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Endocrine Research Unit and Department of Biochemistry (F.G., L.C.H., S.K., B.L.R.) and Molecular Biology (T.C.S.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and Amgen, Inc. (C.R.D.), Thousand Oaks, California
Address all correspondence and requests for reprints to: B. Lawrence Riggs, M.D., Mayo Clinic, 200 First Street SW, Plummer North 6, Rochester, Minnesota 55905. E-mail: Riggs.Lawrence{at}Mayo.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanism(s) responsible for the coordinated sequence
(coupling) of osteoclastogenesis and osteoblastogenesis during the bone
remodeling sequence remains unclear. Traditionally, this linkage
between bone formation and bone resorption has been attributed to
osteoclast-to-osteoblast signaling, possibly mediated by the release of
growth factors from bone matrix during bone resorption (6, 7). However, clearly osteoblast-to-osteoclast signaling also
occurs. Indeed, there are several reasons for believing that the
differentiation of osteoclast precursors is linked to the
differentiation of the osteoblast precursors. First, cell-to-cell
contact of osteoblast lineage cells with preosteoclasts may be required
for differentiation of osteoclast precursors into multinucleated
osteoclasts (4, 8). Second, when osteoblastogenesis is
impaired, as in the SAMP6 mouse, osteoclastogenesis also is impaired
(9), and when osteoblastogenesis is absent, as in the
Osf2/Cbfa1 knock-out mouse, osteoclastogenesis is absent
(10, 11, 12, 13). Third, orchiectomy fails to stimulate
osteoclastogenesis in the SAMP6 mouse (14). Finally, the
two factors that are essential for regulating osteoclastogenesis—M-CSF
and RANK (receptor activator of NF
B) ligand
(RANK-L)3 (15, 16)—as well as
the soluble decoy receptor for RANK-L, osteoprotegerin
(OPG)4 (17, 18), which
regulates the biological availability of RANK-L in the bone
microenvironment, are produced by osteoblasts.
Recently, OPG was identified independently by three groups (17, 18, 19) as a novel member of the tumor necrosis factor (TNF) receptor family that is produced by osteoblast lineage cells. OPG inhibits both the formation and maturation of osteoclasts (17, 18, 19). Mice with targeted deletion of the OPG gene manifest severe osteoporosis (20, 21), whereas transgenic mice overexpressing OPG have suppressed bone resorption and increased bone mass (17). Even more recently, RANK-L ligand (RANK-L), the natural ligand of OPG and a member of the TNF family, was identified as a key effector of osteoclast differentiation (15, 16). RANK-L induces osteoclast formation and differentiation (15, 16) and also directly enhances activity of mature osteoclast by 7-fold (22). RANK-L exerts its biological effects by binding to its receptor, RANK5 (23, 24, 25), on osteoclast lineage cells in either a soluble (15) or the membrane-bound (15, 16) form, the latter requiring cell-to-cell contact. Binding of RANK-L to OPG neutralizes its activity (15, 16). Provided permissive levels of M-CSF are present, RANK-L is both necessary and sufficient for osteoclastogenesis (15, 16, 26). Thus, the biological availability of RANK-L is critically dependent on the RANK-L/OPG production ratio in the bone microenvironment.
We recently developed and characterized conditionally immortalized human marrow stromal (hMS) cell lines that can simulate the complete developmental sequence of osteoblast differentiation from uncommitted precursor cells to mature osteoblastic cells that are capable of forming mineralized nodules (27, 28). Here, we report the use of one of these cell lines, hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15), to test the hypothesis that developmental regulation of the RANK-L/OPG production ratio is associated with reciprocal changes in the ability to support osteoclastogenesis. If so, this mechanism could contribute to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
(1,25(OH)2D3),
and radioisotopes were purchased from NEN Life Science Products (Boston, MA). Polytract messenger RNA (mRNA) isolation
system and the Wizard PCR Preps DNA purification system were purchased
from Promega Corp. (Norwalk, CT). The human ß-actin
insert and ExpressHyb solution were obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Kits for the measurement of
human interleukin (IL)-1ß, human IL-6, IL-11, recombinant human
TNF- and recombinant human macrophages-colony stimulating factor
(M-CSF) were purchased from R&D System (Minneapolis,
MN). Kits for the measurement of procollagen protein were gifts from
Metra Biosystems (Mountain View, CA). CH3/HeN 4- to
6-week-old, male mice were obtained from Charles River Laboratories, Inc. Wilmington, MA). Acid Phosphatase Activity
Assay and Leukocyte Acid Phosphatase Assay were purchased from
Sigma (St. Louis, MO).
Cell culture
Conditionally immortalized human marrow stromal (hMS) cell lines
were established in our laboratory by transfecting hMS cells with a
gene coding for a temperature-sensitive mutant TsA58 of SV40 large
Tantigen (SV40 LTA) (27). Incubation of the cells at
the permissive temperature (34 C), when the SV40 LTA is active, results
in an increased rate of cell proliferation but little or no cell
differentiation. Incubation at the restrictive temperature (39.5 C),
when the SV40 LTA is inactive, results in little or no cell division
but rapid induction of cell differentiation. The undifferentiated cells
are bipotential, and, under appropriate conditions can be induced to
differentiate along the osteoblastic or adipocytic pathways (27, 28). All six hMS cell lines that we had previously characterized
in our laboratories have similar phenotypes and are stable until at
least passage 12 (27). For these present studies, we used
the hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cell line from passage 9 to passage 12.
The hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells were cultured in an humidified
atmosphere 5% CO2/95%, and the cell population
was expanded at 34 C in a growth medium of -MEM containing 10%
(vol/vol) heat inactivated (NOREF>HI)-FBS, 0.2 µg/ml geneticin (NOREF>G418), and
1% (vol/vol) of penicillin 10,000 U/ml-streptomycin 10,000 µg/ml.
Medium was changed twice a week. To induce hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
cell differentiation along the osteoblast pathway, both an increase in
the temperature to 39.5 C and exposure to differentiation medium are
required. The osteoblast differentiation medium contained
dexamethasone (Dex) 10-8
M, ß-glycerol-phosphate (ß-GP) 10 mM and
L-ascorbate-phosphate (Asc-P) 100 µM.
Semiquantitative RT-PCR
Cells were plated at density of 1.8 x
105 cells in six-well microtiter plates in growth
medium and cultured for 2 days at 34 C. The cells were then cultured at
39.5 C for 2 days in growth medium (undifferentiated) or in osteoblast
differentiation medium for 2, 7, 14, or 21 days. Total cellular RNA was
isolated using the RNA-STAT kit. Complementary DNA (cDNA) was
synthesized from 1 µg total RNA in a 20 µl reaction mix containing
1x incubation buffer for AMV reverse transcriptase (RT) 5x, 2.5
µM of poly·dT, 1 mM each of dATP, dCTP,
dGTP and dTTP, 20 U of RNase inhibitor and 20 U of AMVRT (avian Moloney
virus reverse transcriptase) for 2 h at 42 C. Aliquots of cDNA
were amplified in a 25 µl PCR mixture which contained 0.2
µM of 5' and 3' oligo-primers, 1x of expanded high
fidelity PCR buffer 10x with 15 mM
MgCl2, 0.1 nM each of dATP, dCTP,
dGTP, and dTTP, 0.25 µl of [-32P] dCTP (10
µCi/µl) and 0.35 U of expanded high fidelity Taq DNA
polymerase. For each assay performed, each cDNA sample was run in
duplicate. Amplification reactions specific for the following cDNAs
were carried out: alkaline phosphatase (AP), type I collagen (Col I)
OPG, RANK-L, and the housekeeping gene
GAPDH. Amplifications were performed in a GeneAmp
9600 thermal cycler (Perkin-Elmer Corp., Norwalk, CT).
Primer sequences and amplification profiles used for
AP, Col I, and RANK-L were
reported previously (27, 29) except for OPG PCR
products. OPG PCR product identity was confirmed by sequence
analysis in an automated DNA sequencer (Perkin-Elmer Corp.). The PCR primer sequences for OPG used in the
studies were: OPG sense 5'-GTGTCTTTGGTCTCCTGCTAA-3' OPG antisense
5'-GGGCTTTGTTTTGATGTTTC-3'. The PCR product size for OPG was 271 bp.
PCR products were analyzed by electrophoresis of 9-µl samples in
1.5% (wt/vol) agarose gels. The amplified DNA fragments were
visualized by ethidium bromide staining and quantified by counting the
radioactivity in gel slices. The quantitative differences between cDNA
samples were normalized to the radioactivity present in the GAPDH PCR
products.
Bone protein assays
hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells were plated in 48-well microtiter
plates at a density of 2 x 104 cells in
standard growth medium. After 2 days at 34 C, the cells were incubated
in standard growth medium at 39.5 C for 2 days (undifferentiated) or in
osteoblast differentiation medium for 2, 7, 14, or 21 days. The AP
activity in cell lysates was quantified at 37 C in assay buffer
containing 0.75 M 2-amino-2-methyl-1-propanol, pH 10.3 for
1 h using p-nitrophenylphosphate as a substrate. The release of
p-nitrophenol was monitored by measuring absorbance at 410 nm
(30). The media were collected, centrifuged, to remove
cell debris, and then used for carboxypeptide of type I procollagen
(PICP) measurement by ELISA. Results of Col I secretion and AP
activity were normalized to total cell protein, as measured by the
Bradford protein assay method.
Measurement of mineralized matrix formation
Cells were plated at 5 x 104
cells/well in 12 -ell microtiter plates in standard growth medium.
After 2 days at 34 C, the cells were incubated in standard growth
medium at 39.5 C for 2 days (undifferentiated) or in osteoblast
differentiation medium for 2, 7, 14, or 21 days. The formation of
mineralized matrix nodules was determined by alizarin red-S staining
(28, 31). Briefly, the cells were fixed in 70% ethanol
for 1 h at room temperature. The fixed cells were washed with PBS
and stained with 40 mM alizarin red-S pH 4.2, for 10 min at
room temperature. The cells were washed with distilled water five times
and rinsed with PBS for 15 min. For quantitative assessment, the
alizarin red-S dye was eluted and measured spectrophotometrically as
previously described (28, 32).
Northern blot hybridization
Poly (A)+ RNA were isolated from hMS
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells cultured at 39.5 C for 2 days in growth
medium (undifferentiated) or in osteoblast differentiation medium for 2
or 21 days, using the RNAase kit and the Polytract mRNA isolation
system. Total RNA and Poly (A)+ RNA were resolved
on a 1% (wt/vol) agarose/formaldehyde gel using continuos buffer
circulation (33) and then transferred to a nylon membrane
(Hybond N+, Amersham Pharmacia Biotech, Arlington Heights,
IL) by capillary blotting (34). Fifty nanograms of the
probe of full-length OPG, RANK-L and ß-actin cDNA probes, which
hybridized to mRNA species of 6.0, 2.9, 2.4, and 2.0 kb respectively,
were radiolabeled with 5 µl [-32P] dCTP to
a specific activity of > 109 cpm/µg DNA
using a random primer DNA labeling kit (35). Hybridization
and stringent washing were carried out as previously reported
(36). Band intensity was quantified by densitometry.
Control hybridization with human ß-actin cDNA verified that equal
amounts of RNA were loaded.
OPG, IL-1ß, IL-6, IL-11, TNF-, and M-CSF secretion
Media from hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells treated at 39.5 C for
2 days in growth medium (undifferentiated) or in osteoblast
differentiation medium for 2, 7, 14, or 21 days, were collected and
then centrifuged to remove cell debris. IL-1ß, IL-6, IL-11, M-CSF,
and TNF- secretion was measured by a sandwich ELISA. OPG was
measured by ELISA as previously reported (17).
In vitro osteoclastogenesis assay
Nonadherent bone marrow stromal cells were obtained from 4- to
6-week-old male CH3/HeN mice and prepared as previously reported
(18). For coculture experiments, hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
cells, at density of 5 x 104/well in
24-well microtiter plates, were cultured at 39.5 C either with
undifferentiated cells for 2 days in growth medium at 39.5 C (Day 0) or
for 2, 7, 14, or 21 days at 39.5 C in osteoblast differentiation
medium. At each of these time points, 1 x
106 nonadherent mouse marrow stromal cells (as
osteoclast progenitor cells) were added to the hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
cells. The cocultured cells were treated at 37 C for 12 days in the
presence of Dex 10-7
M and 1,25
(OH)2D3
10-9 M and in
the presence or in the absence of 30 ng/ml of M-CSF. In parallel
experiments, RANK-L at dose of 25 ng/ml was added to cocultures of hMS
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) treated for 21 days in osteoblast differentiation
medium and nonadherent mouse marrow stromal cells. New medium
containing fresh reagents was added every 3–4 days. Osteoclast
formation was measured by quantitating the presence of TRAP
multinucleated positive cells (more than three nuclei) using
cytochemical staining.
Experimental protocol
Figure 1
schematically outlines
the standard experimental protocol for evaluating changes in RANK-L and
OPG and osteoblast to osteoclast signaling during osteoblast
differentiation. Undifferentiated hMS cells are plated and allowed to
attach at 34 C in growth medium for 2 days. The temperature is then
increased to 39.5 C and the cells are maintained in the growth medium
for an additional 2 days. Under these culture conditions, as we have
previously demonstrated (27), the hMS cells will maintain
their undifferentiated state short-term, and, thus, this initial 2-day
culture period serves as a baseline for assessing subsequent changes
occurring during differentiation. To initiate differentiation, the
temperature is maintained at 39.5 C, and the growth medium is replaced
with differentiation medium. Experimental measurements are made at
baseline (0 days exposure) and after 2, 7, 14, and 21 days of exposure
to the differentiation medium. Other hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells
that had been exposed to differentiation medium for each of these
intervals were then cocultured for 12 days with mouse marrow cells to
assess osteoclast formation as described in the previous section.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the mRNA levels for
Col I and AP measured by semiquantitative
RT-PCR, demonstrate that, hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells differentiate
along the osteoblast pathway when cultured in osteoblastic
differentiation medium. The mRNA expression for Col I was
maximal (60% increase over values observed in undifferentiated cells)
during the first 2 days of treatment with the osteoblastic
differentiation medium (P < 0.005 by multiple measures
ANOVA). Maximal AP mRNA levels (320% over values of
undifferentiated cells) occurred at 14 days (P <
0.0001 by multiple measures ANOVA). Consistent with the increased
Col I and AP mRNA levels, culture in osteoblast
differentiation medium for 2, 7, 14, or 21 days induced increases in
type I procollagen and AP activity of up to 8-fold and 28-fold,
respectively, over baseline values in undifferentiated cells, at 14
days and 21 days, respectively (P < 0.001 for COL-1
secretion and P < 0.0001 for AP activity by multiple
measures ANOVA). (Fig. 2B) The discrepancy observed in the fold
increase between mRNA and protein levels for both AP and Col I may be
due to a posttranscriptional increase of gene translation. However, our
studies did not directly address this issue.
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OPG mRNA levels increase and RANK-L mRNA levels decrease during
osteoblast differentiation
OPG and RANK-L mRNA levels were first assessed by semiquantitative
RT-PCR (Fig. 2D). During differentiation along the osteoblast pathway,
OPG mRNA levels were increased with a maximal effect of
2.4-fold over values of undifferentiated cells at 21 days. In contrast,
over the same time interval the RANK-L mRNA levels were decreased by
2-fold over values observed in undifferentiated cell. When mRNA levels
were assessed by Northern blot analysis, the relative changes in
steady-state mRNA were even greater. As assessed by densitometry, gene
expression of RANK-L was decreased by 5-fold and
that for OPG was increased by 7-fold at 21 days,
representing an apparent change in the RANK-L/OPG ratio of
approximately 35-fold with the acquisition of the osteoblast phenotype
(P < 0.0001 for changes from baseline for RANK-L, OPG
or RANK-L/OPG ratio) (Fig. 3).
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, IL-6, IL-11, and M-CSF, cytokines known to increase bone
resorption (3, 4, 5) (Fig. 4). Cytokine levels in conditioned
media were relatively high for IL-6, but were 28-fold and 170-fold
lower for IL-11 and M-CSF, respectively. Neither IL-1ß nor TNF-
were detectable in the conditioned media. Acquisition of a mature
osteoblast phenotype was associated with a decrease in IL-6 and IL-11
secretion with a nadir compared with values of undifferentiated cells
at 21 days of 25- and 45-fold, respectively. Interestingly, M-CSF
secretion was increased by 8-fold over values in undifferentiated cells
by 7 days and then decreased. All changes were P <
0.001 by multiple measures ANOVA.
Undifferentiated, but not differentiating or fully differentiated,
hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells can support osteoclastogenesis
To assess whether the decrease in the apparent RANK-L/OPG
production ratio observed during osteoblast differentiation was
associated with the decreased capability to support osteoclastogenesis,
hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) were cocultured with mouse marrow cells, as a
source for osteoclast precursors. As shown in Fig. 5A, coculture with undifferentiated (Day
0) hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells supported the formation of TRAP
positive multinucleated cells. When hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells were
exposed to the osteoblast differentiation medium for 2, 7, 14, or 21
days, before being cocultured for an additional 12 days with mouse
marrow stromal cells, TRAP positive, multinucleated osteoclasts were
not detected (Figs. 5 and 6, B–E).
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and 6F). Similar results from experiments
performed in the absence of M-CSF (data not shown) demonstrated that
this cytokine is not required for osteoclast formation in our coculture
system. Presumably, the permissive concentration of M-CSF required for
osteoclastogenesis was supplied by the hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells.
The development of multinucleated, TRAP+ cells in cultured bone marrow
was associated with large increases in mRNA for calcitonin receptor as
assessed by RT-PCR (data not shown). |
Discussion |
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When cultured in a differentiation medium, the hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
cells differentiate over a 21-day interval and acquire the mature
osteoblast phenotype characterized by increased mRNA and protein for
two characteristic osteoblast genes, type I collagen and alkaline
phosphatase, and the ability to form mineralized nodules. As have
others, we find that glucocorticoids, ß-glycerol-phosphate, and
L-ascorbate-phosphate are required for differentiation of
bone marrow stromal cells into mature osteoblasts in vitro
(27, 37, 38, 39, 40, 41). The other characteristic osteoblast
phenotype gene is osteocalcin. However, osteocalcin expression requires
1,25(OH)2D3 treatment,
which we could not include because its presence would permit some of
the uncommitted precursor cells to differentiate along the adipocytic
pathway (28).
When the early undifferentiated hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells are cocultured with mouse bone marrow cells, the murine preosteoclasts differentiated to TRAP-positive multinucleated osteoclast-like cells, but when they were cocultured with cells cultured in osteoblast differentiation medium for 2, 7, 14, or 21 days they were unable to do so. Thus, we answered the first question affirmatively: in an in vitro model, early undifferentiated stromal-osteoblast cells support osteoclastogenesis, but, as sequential osteoblastic differentiation occurs, the ability to support osteoclast formation is lost. The ability to support osteoclastogenesis was restored by the addition of only RANK-L.
We next investigated the molecular mechanism(s) mediating these changes. Because RANK-L is an obligate requirement for osteoclastogenesis and is produced by osteoblasts, we hypothesized that signaling is dependent on its biological availability; this, in turn, is inversely proportional to the local concentration of the soluble neutralizing receptor, OPG, that competes with RANK for binding to RANK-L. Over 21 days of exposure to osteoblast differentiation medium, there was a steep decreasing gradient in the apparent RANK-L/OPG mRNA ratio: steady-state levels of RANK-L mRNA were high in the undifferentiated cells and during osteoblast differentiation decreased by 5-fold, whereas OPG mRNA and protein increased by 7- and 6-fold, respectively. Unfortunately, because sensitive antibodies were not available, we could not measure RANK-L protein and, thus, could not determine the RANK-L/OPG protein ratio directly. However, the decrease at the mRNA level suggests that, RANK-L protein probably was also decreased.
In addition, the developmental changes in the apparent RANK-L/OPG
gradient correlate with the results of the osteoclastogenesis assay
during the coculture experiments; the early undifferentiated cells
(that are associated with a high RANK-L/OPG expression ratio) can
support osteoclastogenesis, whereas the partially or completely
differentiated cells (with a progressively lower ratio) cannot.
Moreover, treatment of the late differentiated cells with RANK-L
restores their ability to support osteoclastogenesis. In the
osteoclastogenesis assays, similar results occurred in the absence or
in the presence of M-CSF indicating that the hMS cells make sufficient
M-CSF to allow osteoclast to form in coculture. Thus, our data also
answer the second question: a high RANK-L/OPG concentration ratio in
the bone microenvironment is the main molecular signaling mechanism
determining osteoclastogenesis. This mechanism is also supported by the
recent report of Nagai and Sato (42). These workers
assessed the steady-state levels of RANK-L and OPG mRNA by RT-PCR in
several stromal or osteoblastic cell lines that either supported or
failed to support osteoclastogenesis, both with and without treatment
with 1,25(OH)2D3 and
prostaglandin E2. They found that the ability of
the human leukemic HL60 cell line to support osteoclast formation was
critically dependent on the apparent RANK-L/OPG ratio. However, the
HL60 cell line does not require the presence of M-CSF to
differentiate into osteoclast cells, suggesting that it already is
partially differentiated toward osteoclasts.
Hofbauer et al. (29) found that 48 h exposure of the hMS cells to dexamethasone directly increased RANK-L mRNA and decreased OPG mRNA expression and protein production in hMS cells. However, in these studies, early, immature hMS cells were treated acutely with dexamethasone in a culture medium without either FCS or other differentiating compounds. This direct effect is consistent with the presence of a glucocorticoid response element in the RANK-L promoter (43). However, glucocorticoids are also required for hMS differentiation, and 21- day exposure to a differentiation medium including dexamethasone, L-ascorbate-phosphate, and FCS resulted in a progressive acquisition of the mature osteoblast phenotype osteoblastic features by the hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells. Under these conditions, the ratio of RANK-L/OPG decreased progressively as the cells differentiated, a change that was in the opposite direction from the direct effect observed in the early cells. During the 21 days of progressive osteoblastic differentiation, the concentration of dexamethasone in the culture medium remained constant. Thus, the effect of dexamethasone on bone differentiation markers is likely indirect and is due to the differentiation process itself.
A number of proinflammatory cytokines, including IL-1ß, TNF-,
IL-6, and IL-11, stimulate bone resorption. Therefore, we evaluated the
secretion of several of these during the differentiation of the hMS
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells along the osteoblast pathway. The IL-6
secretion was relatively high whereas secretion of IL-11 and,
especially, M-CSF was much lower. IL-1ß and TNF- were
undetectable, an observation that is consistent with their production
occurring mainly in cells of the monocyte-macrophage lineage
(44). As differentiation progressed along the osteoblast
pathway, IL-6 and IL-11 secretion were decreased significantly by 25-
and by 45-fold, respectively. Although IL-6 may not affect gene
expression of RANK-L directly, the high production of this cytokine by
early undifferentiated marrow stromal cells could still enhance bone
resorption by increasing the number of preosteoclasts upon which RANK-L
could act and by enhancing the activity of mature osteoclasts
(45).
Unexpectedly, M-CSF production increased significantly during middle phase of osteoblast differentiation of hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells, although its concentrations were much lower than IL-6. In contrast to RANK-L, only low levels of M-CSF appear to be required for acquisition of osteoclast phenotype (15, 16, 46) and in our studies, exogenous M-CSF was not required for osteoclast formation. Moreover, concentrations of M-CSF were low and varied between 0.1 ng/ml and 0.8 ng/ml during hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cell differentiation along the osteoblast lineage, whereas the concentration of OPG protein was much higher at all time-points and increased from 2 to 15 ng/ml in a time-dependent manner during osteoblast differentiation. Thus, it appears that small changes in M-CSF production that we observed during hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cell differentiation probably are insufficient to override the concurrent progressive decrease in the RANK-L/OPG ratio.
Although observations in vitro cannot necessarily be extrapolated to physiological mechanisms in vivo, we speculate the developmental regulation of the RANKL/OPG ratio that we have observed in vitro may have an in vivo counterpart that contributes to coordination of osteoclast and osteoblast differentiation. Using a specific monoclonal antibody, Weng et al. (47) have recently established that marrow stromal cells are anatomically located close to endosteal and trabecular surfaces and rarely are found elsewhere in bone marrow. In such an anatomical location, they would be ideally situated to attract and activate osteoclast precursors and, thus, to initiate the resorption phase of bones remodeling. As osteoclastogenesis proceeds, the stromal cells differentiate toward the mature osteoblast phenotype and lose their ability to support osteoclastogenesis. Following termination of the resorption phase by apoptosis of osteoclasts (48), the now fully mature osteoblasts would be positioned to respond to chemotactic signals during the brief reversal phase by migrating to the now empty surfaces of the resorption lacunae. They could then initiate the ensuing formation phase by refilling the resorption space with newly formed bone. Further studies in vivo are needed to confirm or refute this hypothesis.
In summary, we have shown that osteoblast differentiation in vitro is associated with a steep gradient ranging from a high RANK-L/OPG production ratio in the early undifferentiated hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells that are able to support osteoclastogenesis to a low ratio in the late differentiated hMS (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) cells that cannot. We speculate that the developmental regulation of this ratio may contribute to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.
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Acknowledgments |
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Footnotes |
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2 Recipient of a postdoctoral fellowship from the Deutsche
Forschungsgemeinschaft (Ho 1875/2–1).
3 Also known as osteoprotegerin ligand
(OPG-L) (15 ) and TNFrelated activation-induced
cytokine, (TRANCE) (16 ).
4 Also known as OPG are osteoclastogenesis
inhibitory factor (13 ) and TNF receptor-like molecule 1
(TRI) (14 ).
5 Also known as the osteoclast
differentiation and activation receptor (ODAR) (25 ).
Received April 7, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. L. Andersen, T. E. Sondergaard, K. E. Skorzynska, F. Dagnaes-Hansen, T. L. Plesner, E. M. Hauge, T. Plesner, and J.-M. Delaisse A Physical Mechanism for Coupling Bone Resorption and Formation in Adult Human Bone Am. J. Pathol., January 1, 2009; 174(1): 239 - 247. [Abstract] [Full Text] [PDF]
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 A. E. Kearns, S. Khosla, and P. J. Kostenuik Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease Endocr. Rev., April 1, 2008; 29(2): 155 - 192. [Abstract] [Full Text] [PDF]
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 A. D Anastasilakis, D. G Goulis, S. A Polyzos, S. Gerou, V. Pavlidou, G. Koukoulis, and A. Avramidis Acute changes in serum osteoprotegerin and receptor activator for nuclear factor-{kappa}B ligand levels in women with established osteoporosis treated with teriparatide Eur. J. Endocrinol., March 1, 2008; 158(3): 411 - 415. [Abstract] [Full Text] [PDF]
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 R. A. Deckelbaum, A. Majithia, T. Booker, J. E. Henderson, and C. A. Loomis The homeoprotein engrailed 1 has pleiotropic functions in calvarial intramembranous bone formation and remodeling Development, January 1, 2006; 133(1): 63 - 74. [Abstract] [Full Text] [PDF]
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 A. Rogers and R. Eastell Circulating Osteoprotegerin and Receptor Activator for Nuclear Factor {kappa}B Ligand: Clinical Utility in Metabolic Bone Disease Assessment J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6323 - 6331. [Abstract] [Full Text] [PDF]
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 S. Lossdorfer, W. Gotz, and A. Jager PTH(1-34) Affects Osteoprotegerin Production in Human PDL Cells in vitro Journal of Dental Research, July 1, 2005; 84(7): 634 - 638. [Abstract] [Full Text] [PDF]
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 X.-H. Liu, A. Kirschenbaum, S. Yao, and A. C. Levine Cross-Talk between the Interleukin-6 and Prostaglandin E2 Signaling Systems Results in Enhancement of Osteoclastogenesis through Effects on the Osteoprotegerin/Receptor Activator of Nuclear Factor-{kappa}B (RANK) Ligand/RANK System Endocrinology, April 1, 2005; 146(4): 1991 - 1998. [Abstract] [Full Text] [PDF]
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 S. M. Krane Identifying genes that regulate bone remodeling as potential therapeutic targets J. Exp. Med., March 21, 2005; 201(6): 841 - 843. [Abstract] [Full Text] [PDF]
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E. Gazzerro, R. C. Pereira, V. Jorgetti, S. Olson, A. N. Economides, and E. Canalis Skeletal Overexpression of Gremlin Impairs Bone Formation and Causes Osteopenia Endocrinology, February 1, 2005; 146(2): 655 - 665. [Abstract] [Full Text] [PDF]
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M. Heim, O. Frank, G. Kampmann, N. Sochocky, T. Pennimpede, P. Fuchs, W. Hunziker, P. Weber, I. Martin, and I. Bendik The Phytoestrogen Genistein Enhances Osteogenesis and Represses Adipogenic Differentiation of Human Primary Bone Marrow Stromal Cells Endocrinology, February 1, 2004; 145(2): 848 - 859. [Abstract] [Full Text] [PDF]
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V. Viereck, C. Grundker, S. Blaschke, B. Niederkleine, H. Siggelkow, K.-H. Frosch, D. Raddatz, G. Emons, and L. C. Hofbauer Raloxifene Concurrently Stimulates Osteoprotegerin and Inhibits Interleukin-6 Production by Human Trabecular Osteoblasts J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4206 - 4213. [Abstract] [Full Text] [PDF]
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 O. Sezer, U. Heider, I. Zavrski, C. A. Kuhne, and L. C. Hofbauer RANK ligand and osteoprotegerin in myeloma bone disease Blood, March 15, 2003; 101(6): 2094 - 2098. [Abstract] [Full Text] [PDF]
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 V. Katavic, I. K. Lukic, N. Kovacic, D. Grcevic, J. A. Lorenzo, and A. Marusic Increased Bone Mass Is a Part of the Generalized Lymphoproliferative Disorder Phenotype in the Mouse J. Immunol., February 1, 2003; 170(3): 1540 - 1547. [Abstract] [Full Text] [PDF]
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 E M Gravallese Bone destruction in arthritis Ann Rheum Dis, November 1, 2002; 61(90002): ii84 - 86. [Abstract] [Full Text] [PDF]
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 K. Janssens and W. Van Hul Molecular genetics of too much bone Hum. Mol. Genet., October 1, 2002; 11(20): 2385 - 2393. [Abstract] [Full Text] [PDF]
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 V. Geoffroy, M. Kneissel, B. Fournier, A. Boyde, and P. Matthias High Bone Resorption in Adult Aging Transgenic Mice Overexpressing Cbfa1/Runx2 in Cells of the Osteoblastic Lineage Mol. Cell. Biol., September 1, 2002; 22(17): 6222 - 6233. [Abstract] [Full Text] [PDF]
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S. Khosla Minireview: The OPG/RANKL/RANK System Endocrinology, December 1, 2001; 142(12): 5050 - 5055. [Abstract] [Full Text] [PDF]
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B. Burguera, L. C. Hofbauer, T. Thomas, F. Gori, G. L. Evans, S. Khosla, B. L. Riggs, and R. T. Turner Leptin Reduces Ovariectomy-Induced Bone Loss in Rats Endocrinology, August 1, 2001; 142(8): 3546 - 3553. [Abstract] [Full Text] [PDF]
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) or
AP (•) mRNAs were expressed as ratio to
GAPDH levels. B, Bone protein secretion. Col I secretion
(
), and RANK-L over 21
days of differentiation as assessed by semiquantitative RT-PCR (•).
Results were expressed as ratio to GAPDH levels and values at day 0
were set at 100%. The data are representative of at least three
separate experiments. The results are the means ± SEM
of quadruplicates. P < 0.0001 for
AP mRNA and activity and P < 0.005
for Col I mRNA and P < 0.001 for
Col I secretion and P < 0.0001 for alizarin red S
compared with the corresponding control values as assessed by multiple
measures ANOVA.
