Endocrinology Vol. 140, No. 7 2954-2961
Copyright © 1999 by The Endocrine Society
Characterization of Immortalized Osteoclast Precursors Developed from Mice Transgenic for Both bcl-XL and Simian Virus 40 Large T Antigen1
Teuvo A. Hentunen,
Sharon H. Jackson,
Hoyeon Chung,
Sakamuri V. Reddy,
Joseph Lorenzo,
Sun Jin Choi and
G. David Roodman
Department of Medicine, Division of Hematology (T.A.H., H.C.,
S.V.R., S.J.C., G.D.R.), University of Texas Health Science Center, San
Antonio, Texas 78284; the National Institutes of Health (S.H.J.),
Bethesda, Maryland 20892; the Department of Research, Veterans
Administration Medical Center (J.L.), Newington, Connecticut 06111; and
the Audie Murphy Veterans Administration Hospital (G.D.R.), San
Antonio, Texas 78284
Address all correspondence and requests for reprints to: G. David Roodman, M.D., Ph.D., Research Service (151), Audie Murphy Veterans Administration Hospital, 7400 Merton Minter Boulevard, San Antonio, Texas 78284. E-mail: roodman{at}uthscsa.edu
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Abstract
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We recently developed an immortalized osteoclast (OCL) precursor cell
line that forms large numbers of OCLs. This cell line was derived from
mice doubly transgenic for bcl-XL and large
T antigen that was targeted to cells in the OCL lineage
(bcl-XL/Tag cells). We have now
characterized these cells in terms of their surface and enzymatic
phenotype, responsiveness to osteotropic factors, and differentiation
potential. The bcl-XL/Tag cells expressed
interleukin-1 receptors 1 and 2, gelatinase B (MMP9), as well as Mac-1,
CD16/CD32 (Fc
receptors), CD45.2 (common leukocyte marker), CD86
(costimulatory molecule expressed on B cells, follicular dendritic
cells, and thymic epithelium), major histocompatibility complex I, and
nonspecific esterase when cocultured with MC3T3E1 cells. However, they
did not express the antigens for F4/80 (mature macrophage/dendritic
cell marker) by immunostaining. Treatment of
bcl-XL/Tag cells, cocultured with MC3T3E1
cells, with the combination of 1,25-dihydroxyvitamin D3 and
dexamethasone induced high levels of OCL formation. The
bcl-XL/Tag cells formed large numbers of
OCLs when cultured with RANK ligand and macrophage colony-stimulating
factor in the absence of feeder cells. In the absence of RANK ligand
and a feeder cell layer, 100% of the cells differentiated into
F4/80-positive cells. However, neither PTH nor PTH-related protein
enhanced OCL formation by bcl-XL/Tag cells
even when they were cocultured with primary osteoblasts, suggesting
that they differ from primary mouse bone marrow cells in their
responsiveness to PTH/PTH-related protein. Thus,
bcl-XL/Tag cells have many of the properties
of primary mouse OCL precursors and should be very useful for studies
of OCL differentiation and divergence of OCL precursors from the
macrophage lineage.
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Introduction
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OSTEOCLASTS (OCLs) are multinucleated cells
(MNC) in bone that are the primary cells responsible for bone
resorption. They are derived from hematopoietic precursors that
proliferate and differentiate into OCLs. The final differentiation of
OCLs occurs in bone, where mononuclear OCL progenitors in close contact
with osteoblasts fuse and form bone-resorbing OCLs. OCLs are rare cells
that are difficult to isolate in large numbers for cellular and
molecular biology studies. Therefore, we developed immortalized OCL
precursors from transgenic mice by targeting transforming genes and
antiapoptosis genes into cells of the OCL lineage using the
tartrate-resistant acid phosphatase (TRAP) promoter. In our first
attempts to produce immortalized OCL precursors, we developed
transgenic mice in which simian virus 40 large T antigen (Tag) was
targeted to OCLs. However, we were unable to develop immortalized OCL
precursors from these mice, probably because apoptosis increased in
cells of the osteoclast lineage (1). To overcome this problem, we
targeted an antiapoptosis gene, bcl-XL, to cells
of the OCL lineage using the same techniques. The
bcl-XL gene is a bcl-2-related gene
that is expressed in a wide range of cell types and prevents apoptosis
both in vivo and in vitro (2, 3, 4, 5, 6). By crossing
mice transgenic for Tag and bcl-XL, we obtained
doubly transgenic mice and succeeded in immortalizing OCL precursors
(7). These cells formed OCLs with high efficiency, expressed calcitonin
receptors, as shown by RT-PCR and autoradiography, and resorbed bone
when cultured on dentine slices. The OCL formation capacity of the
bcl-XL/Tag cells was approximately 500 times
greater than that of normal mouse marrow. In the current work we
characterized this cell line further by studying its surface and
enzymatic phenotype, its responsiveness to different osteotropic
factors, and its differentiation potential. These data demonstrate that
bcl-XL/Tag cells form OCL-like cells that
fulfill the phenotypic characteristics of OCLs and show their utility
for studies of the molecular events involved in commitment of bipotent
precursors to OCLs.
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Materials and Methods
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Cytokines and osteotropic hormones used
Bovine PTH, dexamethasone, PGE2, and estrogen
(ß-estradiol) were purchased from Sigma Chemical Co.
(St. Louis, MO). 1,25-Dihydroxyvitamin D3
[1,25-(OH)2D3] was provided by Dr. M.
Uskokovic (Hoffmann-La Roche, Inc., Nutley, NJ). Human
PTH-related protein-(1–34) [PTHrP-(1–34)] was purchased from
Bachem Bioscience, Inc. (King of Prussia, PA). Murine
granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage
colony-stimulating factor (M-CSF), tumor necrosis factor-
(TNF),
and human interleukin-1ß (IL-1ß) were purchased from R&D Systems
(Minneapolis, MN). Soluble human RANK ligand was a gift from
Immunex Corp. (Seattle, WA).
Antibodies
FITC-conjugated monoclonal antibodies (mAB) against Mac-1,
CD45.2, and Ly6G (Gr-1) and phycoerythrin-conjugated mAB against CD80,
CD86, CD14, CD11c, Mac-3, CD40, and CD16/CD32 were purchased from
PharMingen (San Diego, CA). FITC-conjugated mAB against
F4/80 and I-Ab for C57BL/6 major histocompatibility complex type II
(MHC II) were purchased from Caltag Laboratories, Inc.
(Burlingame, CA).
Culture of bcl-XL/Tag cells
The bcl-XL/Tag cells were prepared as
described previously (7) and maintained with MC3T3E1 cells in MEM
containing 10% FCS. To determine the growth characteristics of the
cells, a fixed number of bcl-XL/Tag cells
cultured with feeder cells were plated in four-well plates and cultured
for 2, 24, 48, 72, and 120 h. Cells were fixed with
acetone-methanol (1:1, vol/vol) for 10 min (8) and stained for Mac-1 as
described below, which is only expressed on
bcl-XL/Tag cells. In selected experiments,
bcl-XL/Tag cells were purified as previously
described (7). Briefly, MC3T3 cells were lysed using a polyclonal
antibody prepared against mouse fibroblasts, which also lyses MC3T3
cells. The cells were further enriched by immunoadherence techniques
using the Mac-1 antibody (9). After these purification stages,
bcl-XL/Tag cells were more than 98% pure, as
assessed by Mac-1 staining.
Messenger RNA (mRNA) preparation from osteoclasts formed by
bcl-XL/Tag cells
The bcl-XL/Tag cells were cocultured with
MC3T3E1 cells in six-well plates with or without
1,25-(OH)2D3 (2 x 10-9
M) and dexamethasone (10-7 M) for
7 days to induce OCL-like cell formation. Total RNA was extracted with
RNAzol (3 ml/plate; Biotex Laboratories, Inc., Houston,
TX) by incubating the cells over ice for 5 min, the lysates were
transferred to 15-ml tissue culture tubes, and then 600 µl chloroform
were added to the tubes. The tubes were shaken vigorously and then
centrifuged for 10 min at 4 C for 9000 x g. The upper
aqueous phase was collected, and an equal volume of isopropanol was
added. The tubes were incubated at -20 C for 30 min and then
centrifuged for 15 min at 4 C at 9000 x g. The
supernates were discarded, and the pellets were washed with 500 µl
80% ethanol-20% water. RNA was pelleted by centrifugation for 3 min
at 4 C in a microfuge and completely air-dried. Finally, the RNA
pellets were resuspended in diethylpyrocarbonate-water and stored at
-80 C until needed.
RT-PCR analysis of bcl-XL/Tag cells for
expression of MMP9, IL-1 receptor 1 (IL-1R1), and IL-1R2 mRNAs
Total RNA was isolated from highly purified
bcl-XL/Tag cells or MC3T3 feeder cells by the
guanidinium isothiocyanate method using RNAzol reagent (Biotex Laboratories, Inc.) following the manufacturer’s protocol.
Approximately 3 µg total RNA from each sample were denatured at 65 C
for 10 min and reverse transcribed using murine Moloney leukemia virus
reverse transcriptase. The reaction volume was 20 µl and contained 5
mmol/liter MgCl2, 50 mmol/liter KCl, 10 mmol/liter Tris-HCl
(pH 8.3), 1 mmol/liter of each deoxy-NTP, 2.5 µmol/liter random
hexamers, and 1 U ribonuclease inhibitor. The reaction was incubated
for 15 min at 42 C, followed by inactivation of reverse transcriptase
by heating at 94 C for 5 min. The complementary DNA products obtained
were subjected to PCR in a reaction mixture of 100 µl containing 2
mmol/liter MgCl2, 50 mmol/liter KCl, 10 mmol/liter Tris-HCl
(pH 8.3), 0.2 mmol/liter of each deoxy-NTP, 2.0 U AmpliTaq DNA
polymerase, and 0.1 µmol/liter of sense and antisense primers of the
following murine gene-specific primers: MMP9 sense primer, 5'-TCT GAG
GCC TCT ACA GAG TCT-3'; antisense primer, 5'-CTC ATG GTC CAC CTT GTT
CAC-3'; IL-1R1 sense primer, 5'-GAG TTA CCC GAG GTC CAG-3'; antisense
primer, 5'-GAA GAA GCT CAC GTT GTC-3'; and IL-1R2 sense primer, 5'-TGC
CAG CAG TGC AGC AAG ACTC-3'; antisense primer, 5'-GGA TGG GTT CCG TGG
TTG TTCC-3'. The PCR reaction was carried out by incubating the samples
at 94 C for 1 min followed by 35 cycles at 94 C for 1 min and 60 C for
1 min, with a final extension for 5 min at 60 C. The amplified products
were electrophoresed on a 1.5% agarose gel with a 123-bp DNA ladder
(BRL, Gaithersburg, MD) as a size marker. Bands were visualized by
ethidium bromide staining.
Immunostaining of osteoclast precursors for Mac-1 and F4/80
antigen
The bcl-XL/Tag cells were cultured in the
presence or absence of MC3T3E1 cells for 3 days and then fixed either
with acetone-methanol (1:1) for 10 min or with 2% glutaraldehyde in
PBS for 20 min to stain cells for Mac-1 or F4/80, respectively. Rat
monoclonal antibody against mouse Mac-1 antigen was purchased from
Boehringer Mannheim (Indianapolis, IN) and the monoclonal
antibody against F4/80 antigen from Serotec (Oxford, UK).
After fixation, the cultures were incubated in 1% BSA-PBS overnight at
4 C. The cultures were then incubated with the primary antibody (1:100
dilution) for 1 h at room temperature. Cultures were washed four
times with PBS and incubated for an additional hour at room temperature
with a secondary antibody (biotin-labeled rabbit antirat IgG,
DAKO Corp., Glostrup, Denmark) at a 1:100 dilution. After
washing with PBS, the cultures were incubated for 1 h at room
temperature with peroxidase-conjugated ExtrAvidin (Sigma Chemical Co.,) at a dilution of 1:100. All dilutions were performed using
PBS containing 0.1% BSA. Finally, the antigen-antibody conjugates were
visualized with diaminobenzidine (Sigma Chemical Co.) by
incubating the cultures at room temperature for 15 min.
Flow cytometric analysis
Before staining, the cell suspensions were filtered through a
40-mm nylon cell strainer (Becton Dickinson and Co.,
Franklin Lakes, NJ) to remove clumps. Preparations of 5 x
105 cells were preincubated with purified rat antimouse
CD16/CD32 Fc Block (PharMingen, San Diego, CA) or 5%
mouse serum (Sigma Chemical Co.) to minimize nonspecific
binding. After the initial incubation, cells were stained with the
designated fluorescein isothiocyanate- or phycoerythrin-conjugated mAB
or subclass control for 30 min at 4 C and then washed with HBSS
(BioWhittaker, Inc., Walkersville, MD). All cells were
resuspended in Sorter Medium (Quality Biological, Gaithersburg, MD) for
analysis. Cultured cells stained with fluorescent-conjugated antibodies
were titrated (0.5–1 µg/1 x 106 cells) with the
appropriate subclass controls. Samples were analyzed by flow cytometry
using a FACSort (Becton Dickinson and Co.) with CellQuest
software (Becton Dickinson and Co.). Five parameters were
analyzed: forward scatter, side scatter, and up to three-color
fluorescence intensity. Regions were set using forward
vs. side scatter. Propidium iodide was used to gate out the
dead cells for single staining analyses. Ten thousand events of list
mode data were collected.
Osteoclast formation assay
The bcl-XL/Tag cells (2000/well) were
cultured with or without MC3T3 cells or PA6 stromal cells (5000/well)
in 48-well plates for 7 days. Half of the medium was changed after 3
days. Various concentrations of osteotropic hormones and growth factors
were added at the beginning of the culture and at the time of medium
change. On day 7, cells were fixed with 2% glutaraldehyde in PBS for
20 min, and the cells were stained for TRAP using an
immunohistochemical kit (Sigma Chemical Co.).
TRAP-positive MNC containing at least three nuclei were counted as
OCL-like cells.
Pit formation assay
PA6 stromal cells or MC3T3 cells (10,000/dentine slice) were
plated on dentine slices in a 24-well plate containing 1 slice/well. In
some cultures, no stromal cells were added to the dentine slice. On the
following day, 5,000 bcl-XL/Tag cells were added
to each dentine slice, and the cells were cultured for 7 days in the
presence of 1,25-(OH)2D3 (2 x
10-9 M) and dexamethasone (10-7
M). Half of the medium was changed every third day. After 7
days of culture, the medium was removed, and fresh medium containing
various cytokines or hormones, e.g. IL-1ß, TNF, and
PTHrP, were added to the cultures to test their effects on the
resorption activity of MNC formed on dentine slices. After 24–48 h,
the cells were fixed with 2% glutaraldehyde in PBS for 20 min and
stained for TRAP using an immunohistochemical kit (Sigma Chemical Co.). TRAP-positive MNC containing at least three nuclei were
counted as OCL-like cells. The cells were then removed from the dentine
by brushing, and the resorption lacunae were stained with 1% toluidine
blue (10). The number of resorption lacunae and the total resorption
areas were measured using a phase contrast microscope and image
analysis software (Bioquant, Nashville, TN) as described previously
(11).
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Results
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Surface phenotype of bcl-XL/Tag cells
Immunostaining of bcl-XL/Tag cells with the
mAB against Mac-1 showed strong reactivity with these cells (Fig. 1A
). In addition, MNC formed by
bcl-XL/Tag cells in the presence of
1,25-(OH)2D3 and dexamethasone also stained
positively for Mac-1. In contrast, bcl-XL/Tag
cells did not react with a mAB toward the F4/80 antigen (a mature
macrophage/dendritic cell marker; Fig. 1B). The
bcl-XL/Tag cells cultured for 7 days in the
presence of either GM-CSF (1 ng/ml) or M-CSF (25 ng/ml) did not express
the F4/80 antigen (data not shown). When highly purified
bcl-XL/Tag cells were cultured on plastic for 4
days with or without M-CSF (1–20 ng/ml) in the absence of feeder
cells, bcl-XL/Tag cells became larger, adhered
tightly to plastic, and expressed the F4/80 antigen (Fig. 1C). GM-CSF,
but not M-CSF, at 1 ng/ml stimulated the proliferation of
bcl-XL/Tag cells approximately 3-fold compared
with control cultures, as determined by counting the number of
Mac-1-positive bcl-XL/Tag cells on the top of
feeder cells at the beginning and after 3 days of culture. The OCL
formation capacity in these cultures was increased in a parallel manner
as cell proliferation (data not shown), suggesting that GM-CSF did not
induce any changes in the OCL differentiation capacity of
bcl-XL/Tag cells. However, when highly purified
bcl-XL/Tag cells were treated with RANK ligand,
1,25-(OH)2D3, and dexamethasone in the absence
of a feeder layer, they formed large numbers of OCLs (Table 1 and Fig. 2).
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Figure 1. Immunostaining of
bcl-XL/Tag cells for Mac-1 and F4/80
antigens. The bcl-XL/Tag cells were cultured
for 3 days with MC3T3E1 cells as described in Materials and
Methods and then fixed and stained with anti-Mac-1 (A) or F4/80
(B). The bcl-XL/Tag cells reacted with
Mac-1, but did not react with F4/80. The
bcl-XL/Tag cells were purified by immune
lysis and Mac-1 panning and then cultured in the absence of MC3T3E1
cells for 3 days (A–C) or 7 days (D) as described in Materials
and Methods. All bcl-XL/Tag cells
reacted with F4/80 when cultured in the absence of feeder cells (C). D,
Mac-1 staining of osteoclast-like cells formed in cultures treated with
1,25-(OH)2D3 (10-8 M).
MC3T3 feeder cells are shown by arrowheads in A and B.
Magnification, x40.
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Figure 2. Comparison of osteoclasts formed in cultures of
normal mouse marrow or highly purified
bcl-XL/Tag cells treated with RANK ligand,
1,25-(OH)2D3, and dexamethasone. Magnification,
x100.
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Immunofluorescent staining followed by flow cytometry was used to
analyze the surface antigens expressed on
bcl-XL/Tag cells. For each analysis we compared
staining of MC3T3E1 feeders alone to that of
bcl-XL/Tag cultured over feeder layers. Figure 3 shows the surface phenotype of
bcl-XL/Tag cells. The cells expressed the common
leukocyte antigen CD45.2 and the mouse FcIII and FcII (CD16/CD32)
receptors. Staining with a panel of myeloid markers was variable. The
bcl-XL/Tag cells expressed CD11b (Mac-1), but
were negative for Mac-3 and CD11c. A very small subpopulation of the
cells (
10%) expressed low levels of F4/80 and CD14. The cells were
also negative for the granulocyte marker Gr-1. Interestingly,
bcl-XL/Tag cells were stained with mAB against
costimulatory molecule B7–2 (CD86), but were negative for B7–1 (CD80)
and CD40. The bcl-XL/Tag cells were also
positive for MHC I, but not for MHC II (data not shown).
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Figure 3. Cell surface immunophenotype of
bcl-XL/Tag cells. The expression of myeloid
surface markers or bcl-XL/Tag cells cultured
with MC3T3E1 cells and MC3T3E1 cells by themselves were compared as
described in Materials and Methods using flow cytometric
analysis. Data are represented as the fluorescence profiles of
Bcl-XL/Tag cells (thick solid line) and
profiles of feeder cells (solid line) and isotype
control antibody (dashed line). Staining was performed
simultaneously and is representative of two comparable analyses.
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The doubling time of bcl-XL/Tag cells cultured
over MC3T3E1 cells was approximately 24 h, based on the increase
of Mac-1-positive cells in the cocultures (Fig. 4). A cell concentration of 1 x
105 Mac-1-positive cells/ml was optimal for growth.
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Figure 4. Growth curve of the osteoclast precursor cell line
bcl-XL/Tag. The
bcl-XL/Tag cells were cultured with MC3T3
cells at 105 bcl-XL/Tag
cells/ml. Aliquots were taken at the time points indicated, and the
number of Mac-1 cells in the culture was determined. Results represent
the mean of duplicate determinations.
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RT-PCR analysis showed that MC3T3E1 cells (feeders) alone and highly
purified bcl-XL/Tag cells cultured in the
absence of feeders and treated with or without
1,25-(OH)2D3 and dexamethasone for 7 days
expressed the type 1 IL-1R bcl-XL/Tag cells
expressed the type 2 IL-1R only after treatment with
1,25-(OH)2D3 and dexamethasone. Feeder cells
with or without 1,25-(OH)2D3 and dexamethasone
did not express IL-1R2. The bcl-XL/Tag cells and
feeder cells both expressed MMP9 mRNA (Fig. 5).
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Figure 5. Expression of mRNA for IL-1R1, IL-1R2, and MMP9 by
vitamin D3 (10-8 M)- and
dexamethasone (10-7 M)-stimulated and
nonstimulated Bcl-XL/Tag cells. RT-PCR analysis was
performed as described in Materials and Methods. 1)
Unstimulated bcl-XL/Tag cells in the absence
of feeder cells; 2) 1,25-(OH)2D3- and
dexamethasone-treated bcl-XL/Tag cells in
the absence of feeder cells; 3) unstimulated feeder cells; 4)
1,25-(OH)2D3- and dexamethasone-treated feeder
cells. The PCR products were 550, 1150, and 423 bp, corresponding to
MMP9, IL-1R1, and IL-1R2, respectively, as shown by
arrows. ß-Actin transcripts (300 bp) amplified in
these samples served as a positive control.
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Osteotropic factor responsivity of
bcl-XL/Tag cells
1,25-(OH)2D3 (10-9 and
10-8 M) by itself stimulated TRAP-positive MNC
formation 14- to 20-fold by bcl-XL/Tag cells
grown in the presence of MC3T3 cells or PA6 cells compared with that in
control cultures not treated with 1,25-(OH)2D3,
whereas dexamethasone (10-7 M) by itself
increased OCL-like cell formation 7-fold (Fig. 5
). However, when these
two factors were combined, TRAP-positive MNC formation was stimulated
almost 200-fold compared with that in control cultures. In contrast,
human IL-1ß (10 ng/ml) only stimulated OCL-like cell formation in the
presence of PA6 cells, and this increase was 7-fold compared with that
in control cultures. Interestingly, when dexamethasone
(10-7 M) was combined with human IL-1ß (10
ng/ml), TRAP-positive MNC formation was significantly enhanced 11- to
15-fold with or without PA6 stromal cells (data not shown).
Surprisingly, PTH/PTHrP (1–100 ng/ml) and PGE2
(10-8–10-6 M) did not have any
effect on TRAP-positive MNC formation (Fig. 6). The effects of PTHrP on OCL formation
by bcl-XL/Tag cells were also tested in the
presence of primary rat osteoblasts (5000 cells/well in 48-well
plates), but there were no significant effects on MNC formation (data
not shown). This result was not due to a lack of PTHrP responsiveness
of the fetal rat calvarial cells used in these assays. Treatment of
these fetal rat calvarial cells with 100 µg/ml PTHrP for 24 h
increased cAMP production 5-fold from 3.75 to 18.75
mM/5 x 104 cells plated. Furthermore,
PTHrP induced OCL formation in cocultures of mouse spleen cells with
these fetal rat calvarial cells (data not shown). Calcitonin (10–100
mU/ml) dose dependently inhibited TRAP-positive MNC formation in
1,25-(OH)2D3-stimulated cultures with or
without PA6 stromal cells (Fig. 7).
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Figure 6. Effects of osteotropic hormones and growth factors
on OCL-like cell formation by bcl-XL/Tag
cells. The bcl-XL/Tag cells were cultured
with MC3T3 cells or PA6 cells in the presence of different osteotropic
factors as described in Materials and Methods. For these
experiments, 50 ng/ml calcitonin were used. Results represent the
mean ± SEM from four determinations. A similar
pattern of results was seen in four independent experiments.
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Figure 7. Effect of calcitonin on
1,25-(OH)2D3-induced OCL formation by
bcl-XL/Tag cells. The
bcl-XL/Tag cells were cultured with
1,25-(OH)2D3 (10-9 M)
and varying concentrations of salmon calcitonin for 7 days with or
without PA6 stromal cells. Results represent the mean ±
SEM for four determinations. A similar pattern of results
was seen in three independent experiments.
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Bone-resorbing capacity of bcl-XL/Tag
cells
The bcl-XL/Tag cells (5000 cells) formed
approximately 255 ± 25 OCLs/dentine slice when cultured for 10
days in the presence of 1,25-(OH)2D3 and
dexamethasone and MC3T3E1 or PA6 cells, and the resorption area per OCL
was approximately 836 ± 144 µm2. IL-1ß, TNF
and PTHrP did not enhance pit formation by these cells when added after
7 days of culture (data not shown). No bone resorption occurred in the
absence of feeder cells unless the bcl-XL/Tag
cells had been treated with RANK ligand and
1,25-(OH)2D3/dexamethasone. Under these
conditions, 0.2 pits were formed/OCL.
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Discussion
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We have developed an immortalized OCL precursor cell line by
targeting the simian virus 40 large T antigen and
bcl-XL genes to OCL precursors using the TRAP
promoter. These cells express an OCL phenotype and form high numbers of
OCLs when treated with 1,25-(OH)2D3 and
dexamethasone. The OCL formation of bcl-XL/Tag
cells was greater than 500 times that reported for normal mouse marrow
cultures (12). A high level of OCL formation was seen when the cells
were grown over MC3T3E1 cells or PA6 cells. MC3T3E1 cells have been
reported not to support OCL formation (13), but in our hands, these
cells supported the growth of bcl-XL/Tag cells
without inducing their differentiation. In contrast, PA6 cells induce
the differentiation of bcl-XL cells and did not
support their continued growth (7). These data demonstrate an important
role for marrow stromal cells and osteoblastic cells in supporting the
growth of bcl-XL/Tag OCL precursors.
Attempts to grow bcl-XL/Tag cells in the absence
of MC3T3E1 or osteoblasts induced the formation of F4/80-positive
cells that did not form TRAP-positive multinucleated cells when treated
with 1,25-(OH)2D3 and dexamethasone. Highly
purified bcl-XL/Tag cells only formed
TRAP-positive MNC when they were treated with RANK ligand,
1,25-(OH)2D3, and dexamethasone or M-CSF. RANK
ligand is a recently described factor that is induced on osteoblasts
and marrow stromal cells by PTH, PTHrP,
1,25-(OH)2D3, or IL-11 and is absolutely
required for OCL formation when marrow or spleen cells are cocultured
with osteoblasts or stromal cells (14). Furthermore, in the absence of
any feeder cell layers and RANK ligand, all of the
bcl-XL cells expressed the macrophage/dendritic
cell marker, F4/80, suggesting that bcl-XL/Tag
cells without feeder cells and a source of RANK ligand terminally
differentiate toward mature monocyte-macrophages. These data confirm
the important role of RANK ligand and marrow stromal cells in OCL
precursor differentiation. In addition, they suggest that the
bcl-XL/Tag cells are a model of an intermediate
OCL precursor, which is bipotent and can form either OCLs or mature
monocyte-macrophages. These data further suggest that osteoblast-like
cells play a critical role in supporting OCL precursor growth and
differentiation, as neither GM-CSF nor M-CSF induced expression of the
F4/80 antigen on bcl-XL/Tag cells that were
grown in the presence of osteoblastic cells.
Calcitonin (10–100 mU/ml) dose dependently inhibited OCL-like cell
formation by 30–60% in bcl-XL/Tag cells that
were treated with 1,25-(OH)2D3. This is the
same degree of inhibition of OCL formation (64% with calcitonin, 100
mU/ml) induced by calcitonin in normal marrow cultures (12).
The bcl-XL cells differ from OCL precursors in
normal marrow in several ways. PTH or PTHrP did not stimulate OCL
formation by bcl-XL/Tag cells regardless of they
were grown over PA6 cells or primary rat osteoblasts. Udagawa and
co-workers (13) have shown that PA6 cells do not respond to PTH or
PTHrP and that the effects of PTH or PTHrP on OCL formation are
indirect, most likely by induction of RANK ligand. It was not
surprising that PTH and PTHrP had no effect on
bcl-XL/Tag cells when they were grown over PA6
cells or MC3T3E1 cells, as neither cell type responds to PTHrP.
However, the failure of these cells to form OCLs in response to PTHrP
when cocultured with primary rat osteoblasts was unexpected. We have
shown in preliminary studies that bcl-XL/Tag
cells constitutively express the RANK receptor. Thus, the exact
mechanism of why these cells fail to respond to PTH when cocultured
with primary rat osteoblasts remains to be determined.
Surface phenotype analysis of bcl-XL/Tag cells
confirms that they are derived from the monocyte-macrophage lineage.
Miyamato and co-workers (15) recently developed and characterized a
macrophage cell line (C7) that had some osteoclastic characteristics.
However, only 4–7% of these mononuclear cells were able to
differentiate into OCLs. This cell line expressed several macrophage
surface markers, such as Mac-1, F4/80, c-Fms, CD14, and integrins
4, 5, and ß1. The
bcl-XL/Tag cells, on the other hand, did not
express CD11c (monocyte/granulocyte marker), Mac-3 (monocyte marker),
Gr-1 (granulocyte marker), CD40 (B cell marker), or CD80 (B cell
marker) surface markers. Only a very small subpopulation of the cells
expressed F4/80 and CD14, suggesting that these two cell lines differ
from each other. Interestingly, our cell line expressed CD86 antigen,
which is expressed on the surface of B cells. Importantly, the OCL
formation capacity of our cell line (50%) was much higher than that
of the C7 macrophage cell line. Another macrophage cell line, BDM-1,
has been reported that has a 5–9% efficiency in OCL formation
capacity (16). Chambers and his co-workers (17) also used a transgenic
mouse approach for developing an OCL cell line. They produced mice
transgenic for temperature-sensitive simian virus 40 large T antigen
gene driven by an interferon-inducible MHC complex H-2Kb
promoter. This cell line was able to differentiate to both macrophages
and OCLs, and its initial OCL formation capacity was approximately
2.5% (Chambers, T. J., personal communication). Recently, Chen
et al. (18) reported the generation of a murine
osteoclastogenic cell line (MDCP-5) by targeting large T antigen to
marrow cells cocultured over an OCL-inductive marrow stromal cell line.
This cell line also expressed macrophage markers and formed OCLs at a
high efficiency, analogous to our results.
The bone-resorptive capacity of OCLs formed by
bcl-XL/Tag cells was similar to that reported
for primary rat OCLs. The resorption area per mature rat OCL after
24 h of culture has been reported to be 968 ± 145
µm2. There were approximately 220 ± 21 OCLs/bone
slice (19). These data are very similar to the 255 OCLs and 836 ±
144 µm2 resorption area/OCL formed by
bcl-XL/Tag cells.
In summary, the bcl-XL/Tag cells respond
appropriately to most osteotropic hormones and cytokines, although they
do not respond to PTH or PGE2, which are potent stimulators
of normal OCL formation (Table 2). Aside
from these caveats, bcl-XL/Tag cells appear to
be an appropriate model to study interactions between osteoblast and
OCL precursors as well as to examine the molecular events associated
with OCL differentiation and bone resorption. Furthermore, in
preliminary studies, we demonstrated that
bcl-XL/Tag cells can be transfected by
adenoviral or retroviral vectors with a transfection efficiency of
20–40%, respectively. These data suggest that
bcl-XL/Tag cells can be used to examine
differences at the molecular level that occur with commitment to the
OCL lineage or terminal differentiation to the macrophage lineage. Such
studies should provide important insights into the molecular events
controlling OCL formation and activity.
|
Acknowledgments
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The authors thank Bibi Cates for excellent preparation of this
manuscript.
|
Footnotes
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1 This work was supported by the Academy of Finland. 
Received November 10, 1998.
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Top
Abstract
Introduction
