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Endocrinology, doi:10.1210/en.2003-0147
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Endocrinology Vol. 144, No. 10 4536-4543
Copyright © 2003 by The Endocrine Society

The Role of Calmodulin in the Regulation of Osteoclastogenesis

Liang Zhang, Xu Feng and Jay M. McDonald

Department of Pathology (L.Z., X.F., J.M.M.), University of Alabama at Birmingham, Birmingham, Alabama 35294; and Veterans Administration Medical Center (J.M.M.), Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Jay M. McDonald, M.D., Department of Pathology, University of Alabama at Birmingham, Lyons-Harrison Research Building, Room 509, 701 South 19th Street, Birmingham, Alabama 35294-0007. E-mail: mcdonald{at}path.uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calmodulin plays an important role in regulating the function of mature osteoclasts. However, its role in osteoclastogenesis has not been investigated. In the present study, we examined the role of calmodulin in osteoclastogenesis using in vivo and in vitro systems. Calmodulin antagonists, trifluoperazine (TFP), W7, and tamoxifen, dose-dependently inhibited osteoclast formation, which occurred only in the last 24 h of a 4-d osteoclastogenesis culture using mouse bone marrow macrophages. Inhibitory effects were quantitated by measuring tartrate-resistant acid phosphatase activity and counting osteoclast numbers. In contrast, bis indolylmaleimide, a protein kinase C inhibitor, showed no such inhibitory effect even when applied at a concentration that was 10-fold greater than its IC50. Overexpressing calmodulin by recombinant retrovirus reversed the inhibitory effect of TFP on osteoclast-like differentiation in RAW264.7 cells. Furthermore, administration of TFP to mice was as effective as estrogen in abolishing the ovariectomy-induced increment of osteoclastogenesis as determined by quantitative assessment of tartrate-resistant acid phosphatase activity in tibias, which led to the recovery of the ovariectomy-induced decrement in trabecular bone volume. To investigate potential cellular and molecular mechanisms by which calmodulin antagonists inhibit osteoclastogenesis, Z-VAD-FMK, a broad caspase inhibitor, failed to block the inhibitory effect of TFP on mouse osteoclast formation, indicating that apoptosis is not the underlying mechanism. Pretreatment of RAW264.7 cells with different concentrations of TFP dose-dependently inhibited receptor activator of nuclear factor {kappa}B ligand-stimulated phosphorylation of c-Jun N-terminal kinase and inhibitory {kappa}B{alpha} but not that of p38. Taken together, our data indicate that calmodulin mediates osteoclast differentiation, possibly via modulating specific receptor activator of NF-{kappa}B-signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS ARE LARGE, multinucleated, highly specialized bone-resorbing cells derived from the hematopoietic monocyte-macrophage lineage. The mechanism of osteoclastogenesis remained a puzzle in modern biology until several years ago. At that time, receptor activator of NF-{kappa}B (RANK) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were identified as two key factors responsible for osteoclastogenesis (for a review, see Ref. 1). RANKL, a member of the TNF family, is produced by osteoblasts, stromal cells, and B and T cells (2, 3, 4, 5, 6). RANKL stimulates osteoclast precursors to differentiate via binding to the receptor, RANK (7). RANKL-deficient mice develop severe osteopetrosis because of a deficiency in osteoclastogenesis (8). M-CSF, a growth factor secreted by stromal cells and osteoblasts in the bone microenvironment, controls the survival and proliferation of osteoclast precursors via its receptor, c-fms (for a review, see Ref. 9).

These discoveries have not only greatly advanced our understanding of bone biology but also provided potential new therapeutic targets in the management of osteoporosis and bone metastasis. In addition, these advances have led to the establishment of a model for generating osteoclasts in vitro using RANKL and M-CSF in the absence of osteoblasts, simplifying the troublesome coculture system that was used previously (10) and providing a much purer preparation for osteoclast studies. However, an increasing number of other factors such as IL-1, interferon (IFN)-{alpha}, TGF-ß, and TNF-{alpha} are found to directly regulate the differentiation of osteoclasts (11, 12). There exist other unknown serum factors required for osteoclast differentiation because the system requires not just serum but specially selected and screened serum for osteoclastogenesis, in addition to M-CSF and RANKL.

Despite considerable progress in elucidating the pathways of M-CSF and RANKL signaling (for a review, see Ref. 11), a possible role for Ca2+/calmodulin signaling has not been rigorously tested in osteoclastogenesis. Calmodulin is an intracellular 17-kDa calcium-sensing protein with a highly conserved amino acid sequence ubiquitously expressed in virtually every eukaryotic cell type. It translates the Ca2+ signal into 40 different intracellular signaling pathways and thus regulates biological processes as diverse as muscle contraction, fertilization, cell proliferation, vesicular fusion, and apoptosis (for a review, see Ref. 13). Of particular interest to us, intracellular Ca2+ is tightly linked to the regulation of osteoclast activity (14, 15, 16). Previous work in this laboratory has shown that calmodulin is present in high concentrations at osteoclast ruffled membranes (17), and its expression is increased when osteoclasts attach to bone (18). It has also been shown that calmodulin antagonists inhibit acid transport in isolated ruffled membrane vesicles and inhibit bone resorption in avian osteoclasts (19). These data established the role of calmodulin in the regulation of mature osteoclast function. However, there is no information regarding any role for calmodulin in osteoclastogenesis.

A recent report showed that RANKL-RANK activation led to elevation of cytosolic and nuclear calcium levels (20). Even though this study was done in mature osteoclasts, one would expect involvement of calmodulin in the RANKL-RANK-Ca2+-signaling pathway, in the context of osteoclast differentiation, because RANKL is a key factor in osteoclastogenesis. Thus, in the present study, using both in vivo and in vitro models, we tested the hypothesis that calmodulin plays a role in the regulation of osteoclastogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The glutathione-S-transferase (GST)-RANKL vector was a generous gift from Dr. S. L. Teitelbaum (Washington University, St. Louis, MO). GST-RANKL was expressed and used to generate osteoclasts, as described by McHugh et al. (21). Briefly, bone marrow macrophages were isolated from the whole marrow of 4- to 8-wk-old C57BL/6 mice (Charles River Laboratories, Wilmington, MA) and cultured overnight in {alpha}-MEM (plus penicillin and streptomycin) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT; a lot selected after screening several lots) and 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN). The nonadherent cells were collected and subjected to Ficoll-Hypaque gradient purification. Cells at the gradient interface were collected and cultured on 24-well tissue culture plates in {alpha}-MEM with heat-inactivated fetal bovine serum (10%), RANKL (50 ng/ml), and M-CSF (10 ng/ml). Culture media and indicated supplement(s) were changed every 2 d for a total of 4 d. When using RAW264.7 cells, the culture conditions were similar to those used for primary cells except that no M-CSF was added to the culture medium (22).

Western analysis
Total cell lysate proteins (25 µg) were separated by routine SDS-PAGE and then transferred to polyvinyl difluoride membranes (23). For detection of calmodulin, 0.8% glutaraldehyde (Sigma, St. Louis, MO) was used to fix the membrane for 45 min before blocking. The calmodulin antibody is produced in our lab as described previously (24). Specific antibodies against phosphorylated c-Jun N-terminal kinase (JNK), inhibitory {kappa}B (I{kappa}B){alpha}, p38, and total p38, were purchased from Cell Signaling Tech (Beverly, MA) and used as described by the manufacturer.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining
For in situ detection of nuclear DNA fragmentation, TUNEL staining was performed (25).

TRAP (tartrate resistant acid phosphatase) staining
For cell culture experiments, cells were fixed and then stained for the presence of TRAP using a commercial kit (Sigma). For whole bones, the proximal half of the tibia was stripped of soft tissue and fixed in 10% neutral buffered formalin for 3–5 d at room temperature and then decalcified in a 10% solution of EDTA for 10–14 d, paraffin-imbedded, and sectioned into 5-µm thickness. The bone sections were TRAP stained using a commercial kit (Sigma).

Quantification of TRAP activity and trabecular bone volume in bone sections by computer-assisted imaging assay
The TRAP-positive area in the primary spongiosa under the growth plate of the tibia was measured using a computer-assisted image analysis system (Bioquant system, R&M Biometrics, Nashville, TN). The percent TRAP-positive area in a defined total area was obtained for each sample. Each sample was measured twice and a mean value obtained. This method is reproducible with a coefficient of variation of 7.1% for different sections from the same tibia or different tibia from the same mouse. Trabecular bone volume was measured in the secondary spongiosa following the methods of Parfitt et al. (26). Bone sections were hematoxylin and eosin (H&E) stained, and then the regions of interest were selected that were exactly 1 mm distal to the growth plate and extending 2 mm downward through the metaphysis of tibias using the Bioquant system (R&M Biometrics). Trabecular bone volume was calculated by dividing the bone area by the total tissue area.

TRAP activity assay in cell lysates
Cell lysates were obtained, and TRAP activity was assayed using a commercial kit (Sigma). Results are expressed as OD reading at 405 nM.

Construction and production of recombinant retrovirus
Recombinant retrovirus was constructed and produced using the U3nlsLZ retroviral system (27), a gift from Dr. Dan Ory of Washington University and Dr. Richard Mulligan of Harvard University. The full length human calmodulin gene (cDNA vector, American Type Culture Collection, Manassas, VA) was amplified by PCR and cloned into the viral vector. Clones were screened and verified by DNA sequencing. Then virus was produced in 293GPG cells after transient transfection. The culture media containing virus was collected at 48, 72, and 96 h post transfection.

Characterization of the recombinant virus
Recombinant retrovirus was used at 1:1 dilution to infect cells, in the presence of 8 µg/ml polybrene (Sigma) for 24 h without antibiotic selection (28). Transduced cells were grown in regular culture medium for an additional 48 h. To assess the efficiency of viral infection, cells infected with control virus containing the ß-galactosidase (ß-gal) gene were fixed for ß-gal staining. Cells infected with viruses containing the calmodulin gene were collected for Western blotting to determine the level of calmodulin expression.

Treatment of ovariectomized (OVX) mice with trifluoperazine and estrogen
OVX and sham-operated female mice 6 wk of age were purchased from Harlan (Indianapolis, IN) and treated with either trifluoperazine (TFP) (Sigma) or 17ß-estradiol (E2) (Sigma). TFP was dissolved in drinking water (0.06 mg/ml), and the drinking water was changed twice per week. E2 (3.7 µmol/kg) was injected into mice sc twice per week. All treatments lasted for 5 wk. All animal protocols were approved by the UAB Institutional Animal Care and Use Committee.

Measurement of TFP serum levels in OVX mice
Serum samples were collected from mice at the end of the experiment, and the TFP concentration was determined by mass spectrometry (Medtox Labs, St. Paul, MN).

Statistical analysis
In the studies of the effects of calmodulin and protein kinase C antagonists on osteoclastogenesis in vitro, we compared the OD values at 405 nm and osteoclast numbers between the drug treatment groups and the control using the unpaired t test in Excel program (Microsoft Windows 2000, Microsoft, Redmond, WA). P < 0.05 and P < 0.01 were deemed statistically significant and highly significant, respectively. The raw values in the drug treatment groups were expressed into the percentiles of the mean of the control group, and plotted in Figs. 2Go and 3Go, with bars representing means ± SEM. The same statistical method was used for other experiments but raw values were used.



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  		FIG. 2. Quantification of the inhibitory effects of calmodulin antagonists on osteoclastogenesis. Purified mouse bone marrow macrophages were cultured for 3 d in the presence of RANKL and M-CSF and then were treated with varying concentrations of different calmodulin antagonists (TFP, W7, TMX) for 24 h. A, Cell lysates from cultured cells were obtained and TRAP activity was assayed (n = 9). B, Parallel cultured cells were fixed, TRAP stained, and osteoclasts (TRAP positive with three or more nuclei) counted microscopically (n = 9). MOC, Mouse osteoclast. *, P < 0.05, **, P < 0.01, compared with control.

 


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  		FIG. 3. Protein kinase C is not involved at the late stage of osteoclast formation. Mouse bone marrow macrophages were prepared as Figs. 1Go and 2Go, but instead of treating with calmodulin antagonists, different concentrations of bis indolylmaleimide I, a protein kinase C inhibitor, were added to the cultures on d 3 of culture for 24 h. A total of nine samples for each condition were collected for either counting mouse osteoclasts (MOC) number or measuring TRAP activity.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Calmodulin antagonists inhibit mouse osteoclastogenesis in vitro in a time- and dose-dependent manner
To determine the involvement of calmodulin in the regulation of osteoclastogenesis, calmodulin antagonists TFP (IC50 {cong} 1 µM), W7 (IC50 {cong} 28 µM), and tamoxifen (TMX, IC50 {cong} 1 µM) were added to the cell culture (IC50 information for W7 was obtained from the manufacturer, others from Ref. 20), and the effects were monitored by either counting TRAP-positive, multinucleated (>=3 nuclei) osteoclasts or measuring TRAP activity. All calmodulin antagonists dose-dependently inhibited osteoclast formation with a very sensitive time window on d 3 of culture. Figure 1Go shows an example using TFP. Osteoclasts are indicated by the large, red TRAP-positive cells. There was no inhibitory effect on osteoclastogenesis when TFP was withdrawn on d 3 of culture, regardless of the dose used, even though the cells had been treated with the drug for the first 3 d of culture (Fig. 1Go, WD). In contrast, a dose-dependent inhibitory effect was observed in cells, either treated beginning on d 3 for only 24 h (Fig. 1Go, 24HR) or continuously throughout the 4 d of culture (Fig. 1Go, CONT). These data established d 3 as the most sensitive time point for osteoclastogenesis to be inhibited by TFP. It should be noted here that it requires 6–7 d for complete differentiation of osteoclasts from primary bone marrow macrophages in culture. To study the role of calmodulin in osteoclastogenesis, we chose to study the first 4 d of culture including d 3 of culture when cell fusion is taking place.



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  		FIG. 1. TFP inhibits osteoclastogenesis in vitro in a time- and dose-dependent manner. Purified mouse bone marrow macrophages were used for osteoclast differentiation and were stained for TRAP on d 4. Different concentrations of TFP were added to the cultures as indicated. Three different treatments patterns were used. For WD (withdrawal on d 3) and CONT (continuous) treatment patterns, the treatments were started on d 0. For the withdrawal on d 3 (WD), fresh media were added without addition of new drug. For the continuous (CONT) treatment, both fresh media and fresh drug were added on d 3. For the 24 h on d 3 group (24HR), treatment was started on d 3 and lasted for 24 h. Magnification, x40.

 
To quantify the inhibitory effects of calmodulin antagonists, we measured TRAP activity in cell lysates after 24 h of treatment beginning on d 3 of cell culture; IC50s for TFP, W7, and TMX are approximately 3 µM, 5 µM, and 1 µM, respectively (Fig. 2AGo). Maximum inhibition reached 50% at 3 µM TFP, 90% for 10 µM W7, and 80% for 3 µM TMX (Fig. 2AGo). The inhibitory effects were even more pronounced by quantifying osteoclast numbers counted microscopically; IC50s for TFP, W7, and TMX are approximately 1 µM, 3.5 µM, and 0.8 µM, respectively (Fig. 2BGo). Very few osteoclasts were seen after the treatment with 3 µM TFP, 5 µM W7, or 3 µM TMX (Fig. 2BGo). Together, these data demonstrate that the late stage of osteoclastogenesis is very sensitive to all three calmodulin antagonists.

Because TMX could also be a partial estrogen agonist/antagonist in osteoclasts, we tested ICI 182780, a pure estrogen antagonist, for its effect on osteoclastogenesis. When used at 0.01 and 0.1 µM (known concentrations that antagonize estrogen receptor), ICI 182780 had no significant effect on osteoclastogenesis. But at 1 µM, which is known to inhibit calmodulin (19), it exhibited a 35 ± 3.7% inhibition of osteoclastogenesis. Taken together, these data are consistent with both TMX and ICI 182780 inhibiting osteoclastogenesis via calmodulin antagonism rather than estrogen receptor antagonism. Another issue is that TMX may act as an agonist of estrogen, which is known to inhibit osteoclastogenesis both in vivo and in vitro. ICI 182780 at appropriate concentrations was used to test its capacity in blocking the inhibitory effect of TMX on osteoclastogenesis. Treatment with TMX (0.1 µM) alone or together with ICI 182780 (0.1 µM) showed complete inhibition of osteoclast formation as measured by osteoclast counting after TRAP staining (data not shown). Thus, the inhibitory effect of TMX on osteoclastogenesis is not through stimulating the estrogen receptor.

Protein kinase C is not involved in the regulation of osteoclast formation at the late stage of osteoclast differentiation
TFP and other calmodulin antagonists may also inhibit protein kinase C (29). To evaluate the involvement of protein kinase C at the late stage of osteoclastogenesis, bis indolylmaleimide I, a protein kinase C inhibitor (IC50 {cong} 10 nM), was used on d 3 of cell culture for 24 h. No inhibitory effect was observed even at a concentration that was 10-fold greater than its IC50 (Fig. 3Go). Together with the data shown above, these data support the concept that the inhibitory effect of TFP on osteoclastogenesis is through antagonizing calmodulin not protein kinase C.

Overexpressing the calmodulin gene protects RAW264.7 cells against the inhibitory effect of TFP on osteoclast-like differentiation
To investigate whether overexpression of the calmodulin gene would be protective against the inhibitory effects of calmodulin antagonists on osteoclastogenesis, we used RAW264.7 cells, a mouse macrophage cell line that can be differentiated into osteoclast-like cells in the presence of RANKL alone (22). We generated recombinant retrovirus using the U3nlsLZ retroviral system (27, 28). We first determined whether the ß-gal gene can be introduced into RAW264.7 cells by this method. After culturing for 48 h post infection, approximately 30% of cells were positive for ß-gal (data not shown). Then the full length of the human calmodulin gene was cloned into the recombinant retroviral vector and the authenticity of calmodulin-recombinant viral vector was confirmed by DNA sequencing (data not shown). It should be noted here that human and mouse calmodulin share identical amino acid sequences. Overexpression of calmodulin in RAW264.7 cells was verified by Western blotting (Fig. 4AGo). Overexpression of calmodulin in RAW264.7 cells partially protected these cells from the inhibitory effect of TFP on osteoclast-like differentiation, increasing TRAP activity significantly, compared with ß-gal virus added (Fig. 4BGo). Considering an infection rate of 30%, this effect would appear to represent a complete reversal of the TFP effect. Thus, overexpressing calmodulin in RAW264.7 cells does prevent the inhibitory effect of TFP on osteoclast-like differentiation. Because RAW264.7 cells express a much higher level of calmodulin than mouse bone marrow macrophages (data not shown), a higher concentration of TFP (5 µM) was required to obtain a significant inhibitory effect in these cells.



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  		FIG. 4. Overexpressing calmodulin protects cells from the inhibitory effect of TFP. RAW264.7 cells were infected with recombinant retrovirus that contains either the ß-gal gene (LaZ-RV) or the human calmodulin gene (CaM-RV) and then differentiated in the presence of RANKL (50 ng/ml) for 4 d. A, Western blotting using an anticalmodulin antibody (ANTI-CaM) shows increased calmodulin expression after the calmodulin gene was introduced (lane CaM-RV), compared with ß-gal gene expression (lane LaZ-RV) from parallel cell lysates. B, TFP (5 µM) was added on d 3 of culture and cell lysates were obtained for a TRAP activity assay 24 h later (n = 3). *, P < 0.05, comparing to LaZ-RV. Probability values on top were obtained by comparing CaM-RV with Laz-RV when both were treated with TFP.

 
TFP inhibits osteoclastogenesis in OVX mice
Because osteoclastogenesis is increased in OVX mice, we had used these mice to test the effect of TFP on osteoclastogenesis in vivo. As predicted, there was a significant increase of TRAP activity, an indication of enhanced osteoclastogenesis, in the bones of OVX mice, compared with sham-operated control mice (Figs. 5Go and 6Go). Figure 5Go shows representative tibial sections. The red color indicates the location of osteoclasts. TRAP staining is most intense in the OVX mice and appears to be considerably less in the samples from the control, estrogen-treated (OVX + E2) and TFP-treated (OVX + TFP) mice. TRAP was quantitated using a computer-assisted imaging assay (Fig. 6Go). Treatment with either estrogen or TFP abolished the increase in osteoclastogenesis induced by ovariectomy (Fig. 6Go). TFP was administrated in drinking water (0.06 mg/ml), yielding an average serum level of 93 ± 2.7 nM, which is comparable to the therapeutic range in humans (8–83 nM).



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  		FIG. 5. A calmodulin antagonist (TFP) inhibits osteoclastogenesis in OVX mice. Tibial sections obtained from sham-operated control mice, OVX mice with or without treatments with either estrogen (OVX+E2) or TFP (OVX+TFP) for 5 wk), and then TRAP stained (osteoclasts are stained red). Representative sections are shown. Magnification, x20.

 


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  		FIG. 6. Quantification of the effect of TFP on TRAP activity in OVX mice. TRAP-positive areas were measured using a computer-assisted imaging assay in the tibial sections obtained from sham-operated control mice, OVX mice with or without treatment with either estrogen or TFP for 5 wk. *, P < 0.05, compared with sham-operated control.

 
TFP recovers bone loss in OVX mice
To determine whether the inhibitory effects of TFP on ovariectomy-induced osteoclastogenesis would lead to a protection of bone mass in these mice, parallel bone sections were H&E stained, and trabecular bone volumes were measured by a computer-assisted imaging assay. As predicted, ovariectomy caused a significant 60% reduction in trabecular bone volume, compared with sham-operated mice, which was totally reversed by estrogen supplementary treatment (Fig. 7Go). TFP treatment, like estrogen, protected against ovariectomy-induced bone loss (Fig. 7Go). Compared with OVX mice treated with vehicle, there were 219% and 179% increases in trabecular bone volume in the mice treated with estrogen and TFP, respectively.



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  		FIG. 7. Quantification of the effect of TFP on trabecular bone volume in OVX mice. Trabecular bone volumes were measured using a computer-assisted imaging assay in H&E-stained parallel tibial sections obtained from sham-operated control mice, OVX mice with or without treatments with either estrogen or TFP for 5 wk as indicated in Fig. 6Go. *, P < 0.05, compared with sham-operated control, OVX + E2 and OVX + TFP.

 
Apoptosis is not responsible for the TFP inhibitory effect on mouse osteoclastogenesis in vitro
One hypothesis to explain the inhibitory effect of calmodulin antagonists on osteoclastogenesis is that it is due to a selective killing of newly formed osteoclasts. To investigate the possible involvement of apoptosis in the process, we used a broad caspase inhibitor, Z-VAD-FMK (30), which provided no protection against treatment with 1 µM TFP as shown in Fig. 8Go. We also performed TUNEL assay after 24 h treatment with TFP and found no increase of TUNEL-positive cells (data not shown). Thus, apoptosis is not responsible for the inhibitory effects of TFP on osteoclastogenesis.



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  		FIG. 8. Apoptosis is not responsible for the inhibitory effects of TFP on osteoclastogenesis. On d 3 of mouse bone marrow macrophage culture and after an hour of pretreatment with Z-VAD-FMK (75 µM, shortened as Z-VAD), a broad caspase inhibitor, cells were treated with 1 µM TFP for 24 h. Then cells were fixed and TRAP stained (examples shown in lower panel; magnification, x40). The number of osteoclasts (MOC) were counted and expressed as means ± SEM in the upper panel (**, P < 0.01, comparing with control; n = 6).

 
TFP inhibits RANK signaling
RANKL is a key factor in the regulation of osteoclast differentiation; thus, it is logical to determine whether calmodulin modulates RANK signaling. As expected, RANKL stimulated robust increases in the phosphorylation levels of p38, JNK, and I{kappa}B{alpha} in nondifferentiated RAW264.7 cells (Fig. 9Go). Interestingly, the phosphorylation levels of JNK and I{kappa}B{alpha} were decreased by TFP in a dose-dependent manner (Fig. 9Go). However, RANKL-stimulated phosphorylation of p38 was not changed even at the highest concentration of TFP (10 µM). Thus, it appears that TFP interferes only with the RANKL-stimulated phosphorylation of JNK and I{kappa}B{alpha} but not that of p38. Interestingly, RANKL-stimulated degradation of I{kappa}B{alpha} was not fully recovered by TFP treatment. In fact, TFP itself also induced degradation of I{kappa}B{alpha} but not its phosphorylation (Fig. 9Go).



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  		FIG. 9. TFP concentration-dependently inhibits RANKL-stimulated phosphorylation of JNK and I{kappa}B{alpha} but not p38. Nondifferentiated RAW264.7 cells were pretreated with 2.5, 5, 10 µM TFP for 20 min, followed by a challenge with RANKL (100 ng/ml) for an additional 20 min. Then cell lysates were obtained and immunoblotted for phosphorylation levels of JNK, I{kappa}B{alpha}, and p38. Total levels of p38, JNK, and I{kappa}B{alpha} were also measured. A representative blot from three independent experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data from both in vivo and in vitro studies presented here demonstrate an important role for calmodulin in osteoclastogenesis. To our knowledge, this is the first report that implicates calmodulin in the regulation of osteoclast differentiation, even though calmodulin has been reported to participate in modulating the differentiation of other types of cells such as glioma and mammary gland cells (31, 32). The inhibitory effects of calmodulin antagonists occur on d 3 of the 4-d preosteoclast culture (Fig. 1Go), at a time when preosteoclasts start fusing into multinucleated osteoclasts. This suggests that calmodulin may participate in the regulation of osteoclast differentiation at this stage. All three calmodulin antagonists (TFP, W7, and tamoxifen) used here were able to inhibit osteoclastogenesis in a dose-dependent manner (Fig. 2Go). Tamoxifen, known to most investigators as a potent estrogen antagonist, is also a calmodulin antagonist and an effective modulator of osteoclast function at the concentrations used here (19). ICI 182780, a pure estrogen antagonist, showed no effect on osteoclastogenesis when used at low concentrations that are known to antagonize only the estrogen receptor but did inhibit osteoclastogenesis when applied at a higher concentration (1 µM), known to antagonize calmodulin (19). Thus, both tamoxifen and ICI 182780 can inhibit osteoclastogenesis only at concentrations that antagonize calmodulin. In addition, low concentrations of ICI 182780 are unable to block the inhibitory effect of tamoxifen on osteoclastogenesis, indicating that the effects of tamoxifen were not due to estrogen agonist action in our system (data not shown).

Although TFP and other calmodulin antagonists have been reported to act as protein kinase C inhibitors in some cell systems (29), bis indolylmaleimide I, a specific protein kinase C inhibitor, failed to inhibit osteoclastogenesis in our system, indicating that inhibition of protein kinase C is not the underlying mechanism here (Fig. 3Go). It is worth noting here that protein kinase C has been reported to modulate mature avian osteoclast function (33) and also participate in the regulation of osteoclastogenesis via modulation of RANKL expression in osteoblasts (34), an indirect mechanism. A recent report indicated that protein kinase C mediated the survival signal of RANKL in mature osteoclasts isolated from rats (35). However, our data suggest that protein kinase C does not play a direct role in the latter stages of osteoclastogenesis. We speculate that this discrepancy may arise from differences between mature osteoclasts and preosteoclasts. Additionally, overexpression of calmodulin by recombinant retroviruses protected osteoclast-like differentiation in RAW264.7 cells from the inhibitory effect of the calmodulin antagonist, TFP (Fig. 4Go). These data point to a specific role of calmodulin in osteoclastogenesis in vitro.

To further confirm the role of calmodulin in osteoclastogenesis, we showed that administration of TFP to OVX mice reduced osteoclastogenesis as effectively as estrogen, as determined by TRAP activity in tibias (Figs. 5Go and 6Go). Consistent with that, TFP almost completely recovered the loss of trabecular bone in OVX mice (Fig. 7Go). Thus, calmodulin appears to modulate osteoclast differentiation in both in vivo and in vitro systems. TFP is a prescribed drug in humans (brand name Stelazine) for the treatment of nonpsychotic anxiety. Therapeutic serum concentrations in humans are comparable with those presented here in mice. Because it has been reported that bone remodeling is also regulated by the central nervous system (36) and there is a high level of calmodulin expressed in the brain (37), we cannot rule out indirect effects of TFP on brain in intact animal studies. In addition, nonosteoclast (osteoblast or stromal cell) mechanisms in the bone microenvironment could also play a role. Despite all these, considering the dramatic effects of calmodulin antagonists on osteoclastogenesis in vitro at low concentrations and the protective effects of TFP on bone mass in OVX mice, a direct effect of TFP on preosteoclasts likely occurs in vivo.

To evaluate the cellular and molecular mechanisms by which calmodulin modulates osteoclastogenesis, we asked whether apoptosis is involved in the inhibitory effects of calmodulin antagonists. A decrement in osteoclast number could result from either a decreased formation rate or an increased death rate of newly formed osteoclasts. Z-VAD-FMK, a broad caspase inhibitor (30), failed to block the inhibitory effect of TFP on cultured cells (Fig. 5Go), indicating that apoptosis is unlikely to be responsible for the mechanisms by which calmodulin modulates osteoclastogenesis. An alternative hypothesis is that calmodulin antagonists interfere with the fusion process in preosteoclasts because d 3 in culture is when preosteoclast fusion begins to occur and also when TFP inhibits osteoclast formation (Fig. 1Go). The molecular mechanisms of preosteoclast fusion remain underinvestigated and have not been fully elucidated. The macrophage fusion receptor (MFR) has been suggested to be a key molecule in governing this process (38, 39, 40). However, it remains to be seen how applicable this is to osteoclasts because the macrophage fusion receptor data were obtained from lung macrophages (38, 39, 40). In other experimental systems, such as Dictyostelium discoideum (41), chicken embryonic muscle cells (42), and secretory vesicular fusion in the pituitary (43), calmodulin has been reported to be a mediator of cell and or membrane fusion processes. Thus, the experimental systems presented here represent a potentially useful tool for dissection of the detailed mechanisms of the preosteoclast fusion process, a crucial step in the formation of functional mature osteoclasts.

RAW264.7 cells require only the presence of RANKL to differentiate into osteoclasts (22). This process can be inhibited by TFP, which in turn, can be reversed by overexpression of calmodulin (Fig. 4Go). Thus, we speculate that calmodulin may modulate RANK signaling. In support of this, a recent report showed that RANKL evokes Ca2+ oscillations in preosteoclasts that mediated osteoclast differentiation (44). TFP concentration-dependently inhibited RANKL-stimulated phosphorylation of JNK and I{kappa}B{alpha} but not that of p38 (Fig. 9Go). Even though all three signaling pathways are known to be critical in osteoclast differentiation (for a review, see Ref. 11), they are not equally modulated by calmodulin. It is interesting to note that TFP itself also induced I{kappa}B{alpha} degradation but without causing the phosphorylation of I{kappa}B{alpha}, which may be the reason it failed to recover the I{kappa}B{alpha} degradation stimulated by RANKL (Fig. 9Go). Thus, further investigation of the effect of TFP on the mechanism of RANKL-stimulated NF{kappa}B is warranted. Another line of research has been to identify downstream targets of calmodulin signaling that may be involved in the process. We detected no change in calmodulin expression at the protein level during the course of osteoclastogenesis in vitro (data not shown). Preliminary data suggest that calmodulin-dependent kinase II but not calcineurin participates in the late stage of preosteoclast fusion as determined by experiments using specific inhibitors of calmodulin-dependent kinase II and calcineurin (data not shown). These data do not agree with recent reports claiming that calcineurin mediates osteoclastogenesis (44) and modulates RANKL-stimulated NF-{kappa}B activity in mature osteoclasts (35). These discrepancies must be clarified before the precise targets for calmodulin antagonists in osteoclastogenesis can be fully elucidated. For example, calmodulin antagonists could act at the very beginning of RANK signaling, the elevation of cytoplasmic Ca2+ level, which is also under investigation.

In conclusion, these studies show that calmodulin plays a specific role in regulating osteoclast differentiation, possibly via modulating RANK signals and may provide a potential novel therapeutic target for inhibiting bone resorption in bone diseases in which osteoclast activity is prevalent, such as osteoporosis and lytic bone metastases.


   Acknowledgments
 
The authors thank Dr. Steven L. Teitelbaum for the GST-RANKL vector, Drs. Dan Ory and Richard Mulligan for the U3nlsLZ retroviral vector and packaging cells, Dr. H. K. Tiwari for statistical consultation, Ms. Margaret A. McKenna for critical reading of the manuscript, and Mrs. P. Lott and Mr. N. Clark for performing bone histomorphometry.


   Footnotes
 
This work was supported by NIH Grants AR43225, AR46031, and AR47512 (to J.M.M.). L.Z. is currently supported by NIH Research Award 1T32AR47512.

Abbreviations: E2, 17ß-Estradiol; ß-gal, ß-galactosidase; GST, glutathione-S-transferase; H&E, hematoxylin and eosin; I{kappa}B, inhibitory {kappa}B; JNK, c-Jun N-terminal kinase; M-CSF, macrophage colony-stimulating factor; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand; OVX, ovariectomized; TFP, trifluoperazine; TMX, tamoxifen; TRAP, tartrate resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.

Received January 29, 2003.

Accepted for publication June 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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