskeletal reorganization (23). Thus, regulation of this heterodimer
is an issue of importance. In contrast to
vß3, however, little is known concerning
the function of
vß5 in osteoclast
biology.
Our initial observation of reciprocal
vß3
and
vß5 expression in a murine model of
osteoclast formation prompted us to determine whether any of a wide
range of cytokines mediates changes in integrin appearance during
precursor differentiation. While other cytokines are capable of
decreasing ß5-mRNA, only GM-CSF, which is produced by
macrophages and both endothelial and stromal cells (24), all components
of the bone marrow, prompts a simultaneous rise in ß3.
Our data demonstrate GM-CSF alters
vß3 and
vß5 expression in osteoclast precursors by
different mechanisms. Whereas changes in ß5 mRNA levels,
and hence expression of
vß5, are rapid and
transcription dependent, those relating to
vß3 are slow and reflect enhanced
ß3 mRNA stability. Similar to GM-CSF, osteoclastogenesis
is accompanied by a decline in ß5 transcription. In
contrast to marrow macrophages exposed to the cytokine, however,
enhancement of ß5 in osteoclast formation is the product
not of ß3 message stabilization, but of accelerated
transcription. Thus, while GM-CSF is a candidate regulator of
vß5 in murine osteoclast precursors as
they differentiate, other molecules must stimulate surface appearance
of
vß3.
The integrins
vß3 and
vß5, while structurally related, appear to
serve different functions, which may also be cell type specific. This
is true in cells as diverse as human foreskin fibroblasts (25), smooth
muscle cells (26), pancreatic carcinoma cells (27), hamster melanoma
cells (28), or rabbit mesothelial cells (29). Finally, and of greater
relevance, on human monocytes, the integrin
vß5 is involved in spreading on
vitronectin, while
vß3 plays a role in
migration on the same substrate (13).
Essential components of the osteoclast phenotype include
multinucleation and formation of the cells resorptive organelle, its
ruffled membrane. Because induction of both features requires cell
matrix recognition, the manner in which precursors interact with
substrate is critical to their progression into the mature resorptive
cell (30). We find that osteoclast precursors expressing
vß5, but not
vß3, can both bind and spread on the
RGD-containing protein vitronectin, and that these processes are
blocked by a small molecule
v-integrin antagonist. Given
that early osteoclast precursors express only
vß5, this integrin may play an important
role in their initial attachment of precursors to bone matrix, an event
critical to subsequent differentiation and fusion. Furthermore, our
data indicate that these two integrins are expressed at different
stages of osteoclast maturation. These findings, when taken
together, suggest that, as in other cells,
vß5 and
vß3
play separate roles in osteoclasts and/or their precursors.
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MATERIALS AND METHODS
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Isolation and Culture of Osteoclast Precursors
Except for cell attachment studies, which required use of cells
in suspension, all other experiments were performed using adherent bone
marrow macrophages, generated by culture of nonadherent precursors.
These procedures have been reported elsewhere in detail (16, 31).
Briefly, bone marrow was flushed from the tibiae and femurs of
6-week-old male C3H mice with
-modified Eagles medium (
-MEM).
Cells were cultured for 24 h in
-10 (
-MEM supplemented with
10% FBS) in the presence of 500 U/ml macrophage colony stimulating
factor (M-CSF). The nonadherent population was collected and
mononuclear cells were isolated by Ficoll-hypaque gradient
centrifugation (500 x g, 15 min). The
macrophage-enriched population was recovered by centrifugation
(500 x g, 7 min) and cultured for 34 days at 5
x 106 cells per 100-mm plates in
-10 supplemented daily
with 500 U/ml M-CSF. This procedure yields a pure population of
M-CSF-dependent, adherent osteoclast precursors. To generate
osteoclasts, adherent bone marrow macrophages and the murine stromal
line ST-2 were cocultured at a ratio of 10:1. Cultures were fed every
third day, at which time fresh steroids [10 nM
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3) and 100 nM
dexamethasone] were added. Osteoclasts and their precursors were freed
of ST-2 cells by treatment with 0.1% collagenase, 0.1% BSA in
-MEM
for 2 h at 37 C. In selected studies the same adherent osteoclast
precursors, maintained in the presence of 500 U/ml M-CSF throughout,
were treated with or without the indicated doses of GM-CSF for varying
periods. We have demonstrated (32) that the procedure described for
isolation of osteoclasts, or their precursors, does not lead to
detectable contamination by the ST2 cells used during the coculture
period. Thus, our findings of alterations in ß5 mRNA
levels cannot be attributed to the presence of other cell types and
must reflect changes in ß5 expression in the osteoclast
precursors themselves.
Mouse Integrin ß5 cDNA Cloning
A partial mouse integrin ß5 cDNA was generated by
RT-PCR. Total RNA was isolated from 6-week-old mice kidney with TRIzol
(GIBCO-BRL, Gaithersburg, MD), using the manufacturers instructions.
Total RNA was reverse transcribed with a cDNA cycler kit (Invitrogen,
San Diego, CA), using oligo dT as primer. To perform PCR, two
oligonucleotides were synthesized for use as forward
(5'-ATCCGGAGCCTGGGCACCAAGCT-3') and reverse
(5'-CAGGAGAAGTTGTCGCACTCACA-3') primers. The sequences used were based
on a short mouse integrin ß5 cDNA sequence (forward
primer provided by Dr. Rick Brown, Department of Medicine, Washington
University School of Medicine) and the published human integrin
ß5 sequence (33). After denaturation for 3 min at 94 C
denaturation, PCR was carried out for 40 cycles as follows: 94 C, 1
min; 50 C, 2 min; 72 C, 3 min 30 sec; followed by incubation at 72 C
for 7 min. The PCR product was cloned directly into pCRII vector (TA
cloning kit, Invitrogen) and sequenced at both ends using standard
methods.
Northern Blot Analysis and mRNA Stability Assay
Osteoclast precursors were treated with either vehicle or
cytokine (GM-CSF) for the indicated times. Total cellular RNA was
isolated with TRIzol, and equal amounts (5 µg per lane) were
electrophoresed in 1% agarose gel containing formaldehyde and
transferred to Hybond N (Amersham, Arlington Heights, IL) with a vacuum
blotter. The membrane was prehybridized (5x SSPE, 5x
Denhardts solution, 50% formamide, 0.1% SDS, 1x Background
Quencher (Tel-Test Inc., Friendswood TX)) for at least 2 h at 42
C. Northern analysis was performed using the partial mouse integrin
ß5 cDNA, a mouse integrin ß3 cDNA, which
was cloned in our laboratory (34), or human
v cDNA
kindly provided Dr. Eric Brown, Washington University School of
Medicine). All probes were 32P labeled by the random primer
method, using a kit (Boehringer Mannheim, Indianapolis, IN). After
16 h hybridization, membranes hybridized with ß5 or
ß3 cDNAs were washed with 1x SSPE, 0.1% SDS for 15 min
at 50 C twice, 0.1x SSPE 0.1% SDS for 15 min at 50 C twice, while
probing with
v cDNA was followed by washing with 2x
SSPE 0.1% SDS for 20 min at room temperature twice, 2x SSPE 0.1% SDS
for 20 min at 42 C. For reprobing, membranes were stripped by
submerging in 0.1% SDS at 100 C. To normalize for RNA loading, blots
were finally reprobed with an end-labeled oligonucleotide specific to
18S RNA, as previously described (35). For determination of mRNA
stability, cells in 100-mm dishes were cultured with or without GM-CSF
for 4 days. Actinomycin D (5 µg/ml) was added to all plates and total
RNA was isolated 0, 4, 8, or 12 h later. To compare
ß3-integrin subunit mRNA stability between osteoclasts
and their precursors, cells were cultured for 7 days either alone in
the presence of 500 U/ml M-CSF, or in coculture with ST2 cells and
steroids. ST2 cells were removed from cocultures by collagenase
treatment, and actinomycin D (5 µg/ml) was added to all cells. At
zero time and 7 h later, total RNA was isolated for analysis. In
all instances, 30 µg per lane of total RNA was fractionated in
agarose, Northern analysis was performed using ß3 or
ß5 cDNA, as appropriate, and the results were quantified
with a phosphorimage analyzer, using levels of 18S and 28S RNA to
normalize the data.
Nuclear Run-on Transcription Assay
For studies involving GM-CSF treatment, adherent bone marrow
macrophages were cultured for 48 h with or without cytokine (10
ng/ml) and washed twice with ice-cold PBS. For comparison of the rates
of gene transcription in osteoclast precursors and during
osteoclastogenesis, the same adherent precursors were cultured for 7
days either alone, with the addition every third day of 500 U/ml M-CSF,
or together with ST2 cells and optimal concentrations of
1,25-(OH)2D3 and dexamethasone
(10-9 and 10-8 M, respectively).
To isolate osteoclast-derived nuclei, ST2 cells were removed totally
from cocultures by collagenase treatment. In all instances, isolation
of nuclei and in vitro transcription were performed
essentially as previously described (36). Briefly, cells were extracted
with lysis buffer and nuclei were obtained by low-speed centrifugation.
Isolated nuclei were resuspended in suspension buffer and mixed with a
reaction mixture containing ATP, CTP, GTP, and
[32P]UTP (3000 Ci/mmol). After collection
of nuclei by centrifugation, RNA was isolated with TRIzol. Equal
amounts of freshly transcribed RNA (determined by trichloroacetic
acid-precipitable counts) were hybridized to denatured DNA [5
µg/slot murine ß5 or ß3 cDNA, vector DNA,
or human GAPDH (purchased from CLONTECH, San Diego, CA)] in a slotblot
device. After 48 h, membranes were washed with 1x SSPE 0.1% SDS
for 20 min at 50 C, 0.1x SSPE 0.1% SDS for 20 min at 60 C twice and
exposed to x-ray film. Quantitation was performed using a
phosphorimage analyzer.
Immunoprecipitation of Surface-Expressed Integrins
Cells were washed with PBS and surface-labeled with
125I as described previously (36). Cells were lysed with a
buffer containing 2% Renex 30, 10 mM Tris, pH 8.5, 150
mM NaCl, 1 mM CaCl2, 1
mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.02%
NaN3. Each lysate, containing equal numbers of
trichloroacetic acid-precipitable counts, was incubated with
Gammabind (Pharmacia Biotech, Inc., Piscataway, NJ) and precleared
again with Gammmabind plus whole rabbit serum (for ß5) or
class-matched monoclonal antibody (for ß3). The
polyclonal antibody was raised against the peptide sequence of the
human ß5 cytoplasmic tail. Given the homology between the
murine and human ß5 tail sequences (X. Feng, unpublished
data), it is not surprising this antibody recognizes murine
ß5. The precleared lysate was immunoprecipitated with a
polyclonal rabbit anti-ß5-integrin subunit, kindly
provided by Dr. Louis Reichardt, University of California, San
Francisco, or hamster anti-ß3 integrin subunit
(PharMingen, San Diego, CA). The immune complex was bound to excess
Gammabind, the precipitate recovered by boiling the beads in
electrophoresis sample buffer and subject to 7% SDS-PAGE) under
nonreducing conditions. The gels were dried and subject to
autoradiography.
Cell Attachment Assay
Ninety six-well microtiter plates were coated overnight at 4 C
with collagen (10 µg/ml) or vitronectin (10 µg/ml). The plate was
washed with PBS (BSA wells) or blocked with 1% BSA, followed by PBS
washing (vitronectin wells) before use. To each well was added, in 100
µl of
-MEM supplemented with 0.1% BSA, a total of 4 x
105 cells, previously maintained in Teflon-coated plates
with or without GM-CSF (10 ng/ml) for 96 h. Plates were incubated
for 45 min at 37 C, after which they were rinsed three times with PBS.
Cells were fixed with 2% formaldehyde for 20 min, rinsed twice with 10
mM borate buffer, pH 8.4, and stained with 1% methylene
blue for 10 min. After washing three times with tap water, each well
was filled with 100 µl of 0.1 M HCl and incubated for
1 h at room temperature, and absorbance at 650 nm was measured
using a microtiter reader.
Cell Morphology
Cells were cultured in 48-well plates in the presence of 500
U/ml M-CSF. When cells reached subconfluency, wells were treated with
GM-CSF (10 ng/ml) or vehicle (PBS). In selected wells a small integrin
peptidomimetic (SC56331, Searle Corp., Skokie, IL), known to inhibit
v-integrin function (12), was added daily to a final
concentration of 10 µM. After 3 days, cells were fixed
with 2% paraformaldehyde and photographed with a light microscope.
Experimental Animals
All animal experimentation described herein was conducted in
accord with the highest standards of humane animal care as outlined in
the Guidelines for the Care and Use of Experimental Animals.
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ACKNOWLEDGMENTS
|
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The authors would like to thank Dr. Louis Reichardt, for the
anti-ß5 polyclonal antibody, Dr. Eric J. Brown, for the
murine ß5 cDNA sequence, and Dr. Allen Nickols
(Monsanto/Searle), for providing the peptidomimetic SC56631.
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FOOTNOTES
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Address requests for reprints to: F. Patrick Ross, Ph.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail:
rossf{at}medicine.wustl.edu
Supported by NIH Grants AR42404 (F.P.R.), DE05413, and AR32788, and a
grant from the Shriners Hospital for Crippled Children (St. Louis Unit)
(S.L.T.).
Received for publication March 6, 1998.
Revision received September 15, 1998.
Accepted for publication September 16, 1998.
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