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v-Associated Integrins on Murine Osteoclast Precursors
Department of Pathology Washington University School of Medicine St. Louis, Missouri 63110
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 integrin’s 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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). 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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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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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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vß5 Is Functional
in Osteoclast Precursorsvß5 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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).
Given the failure of GM-CSF to impact ß3 transcription in
the face of increased steady-state ß3 mRNA, we measured
the cytokine’s 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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) 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). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 cell’s 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 3–4 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 manufacturer’s 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
Denhardt’s 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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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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vß3, 2ß1 and
vß1 integrins. J Cell Biol 268:16737–16745
vß3 potentiate bone resorption. J
Biol Chem 268:9901–9907vß3 integrin inhibits bone resorption
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v
integrin expression on cultured human macrophages. Proc Natl Acad Sci
USA 90:2517–2521vß3 integrin stimulates immediate cell
signals in osteoclasts. J Biol Chem 266:20369–20374vß3-integrin ligands raise
[Ca2+]i in rat osteoclasts and mouse-derived
osteoclast-like cells. Am J Physiol 266:C376–C381
v ß5. J Clin Invest 96:1987–2001
vß3 via transactivation of the
ß3 gene. J Biol Chem 270:4115–4120This article has been cited by other articles:
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  T. J. de Vries, T. Schoenmaker, B. Hooibrink, P. J. M. Leenen, and V. Everts Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts J. Leukoc. Biol., June 1, 2009; 85(6): 919 - 927. [Abstract] [Full Text] [PDF]
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 S. L. Teitelbaum Osteoclasts: What Do They Do and How Do They Do It? Am. J. Pathol., February 1, 2007; 170(2): 427 - 435. [Abstract] [Full Text] [PDF]
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 S. J. Bakewell, P. Nestor, S. Prasad, M. H. Tomasson, N. Dowland, M. Mehrotra, R. Scarborough, J. Kanter, K. Abe, D. Phillips, et al. Platelet and osteoclast {beta}3 integrins are critical for bone metastasis PNAS, November 25, 2003; 100(24): 14205 - 14210. [Abstract] [Full Text] [PDF]
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