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Department of Medicine, Emory University, and Veterans Affairs Medical Center, Atlanta, Georgia 30033; and the Department of Pathology, University of Utah (S.P.), Salt Lake City, Utah 84132
Address all correspondence and requests for reprints to: Dr. J. Rubin, Veterans Affairs Medical Center-151, 1670 Clairmont Road, Decatur, Georgia 30033. E-mail: jrubi02{at}emory.edu
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Abstract |
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Introduction |
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The complexity of the culture environment required for osteoclast differentiation has made study of the stimulatory effects of glucocorticoids on osteoclastogenesis most difficult. The osteoblast is intimately involved in the recruitment of osteoclasts; osteoclast progenitor cells must, in fact, physically contact the underlying stromal environment to allow differentiation of the multinuclear osteoclast cells (9, 10). Many factors that affect osteoclast recruitment, therefore, may regulate functions of the supporting stromal or osteoblast cells rather than directly affecting cells of the osteoclast progenitor lineage.
Macrophage colony-stimulating factor (MCSF) is critical for osteoclastogenesis (11). In the op/op mouse model, osteopetrosis arises as a result of a dysfunctional MCSF (12, 13). In the marrow model of osteoclastogenesis as well, MCSF is critical, and blockade of this growth factor prevents osteoclast formation (14) by preventing expansion of the osteoclast progenitor pool (15). Delineating the effects of this growth and differentiative factor is complicated by the presence of at least two distinct isoforms that arise out of posttranscriptional processing. The deletion of a large part of exon 6 generates a shorter protein that is more stably expressed on the membrane of the cell, whereas the longer protein is rapidly secreted (16, 17). The membrane and soluble forms of MCSF (mMCSF and sMCSF, respectively) may have different roles in bone. For instance, high levels of the secreted form of MCSF can have a negative effect on osteoclastogenesis: sMCSF promotes the progression of the monocytic precursor into the macrophage lineage, rather than the osteoclast (18, 19). Alternatively, the importance of the membrane-bound MCSF, which is displayed on the membranes of stromal and osteoblast cells (17, 20), is not known. Very low levels of mMCSF, at levels less than 5% of that secreted into the medium, can support expansion of the osteoclast precursor pool on glutaraldehyde-treated stromal layers (20). A specific role of mMCSF during osteoclastogenesis is unknown, but is suggested by the fact that this necessary growth factor is accessible to the progenitor cells that physically contact the stromal cell layer.
In this work we have investigated the possibility that Dex alters the expression of mMCSF by stromal and osteoblast cells capable of supporting the osteoclastogenesis of spleen cells in the presence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Our results show that Dex increases the expression of the mMCSF transcript and protein in osteoblast-like cells, and exerts regulatory control on the expression of the sMCSF protein as well.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEM-10% FCS in 12-well plates or
200,000 cells were placed in 25-cm2 flasks (Corning,
Corning, NY) and grown for 4 days to confluence. Treatments were added
in fresh medium at confluence. Murine osteoblast cells were obtained
from the fetal calvariae of C57BL/6 mice as described previously (17);
treatment protocols were the same as those for ST2 cells. Cell counts
were determined with a Coulter counter (Coulter Co., Hialeah, FL).
Spleen/ST2 cocultures
Spleens collected from male C57BL/6 mice less than 4 weeks of
age were washed with Hanks’ Buffered Salt Solution (HBSS; pH 7.4),
minced and suspended in HBSS. Erythrocytes were eliminated by adding
0.83% ammonium chloride in 10 mM Tris buffer. The cells
were washed three times in HBSS and resuspended in MEM-10% FBS.
Subsequently, 3 million spleen cells were plated over 30,000 ST2 cells
in MEM-10% FBS and grown for 9–10 days (5).
1,25-(OH)2D (10 nM) was added to support
osteoclastogenesis in all cultures. Varying doses of Dex were added, as
stated in the figure legends.
MCSF bioassay
MCSF activity was determined by measuring the proliferation of
the MCSF-dependent M-NFS-60 cell line (American Type Tissue Collection,
Rockville MD); 10,000 cells/well were cultured with test samples in a
final volume of 100 µl as previously described (17). M-NFS-60 cell
proliferation was assessed using a colorimetric assay to detect
mitochondrial dehydrogenase levels (17). Purified murine MCSF (5
x 107 colony-forming units/mg; from Dr. G. Gilmore,
Western Pennsylvania Cancer Institute, Pittsburgh, PA) was used as the
standard. A standard curve using recombinant human MCSF (6.94 x
107 colony-forming units/mg; Cetus, now Chiron Co.,
Emeryville, CA) produced bioassay results similar to those established
with murine MCSF, allowing us to report here, and previously, that MOB
cells secrete on the order of 500 U/100,000 cells over 3 days (17).
MCSF secretion from ST2 cells was similar and was comparable to that of
murine stromal cells measured by a similar bioassay in another
laboratory (21).
Extraction of mMCSF
mMCSF was released by trypsin. Cells were cultured in
25-cm2 flasks for 3 days and washed twice with PBS.
Matrix-bound MCSF was removed by incubation in hypertonic salt solution
(3.5 ml 2 M NaCl-10 mM HEPES, pH 7.4, per
flask) and washing with HEPES at room temperature for 30 min (20). Cell
layers were then equilibrated with HBSS at room temperature for 30 min
before treating with 1.5 ml trypsin solution (1 mg/ml) at 37 C for 10
min. Adding FBS to a final concentration of 15% stopped the reaction.
The supernatants were collected after centrifugation of cell
suspensions at 10,000 x g for 10 min and were stored
at -20 C.
MCSF ELISA
For assay of medium samples containing glucocorticosteroids, a
sandwich ELISA was used. The rat monoclonal antibody D24 (mAb D24) was
isolated from ascites of the Lou/M rat using D24 rat hybridoma (from
Dr. H. S. Lin, Washington University, St. Louis, MO) (15). A
neutralizing goat antimurine MCSF polyclonal antibody was used (R&D
Systems, Minneapolis, MN; 1 µg/ml in PBS) to coat 96-well ELISA
plates (Corning, Cambridge, MA) overnight at room temperature. The
plates were washed three times with PBS (pH 7.4) containing 0.05%
Tween-20 (PBST) and blocked with 1% BSA-0.05% Tween-20 in PBS at room
temperature for 1 h. The plates were washed three times with PBST
before addition of samples or murine MCSF standards. The samples were
incubated at room temperature for 2 h and washed three times with
PBST. The plates were incubated with mAb D24 (Bt-D24), which was
biotinylated by using an NHS-LC-biotinylation kit (Pierce Chemical Co.,
Rockford, IL), diluted to 250 ng/ml in Tris-buffered saline (20
mM Trizma base and 150 mM NaCl, pH 7.3)
containing 0.1% BSA and 0.05% Tween-20 (diluent). After 1.5-h
incubation at room temperature, plates were washed three times with
PBST and incubated with streptavidin-horseradish peroxidase (Kpl,
Gaithersburg, MD) for 45 min. After three washes with PBST, the
substrate solution (1:1 of TMB peroxidase-peroxidase B, Kpl) was
added. After 30-min incubation in the dark, 1 M
H3PO4 was added to stop the reaction. Optical
densities of each well were read with a microplate autoreader at 450 nm
(Bio-Tek, Winooski, VT) and analyzed against a standard curve created
using known quantities of purified murine MCSF. This method detected
MCSF levels as low as 0.21 ng/ml. Our enzyme-linked immunosorbent assay
(ELISA) was not sensitive enough to measure the small amounts of MCSF
found in the membrane-associated fraction.
ELISA of the same MCSF samples revealed a 35-fold reduction in reported units compared with bioassay results. Therefore, a MCSF reading of 1000 U in our bioassay would be about 30 U using our ELISA. The relative increases and decreases in either referent assay were, importantly, identical. We here report results for sMCSF as 100% of control values, with both ELISA and bioassay control values, as units per ml/100,000 cells, as noted in the figure legends.
RT-PCR
Medium was removed from treated cultures, and adherent cells
were lysed in TRIZOL (Life Technologies, Gaithersburg, MD). For
detection of transcripts encoding the membrane-bound isoform,
semiquantitative RT-PCR was used as reported previously (17, 22), which
was rectified by concurrently assaying for glyceraldehyde-phosphate
dehydrogenase (GAP) in each sample. For quantitation of all PCR
products, forward primers were [32P]ATP end labeled using
T4 kinase (Life Technologies) and used in a 1:4 ratio with unlabeled
primers for each PCR reaction. PCR was performed on a Perkin-Elmer
thermocycler 4486 (Norwalk, CT) for 20 or 23 cycles (except in
experiments to determine plateau range) at 15 sec each for melting at
94 C, annealing at 68 C, and extension at 72 C. The samples were run on
a 15% polyacrylamide gel and stained using a Bio-Rad silver staining
kit (Bio-Rad, Emeryville, CA) to assess size compared with DNA
standards. Densitometry of the 32P signal on fixed and
dried gels images was captured by a Molecular Dynamics PhosphorImager
(Sunnyvale, CA).
For semiquantitative measurement of carbonic anhydrase II messenger RNA (mRNA) we used primers that have been detailed previously (10, 19) and the GAP primers referred to above (and in Refs. 17 and 22).
Northern analysis
Total RNA made as described above was chromatographed on a 12%
formaldehyde gel and capillary transferred to Nytran paper (Schleicher
and Schuell, Keene, NH) as previously described (23). A 3.9-kilobase
murine MCSF probe (courtesy of Dr. W. Hofstetter, University of Bern,
Bern, Switzerland) was random hexamer labeled with
[32P]CTP followed by a standard hybridization protocol.
mRNA stability was assayed by adding actinomycin D (5 µg/ml) 10, 5,
and 2.5 h before RNA extraction. The 18S band on the ethidium
bromide-stained gel was quantitated by densitometry, and data were
analyzed as the density of MCSF/18S in arbitrary units.
Statistics
Significance between groups was assessed with the Bonferroni
ANOVA using Systat (Evanston, IL) software for DOS-based machines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), an effect
similar to that of 1,25-(OH)2D. Together,
1,25-(OH)2D and Dex did not significantly further increase
the measured levels. In results obtained from ELISA measurements, Dex
inhibited 1,25-(OH)2D-induced secretion of sMCSF in ST2
cells. 1,25-(OH)2D increased the amount secreted to
151 ± 5% of that in control cultures, whereas in Dex-treated
cultures no change from the control value was seen (100 ± 5%).
When both were added together, the predominant effect was that of Dex,
with sMCSF levels not significantly different from control levels
(115 ± 7%).
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). At 1
nM, a dose that minimally supports osteoclastogenesis in
cocultures with spleen cells (see Table 1), there was no inhibitory effect on
1,25-(OH)2D’s stimulation of sMCSF secretion. At 100
nM Dex, 1,25-(OH)2D’s induction of sMCSF
secretion was completely prevented. The effect of Dex alone on mMCSF
measured by bioassay was also dose dependent; 10 and 100 nM
caused greater increases in mMCSF than 1 nM (1
nM Dex caused a 120% increase over control values; 10
nM caused a 121% increase; 100 nM caused a
154% increase; P < 0.05 for a difference between 1
and 100 nM).
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. mMCSF increased to 148 ± 8%
that of the control value in the presence of 100 nM Dex and
increased further when 1,25-(OH)2D was added concurrently
(192 ± 18%). The effects of either steroid on sMCSF were not
significant (by ANOVA). The lack of effect of 1,25-(OH)2D
on sMCSF in these experiments was in contrast to those we have
previously reported. However, in the previous studies FCS was decreased
to 1% 24 h before and during treatment with
1,25-(OH)2D (17).
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is a compilation of six experiments
that were performed in duplicate, and the results shown are expressed
as a ratio with respect to the control mMCSF/GAP RT-PCR product. Figure 4b shows the phosphorimage of a representative experiment, with
densitometry presented below the lanes. Dex alone increased the mRNA
isoform encoding mMCSF (170 ± 11% compared with the control), an
effect that was additive with that of 1,25-(OH)2D (310
± 18% of control with both agents included). RT-PCR for mMCSF further
showed that Dex’s effect in the presence of 1,25-(OH)2D
(Fig. 5) was dose dependent, paralleling
the effect on osteoclast phenotype as shown in Table 1
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Northern analysis for sMCSF in the presence of Dex
To measure the RT-PCR product for sMCSF, primers that required the
presence of exon 6 were used (17). RT-PCR in ST2 cells showed that the
isoform encoding sMCSF was increased in the presence of Dex
[1,25-(OH)2D, 131 ± 32%; Dex, 165 ± 31%;
1,25-(OH)2D plus Dex, 219 ± 53%; n > 6 for
each condition; the last two groups significantly different from the
control, P < 0.05]. This effect was confirmed using
Northern analysis where the steady state mRNA coding for the secreted
form was quantitated by densitometry, as shown in Fig. 6. 1,25-(OH)2D and Dex
increased MCSF mRNA, and the effect of both together was additive.
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MCSF mRNA stability was increased by Dex, as shown in Fig. 7. The half-life for sMCSF mRNA in ST2
cells was about 2.8 h in control cells, increasing to 7.5 h
in Dex-treated cells.
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shows that Dex has a dose-dependent effect on the
expression of the osteoclast phenotype in cocultures containing ST2
cells and osteoclast progenitor cells from spleen. Both the appearance
of tartrate-resistant acid phosphatase-positive cells and the
expression of carbonic anhydrase II, an enzyme that rises in these
cultures in concert with the osteoclastogenic potential (19), were
measured. Both values increased significantly at 1 nM Dex,
but greater increases were seen with 10 and 100 nM Dex. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of mMCSF in osteoclastogenesis is not fully appreciated. Amounts of MCSF associated with cells in culture represent less than 5% of the total MCSF present, as we and others (20) have found. However, this small amount of protein, when bound to the membranes of cells, can support macrophage clonal expansion in culture (20). It is possible that presentation of MCSF to responsive cells in the local environment (i.e. cells of monoblastic lineage bearing receptors for MCSF) differentiates the signal from that transmitted by soluble MCSF. In fact, it is likely that the binding of mMCSF to receptor-bearing cells allows adhesion events critical to the accessory role of stromal cells (25, 26). The differential response of cells with respect to soluble and membrane-bound MCSF may help to explain the ability of killed cell layers to sustain hematopoiesis, whereas relatively high levels of soluble MCSF, as much as 500 U/ml, are required to similarly maintain growth and differentiation in these systems (27).
The processes of bone formation and resorption are tightly coupled, such that disentangling effects on one or the other is difficult. Stimuli to bone resorption frequently inhibit bone formation. Glucocorticoids are an excellent example of this, as they clearly diminish the growth and function of osteoblasts (28, 29, 30) as well as promote osteoclastogenesis, as is evidenced by their widespread use as enhancing agents in osteoclast-generating systems (5, 6, 31). The list of osteoblast functions regulated by Dex includes inhibition of insulin-like growth factor I (29) and collagen synthesis (30), and stimulation of collagenase expression (32), all effects that would lead to a decrement in bone formation. Specific effects of Dex on the differentiation of osteoclasts, beyond adjusting the coupling mechanism away from bone formation, are less well defined. Our data highlight a previously unknown effect of glucocorticoids on a factor critical for osteoclast differentiation: Dex promotes the expression of mMCSF. Interestingly, a decrease in mMCSF may, in turn, inhibit osteoclast formation. Physical force delivered as increased hydrostatic pressure causes a decrease in osteoclast recruitment and has been linked to decreased expression of mMCSF by accessory cells in culture (22). Another effect of Dex on osteoclast recruitment is to increase the expression of interleukin-6 (IL-6) receptors by osteoblasts (7), as IL-6 has been shown to be important in postmenopausal osteoporosis (21). Other effects of Dex on accessory cells that enhance their ability to promote osteoclast differentiation are possible as well.
Dex inhibits the protein expression of sMCSF by both MOB and ST2 cells, despite increasing the steady state mRNA encoding this species. Dex also inhibits 1,25-(OH)2D-stimulated increases in sMCSF while acting additively to increase mRNA levels. Therefore, despite positive effects on mRNA, the presence of Dex results in lower realized sMCSF levels. The mechanism by which this occurs is unknown. We and others have shown that exogenously added MCSF can inhibit osteoclastogenesis in some culture systems (19, 18). This effect appears to be due to the promotion of macrophage differentiation at the expense of monoblast entry into the osteoclast lineage (19). If this situation pertains in vivo, the effect of glucocorticosteroid to repress sMCSF secretion, coincident with an increase in presented mMCSF, may further enhance the osteoclastic potential of the progenitor pool.
Our use of both ELISA and bioassay to quantitate soluble MCSF highlights a difficult issue in this field: the reporting of MCSF as units of potency. A review of the literature shows that units of MCSF can be taken to represent colonies formed in agar, conversion from gram quantities of protein, or units generated with respect to a standard in the M-NFS-60 proliferation assay. A comparison of MCSF across studies, therefore, is difficult. Furthermore, purified MCSF may generate different results than recombinant protein in assays when tested carefully. A recent international collaborative study suggested that antibodies used in immunoassays might not recognize different natural forms of some cytokines (33). As well, currently available ELISAs are not as sensitive as bioassay, and ours, in fact, could not resolve the small amounts present in the membrane fraction. Levels of MCSF secreted by cells in culture reported by bioassay in the range of 500-5000 U may only form 10–100 colonies in semisolid agar, or represent 100-1000 pg. It is uncertain whether the discrepancy noted between ELISA and bioactivity assays reflects inherent differences in methodological sensitivity (i.e. all bioactive MCSF may not be detectable immunologically in an ELISA) or if conversion of weight in picograms may be directly applied to bioactivity with variable glycosylation and posttranslational modifications. We have endeavored to address these difficulties by comparing values in both assays.
The mechanism by which Dex increases the steady state levels of MCSF
appears at least partly to involve changes in mRNA stability. The
stability of MCSF message has indeed been shown to be regulated by
labile destabilizing proteins (34). Our results indicate that Dex
increases the half-life of the message from 2.8 h to about
7.5 h. Other agents, including PTH (35), tumor necrosis factor-
(36), and transforming growth factor-ß (37), have been shown to
increase MCSF expression at the level of gene transcription. The
promoter region of the gene includes putative cis-acting
elements for AP1, PU.1, and IL-6, all which could be directly or
indirectly affected by glucocorticosteroid (38, 39) in addition to its
effects on stability. Further experiments will be necessary to confirm
that transcription of the MCSF gene is not also stimulated by
glucocorticoids.
The effects of glucocorticoid on MCSF isoforms are similar in both ST2 stromal cells and calvarial osteoblasts (MOBs). MOBs support osteoclastogenesis of spleen cells in the absence of Dex (14, 40), although glucocorticoids further enhance osteoclast numbers (data not shown). ST2 cells, representing a less osteoblastic, or differentiated, phenotype, require the addition of Dex before they can act as accessory cells for osteoclast formation (7). Dex dose dependently increases the expression of both tartrate-resistant acid phosphatase-positive cells and carbonic anhydrase II in spleen/ST2 coculture. These findings suggest that MCSF, which is basally expressed in both types of cells, is not the only osteoclastogenic factor impacted by glucocorticoids. However, the fact that Dex increases mMCSF while decreasing sMCSF suggests that this regulation is an important event in glucocorticoid-stimulated osteoclast formation. Furthermore, the expression of the stable mMCSF isoform may be a dominant signal promoting the selection of osteoclast lineage by potential osteoclast progenitors.
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Acknowledgments |
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Footnotes |
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Received June 2, 1997.
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B-like protein in induction of the MCSF gene by tumor necrosis
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control, b 
