Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells

X. Fan, D. Fan, H. Gewant, C. L. Royce, M. S. Nanes, and J. Rubin

Department of Medicine, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required >= 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.

tetracycline regulation; osteoclast differentiation factor; TRANCE; colony stimulating factor-1; bone marrow


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OSTEOBLASTS PLAY AN IMPORTANT ROLE in the regulation of osteoclastogenesis. Macrophage colony-stimulating factor (MCSF or CSF-1), released by osteoblasts and stromal cells in the bone microenvironment, appears to be essential for the proliferation of the early osteoclast progenitor as well as for the survival of the mature osteoclast (4). Posttranscriptional modifications of MCSF mRNA determine whether the final protein is destined for secretion (secreted MCSF, or sMCSF) or insertion into the cell membrane (membrane-bound MCSF, or mMCSF). Although <5% of the total MCSF made by cultured osteoblasts and stromal cells is membrane bound (7, 24), mMCSF can support macrophage proliferation (7) as well as formation of osteoclast-like cells (33). Because mMCSF is anchored and displayed by cells within bone, this protein isoform might represent a local regulatory signal by which osteoclast development is controlled. However, differentiating the effects of the membrane-bound from the secreted form of MCSF has posed a problem: sMCSF is rapidly removed from the microenvironment by MCSF receptor-positive cells, whose numbers are directly proportional to the amount of MCSF available. Thus the role of mMCSF in regulating osteoclast formation has been difficult to ascertain in the presence of significantly larger amounts of the secreted form.

Recently another factor critical to osteoclast maturation has been identified: osteoprotegerin ligand (OPGL/ODF/TRANCE), a member of the membrane-associated tumor necrosis factor (TNF) ligand family (15, 34). OPGL is displayed by osteoblast and/or stromal cells in bone and is a critical factor allowing entry of progenitors into the osteoclast differentiation pathway. When spleen cells (34) or nonadherent bone marrow cells (15) are cultured in the presence of MCSF and OPGL in the absence of osteoblasts or stromal cells, osteoclasts are formed. Thus MCSF and OPGL, both existing in forms expressed on cell membranes, are necessary for the process of osteoclastogenesis.

These findings beg the question of which factor, MCSF or OPGL, is dominant, sufficient, or regulatory. Several studies have suggested that MCSF levels might regulate the rate of osteoclastogenesis. Because we and others have shown that high levels of sMCSF are inhibitory to osteoclast formation because of increased entry of precursors into nonosteoclast lineages (6, 20), the possibility that the membrane-bound form of MCSF functions as a critical regulatory signal in bone is raised. In fact, Lea et al. (17) found large changes in bone marrow mMCSF after ovariectomy, a condition that is associated with increased osteoclast differentiation and subsequent bone resorption. In this study we explore the effects of mMCSF during osteoclast generation. Our findings provide the first definitive evidence that mMCSF serves a permissive role during bone marrow cell selection of osteoclast lineage. Whereas osteoclastogenesis is dependent on and sensitive to the amount of OPGL available, increasing mMCSF 20-fold, i.e., to levels surpassing in vivo levels of expression, it does not increase osteoclast formation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. 1,25-Hydroxyvitamin D3, or 1,25(OH)2D3, was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). C57BL/6 male mice were purchased from the Frederick Cancer Center (Frederick, MD). Fetal bovine serum (FBS) was obtained from Atlanta Biological (Atlanta, GA). Mouse OPGL was obtained from Amgen (Thousand Oaks, CA). Other chemicals and media were obtained from Sigma (St. Louis, MO) except as noted.

Fixed ST-2 cell culture systems. ST-2 cells (Riken Cell Bank, Tsukuba Science City, Japan), original clones from murine bone marrow stromal cells, were plated in 12-well plates overnight and treated with 10 nM 1,25(OH)2D3 (1,25D) and 100 nM dexamethasone (Dex) for 2 days. ST-2 cell layers were fixed in 2.5% glutaraldehyde for 1 min, followed by the addition of 1.5% glycine. The cell layers were washed with PBS three times and covered with alpha -MEM containing 10% FBS (33). Nonadherent bone marrow cells (BMMs) were isolated from 3- to 4-wk-old C57BL/6 male mice. Briefly, BMMs were collected from mouse tibia and forma by flushing bone marrow cavities. The cells were incubated in alpha -MEM containing 10% FBS at 37°C overnight. On the next day, nonadherent BMMs were collected and overlaid on prefixed ST-2 cell layers at a density of 5.5-6 million/well. After 7 days of culture, the cultures were fixed with ethanol-acetone and stained for tartrate-resistant acid phosphatase (TRAP; Sigma, St. Louis, MO). A TRAP-positive cell containing three or more nuclei was counted as a TRAP-positive multinuclear cell (TRAP+MNC).

mMCSF extraction. Live cell layers were washed with PBS and then treated for 10 min at 37°C with trypsin solution (1 mg/ml) to release mMCSF. Adding FBS for a final concentration of 15% stopped the reaction. The cell suspensions were centrifuged at 1,500 g for 5 min, and supernatants were stored at -30°C until bioassay (7, 24, 30).

MCSF bioassay. MCSF activity was determined by measuring the proliferation of 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 (24). M-NFS-60 cell proliferation was assessed using MTT, a colorimetric assay to detect mitochondrial dehydrogenase levels. Recombinant human MCSF (6.94 × 107 CFU/mg; Cetus, Emeryville, CA) was used as the standard.

Blocking mMCSF. ST-2 cells were plated in 24-well plates at a density of 15,000/well and cultured with 10 nM 1,25D and 100 nM Dex for 2 days. ST-2 cell layers were treated at 50% confluence with 10 µg/ml of goat anti-mouse MCSF neutralizing polyclonal antibody (pAb; R&D System, Minneapolis, MN) or 10 µg/ml goat IgG at 37°C for 2 h before glutaraldehyde fixation. To determine the dose of blocking pAb, 0.1, 1, 10, or 100 µg/ml of pAb or IgG were added to ST-2 cells before fixation. Spleen cells were plated over fixed layers, and [3H]thymidine incorporation was determined. pAb at a dose of 1 µg/ml blocked one-half of the mMCSF-dependent proliferation, and complete blocking was seen by 10 µg/ml (no further decrease by 100 µg/ml).

Preparation of spleen cells. Spleen cells were isolated from 3- to 4-wk-old C57BL/6 male mice. Briefly, spleens were washed, minced, and suspended in PBS. Contaminating erythrocytes were eliminated from the cell pellet by adding 0.83% NH4Cl in 10 mM Tris buffer (pH 7.4). The cells were washed three times with PBS, suspended in alpha -MEM containing 10% FBS, and cultured in 75-cm2 flasks overnight. The nonadherent spleen cells were collected and overlaid on prefixed ST-2 or CHO-MCSF Tet-OFF cell layers.

TRAP assay. After culture for 7 days, the cell layers were washed twice with PBS and lysed with 0.05% Triton X-100. The cell lysates were centrifuged at 10,000 g for 15 min. The supernatants were stored at -30°C. For assay, 20 µl of cell lysate and 180 µl of substrate buffer [0.48 M acetate buffer pH 5, 2 mM methylumbelliferyl phosphate (MUP), 83 mM tartaric acid] were added to each well in Microtiter 96-well white plates (Dynex Technologies, Chantilly, VA). After incubation at 37°C for 30 min in the dark, 100 µl of 0.5 M glycine solution containing 50 mM EDTA (pH 10.4) were added to stop the reaction. Fluorescence was measured on an LB 50 Plate Reader (Perkin Elmer, Buckinghamshire, England) at excitation wavelength 366 nm and emission wavelength 456 nm. A serial dilution of methyl-umbelliferone (0-800 µM) was used to generate a standard curve. The enzyme activity was represented as micromoles of MUP hydrolyzed per milligram of protein per min.

Cell proliferation. [3H]thymidine (2 µCi/well) was added to cultures on the specific day. After 24 h, cells were lysed in 20 mM NaOH containing 1% SDS, transferred to scintillation vials, and counted by a PACKARD 2500 TR Liquid Scintillation Analyzer (Downers Grove, IL).

RT-PCR. Total RNA was prepared in TRIzol (Life Technologies, Gaithersburg, MD). For measurement of cathepsin K, 0.5 µg of total RNA was added to an RT reaction containing 1 mM dNTP, 0.5 µM forward primer, 100 U of MMLV reverse transcriptase, and 20 U of RNAsin. The RT reaction was incubated for 30 min at 37°C. For quantitation of PCR products, 1 µCi of alpha -[32P]dCTP was used in each standard PCR reaction (cathepsin K PCR forward primer 5'-CCAGTGAA-GAAGTGGTTCAG, reverse primer 5'-TATCCTTTCTTTCGATAGTCG). PCR products were chromatographed and phosphorimages captured on a Molecular Dynamics instrument. The cathepsin K density was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) density obtained from the same sample.

Tet-OFF system. The mMCSF cDNA (1.6 kb) plasmid was the generous gift of Dr. Martine Roussel (St. Jude's Children Research Hospital, Memphis, TN). The region coding for mMCSF (i.e., - exon 6) was obtained by PCR with adapters added to mutate two base pairs on each PCR primer (forward primer 5' CCC GGC CGC GGC CCA GCT GCC CGT ATG ACC G 3' and reverse primer 5' GAG AGA TCT TAG AAT TCC CTC TAC ACT GGC 3') to create Sac II and Bgl II sites, respectively. PCR was performed by adding 50 ng of 1.6 kb mMCSF cDNA plasmid with 100 ng each of primers into 50 µl of 200 µM dNTP, 2.2 µM Mg2+, and 10 units of Pfu DNA polymerase (Stratagene, San Diego, CA). Twenty-five cycles of PCR were used at the following parameters: denature at 94°C for 30 s, anneal at 60°C for 30 s, and extend at 72°C for 2 min. The PCR product was digested with Sac II and Bgl II, and the appropriate band was purified from 1% agarose using a gel extraction kit (Qiagen, Valencia, CA). The pTRE vector obtained from Tet-OFF gene expression system (Clontech, Palo Alto, CA) was opened with Sac II and BamH I and then extracted and purified. The TRE-MCSF construct was generated by ligating the above insert and vector, because BamH I and Bgl II have compatible ends. Sequencing confirmed that the entire coding region of mMCSF was inserted into the TRE response vector. CHO cells purchased from Clontech had appropriate tetracycline-regulatory protein (Tet-R) expression. CHO cells cultured in alpha -MEM containing 10% Tet-free FBS were cotransfected with pTRE-MCSF and a vector carrying resistance to hygromycin (pTK-Hyg) at a 10:1 ratio. Stable cell lines were selected under 400 µg/ml G418 and 200 µg/ml hygromycin (Sigma). Thirty-six clones were isolated and tested by MCSF bioassay in the presence and absence of doxycycline (Dox). Clone 4 produced the highest mMCSF expression in the absence of Dox vs. low expression with 1 µg/ml Dox. The doubly stable CHO-mMCSF cell line was maintained under 200 µg/ml G418, 100 µg/ml hygromycin, and 1 µg/ml Dox constraints.

Statistical analysis. Data were analyzed by Tukey ANOVA by use of Prism software.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

mMCSF stimulates bone marrow macrophage and spleen cell proliferation. ST-2 cells were plated at densities from 7,500 to 120,000 per well. After culture with 1,25D and Dex for 2 days, cell number and mMCSF were assayed. The ST-2 cell doubling time of 28 h was highly reproducible, as was the final mMCSF level achieved. Figure 1A shows that mMCSF levels as measured by bioassay before fixation increased from 3.5 to 138 U/well as final cell numbers increased from 56,000 to 266,000/well. Glutaraldehyde treatment at this time can thus ensure a controlled amount of mMCSF for study.


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Fig. 1.   Membrane-bound macrophage colony-stimulating factor (mMCSF) is directly proportional to ST-2 cell number. Different numbers (15,000-80,000) of ST-2 cells were plated in 12-well plates and treated with 1,25-hydroxyvitamin D3 (1,25D) + dexamethasone (Dex) for 2 days. A: mMCSF concentration was measured by bioassay. Data show that mMCSF expression is directly proportional to ST-2 cell number. B: bone marrow cells (BMMs) were cultured on fixed ST-2 cell layers for 4 days with varying numbers, and mMCSF was induced. [3H]thymidine was added to cultures and thymidine uptake measured after 24 h. BMM proliferation increased in a mMCSF dose-dependent manner. Values are means ± SE (n = 3) and representative of 3 independent experiments.

We next evaluated the ability of the glutaraldehyde-fixed mMCSF units in each well to support dose-dependent proliferation of MCSF-responsive BMMs. Nonadherent BMMs were cultured on fixed ST-2 cell layers containing different amounts of cells with variable units of mMCSF in proportion to the cell number. [3H]thymidine incorporation assay showed that cell proliferation increased threefold in those wells with the highest levels of mMCSF (93 ± 6 U/well) compared with wells containing one-third as many cells (36 ± 3 U/well) (Fig. 1B), demonstrating as well that functional mMCSF remained in ST-2 cell layers after glutaraldehyde fixation. Increasing ST-2 cell number was thus associated with proportional increases in both mMCSF expression and proliferation of bone marrow macrophages.

To verify that bone marrow cell proliferation on fixed ST-2 cells was dependent on mMCSF, mMCSF was blocked with goat anti-MCSF pAb before fixing ST-2 cells. As shown in Fig. 2, spleen cell proliferation decreased to 57.8 ± 7% (P < 0.05) after treatment with pAb compared with ST-2 cultures pretreated with a control IgG. pAb to murine MCSF added to the media with spleen cells did not further decrease proliferation in cultures pretreated with anti-MCSF pAb, but it did in the IgG-pretreated cultures. These data indicate that mMCSF on fixed ST-2 cell layers was fully blocked by the pAb before fixation and that MCSF was responsible for more than one-half of the ensuing proliferation. The mMCSF-independent proliferation indicated that BMMs were able to respond to factors other than mMCSF enmeshed in the glutaraldehyde-fixed cell layer or the extracellular matrix, e.g., vascular endothelium growth factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-beta (TGF-beta ) (19, 28).


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Fig. 2.   Progenitor proliferation on fixed ST-2 layers is diminished by pretreatment of cell layers with anti-MCSF antibody. ST-2 cells were treated with goat anti-murine MCSF neutralized polyclonal antibody (pAb) or goat IgG before fixation. Spleen cells were cultured on the fixed ST-2 layers for 6 days, and [3H]thymidine (2 µCi/well) was added for the final 24 h of culture. Cells were harvested and [3H]thymidine uptake was counted. [3H]thymidine uptake by spleen cells decreased to 57% in the presence of blocking antibodies to mMCSF (n = 3, P < 0.05). Addition of pAb (10 µg/ml) to the culture medium decreased proliferation by 50% in the IgG-pretreated group but had no further effect in groups pretreated with pAb. Data are means ± SE from 3 separate experiments.

We next established a CHO-mMCSF Tet-OFF system that allowed control of mMCSF expression in cells that did not normally express mMCSF. CHO cells constitutively expressing the tetracycline repressor (Tet-R) were transfected with a cDNA where a tetracycline response element served as the promoter for murine mMCSF. A clone (clone 4 of 36) was selected that expressed high levels of mMCSF in the absence of Dox and low levels in the presence of the antibiotic. CHO-mMCSF cells or control CHO cells were cultured with different doses of Dox (0-2 µg/ml) in 12-well plates, and after 3 days, mMCSF was assayed. Dox caused a dose-dependent inhibition of mMCSF expression (Fig. 3A): mMCSF decreased 22-fold in CHO-MCSF cells treated with 2 µg/ml Dox (21 ± 7 U/well) compared with the group without Dox (466 ± 89 U/well). The highest dose of Dox caused mMCSF expression consistent with the background bioassay mMCSF level in nontransfected CHO cells (13 U/well). CHO cell numbers were not affected by the Dox treatment (data not shown). The CHO-mMCSF cells were thus treated with Dox for 3 days before the glutaraldehyde fixation procedure. As shown in Fig. 3B, the proliferation of spleen cells plated over these fixed CHO-mMCSF cell layers decreased in a dose-dependent fashion as Dox shut off mMCSF expression.


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Fig. 3.   mMCSF expression in Chinese hamster ovary (CHO)-mMCSF Tet-OFF cells. CHO cells were doubly transfected with pTRE-MCSF and tetracycline regulator plasmids. Stable cell lines were selected in the presence of G418 and hygromycin. CHO-mMCSF cells were treated with doxycycline (Dox: 0-2 µg/ml) for 3 days. A: mMCSF on cell surface was released by trypsin and measured by MCSF bioassay. mMCSF expression in the absence of Dox was >10-fold higher than that in the presence of maximal 2 µg/ml Dox treatment. B: spleen cells were grown over fixed CHO-mMCSF cell cultures for 5 days. [3H]thymidine was added to cultures during the last 24 h. mMCSF-dependent proliferation can be seen in the presence of decreasing Dox. Values are means ± SE (n = 3) and are representative of >= 3 experiments.

OPGL regulation of osteoclast formation is dose dependent. ST-2 cells were treated for 2 days with 1,25D (10 nM) and Dex (100 nM), which induces maximal expression of both mMCSF (22) and OPGL (10, 34). At this time, cells were fixed with 2.5% glutaraldehyde. To test whether the fixed ST-2 cells were able to support osteoclastogenesis, nonadherent BMMs were plated over the fixed ST-2 layers in the presence or absence of murine sOPGL (0-10 ng/ml). After 7 days of culture, no TRAP+MNCs appeared when BMMs were cultured over ST-2 cells alone, but they were induced with the addition of exogenous OPGL. Because ST-2 cells are well known to display OPGL and support osteoclastogenesis under the culture conditions used here (34), the glutaraldehyde fixation procedure blocked or destroyed the OPGL molecule on the cell surface. In the presence of exogenous OPGL, however, glutaraldehyde-fixed layers were able to support osteoclast formation. This result suggested that the process of glutaraldehyde fixation removed the contribution of endogenous OPGL and allowed us to study the effects of mMCSF availability on osteoclast formation.

Osteoclast formation increased in an sOPGL dose-dependent fashion as seen both visually (Fig. 4A) and by counting TRAP+MNCs (Fig. 4B). As a corroborative measure of increase in the osteoclast phenotype, TRAP activity was measured using a sensitive methylumbilliferone assay. In Fig. 4C, TRAP activity dose dependently increased in proportion to the added sOPGL. ST-2 cells express mMCSF in the absence of 1,25D or Dex, as we have previously shown (11). To assure that a lower level of mMCSF expression also supported osteoclastogenesis, sOPGL was added to cocultures of ST-2 cells and BMMs in the absence of 1,25D or Dex. sOPGL caused a dose-dependent increase in osteoclast numbers as follows: sOPGL at 0 ng/ml = 6 ± 2 osteoclasts, at 2.5 ng/ml = 74 ± 7 osteoclasts, at 5 ng/ml = 353 ± 16 osteoclasts, and at 10 ng/ml = 783 ± 43 osteoclasts. Although these data cannot be compared directly with the results obtained when ST2 cells are incubated in the presence of 1,25D and Dex, they serve to show that basal levels of mMCSF are adequate to support osteoclastogenesis in the presence of sOPGL.


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Fig. 4.   Secreted osteoprotegerin ligand (sOPGL) dose dependently increases osteoclastogenesis. ST-2 cells (30,000/well) were plated into 12-well plates and treated with 1,25D + Dex for 2 days. After ST-2 cell layers were fixed, BMMs (5.5-6 million/ml) were plated over the fixed cells and cultured with different concentrations of sOPGL for 7 days, as shown. Tartrate-resistant acid phosphatase (TRAP) staining (A), numbers of TRAP-positive multinuclear cells (TRAP+MNC, B), and TRAP activity (C) showed the dose-dependent increase in osteoclast formation as sOPGL concentration increased (B). This experiment is representative of >= 3 separate experiments.

Cathepsin K has been previously used as a positive measure of osteoclast phenotype (1). Both BMMs and spleen cells express cathepsin K in the absence of OPGL in our culture system, decreasing the specificity for assaying osteoclast transformation of the semiquantitative RT-PCR assay. Thus, although sOPGL dose dependently increased cathepsin K mRNA expression, the dynamic range was very poor (the ratio of cathepsin K to GAPDH was 0.054 ± 0.0001 in the presence of 8 ng/ml sOPGL compared with 0.031 ± 0.001 in the absence of sOPGL). These data showed that cathepsin K mRNA measurement was neither specific nor sensitive enough to evaluate osteoclast formation in this culture system.

Role of mMCSF in regulation of osteoclast formation from bone marrow progenitors. To evaluate whether mMCSF might play a dominant role in regulating osteoclast precursor differentiation, we varied concentrations of the presented growth factor before fixation. In the first system, the ST-2 cell-plating density regulated mMCSF availability. In the second, mMCSF expression was regulated in the tetracycline-sensitive CHO-mMCSF clone with Dox.

The range of mMCSF in fixed ST-2 cell layers was between 35 and 120 U/well. Soluble OPGL was added to stimulate osteoclastogenesis: 5 ng/ml sOPGL represented the ED50 for sOPGL-induced osteoclast formation in the fixed ST-2 culture system (see Fig. 4). After 7 days of culture with BMMs, cultures were fixed and stained for TRAP. As shown in Fig. 5A, TRAP+MNCs did not increase when mMCSF availability rose from 34 to 120 U/well. The sensitive TRAP activity assay also revealed no changes as mMCSF units increased, as shown in Fig. 5 as TRAP activity. There was even a trend toward decreased osteoclast formation at the higher mMCSF availability. In addition, experiments were performed with 2.5 ng/ml sOPGL, which confirmed that increasing mMCSF did not increase TRAP activity (data not shown).


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Fig. 5.   Increasing mMCSF does not regulate osteoclastogenesis in BMM cultures. A: ST-2 cells were plated at 15,000-80,000/well in 12-well culture dishes. After treatment of 1,25D + Dex for 2 days, cell layers were fixed. BMMs were cultured with 5 ng/ml sOPGL over fixed ST-2 cell layers and stained for TRAP after 7 days. Under photograph, mMCSF concentrations from 3 experiments are shown, and TRAP+MNCs and TRAP activity are presented. There was decrease in nos. of TRAP+MNCs as the mMCSF levels increased. a = Mean value significantly different from b (n = 3, P < 0.05). The experiment was repeated twice. B: mMCSF expression in CHO-mMCSF cells under Dox regulation ranged from 21 to 466 U/well. After 3 days, cell layers were fixed, and BMMs were cultured with sOPGL 2.5 ng/ml on fixed cells for 7 days. mMCSF levels shown beneath figure (averaged from 3 experiments) were decreased 20-fold by maximal Dox treatment without effect on TRAP-positive cell no. (n = 4, P > 0.05, data from 2 experiments). TRAP+MNC nos. in the group without Dox treatment are ~100-200/well.

To confirm the observations with ST-2 cell layers, BMMs were plated over tetracycline-sensitive CHO-mMCSF Tet-OFF cell layers. In these cultures, the addition of Dox from 0.01 to 2,000 ng/ml reduced mMCSF from nearly 500 to <25 U/well as measured by bioassay (see Figs. 3A and 5B). In the presence of sOPGL, TRAP+MNC numbers were not significantly changed despite the >20-fold increase in mMCSF. Thus raising mMCSF had no apparent effect on the number of cells entering the osteoclast lineage in this unique culture system.

Requirements for MCSF and OPGL during spleen progenitor to osteoclast differentiation. Spleen cells can proliferate with added MCSF and differentiate into the osteoclast lineage when sOPGL is added to the culture medium in the absence of accessory stromal cells (34). We were not able to generate osteoclasts from spleen cells over fixed ST-2 cell layers; however, the CHO-mMCSF-Tet-OFF system, which displays higher amounts of mMCSF, was successful. TRAP+MNCs were present at the highest doses of fixed mMCSF (632 and 320 U/well), disappearing when Dox decreased mMCSF expression below 100 U/well (Fig. 6). Figure 6, which compiles two separate experiments, did show a statistical trend toward an increase in osteoclast numbers when mMCSF rose above 300 U/well, suggesting that spleen cells respond differently than do BMMs.


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Fig. 6.   Osteoclast formation on fixed CHO-mMCSF cells. mMCSF expression in CHO-mMCSF cells was controlled by Dox (0-2 µg/ml). Control CHO cells (not transfected with pTRE-MCSF) were also plated at the same density. After 3 days of culture, mMCSF was measured, and the remaining CHO-MCSF cells were fixed by glutaraldehyde. Spleen cells (4 million/well) were cultured with 10 ng/ml secreted osteoprotegerin ligand (sOPGL) on fixed CHO-mMCSF cells for 7 days. TRAP+MNCs in (0) Dox treatment group (mMCSF 633 U) was 144 ± 47/well vs. 2.5 ± 0.5/well in 1 ng/ml Dox-treated group (mMCSF 18 U). Control CHO cells with 10 ng/ml sOPGL did not form TRAP+MNC. Means significantly different from other groups (n = 4, *P < 0.05). Data are representative of 2 independent experiments.

Although it is not possible to compare amounts of membrane-bound and soluble ligands directly, spleen cell resistance to MCSF during osteoclast differentiation can also be demonstrated using sMCSF. BMM and spleen cell cultures were set up in standard fashion to promote osteoclastogenesis, and soluble MCSF (sMCSF; 125-2,000 U/ml) and sOPGL were added. As shown in Fig. 7 in spleen cell cultures, addition of 250 U/ml of sMCSF was required before the appearance of TRAP+MNCs. TRAP+MNC numbers reached a plateau at 1,000 U/ml sMCSF. In comparison, BMMs cultured with sOPGL formed osteoclasts even in the absence of added sMCSF, reaching plateau immediately upon addition of 200-250 U/ml of sMCSF. Interestingly, sMCSF above 500 U/ml inhibited osteoclast formation, as we have previously reported (6). Although it is not possible to compare osteoclast numbers directly between the BMM and spleen cell cultures, Fig. 7 demonstrates that the MCSF requirement for osteoclastogenesis is greater in spleen cell cultures. These results also indicate that the inability of fixed ST-2 cell layers to support spleen cell-to-osteoclast transformation is due to inadequacy of mMCSF.


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Fig. 7.   Addition of exogenous soluble MCSF in BMM and spleen cell cultures. BMMs (2 million/well) were plated in 24-well plates and treated with 2.5 ng/ml OPGL and recombinant human MCSF (rhMCSF, range 0-2,000 U/ml) for 7 days. TRAP+MNC nos. reach peak level in 250 U/ml rhMCSF and decrease significantly at addition of 2,000 U/ml rhMCSF. a = Mean value significantly different from group without rhMCSF treatment (n = 4, P < 0.01). For spleen cell cultures, 1.5 million spleen cells were plated in 24-well plates, treated with 10 ng/ml sOPGL and rhMCSF (0-2,000 U/ml), and stained for TRAP after 7 days. TRAP+MNC numbers reached plateau in the presence of rhMCSF above 1,000 U/ml. b = Mean value significantly different from that of cells without rhMCSF treatment (n = 4, P < 0.01).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the work presented here, we wished to clarify whether mMCSF levels could regulate the level of osteoclast formation. During the past decade, MCSF elaborated by stromal cells has been recognized to be an essential factor for osteoclast precursor proliferation and differentiation and osteoclast survival (27). Stromal cells from op/op mice that are deficient in MCSF do not support osteoclastogenesis in the absence of exogenous MCSF (32) Antibodies that completely block the MCSF receptor inhibit the development of osteoclasts in vitro (9). Furthermore, even with the addition of substantial amounts of sOPGL, monocytic precursors do not appear to generate osteoclasts in the absence of MCSF (3, 15, 34).

Osteoclastogenesis is known to be modulated by systemic hormones and local stimulatory factors, such as 1,25D, parathyroid hormone (PTH), interleukins, TNF-alpha , and TGF-beta , among others (8, 19, 25, 27). Many of these factors stimulate the expression of MCSF, leading to the hypothesis that the level of MCSF present within the local bone environment might dose dependently direct osteoclast formation. For instance, 1,25D, TNF-alpha , and PTH, all of which potently induce osteoclast formation, cause increased secretion of sMCSF (11, 24, 31). Additionally, several investigators have suggested that estrogen deficiency might promote osteoclastogenesis by increasing the production of sMCSF by bone stromal cells (12, 26). However, very high levels of added sMCSF inhibit recruitment of progenitors into the osteoclast lineage despite increased proliferation of the predominant clone (6, 20, 29, 34). Further confounding these issues is a lack of data demonstrating significant transcriptional control within the murine MCSF promoter by osteoclastogenic factors known to affect sMCSF expression (11, 23). We and others have suggested that sMCSF secretion might be controlled posttranscriptionally (5) or posttranslationally via the trafficking of the secreted protein within the stromal cell (22). In sum, the role of sMCSF in regulating osteoclastogenesis, directly or indirectly, has never been firmly established.

Alternatively, mMCSF, which is directed to the cell membrane after posttranscriptional excision of exon 6 (16), might represent a more finely tuned and significant regulatory molecule as expressed by cells within bone. Glucocorticoids, which promote bone resorption, increase the expression of mMCSF in bone stromal cells while decreasing secretion of the soluble form (22). As well, estrogen deficiency may specifically increase the expression of mMCSF, as opposed to sMCSF, within the bone marrow (17). Recent studies have shown that mMCSF is sufficient to support osteoclast formation in vitro (33), suggesting a role for this isoform in regulating osteoclast lineage selection.

To investigate effects of mMCSF on regulation of osteoclastogenesis, we used both fixed ST-2 cells and CHO-MCSF Tet-OFF cells overlaid with osteoclast progenitors. In both systems, proliferation of the osteoclast progenitors was proportional to mMCSF availability, as expected (6). Neither cell system, however, supported osteoclastogenesis in the absence of exogenous OPGL. This was unexpected in the case of the ST-2 cells, which express OPGL under the control of hormones (13), indicating that OPGL was destroyed by our fixation procedure or was fixed in an inactive conformation. In contrast, Kong et al. (14) reported that fixed activated T cells expressing OPGL could trigger osteoclastogenesis; this difference may be due to differences in fixation or in aspects of lymphocyte presentation of the molecule. Nonetheless, our system allowed investigation of the role of mMCSF in directing osteoclast formation.

Our experiments showed that BMMs cultured on fixed ST-2 cell layers displaying varying amounts of mMCSF (34-120 U) showed no increase in osteoclast formation as mMCSF availability increased. The concentration of sOPGL, on the other hand, was intrinsically related to the amount of osteoclastogenesis. In confirmation of this finding, experiments using fixed CHO-mMCSF cell layers showed that BMM osteoclastogenesis was regulatable by sOPGL and that osteoclast formation was not changed as mMCSF was increased 20-fold. These data indicate that increasing mMCSF expression stimulates precursor proliferation but does not enhance osteoclast formation. Thus, whereas mMCSF undoubtedly has an important role in the recruitment of monocytes and may have effects on both osteoclast activity and fusion (2, 18), its regulatory function during osteoclast recruitment from marrow cells appears to be minor. MCSF's ability to enhance the expression of RANK, the receptor for OPGL (3), suggests a competence role for MCSF, rather than one that predicates final selection of the osteoclast phenotype.

Both marrow and spleen cells represent sources of osteoclast precursors. In our experiments, these two cell sources were not interchangeable. Whereas BMMs formed osteoclasts in the presence of minimal amounts of mMCSF (34 U/well) and sOPGL (2 ng/ml), spleen cells required nearly ten times as much mMCSF and 10 ng/ml sOPGL to induce osteoclast formation. This could be due to the differentiation state of the spleen cells, which may harbor fewer cells capable of proliferating and responding to sOPGL, perhaps suggesting that fewer spleen cells express either RANK or MCSF receptors. In addition, spleen cells may not provide other stimulatory factors that enhance osteoclast development; for instance, Sells Galvin et al. (25) reported that TGF-beta had a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with sOPGL/ODF and MCSF. Furthermore, our data did show that increasing mMCSF far above levels that would generally be expressed in bone would further enhance osteoclast formation from spleen progenitors. The source of osteoclast progenitors thus represents a significant variable in studies that evaluate the effects of stimulators and inhibitors of osteoclast development. Because marrow cells, and not spleen or peripheral blood cells, are precursors for bone osteoclasts in most organisms, lower levels of mMCSF are likely adequate to support osteoclastogenesis.

Thus the major limiting factor for osteoclast induction in marrow culture is OPGL. Previous results showing that very high levels of sMCSF were associated with decreased entry of proliferating cells into the osteoclast lineage can now be reinterpreted as being limited by the fixed OPGL potential presented by stromal cells (6, 20). Furthermore, it is unlikely that small changes in mMCSF induced by resorptive factors such as TNF-alpha (33) and 1,25D (24) or physical factors such as hydrostatic pressure (21) have significant effects on osteoclast recruitment. Alternatively, BMMs cultured with increasing amounts of sOPGL on fixed ST-2 cell layers providing equivalent mMCSF showed a clear sOPGL dose dependence in terms of osteoclast formation. In summary, our results suggest that the osteoclastogenic potential of the marrow environment is dominantly regulated by OPGL, whereas mMCSF serves as a competence factor. It is likely that agents regulating osteoclastogenesis do so directly or indirectly through OPGL.


    ACKNOWLEDGEMENTS

This work was supported by the Veterans Administration (Merit and Research Enhancement Award Program) and National Institute of Arthritis and Musculoskeletal and Skin Diseases R01-AR-42360.


    FOOTNOTES

Address for reprint requests and other correspondence: X. Fan, Veterans Affairs Medical Center - 151, Emory Univ. Medical School, 1670 Clairmont Rd., Decatur, GA 30033 (E-mail: xfan{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 February 2000; accepted in final form 26 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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