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PHAGOCYTES
From the Department of Cell Differentiation, Institute
of Molecular Embryology and Genetics, and the Department of Orthopedic
Surgery, Kumamoto University School of Medicine, Kumamoto, Japan; and
the Department of Molecular Biology, Immunex Corporation, Seattle, WA.
Identification of receptor activator of nuclear factor- Osteoclasts are bone-resorbing multinuclear cells
derived from hematopoietic stem cells.1-3 Recent studies
have shown that receptor activator of nuclear factor An adherent environment is essential for osteoclast development, and
several studies have identified an important role of integrins in the
bone resorption function of osteoclasts.23,24 Most
integrins bind ligands that contain the arginine-glycine-aspartic acid
(RGD) tripeptide, and this 3 amino acid motif is found in many
extracellular matrix proteins including fibronectin, vitronectin, fibrinogen, osteopontin, and thrombospondin. Most cells express several
integrins, and individual types of integrins can often bind more than
one ligand.25 The In this study, we have examined the effect of adherence on osteoclast
precursor cell differentiation and proliferation using 2 culture
methods: cultivation in semisolid medium (a condition where the cells
are kept nonadherent) and in liquid medium (where the cells can become adherent).
Fluorescence-activated cell sorter analysis and cell
sorting
Osteoclast differentiation in vitro
For integrin-blocking assay, we added the following to culture
on day 0 or day 1: 0.5 mg/mL glycine-arginine-glycine-aspartic acid-serine (GRGDS) peptide or glycine-arginine-glycine-glutamic acid-serine (GRGES) peptide (American Peptide, Sunnyvale, CA) or
blocking monoclonal antibodies against control-purified immunoglobulin G (IgG) (rat, R35-95; hamster, G235-2356; PharMingen, San Diego, CA) or
against integrins Colony assay Colony assay was performed as described elswhere.19 After 6 days of cultivation, the number of colonies present was scored under a microscope, and single colonies were placed on microscope slides one by one. The cells on the slides were then stained with TRAP solution, and the frequency of TRAP+ cells was calculated. Single colonies derived from c-Fms+RANK cells
cultured with M-CSF alone or both M-CSF and sRANKL in methylcellulose medium were removed and aliquoted for subsequent analyses. First, cells
were stained with TRAP solution, and the frequency of TRAP+
cells was calculated. Second, cells were replated onto
fibronectin-coated 96-well plates or 35-mm culture dishes (Falcon1008)
and further cultured with M-CSF alone or M-CSF and sRANKL in liquid or
methylcellulose medium for 6 days. Cultured cells were stained with
TRAP solution every day. Subsequently, colonies from 6-day
methylcellulose cultures were collected again, and cells derived from
single colonies were subcultured further in methylcellulose or liquid
medium. TRAP staining was performed daily as described above.
To investigate the differentiation of cells in methylcellulose medium, single colonies were collected at first and secondary transfer and incubated with 0.027% fluorescence-labeled latex beads (diameter, 1 µm) (Polyscience, Washington, PA) for 4 hours at 37°C. Free beads were removed by washing twice with cold PBS, and then cells were cultured with M-CSF and sRANKL for 6 days. Cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) and also stained with TRAP solution. Analysis of expression of integrins on
c-Fms+RANK cells cultured with M-CSF and
sRANKL for 6 days in methylcellulose medium were incubated with Fc
block (concentration, 1:200) on ice for 15 minutes. Cells were stained
with biotinylated anti-RANK antibody followed by APC-conjugated
streptavidin (Caltag). To detect expression of integrins, cells were
stained with hamster antimouse  3 (2C9.G2)
 2 (HM-2), and 1 (HM-1-1) integrin
antibodies (brand names in parentheses, all from PharMingen) and
detected by phycoerythrin (PE)-conjugated antihamster cocktail IgG
antibody (G70-204, G94-90.5, PharMingen). To detect the expression of
v integrin, cells were stained with PE-conjugated
anti-v antibody (H9.2B8, PharMingen). For
isotype-matched controls, biotinylated rat IgG2a (R35-95, PharMingen)
or hamster IgG (G235-2356, PharMingen) was used. The expression of
integrins or RANK was analyzed on a FACS Calibur.
Reverse transcriptase-derived polymerase chain reaction analysis Total RNA was isolated using RNeasy mini kit (Qiagen GmbH, Hilden, Germany) from freshly isolated c-Fms+RANK cells or cells cultured with M-CSF
and sRANKL in liquid or methylcellulose medium for 6 days.
Single-strand complementary DNAs (cDNAs) were synthesized with a
reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Clontech
Laboratories, Palo Alto, CA). The cDNAs were amplified using the SYBR
Green PCR System (Perkin-Elmer, Norwalk, CT) in a GeneAmp PCR system
(Model 9700, Perkin-Elmer) for 40 cycles. Each cycle consisted of 30 seconds denaturation at 94°C and 4 minutes annealing and/or extension
at 70°C. G3PDH was used as an internal control. Primers used
for RT-PCR were as follows: v-5';
5'-GCCTGGGATTGTAGAAGGA GGGCAAGTT-3', v-3'; 5'-GCTTGTGCAGTCCGTGTTGCTAATTGGT-3', 3-5';
5'-TGAAGAAACAGAGCGTGTCCCGTAATCG-3', 3-3';
5'-AGGTGGCATTGAA- GGA CAGTGACAGCTC-3', 2-5';
5'-GCTTTGGGGCAAGTGATTCAACT- CTGGT-3', 2-3';
5'-AGCTGTTGGACATCCAGGATCAGGTCAG-3', 1-5';
5'-GTGCACAGGAGTGCT C CC ACTTCAATCT-3', 1-3';
5'-GGCCACAGAGCCCCAAAACTACCCTACT-3', 5-5';
5'-AGAAACGAGAGGCTCCAGGGAGGAGTTC-3', 5-3'; 5'-CATGAGTCTGA GATCAGGAGGGCTCAGG-3', FAK (focal adhesion kinase)-5';
5'-CCACCAGCAGCGAGGAAG- TAACCTTAGC-3', FAK-3';
5'-GCAACCCAATGCTTCAGTATCCACAGGA-3', Pyk2 (proline-rich
tyrosine kinase 2)-5'; 5'-GGAGCTCAGGCGGTTCTTCAAGGATATG-3', Pyk2-3';
5'-ATGAGGTCAGCCATGTTCTCAGCCTCTG-3', CTR (calcitonin receptor)-5'; 5'-TGGTTGAGGTTGTGCCC- AATGGAGA-3', CTR-3';
5'-CTCGTGGGTTTGCCTCATCTTGGTC-3', CD11b-5';
5'-TCCTCTAGAGTCTTCCTGGACCACAGCA-3', CD11b-3';
5'-GATCTCTTTGTTGGGGACGTCACTGGTA-3', c-Src-5';
5'-TG- ATGTTATGAAGAACTGCTCGCACCTG-3', c-Src-3';
5'-TGCCCATTTGCTGGGTACTTTCTTTCTC-3', G3PDH-5';
5'-TGAAGGTCGGTGTGAACGGATTTGGC-3', G3PDH-3'; and 5 '-CATGTAGGCCATGAGGTCCACCAC-3'.
Proliferation analysis We define the proliferative subpopulation of cultured c-Fms+RANK cells by FACS. Freshly sorted
c-Fms+RANK cells (1 × 105)
were plated on a fibronectin-coated 35-mm dish (Iwaki 4000-030; Iwaki,
Chiba, Japan) and cultured in -MEM containing 10% FBS with 100 ng/mL M-CSF alone or together with 25 ng/mL sRANKL for 6 days.
Fibronectin-coated dishes were preincubated with 2% BSA/PBS for 30 minutes at 37°C. After 48 hours of incubation, cells were harvested
and washed twice by cold PBS and suspended in lysing buffer comprising
0.5% TritonX-100 (Sigma), 1% BSA, and 0.2 µg/mL ethylenediamine
tetraacetic acid (EDTA)/PBS. Cells were kept on ice for 15 minutes and
fixed overnight in 85% methanol. Cells were collected and washed twice
by 0.1% TritonX-100/PBS and stained with biotinylated
anti-proliferating-cell nuclear antigen (anti-PCNA) monoclonal
antibody (32552A, PharMingen) for 30 minutes on ice. After 2 washes,
cells were incubated with APC-conjugated streptavidin (Caltag). After a
final wash, cells were suspended with PBS, and ribonuclease (RNase) A
type I-AS (Sigma) was added. After incubation for a few minutes,
propidium iodine (PI) (Sigma) was added, and then FACS analysis was
performed using a FACS Calibur.
Isolation and characterization of osteoclast precursor cells To isolate osteoclast precursor cells from murine bone marrow, cells were stained with monoclonal antibodies against a receptor of M-CSF (c-Fms) and a receptor of RANKL (RANK), and 3 populations were then fractionated by FACS on the basis of the expression of c-Fms and RANK. The frequency of each fraction was 11.5%±2.2% (c-Fms+RANK), 5.9%±1.5%
(c-Fms+RANK+), and 1.5%±0.6%
(c-FmsRANK+) in mononuclear murine bone
marrow cells (Figure 1A).
To elucidate which cell fraction has the highest capacity to
differentiate into osteoclasts, sorted cells were cultured in the
presence of M-CSF and sRANKL, and TRAP activity was examined (Figure
1B). The c-Fms+RANK Multinucleation is a marker for osteoclast maturation along with
positivity for TRAP staining. Multinuclear osteoclasts differentiated from c-Fms+RANK To investigate the proliferative activity in each fraction,
methylcellulose colony assays were performed (Table
1). The
c-Fms+RANK
Analysis of differentiation of osteoclast precursor cells cultured in methylcellulose or liquid medium To examine how adherence affects osteoclast development, isolated osteoclast precursor cells (c-Fms+RANK cells)
were assayed in methylcellulose and liquid cultures. Interestingly, cell fusion was not observed in the methylcellulose cultures containing M-CSF and sRANKL even though the cells were already present in aggregates within each colony, whereas these same cytokines were able
to stimulate multinuclear osteoclast formation in liquid culture.
Approximately 50 colonies derived from
c-Fms+RANK cells cultured in methylcellulose
medium in the presence of M-CSF and sRANKL were removed individually
and stained with TRAP on day 6 (Figure 1Cvi). A large number of
TRAP cells were observed in all colonies, and 2.7% to
87.5% (mean ± SD: 23.4%±17.8%) of cells in each colony
were TRAP+. These results suggest that cell adhesion
together with M-CSF and sRANKL play an important role in osteoclast
development. RT-PCR analysis revealed that messenger RNA (mRNA) for the
calcitonin receptor (CTR), TRAP, and c-Src could be detected in pooled
colony cells as well as in cells produced in liquid culture (data not shown). These results indicate that the cells can commit to the osteoclast lineage in methylcellulose cultures.
Analysis of the differentiation ability of cells cultured in methylcellulose medium If the differentiation was just delayed in methylcellulose medium compared to liquid culture, prolonged culture might induce differentiation of osteoclasts, as observed in the liquid medium. To test this possibility, single colonies derived from osteoclast precursor cells cultured in methylcellulose medium were transferred to secondary liquid or methylcellulose medium in the presence of M-CSF and sRANKL, and the frequency of TRAP+ cells was then determined at daily intervals. This showed that the frequency of TRAP+ cells increased in liquid culture from day 2 and reached 100% by day 5 (Figure 2A). In contrast, only 24%±18% of cells subcultured in the presence of M-CSF and sRANKL in methylcellulose were TRAP+ on day 6. Next, 50 colonies were removed from each dish, and the frequency of TRAP+ cells was determined. When the cells cultured in methylcellulose were transferred into liquid medium, the frequency of TRAP+ cells increased and reached a plateau on day 5 (Figure 2B), and multinuclear osteoclasts were observed in the presence of M-CSF and sRANKL from day 2. In contrast, when the cells cultured in methylcellulose were transferred into methylcellulose medium again, the frequency of TRAP+ cells did not change, and multinuclear cells were not observed in methylcellulose with M-CSF and sRANKL (Figure 2B). To confirm these results, the same experiments were performed using secondary colonies. As expected, the results were the same as the first experiments (data not shown). Moreover, 12- to 18-day culture in methylcellulose culture with M-CSF and sRANKL did not increase the frequency of TRAP+ cells. These results demonstrate that the differentiation of osteoclasts is not delayed in a nonadherent environment, but it is arrested. However, the cells in methylcellulose still have the ability to differentiate into osteoclasts.
When osteoclast precursor cells were cultured in the presence of M-CSF alone, there were no TRAP+ cells observed in either liquid or methylcellulose cultures. When single colonies from methylcellulose cultures containing only M-CSF were transferred to secondary liquid cultures containing M-CSF and sRANKL, the frequency of TRAP+ cells increased, and multinuclear cells were also formed. In contrast, when the cells were transferred into secondary methylcellulose cultures, approximately 30% of cells became TRAP+ in the presence of M-CSF and sRANKL (Figure 2C). Repeated analyses using single colonies demonstrated that an adherent environment is crucial for osteoclast differentiation. Cells cultured with M-CSF alone or with M-CSF and sRANKL in methylcellulose for 12 days were still able to differentiate into multinuclear TRAP+ cells when transferred to liquid cultures containing M-CSF and sRANKL. Comparison of osteoclast differentiation in the presence of leucine zipper sRANKL and sRANKL To exclude the possibility that differentiation of osteoclasts in methylcellulose culture might be suppressed because of insufficient differentiation signals, we added the leucine zipper form of sRANKL (LZ-RANKL), which is thought to facilitate and stabilize oligomerization, and determined the frequency of TRAP+ cells. First, the dose-dependency of LZ-RANKL was examined in liquid culture by comparison with sRANKL (Figure 3A). TRAP activity reached a plateau at a concentration of 1 ng/mL LZ-RANKL, whereas it peaked at 25 ng/mL sRANKL. Second, we examined how LZ-RANKL affects the differentiation of osteoclast precursor cells when sufficient LZ-RANKL is added to methylcellulose culture (Figure 3B). Although, there was no significant difference observed between 25 ng/mL sRANKL and LZ-RANKL in liquid culture, a difference was observed in methylcellulose culture. Although the frequency of TRAP+ cells varied in each colony, it was much higher (63% ± 27% [mean ± SD]) in the presence of 25 ng/mL LZ-RANKL compared with cultures containing 25 ng/mL sRANKL (32% ± 24%). Multinuclear TRAP+ cells (1%) were formed even in methylcellulose medium with LZ-RANKL, but no multinucleation was observed with sRANKL (data not shown).
These results indicate that the RANK signal is reduced in cells maintained under nonadherent conditions compared to when they are adherent, although a signal that is strong enough can support osteoclast differentiation even under nonadherent conditions. In the presence of 1000 ng/mL LZ-RANKL, the frequency of TRAP+ cells increased markedly (85% ± 19%), and the number of multinuclear cells increased proportionately. However, the number of nuclei in the multinuclear TRAP+ cells was still lower in the methylcellulose cultures (nuclei per cell, 7% ± 4%; n = 448 cells) than in the liquid cultures (nuclei per cell, 12% ± 14%; n = 466 cells), and no multinuclear cells possessing more than 25 nuclei were detected (data not shown). Bifurcated differentiation of osteoclast precursor cells in vitro To investigate whether the cells cultured in methylcellulose are able to differentiate into monocyte-macrophages, the ability to phagocytose (which is a characteristic of macrophages) was examined. Colonies cultured with M-CSF alone or M-CSF and sRANKL were collected and cultured with fluorescence-labeled latex beads. After a 4-hour incubation with the beads, most of the cells could be seen to have phagocytosed some beads. Further cultivation was performed with M-CSF and sRANKL to examine whether cells carrying latex beads are able to differentiate into osteoclasts (Figure 4). A large number of cells containing latex beads became TRAP+ after 6 days of cultivation, and some were multinuclear. On RT-PCR analysis, cells cultured in methylcellulose for 6 days expressed not only CTR and c-Src mRNA, which is characteristic of osteoclasts, but also CD11b mRNA, which is characteristic of monocyte-macrophages (data not shown). Thus, it is suggested that once osteoclast precursor cells adhere, they can differentiate into osteoclasts even after they have shown an ability to phagocytose.
Analysis of expression of integrins in cultured osteoclast precursor cells To elucidate whether isolated osteoclast precursor cells express integrins, the expression of various integrins such asv, 3, 2, 1,
and 5 on c-Fms+RANK cells was
analyzed. All tested integrins, especially those that recognize the RGD
motif, were expressed on c-Fms+RANK cells
(Figure 5A, top). Colonies derived from
c-Fms+RANK cells cultured with both M-CSF and
sRANKL in methylcellulose were collected, and the expression of
integrins or RANK was examined (Figure 5A, bottom). Expression of
v, 3, and 5, as well as RANK, were up-regulated during cultivation even in methylcellulose medium, and the expression patterns of the integrins were similar to
those for liquid culture, as revealed by RT-PCR (Figure 5B).
Because integrins were expressed on cells cultured in methylcellulose medium, a ligand of integrin, 0.5 mg/mL GRGDS peptide, was added to the methylcellulose culture. The number of colonies and frequency of TRAP+ cells were not affected by the addition of GRGDS peptide (data not shown). FAK and Pyk2 are known as signal transducers acting downstream of integrins,43,46 and Pyk2 has been reported to be expressed mostly in osteoclasts.45 It is possible that if these molecules were not expressed in cells cultured in methylcellulose, osteoclast differentiation would not be induced in methylcellulose, unlike induced differentiation in liquid culture. However, by RT-PCR analysis, the expression of both FAK and Pyk2 in methylcellulose culture seemed not to differ from that in liquid culture (data not shown). These results clearly indicate that the expression of integrins and their signal molecules are not affected by a nonadherent environment, and a ligand of integrin (GRGDS) does not affect osteoclast differentiation in spite of the expression of integrins on cells cultured in methylcellulose. GRGDS peptide and antibody against cells were plated onto
fibronectin-coated plates and cultured with 0.5 mg/mL GRGDS or GRGES
peptide in the presence of M-CSF and sRANKL. The number of multinuclear
TRAP+ cells was dose-dependently reduced after 6 days of
cultivation by the addition of GRGDS peptide but not GRGES peptide
(Figure 6A). Because GRGDS peptide was
initially added to culture, it might affect initial cell attachment and
inhibit osteoclast proliferation. Even when GRGDS peptide was added 24 hours after starting the cell culture, the number of multinuclear
TRAP+ cells was reduced to 16% of the number seen in
control cultures. Although the multinuclear cells were not seen, 100%
of the cultured cells were TRAP+ in the presence of GRGDS
peptide (Figure 6B). On the other hand, GRGES peptide did not affect
multinuclear osteoclast formation at all (Figure 6A,B). To test the
effect of the GRGDS peptide on cell adhesion,
c-Fms+RANK cells were cultured in the
presence of M-CSF and sRANKL with GRGDS peptides for 8 hours, and then
the frequency of adherent cells was scored. Compared with controls
(no peptide or GRGES peptide), initial attachment was not
affected by the presence of the GRGDS peptide (Figure 6C).
To investigate whether the GRGDS peptide affects cell fusion itself,
larger numbers (104 cells per well) of
c-Fms+RANK
To analyze more directly whether the GRGDS peptide affects cell
proliferation, cultured cells were harvested after a 48-hour exposure
and stained with PI and PCNA (Figure 8).
The proliferative activity of the osteoclast precursor cells was
reduced to 50% by the addition of sRANKL compared to the culture with
M-CSF alone. PCNA expression was markedly reduced by GRGDS peptide
treatment (30% compared with GRGES treatment) in the presence of M-CSF
and sRANKL. These results suggest that integrins that recognize RGD motif play an important role in regulating the proliferation of TRAP+ cells.
To investigate which integrin was critical, blocking antibodies against
In this study we have shown that adhesion is required for osteoclast development, especially during the differentiation of osteoclast precursor cells into TRAP+ multinucleated cells. Osteoclast precursor cells prevented from adhering are unable to differentiate into multinuclear osteoclasts even in the presence of M-CSF and sRANKL, and their terminal differentiation can be arrested at a late stage when the cells may have characteristic features of both osteoclasts and macrophages. However, when transferred to conditions where they become adherent, further differentiation is induced and multinuclear osteoclasts develop. We have also shown that integrins which recognize the RGD motif are involved in the proliferation and multinuclear osteoclast formation. There could be 2 explanations as to how osteoclast precursor cells would be maintained at an undifferentiated stage in methylcellulose medium: (1) Osteoclast precursor cells are not fully stimulated by a differentiation factor or RANKL, or (2) osteoclast precursor cells are delayed in their terminal differentiation in methylcellulose medium. To test the first possibility, trimerized RANKL (designated as LZ-RANKL) was added to methylcellulose medium instead of sRANKL. Ligands of the TNF receptor superfamily are known to form trimers, and trimerized ligands transduce signals more efficiently to the target cells by clustering of receptors.46 Stimulation of LZ-RANKL is more intense than that of the monomer form of sRANKL (D.M.A., unpublished data, 2000). Together with the shorter spacer region of LZ-RANKL, which begins at amino acid 138, compared to sRANKL, which begins at amino acid 76, LZ-RANKL induced the differentiation of TRAP+ cells more strongly than sRANKL in methylcellulose culture because the soluble form of RANKL containing a shorter length of the "spacer" region was more active than that of the longer one.5 Although LZ-RANKL induced the differentiation of TRAP+ cells more strongly than sRANKL in methylcellulose cultures, no pure osteoclast colonies were obtained, and the frequency of TRAP+ cells varied among the colonies. Even in methylcellulose medium with LZ-RANKL, no giant cells containing more than 25 nuclei were detected, in contrast to what was seen in liquid culture with sRANKL. This suggests that in a nonadherent environment, osteoclasts do not differentiate well, even if the osteoclast precursor cells are stimulated by LZ-RANKL. Trimerized RANKL may partially supplement the function of cell adhesion for efficient cell signaling. To examine the second possibility, the effect of prolonged culture under nonadherent conditions on osteoclast differentiation was analyzed using twice serially passaged single colonies. Once replated into conditions that allowed the cells to become adherent, osteoclast precursor cells started to differentiate into TRAP+ cells in the presence of M-CSF and RANKL, and the frequency of TRAP+ cells eventually reached 100%. Although osteoclast precursor cells cultured with M-CSF plus RANKL and with M-CSF alone both generated TRAP+ cells after transfer into liquid cultures, those cells cultured with M-CSF alone took slightly longer to achieve complete differentiation (100% TRAP+ cells). Osteoclast precursor cells were thus maintained in M-CSF and were also able to differentiate into TRAP+ osteoclasts, but only if they were allowed to become adherent. Because osteoclast precursor cells were thought to have the same
ancestor as monocyte-macrophages, phagocytotic ability was examined in
both primary cells and cells cultured in methylcellulose with M-CSF and
sRANKL. Not only primary isolated osteoclast precursor cells but also
cells cultured with M-CSF and sRANKL for 6 days took up
fluorescence-labeled beads and then differentiated into TRAP+ multinuclear osteoclasts. This suggests that
osteoclast precursor cells maintain the potential to differentiate into
macrophages or osteoclasts in methylcellulose medium. Although freshly
isolated c-Fms+RANK Because integrins, especially the RGD-recognizing integrins
It has been reported that adhesion or ligation of integrins is involved in activation of the MAP kinase pathway via activation of growth factor receptors, such as endothelial cell, platelet-derived, or fibroblast growth factor (ECGF, PDGF, or FGF, respectively) receptors, and this activation is involved in cell survival and proliferation.47-49 This may be relevant to the finding here that osteoclast precursor proliferation was reduced in the presence of GRGDS peptide, whereas some other mechanism might be involved in suppression of multinucleation. RGD peptides or peptides containing the RGD motif were also reported to induce the detachment35 or apoptosis50 of cultured cells. Echistatin, which contains the RGD motif, inhibits osteoclast multinucleation by impairing the cell migration but not the fusion process itself.36 We propose a model in which osteoclast differentiation proceeds through
2 stages. The first is represented by bipotential precursor cells that
can proliferate in an adherence-independent manner. The second is
represented by osteoclast-committed precursor cells whose subsequent
differentiation is adherence-dependent. The differentiation of
osteoclast precursor cells is arrested in a nonadherent environment,
and when the cells are transferred into an adherent environment,
differentiation proceeds in order to allow the appearance of mature
osteoclasts. Integrins, especially the RGD-recognizing integrins
Submitted April 19, 2000; accepted August 16, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Toshio Suda, Department of Cell Differentiation, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan; e-mail: sudato{at}gpo.kumamoto-u.ac.jp.
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B ligand
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Abstract
Introduction
B
(RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and
the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of
stromal-cell-free culture systems: a semisolid culture medium (a
nonadherent system) and a liquid culture medium (an adherent system).
Osteoclast precursor cells were not able to differentiate into mature
osteoclasts efficiently in the semisolid culture system. Trimerized
RANKL enhanced osteoclast differentiation in semisolid cultures, but
not to the extent seen when cells were allowed to adhere to plastic.
Initial precursor cells were capable of differentiating into
macrophages or osteoclasts. Once these cells were transferred to
adherent conditions, striking differentiation was induced. Multinuclear
cells were observed even after they had displayed phagocytic activity,
which suggests that cell adhesion plays an important role in the
differentiation of osteoclast precursor cells. Integrins, especially
the arginine-glycine-aspartic acid (RGD)-recognizing integrins
) or methylcellulose medium (
) for 6 days. (B) Colonies cultured with M-CSF and sRANKL in methylcellulose
were replated into liquid (
,
multinuclear TRAP-positive cells; 
, average number of nuclei. (B)
Distribution of the number of nuclei in multinuclear TRAP+
cells. Note that the number of giant cells containing more than 100 nuclei was significantly reduced in the presence of the GRGDS peptide
compared with the control.
