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Abstract
The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2−/−) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2−/− mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2−/− mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2−/− mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.
BONE REMODELING IS a complex process that is regulated by an interplay between circulating hormones and locally produced factors that act in a concerted manner to regulate osteoblast and osteoclast activity (1). Over recent years, there has been increasing interest in the role that the nervous system and neurotransmitters play in the regulation of bone remodeling (2). Reflecting this fact, the endocannabinoid pathway has recently been implicated as are important regulator of bone turnover and bone mass (3, 4, 5, 6).
The endocannabinoid system comprises two known receptors [the type 1 (CB1) and type 2 (CB2) cannabinoid receptors], a family of endogenous ligands, and a molecular machinery for ligand synthesis, transport, and inactivation. Cannabinoid receptors belong to the G protein-coupled receptor superfamily and are highly expressed in the brain (CB1), immune system (CB2), and a number of other tissues (7). A variety of natural and synthetic ligands have been identified that bind to these receptors. Cannabinoid receptor agonists such as Δ9-tetrahydrocannabinol, anandamide, 2 arachinodyl glycerol, CP55,490, and JWH133 bind to the receptors causing inhibition of adenylyl cyclase and activation of ERK kinases and other signaling pathways in target cells (8). Another class of cannabinoid receptor ligands have been synthesized including AM251, AM630, and SR141716A (rimonabant), which block the effects of cannabinoid receptor agonists. These compounds were originally referred to as cannabinoid receptor antagonists, but it has now become clear that these compounds cause activation of the receptors in the absence of agonist binding, with opposite effects on downstream signaling cascades as those of the agonists. Accordingly, these compounds are now referred to as cannabinoid receptor antagonists/inverse agonists (9).
We previously reported that osteoblasts and osteoclasts express cannabinoid receptors and that mice with inactivation of CB1 have increased bone mass and are protected from ovariectomy-induced bone loss (3). We also found that the CB1-selective antagonist/inverse agonist AM251 inhibited osteoclast differentiation and prevented ovariectomy-induced bone loss in vivo, confirming the importance of CB1 as a regulator of bone resorption (3). In another study, Tam and colleagues (5) reported that mice with CB1 deficiency on a CD1 background had high bone mass but surprisingly found that mice with CB1 deficiency on a C57BL/6 background had low bone mass. There is evidence that CB2 also plays a role in regulating bone metabolism. Mice with deficiency of CB2 (CB2−/−) have been reported to develop age-related osteoporosis in association with increased bone turnover (4). The CB2-selective agonist HU308 has also been reported to promote osteoblast differentiation, to inhibit osteoclast differentiation, and to protect against ovariectomy-induced bone loss (4). On the basis of these observations, it has been suggested that an important function of CB2 is to suppress bone turnover and regulate osteoblast-osteoclast coupling (10). Contrasting with these findings, however, we and others have reported that pharmacological agonists of cannabinoid receptors stimulate osteoclast formation and bone resorption in vitro in both mouse and human cells (3, 11). In these studies, however, it was not possible to determine whether the effects were mediated by CB1, CB2, or both receptors. In view of this, the aim of the present study was to clarify the role that CB2 plays in osteoclast function and ovariectomy-induced bone loss using a combination of pharmacological and genetic approaches.
Materials and Methods
Materials
Reagents were obtained from Sigma (Dorset, UK) unless otherwise indicated. The cannabinoid receptor ligands JWH133, AM251, and AM630 were purchased from Tocris Bioscience (Bristol, UK) and HU308 was a kind gift from Dr. R. J. Arends (Organon). The pharmacological properties of the cannabinoid receptor ligands used in the study are summarized in Table 1⇓. Tissue culture medium was obtained from Invitrogen (Paisley, UK), and primary antibodies were purchased from Cell Signaling Technology (Beverly, MA), unless otherwise stated. Human IL-1β was obtained from Roche (London, UK); human macrophage colony-stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK); 1,25-(OH)2-vitamin D3 was obtained from Alexis Biochemicals (Nottingham, UK); and mouse receptor activator of nuclear factor-κB ligand (RANKL) was a gift from Dr. F. P. Ross (Washington University, St. Louis, MO).
Mouse osteoblast-bone marrow cocultures
Osteoblast-bone marrow cocultures were performed essentially as previously described (12). Briefly, osteoblasts were isolated from the calvarial bones of 2-d-old mice by sequential collagenase digestion (type I collagenase; Sigma) and cultured in α-MEM supplemented with 10% fetal calf serum (FCS) and penicillin and streptomycin at 37 C in 5% CO2. Bone marrow cell populations containing osteoclast precursors were isolated from the long bones of 3- to 5-month-old mice, and erythrocytes were removed by Ficoll Hypaque density gradient centrifugation. The cells were washed with PBS and resuspended in culture medium. Osteoblasts and bone marrow cells were plated together at 10 × 103 cells per well and 2 × 105 cells per well, respectively, on dentine slices in 96-well plates in 150 μl α-MEM supplemented with 10% FCS, antibiotics, and 10 nM 1,25-(OH)2-vitamin D3 for 7 d. The medium was replaced with the addition of IL-1β (10 U/ml), test compounds were added, and the cultures were terminated on d 8 for actin-ring identification or d 9 for quantification of bone resorption. At the end of the culture period, dentine slices with adherent cells were fixed in 4% paraformaldehyde, washed with PBS, and incubated with phalloidin at 37 C for 30 min to identify actin rings. The cultures were counterstained for tartrate-resistant acid phosphatase (TRAcP) as previously described (13), and TRAcP-positive cells with three or more nuclei bearing an actin ring were considered to be active osteoclasts. Cells were then removed from the dentine slices by wiping with tissue paper. Resorption pits were visualized by reflected light microscopy, and the area resorbed was quantified by Image Analysis using custom software developed using Aphelion ActiveX objects (ADCIS, Gif sur Yvette, France) as previously described (13).
Osteoclast cultures
Osteoclast formation and survival were studied using RANKL- and M-CSF-generated osteoclasts from bone marrow macrophages as described (14). Briefly, bone marrow cells were flushed from the long bones of 3- to 5-month-old mice, plated onto petri dishes, and incubated for 48 h in the presence of M-CSF (100 ng/ml). Nonadherent erythrocytes were removed, and the adherent cells were washed with PBS and resuspended in culture medium. The resulting M-CSF-dependent bone marrow macrophages were plated onto either 96-well plates (104 cells per well) or 24-well plates (2 × 105 cells per well) in 150 or 400 μl α-MEM supplemented with 10% FCS, antibiotics, M-CSF (25 ng/ml), and RANKL (100 ng/ml). Test agents were added on d 5 and the cultures terminated on d 6. The cells were fixed in 4% paraformaldehyde, washed with PBS and stained for TRAcP as described above. TRAcP-positive cells with more than three nuclei were counted as osteoclasts.
Apoptosis and caspase assays
Adherent and nonadherent cells from the osteoclast cultures were collected, fixed with 4% paraformaldehyde, and cytospun onto glass slides. Apoptosis was identified on the basis of characteristic changes in nuclear morphology using 4′,6-diamidino-2-phenylindole (DAPI) staining (15) and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining (14). An average of six microscopic fields per treatment group was analyzed at ×200 magnification, and the number of apoptotic cells was quantitated in relation to total cell number. Levels of inactive (p-30) and activated (p-19) caspase-3 expression were evaluated by Western blotting. Briefly, both adherent and nonadherent cells were washed in PBS and homogenized in lysis buffer (0.1% wt/vol sodium dodecyl sulfate, 0.5% wt/vol sodium deoxycholate in PBS) supplemented with 2% vol/vol protease inhibitor cocktail (Sigma). The samples were cleared by centrifugation at 12,000 rpm for 10 min at 4 C, and protein concentration was determined using a Bio-Rad (Hercules, CA) protein assay kit. Cell extracts (20 μg/lane) were run on a 10% acrylamide gel and blotted onto a nylon membrane. Activated caspase-3 was detected using a rabbit polyclonal anti-cleaved caspase-3 antibody (1:1000 dilution; Cell Signaling Technology), and inactive caspase-3 was detected using a goat polyclonal anti-caspase-3 antibody (1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). β-Actin was detected using a mouse monoclonal antibody (1:1000 dilution; Santa Cruz Biotechnology). Subsequently, the membranes were washed in Tris-buffered saline, incubated with the appropriate secondary antibodies coupled to horseradish peroxidase, and washed in Tris-buffered saline again, and bands were visualized using chemiluminescence (Amersham, Little Chalfont, UK) on a Bio-Rad Fluomax gel imaging system.
Animals and ovariectomy-induced bone loss
Mice with CB2 deficiency were obtained from Dr. Susana Winfield at the National Institutes of Health and were generated as previously described (17). These mice have previously been crossed with wild-type C57BL/6 mice for at least 10 generations to create a congenic strain on a C57BL/6 background. To minimize the chance of genetic drift, CB2−/− mice used in this study were generated by mating of heterozygote breeding pairs, and all comparisons between CB2−/− and wild-type mice were performed on littermates.
Animal experiments were approved by the ethical review board of the University of Edinburgh and were conducted in accordance with United Kingdom Home Office regulations. Ovariectomy or sham ovariectomy was performed in 9-wk-old adult female mice. Treatment with test agents was commenced 2 d after ovariectomy or sham ovariectomy by ip administration of the drug in corn oil. Controls received vehicle in corn oil. The treatment was continued for 19 d and the experiment terminated on d 21. Bone mineral density was measured at the tibial metaphysis by microcomputed tomography (micro-CT), using a Skyscan 1172 scanner. After bone mineral density scanning, the limbs were embedded and processed for bone histomorphometry as previously described (18). We also performed some in vitro experiments on osteoclasts generated from mice with CB1 deficiency on an ABH background (19) that were a kind gift from Dr. David Baker (University of London).
Statistical analysis
Comparison between groups was by ANOVA followed by Dunnett's post test using SPSS for Windows version 11. A P value of ≤0.05 was considered statistically significant.
Results
Cannabinoid receptor antagonists/inverse agonists inhibit osteoclast formation and bone resorption in osteoblast-bone marrow cocultures
Treatment of mouse osteoblast-bone marrow cocultures for 48 h with the CB2-selective antagonist/inverse agonist AM630 and the CB1-selective antagonist/inverse agonist AM251 inhibited osteoclast formation and bone resorption in a concentration-dependent manner (Fig. 1⇓). The mean ± SEM concentration of AM630 that half-maximally inhibited osteoclast formation and bone resorption (IC50) was 6.3 ± 0.3 and 7.2 ± 0.1 μM, respectively. Corresponding values for AM251 were 5.9 ± 1.3 and 6.6 ± 0.4 μM (Fig. 1⇓, A—D). Exposure of the cultures to these agents for 24 h also inhibited actin ring formation by about 50% (Fig. 1⇓, E and F). Next, we investigated the effects of AM630, AM251, and the CB2-selective agonists JWH133 and HU308 on RANKL- and M-CSF-induced osteoclast formation in vitro (Fig. 2⇓). We found that AM630 and AM251 both significantly inhibited RANKL-induced osteoclast formation in a concentration-dependent manner with 50% inhibition at values of 330 ± 40 nM for AM630 and 870 ± 100 nM for AM251. Conversely, JWH133 and HU308 both stimulated osteoclast formation from about 10 nM with maximal stimulation at about 300 nM. At higher concentrations, HU308 inhibited osteoclast formation, whereas JWH133 stimulated osteoclast formation at all concentrations tested (Fig. 2⇓, A and B). We also studied the effect of JWH133 and HU308 on osteoclast fusion by counting the number of osteoclasts with 20 or more nuclei. As shown in Fig. 2⇓, JWH133 and HU308 significantly increased osteoclast size and nuclearity such that the proportion of cells with more than 20 nuclei rose from about 15% in vehicle-treated cells to 20—25% in JWH133- and HU308-treated cells (Fig. 2B⇓). None of the ligands that we tested significantly affected macrophage growth or viability at concentrations up to 10 μM, excluding a nonspecific toxic effect of the compounds on cell viability (data not shown).
Cannabinoid receptor antagonists/inverse agonists induce osteoclast apoptosis in vitro
To investigate the mechanism of osteoclast inhibition, we studied the effect of AM630 and AM251 on osteoclast apoptosis. These studies showed that both agents induced apoptosis in mature osteoclast at concentrations greater than 1 μM (Fig. 3A⇓) and activated caspase-3 as early as 12 h (Fig. 3C⇓). After 24 h, both agents had induced apoptosis in about 90% of cells, whereas the CB2-selective agonist JWH133 had no significant effect on apoptosis. Representative photomicrographs of osteoclast cultures treated with the cannabinoid receptor ligands JWH133, AM251, and AM630 are shown in Fig. 3B⇓.
Osteoclast inhibition by cannabinoid receptor antagonists/inverse agonists can be mediated through CB1 or CB2
To determine whether the inhibitory effects of the cannabinoid receptor antagonists/inverse agonists were mediated by CB1, CB2, or both receptors, we conducted further studies in M-CSF- and RANKL-generated osteoclasts from CB2−/− and wild-type littermates that were treated with AM630 and JWH133. In wild-type cultures, we found that JWH133 stimulated osteoclast formation and partially reversed the inhibitory effect of AM630 osteoclast formation at an AM630 concentration of 500 nM. However, at the higher AM630 concentration of 1000 nM, JWH133 did not significantly rescue the osteoclast-inhibitory effect of AM630 (Fig. 4A⇓). Cultures prepared from CB2−/− mice were resistant to the inhibitory effects of AM630 at a concentrations of 100 nM (Fig. 4B⇓), but at the higher concentration of 300 nM, AM630 inhibited osteoclast formation to a similar extent as in wild-type cultures, although the remaining osteoclasts were larger in CB2−/− cultures (Fig. 4C⇓). In contrast, both concentrations of AM630 tested inhibited osteoclast formation to a similar extent in CB1−/− cultures. Taken together, these data suggest that low concentrations of AM630 inhibit osteoclast differentiation by a CB2-mediated mechanism but that at higher concentrations, selectivity for CB2 is lost and osteoclast inhibition occurs through interaction with CB1 consistent with the results of previous studies (3, 20, 21, 22).
Cannabinoid receptor antagonists/inverse agonists prevents ovariectomy-induced bone loss
To determine whether cannabinoid receptor antagonists/inverse agonists prevented ovariectomy-induced bone loss in vivo, we studied the effects of AM630 on ovariectomy-induced bone loss in wild-type and CB2−/− mice. Before ovariectomy, there was no significant difference in trabecular bone volume at the tibial metaphysis as assessed by micro-CT in wild-type and CB2−/− mice (Fig. 5⇓, A and B). Analysis of cortical bone and total bone volume similarly showed no difference between the genotypes (data not shown). After ovariectomy, loss of trabecular bone volume (Fig. 5E⇓), trabecular thickness (Fig. 5D⇓), and trabecular number (Fig. 5E⇓) were slightly but significantly less in CB2−/− mice as compared with wild-type littermates. Treatment of wild-type mice with AM630 at a dose of 0.1 mg/kg·d and 1 mg/kg·d completely prevented bone loss, whereas the CB2−/− mice were resistant to the protective effects of AM630 at 0.1 mg/kg but not at 1.0 mg/kg (Fig. 5F⇓). Bone histomorphometry showed that osteoclast numbers and active resorption surfaces were increased after ovariectomy in vehicle-treated wild-type mice, whereas this was prevented in AM630-treated mice. Osteoblast numbers increased after ovariectomy as expected but were unaffected by AM630 treatment (Table 2⇓). These data indicate that AM630 inhibits ovariectomy-induced bone loss by interacting with the CB2 receptor at low concentration, but at higher concentrations, selectivity is lost and prevention of bone loss occurs probably by an effect on CB1.
Discussion
We have previously reported that CB1 is required for ovariectomy-induced bone loss in mice and that AM251, an antagonist/inverse agonist with selectivity toward CB1, causes osteoclast inhibition and prevents ovariectomy-induced bone loss in mice (3). We and others also reported that cannabinoid receptor agonists stimulate osteoclast formation and bone resorption in vitro (3, 11). In contrast, other workers have reported that the CB2-selective agonist HU308 inhibits osteoclast formation in vitro and partially prevents ovariectomy-induced bone loss in vivo (4). In view of this, the aim of the present study was to further investigate the effects of pharmacological activation and blockade of CB2 on osteoclast differentiation and function in vitro and in vivo in wild-type and CB2−/− mice.
We found that the CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and bone resorption in osteoblast cocultures, with similar potency to the CB1-selective antagonist/inverse agonist AM251. The inhibitory effect in bone resorption may have been due in part to disruption of the actin ring formation, but both agents were also found to inhibit osteoclast differentiation and to cause osteoclast apoptosis in RANKL- and M-CSF-generated osteoclast cultures. These data therefore suggest that cannabinoid receptor antagonists/inverse agonists inhibit bone resorption mainly by reducing osteoclast formation but indicate that there may also be an inhibitory effect on the ability of mature osteoclasts to form actin rings and adhere to the bone surface. There was a striking difference in potency in the ability of AM251 and AM630 to cause osteoclast inhibition in RANKL- and M-CSF- generated isolated osteoclast cultures where they were effective in the nanomolar range as opposed to bone marrow osteoblast cocultures where micromolar concentrations were required for osteoclast inhibition. This raises the possibility that cannabinoid receptor antagonists/inverse agonists may influence osteoblast-osteoclast cross talk in a manner that interferes with the direct inhibitory effects of these agents on osteoclasts, but further work will be required to explore the mechanisms responsible. In keeping with the fact that cannabinoid receptor antagonists/inverse agonists caused osteoclast inhibition, the CB2-selective agonists HU308 and JWH133 significantly increased osteoclast formation at concentrations in the nanomolar range. Although HU308 cause osteoclast inhibition at 10 μM, this is approximately 2000 times greater than the concentration of HU308 required for CB2-mediated adenylyl cyclase inhibition (23). This suggests that the inhibitory effects of HU308 at these concentrations may have been nonspecific and mediated by an interaction with pathways other than CB2. The stimulatory effects of HU308 and JWH133 on osteoclast formation are consistent with previous work that has shown that nonselective cannabinoid receptor agonists including anandamide, 2-arachinodyl glycerol, and CP55,940 stimulate osteoclast formation and bone resorption in vitro at nanomolar concentrations (3, 11). The observations reported here differ from those of Ofek and colleagues (4), who found that HU308 caused osteoclast inhibition at concentrations in the nanomolar range. We cannot readily explain this difference except to note that the observations of Ofek were based in part on studies of RAW 264.7 cells rather than authentic osteoclasts. Although Ofek also studied M-CSF- and RANKL-stimulated bone marrow cultures, the numbers of osteoclasts generated were very low (an average of 15 osteoclasts per culture), and this might also have contributed to the differences observed (4).
Because the pharmacological ligands currently available are not completely specific and can interact with both CB1 and CB2, further studies were conducted to determine whether the inhibitory effects of AM630 on osteoclast activity were truly mediated by CB2. Studies in wild-type mice showed that at low concentrations, the osteoclast-inhibitory effect of AM630 was reversed by the CB2-selective agonist JWH133 consistent with a CB2-mediated effect. At higher concentrations of AM630, however, JWH133 did not reverse the osteoclast inhibition, probably because AM630 is known to act as an inverse agonist of CB1 at high concentrations (20, 21, 22). In keeping with this, osteoclasts generated from CB2−/− mice were resistant to the inhibitory effects of AM630 at low concentrations but inhibited osteoclast formation in CB2−/− mice at higher concentrations. In contrast, osteoclasts generated from CB1−/− mice were equally sensitive as wild type to the inhibitory effects of AM630.
To determine whether pharmacological blockade of CB2 can also prevent the bone loss that results from estrogen deficiency, we studied the effects of the CB2-selective antagonist AM630 on ovariectomy-induced bone loss in wild-type and CB2−/− mice. Administration of AM630 to ovariectomized wild-type mice in a dose of 0.1 and 1 mg/kg·d prevented ovariectomy-induced bone loss, with results virtually identical to those observed with the CB1-selective inverse agonist AM251 as previously reported (3). Analysis of bone histomorphometry showed that AM630 blocked the increase in osteoclast numbers and active resorption surfaces that followed ovariectomy, demonstrating that prevention of bone loss with AM630 was due to an inhibitory effect on osteoclasts and bone resorption rather than an effect on osteoblasts and bone formation. We found that CB2−/− mice lost slightly less bone than wild-type mice after ovariectomy, indicating that CB2 deficiency may result in a subtle defect in osteoclastic bone resorption. Treatment with AM630 at a low dose had no significant protective effect on ovariectomy-induced bone loss in CB2−/− mice, but at a higher dose of 1.0 mg/kg·d, AM630 was equally effective at preventing ovariectomy-induced bone loss in CB2−/− mice and wild-type littermates, probably due to an effect on CB1.
In summary, the results reported here indicate that CB2 regulates osteoclast differentiation and function in vitro and ovariectomy-induced bone loss in vivo. We have confirmed that pharmacological inverse agonists of cannabinoid receptors inhibit osteoclast differentiation and bone resorption by a CB2-mediated pathway as well as by interacting with CB1 as previously reported (3). Conversely, it appears that the stimulatory effects of cannabinoid receptor agonists on osteoclast formation and bone resorption that we and others have previously observed (3, 11) can also be mediated by an interaction with either or both receptors. These data suggest that cannabinoid receptor inverse agonists have potential value as antiresorptive drugs that might be of clinical value in the prevention and treatment of diseases such as osteoporosis that are characterized by increased osteoclastic bone resorption. They also raise the possibility that pharmacological or recreational use of cannabinoid receptor agonists might increase the risk of osteoporosis, by causing increased bone resorption.
Source, Graphs and Figures: Regulation of Bone Mass, Osteoclast Function, and Ovariectomy-Induced Bone Loss by the Type 2 Cannabinoid Receptor
The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2−/−) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2−/− mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2−/− mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2−/− mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.
BONE REMODELING IS a complex process that is regulated by an interplay between circulating hormones and locally produced factors that act in a concerted manner to regulate osteoblast and osteoclast activity (1). Over recent years, there has been increasing interest in the role that the nervous system and neurotransmitters play in the regulation of bone remodeling (2). Reflecting this fact, the endocannabinoid pathway has recently been implicated as are important regulator of bone turnover and bone mass (3, 4, 5, 6).
The endocannabinoid system comprises two known receptors [the type 1 (CB1) and type 2 (CB2) cannabinoid receptors], a family of endogenous ligands, and a molecular machinery for ligand synthesis, transport, and inactivation. Cannabinoid receptors belong to the G protein-coupled receptor superfamily and are highly expressed in the brain (CB1), immune system (CB2), and a number of other tissues (7). A variety of natural and synthetic ligands have been identified that bind to these receptors. Cannabinoid receptor agonists such as Δ9-tetrahydrocannabinol, anandamide, 2 arachinodyl glycerol, CP55,490, and JWH133 bind to the receptors causing inhibition of adenylyl cyclase and activation of ERK kinases and other signaling pathways in target cells (8). Another class of cannabinoid receptor ligands have been synthesized including AM251, AM630, and SR141716A (rimonabant), which block the effects of cannabinoid receptor agonists. These compounds were originally referred to as cannabinoid receptor antagonists, but it has now become clear that these compounds cause activation of the receptors in the absence of agonist binding, with opposite effects on downstream signaling cascades as those of the agonists. Accordingly, these compounds are now referred to as cannabinoid receptor antagonists/inverse agonists (9).
We previously reported that osteoblasts and osteoclasts express cannabinoid receptors and that mice with inactivation of CB1 have increased bone mass and are protected from ovariectomy-induced bone loss (3). We also found that the CB1-selective antagonist/inverse agonist AM251 inhibited osteoclast differentiation and prevented ovariectomy-induced bone loss in vivo, confirming the importance of CB1 as a regulator of bone resorption (3). In another study, Tam and colleagues (5) reported that mice with CB1 deficiency on a CD1 background had high bone mass but surprisingly found that mice with CB1 deficiency on a C57BL/6 background had low bone mass. There is evidence that CB2 also plays a role in regulating bone metabolism. Mice with deficiency of CB2 (CB2−/−) have been reported to develop age-related osteoporosis in association with increased bone turnover (4). The CB2-selective agonist HU308 has also been reported to promote osteoblast differentiation, to inhibit osteoclast differentiation, and to protect against ovariectomy-induced bone loss (4). On the basis of these observations, it has been suggested that an important function of CB2 is to suppress bone turnover and regulate osteoblast-osteoclast coupling (10). Contrasting with these findings, however, we and others have reported that pharmacological agonists of cannabinoid receptors stimulate osteoclast formation and bone resorption in vitro in both mouse and human cells (3, 11). In these studies, however, it was not possible to determine whether the effects were mediated by CB1, CB2, or both receptors. In view of this, the aim of the present study was to clarify the role that CB2 plays in osteoclast function and ovariectomy-induced bone loss using a combination of pharmacological and genetic approaches.
Materials and Methods
Materials
Reagents were obtained from Sigma (Dorset, UK) unless otherwise indicated. The cannabinoid receptor ligands JWH133, AM251, and AM630 were purchased from Tocris Bioscience (Bristol, UK) and HU308 was a kind gift from Dr. R. J. Arends (Organon). The pharmacological properties of the cannabinoid receptor ligands used in the study are summarized in Table 1⇓. Tissue culture medium was obtained from Invitrogen (Paisley, UK), and primary antibodies were purchased from Cell Signaling Technology (Beverly, MA), unless otherwise stated. Human IL-1β was obtained from Roche (London, UK); human macrophage colony-stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK); 1,25-(OH)2-vitamin D3 was obtained from Alexis Biochemicals (Nottingham, UK); and mouse receptor activator of nuclear factor-κB ligand (RANKL) was a gift from Dr. F. P. Ross (Washington University, St. Louis, MO).
Mouse osteoblast-bone marrow cocultures
Osteoblast-bone marrow cocultures were performed essentially as previously described (12). Briefly, osteoblasts were isolated from the calvarial bones of 2-d-old mice by sequential collagenase digestion (type I collagenase; Sigma) and cultured in α-MEM supplemented with 10% fetal calf serum (FCS) and penicillin and streptomycin at 37 C in 5% CO2. Bone marrow cell populations containing osteoclast precursors were isolated from the long bones of 3- to 5-month-old mice, and erythrocytes were removed by Ficoll Hypaque density gradient centrifugation. The cells were washed with PBS and resuspended in culture medium. Osteoblasts and bone marrow cells were plated together at 10 × 103 cells per well and 2 × 105 cells per well, respectively, on dentine slices in 96-well plates in 150 μl α-MEM supplemented with 10% FCS, antibiotics, and 10 nM 1,25-(OH)2-vitamin D3 for 7 d. The medium was replaced with the addition of IL-1β (10 U/ml), test compounds were added, and the cultures were terminated on d 8 for actin-ring identification or d 9 for quantification of bone resorption. At the end of the culture period, dentine slices with adherent cells were fixed in 4% paraformaldehyde, washed with PBS, and incubated with phalloidin at 37 C for 30 min to identify actin rings. The cultures were counterstained for tartrate-resistant acid phosphatase (TRAcP) as previously described (13), and TRAcP-positive cells with three or more nuclei bearing an actin ring were considered to be active osteoclasts. Cells were then removed from the dentine slices by wiping with tissue paper. Resorption pits were visualized by reflected light microscopy, and the area resorbed was quantified by Image Analysis using custom software developed using Aphelion ActiveX objects (ADCIS, Gif sur Yvette, France) as previously described (13).
Osteoclast cultures
Osteoclast formation and survival were studied using RANKL- and M-CSF-generated osteoclasts from bone marrow macrophages as described (14). Briefly, bone marrow cells were flushed from the long bones of 3- to 5-month-old mice, plated onto petri dishes, and incubated for 48 h in the presence of M-CSF (100 ng/ml). Nonadherent erythrocytes were removed, and the adherent cells were washed with PBS and resuspended in culture medium. The resulting M-CSF-dependent bone marrow macrophages were plated onto either 96-well plates (104 cells per well) or 24-well plates (2 × 105 cells per well) in 150 or 400 μl α-MEM supplemented with 10% FCS, antibiotics, M-CSF (25 ng/ml), and RANKL (100 ng/ml). Test agents were added on d 5 and the cultures terminated on d 6. The cells were fixed in 4% paraformaldehyde, washed with PBS and stained for TRAcP as described above. TRAcP-positive cells with more than three nuclei were counted as osteoclasts.
Apoptosis and caspase assays
Adherent and nonadherent cells from the osteoclast cultures were collected, fixed with 4% paraformaldehyde, and cytospun onto glass slides. Apoptosis was identified on the basis of characteristic changes in nuclear morphology using 4′,6-diamidino-2-phenylindole (DAPI) staining (15) and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining (14). An average of six microscopic fields per treatment group was analyzed at ×200 magnification, and the number of apoptotic cells was quantitated in relation to total cell number. Levels of inactive (p-30) and activated (p-19) caspase-3 expression were evaluated by Western blotting. Briefly, both adherent and nonadherent cells were washed in PBS and homogenized in lysis buffer (0.1% wt/vol sodium dodecyl sulfate, 0.5% wt/vol sodium deoxycholate in PBS) supplemented with 2% vol/vol protease inhibitor cocktail (Sigma). The samples were cleared by centrifugation at 12,000 rpm for 10 min at 4 C, and protein concentration was determined using a Bio-Rad (Hercules, CA) protein assay kit. Cell extracts (20 μg/lane) were run on a 10% acrylamide gel and blotted onto a nylon membrane. Activated caspase-3 was detected using a rabbit polyclonal anti-cleaved caspase-3 antibody (1:1000 dilution; Cell Signaling Technology), and inactive caspase-3 was detected using a goat polyclonal anti-caspase-3 antibody (1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). β-Actin was detected using a mouse monoclonal antibody (1:1000 dilution; Santa Cruz Biotechnology). Subsequently, the membranes were washed in Tris-buffered saline, incubated with the appropriate secondary antibodies coupled to horseradish peroxidase, and washed in Tris-buffered saline again, and bands were visualized using chemiluminescence (Amersham, Little Chalfont, UK) on a Bio-Rad Fluomax gel imaging system.
Animals and ovariectomy-induced bone loss
Mice with CB2 deficiency were obtained from Dr. Susana Winfield at the National Institutes of Health and were generated as previously described (17). These mice have previously been crossed with wild-type C57BL/6 mice for at least 10 generations to create a congenic strain on a C57BL/6 background. To minimize the chance of genetic drift, CB2−/− mice used in this study were generated by mating of heterozygote breeding pairs, and all comparisons between CB2−/− and wild-type mice were performed on littermates.
Animal experiments were approved by the ethical review board of the University of Edinburgh and were conducted in accordance with United Kingdom Home Office regulations. Ovariectomy or sham ovariectomy was performed in 9-wk-old adult female mice. Treatment with test agents was commenced 2 d after ovariectomy or sham ovariectomy by ip administration of the drug in corn oil. Controls received vehicle in corn oil. The treatment was continued for 19 d and the experiment terminated on d 21. Bone mineral density was measured at the tibial metaphysis by microcomputed tomography (micro-CT), using a Skyscan 1172 scanner. After bone mineral density scanning, the limbs were embedded and processed for bone histomorphometry as previously described (18). We also performed some in vitro experiments on osteoclasts generated from mice with CB1 deficiency on an ABH background (19) that were a kind gift from Dr. David Baker (University of London).
Statistical analysis
Comparison between groups was by ANOVA followed by Dunnett's post test using SPSS for Windows version 11. A P value of ≤0.05 was considered statistically significant.
Results
Cannabinoid receptor antagonists/inverse agonists inhibit osteoclast formation and bone resorption in osteoblast-bone marrow cocultures
Treatment of mouse osteoblast-bone marrow cocultures for 48 h with the CB2-selective antagonist/inverse agonist AM630 and the CB1-selective antagonist/inverse agonist AM251 inhibited osteoclast formation and bone resorption in a concentration-dependent manner (Fig. 1⇓). The mean ± SEM concentration of AM630 that half-maximally inhibited osteoclast formation and bone resorption (IC50) was 6.3 ± 0.3 and 7.2 ± 0.1 μM, respectively. Corresponding values for AM251 were 5.9 ± 1.3 and 6.6 ± 0.4 μM (Fig. 1⇓, A—D). Exposure of the cultures to these agents for 24 h also inhibited actin ring formation by about 50% (Fig. 1⇓, E and F). Next, we investigated the effects of AM630, AM251, and the CB2-selective agonists JWH133 and HU308 on RANKL- and M-CSF-induced osteoclast formation in vitro (Fig. 2⇓). We found that AM630 and AM251 both significantly inhibited RANKL-induced osteoclast formation in a concentration-dependent manner with 50% inhibition at values of 330 ± 40 nM for AM630 and 870 ± 100 nM for AM251. Conversely, JWH133 and HU308 both stimulated osteoclast formation from about 10 nM with maximal stimulation at about 300 nM. At higher concentrations, HU308 inhibited osteoclast formation, whereas JWH133 stimulated osteoclast formation at all concentrations tested (Fig. 2⇓, A and B). We also studied the effect of JWH133 and HU308 on osteoclast fusion by counting the number of osteoclasts with 20 or more nuclei. As shown in Fig. 2⇓, JWH133 and HU308 significantly increased osteoclast size and nuclearity such that the proportion of cells with more than 20 nuclei rose from about 15% in vehicle-treated cells to 20—25% in JWH133- and HU308-treated cells (Fig. 2B⇓). None of the ligands that we tested significantly affected macrophage growth or viability at concentrations up to 10 μM, excluding a nonspecific toxic effect of the compounds on cell viability (data not shown).
Cannabinoid receptor antagonists/inverse agonists induce osteoclast apoptosis in vitro
To investigate the mechanism of osteoclast inhibition, we studied the effect of AM630 and AM251 on osteoclast apoptosis. These studies showed that both agents induced apoptosis in mature osteoclast at concentrations greater than 1 μM (Fig. 3A⇓) and activated caspase-3 as early as 12 h (Fig. 3C⇓). After 24 h, both agents had induced apoptosis in about 90% of cells, whereas the CB2-selective agonist JWH133 had no significant effect on apoptosis. Representative photomicrographs of osteoclast cultures treated with the cannabinoid receptor ligands JWH133, AM251, and AM630 are shown in Fig. 3B⇓.
Osteoclast inhibition by cannabinoid receptor antagonists/inverse agonists can be mediated through CB1 or CB2
To determine whether the inhibitory effects of the cannabinoid receptor antagonists/inverse agonists were mediated by CB1, CB2, or both receptors, we conducted further studies in M-CSF- and RANKL-generated osteoclasts from CB2−/− and wild-type littermates that were treated with AM630 and JWH133. In wild-type cultures, we found that JWH133 stimulated osteoclast formation and partially reversed the inhibitory effect of AM630 osteoclast formation at an AM630 concentration of 500 nM. However, at the higher AM630 concentration of 1000 nM, JWH133 did not significantly rescue the osteoclast-inhibitory effect of AM630 (Fig. 4A⇓). Cultures prepared from CB2−/− mice were resistant to the inhibitory effects of AM630 at a concentrations of 100 nM (Fig. 4B⇓), but at the higher concentration of 300 nM, AM630 inhibited osteoclast formation to a similar extent as in wild-type cultures, although the remaining osteoclasts were larger in CB2−/− cultures (Fig. 4C⇓). In contrast, both concentrations of AM630 tested inhibited osteoclast formation to a similar extent in CB1−/− cultures. Taken together, these data suggest that low concentrations of AM630 inhibit osteoclast differentiation by a CB2-mediated mechanism but that at higher concentrations, selectivity for CB2 is lost and osteoclast inhibition occurs through interaction with CB1 consistent with the results of previous studies (3, 20, 21, 22).
Cannabinoid receptor antagonists/inverse agonists prevents ovariectomy-induced bone loss
To determine whether cannabinoid receptor antagonists/inverse agonists prevented ovariectomy-induced bone loss in vivo, we studied the effects of AM630 on ovariectomy-induced bone loss in wild-type and CB2−/− mice. Before ovariectomy, there was no significant difference in trabecular bone volume at the tibial metaphysis as assessed by micro-CT in wild-type and CB2−/− mice (Fig. 5⇓, A and B). Analysis of cortical bone and total bone volume similarly showed no difference between the genotypes (data not shown). After ovariectomy, loss of trabecular bone volume (Fig. 5E⇓), trabecular thickness (Fig. 5D⇓), and trabecular number (Fig. 5E⇓) were slightly but significantly less in CB2−/− mice as compared with wild-type littermates. Treatment of wild-type mice with AM630 at a dose of 0.1 mg/kg·d and 1 mg/kg·d completely prevented bone loss, whereas the CB2−/− mice were resistant to the protective effects of AM630 at 0.1 mg/kg but not at 1.0 mg/kg (Fig. 5F⇓). Bone histomorphometry showed that osteoclast numbers and active resorption surfaces were increased after ovariectomy in vehicle-treated wild-type mice, whereas this was prevented in AM630-treated mice. Osteoblast numbers increased after ovariectomy as expected but were unaffected by AM630 treatment (Table 2⇓). These data indicate that AM630 inhibits ovariectomy-induced bone loss by interacting with the CB2 receptor at low concentration, but at higher concentrations, selectivity is lost and prevention of bone loss occurs probably by an effect on CB1.
Discussion
We have previously reported that CB1 is required for ovariectomy-induced bone loss in mice and that AM251, an antagonist/inverse agonist with selectivity toward CB1, causes osteoclast inhibition and prevents ovariectomy-induced bone loss in mice (3). We and others also reported that cannabinoid receptor agonists stimulate osteoclast formation and bone resorption in vitro (3, 11). In contrast, other workers have reported that the CB2-selective agonist HU308 inhibits osteoclast formation in vitro and partially prevents ovariectomy-induced bone loss in vivo (4). In view of this, the aim of the present study was to further investigate the effects of pharmacological activation and blockade of CB2 on osteoclast differentiation and function in vitro and in vivo in wild-type and CB2−/− mice.
We found that the CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and bone resorption in osteoblast cocultures, with similar potency to the CB1-selective antagonist/inverse agonist AM251. The inhibitory effect in bone resorption may have been due in part to disruption of the actin ring formation, but both agents were also found to inhibit osteoclast differentiation and to cause osteoclast apoptosis in RANKL- and M-CSF-generated osteoclast cultures. These data therefore suggest that cannabinoid receptor antagonists/inverse agonists inhibit bone resorption mainly by reducing osteoclast formation but indicate that there may also be an inhibitory effect on the ability of mature osteoclasts to form actin rings and adhere to the bone surface. There was a striking difference in potency in the ability of AM251 and AM630 to cause osteoclast inhibition in RANKL- and M-CSF- generated isolated osteoclast cultures where they were effective in the nanomolar range as opposed to bone marrow osteoblast cocultures where micromolar concentrations were required for osteoclast inhibition. This raises the possibility that cannabinoid receptor antagonists/inverse agonists may influence osteoblast-osteoclast cross talk in a manner that interferes with the direct inhibitory effects of these agents on osteoclasts, but further work will be required to explore the mechanisms responsible. In keeping with the fact that cannabinoid receptor antagonists/inverse agonists caused osteoclast inhibition, the CB2-selective agonists HU308 and JWH133 significantly increased osteoclast formation at concentrations in the nanomolar range. Although HU308 cause osteoclast inhibition at 10 μM, this is approximately 2000 times greater than the concentration of HU308 required for CB2-mediated adenylyl cyclase inhibition (23). This suggests that the inhibitory effects of HU308 at these concentrations may have been nonspecific and mediated by an interaction with pathways other than CB2. The stimulatory effects of HU308 and JWH133 on osteoclast formation are consistent with previous work that has shown that nonselective cannabinoid receptor agonists including anandamide, 2-arachinodyl glycerol, and CP55,940 stimulate osteoclast formation and bone resorption in vitro at nanomolar concentrations (3, 11). The observations reported here differ from those of Ofek and colleagues (4), who found that HU308 caused osteoclast inhibition at concentrations in the nanomolar range. We cannot readily explain this difference except to note that the observations of Ofek were based in part on studies of RAW 264.7 cells rather than authentic osteoclasts. Although Ofek also studied M-CSF- and RANKL-stimulated bone marrow cultures, the numbers of osteoclasts generated were very low (an average of 15 osteoclasts per culture), and this might also have contributed to the differences observed (4).
Because the pharmacological ligands currently available are not completely specific and can interact with both CB1 and CB2, further studies were conducted to determine whether the inhibitory effects of AM630 on osteoclast activity were truly mediated by CB2. Studies in wild-type mice showed that at low concentrations, the osteoclast-inhibitory effect of AM630 was reversed by the CB2-selective agonist JWH133 consistent with a CB2-mediated effect. At higher concentrations of AM630, however, JWH133 did not reverse the osteoclast inhibition, probably because AM630 is known to act as an inverse agonist of CB1 at high concentrations (20, 21, 22). In keeping with this, osteoclasts generated from CB2−/− mice were resistant to the inhibitory effects of AM630 at low concentrations but inhibited osteoclast formation in CB2−/− mice at higher concentrations. In contrast, osteoclasts generated from CB1−/− mice were equally sensitive as wild type to the inhibitory effects of AM630.
To determine whether pharmacological blockade of CB2 can also prevent the bone loss that results from estrogen deficiency, we studied the effects of the CB2-selective antagonist AM630 on ovariectomy-induced bone loss in wild-type and CB2−/− mice. Administration of AM630 to ovariectomized wild-type mice in a dose of 0.1 and 1 mg/kg·d prevented ovariectomy-induced bone loss, with results virtually identical to those observed with the CB1-selective inverse agonist AM251 as previously reported (3). Analysis of bone histomorphometry showed that AM630 blocked the increase in osteoclast numbers and active resorption surfaces that followed ovariectomy, demonstrating that prevention of bone loss with AM630 was due to an inhibitory effect on osteoclasts and bone resorption rather than an effect on osteoblasts and bone formation. We found that CB2−/− mice lost slightly less bone than wild-type mice after ovariectomy, indicating that CB2 deficiency may result in a subtle defect in osteoclastic bone resorption. Treatment with AM630 at a low dose had no significant protective effect on ovariectomy-induced bone loss in CB2−/− mice, but at a higher dose of 1.0 mg/kg·d, AM630 was equally effective at preventing ovariectomy-induced bone loss in CB2−/− mice and wild-type littermates, probably due to an effect on CB1.
In summary, the results reported here indicate that CB2 regulates osteoclast differentiation and function in vitro and ovariectomy-induced bone loss in vivo. We have confirmed that pharmacological inverse agonists of cannabinoid receptors inhibit osteoclast differentiation and bone resorption by a CB2-mediated pathway as well as by interacting with CB1 as previously reported (3). Conversely, it appears that the stimulatory effects of cannabinoid receptor agonists on osteoclast formation and bone resorption that we and others have previously observed (3, 11) can also be mediated by an interaction with either or both receptors. These data suggest that cannabinoid receptor inverse agonists have potential value as antiresorptive drugs that might be of clinical value in the prevention and treatment of diseases such as osteoporosis that are characterized by increased osteoclastic bone resorption. They also raise the possibility that pharmacological or recreational use of cannabinoid receptor agonists might increase the risk of osteoporosis, by causing increased bone resorption.
Source, Graphs and Figures: Regulation of Bone Mass, Osteoclast Function, and Ovariectomy-Induced Bone Loss by the Type 2 Cannabinoid Receptor
