The CB1 cannabinoid receptor has been implicated in the regulation of bone remodeling and bone mass. A high bone mass (HBM) phenotype was reported in CB1-null mice generated on a CD1 background (CD1^CB1−/− mice). By contrast, our preliminary studies incb1^−/− mice, backcrossed to C57BL/6J mice (C57^CB1−/− mice), revealed low bone mass (LBM). We therefore analyzed CB1 expression in bone and compared the skeletons of sexually mature C57^CB1−/− and CD1^CB1−/− mice in the same experimental setting. CB1 mRNA is weakly expressed in osteoclasts and immunoreactive CB1 is present in sympathetic neurons, close to osteoblasts. In addition to their LBM, male and female C57^CB1−/− mice exhibit decreased bone formation rate and increased osteoclast number. The skeletal phenotype of the CD1^CB1−/− mice shows a gender disparity. Female mice have normal trabecular bone with a slight cortical expansion, whereas male CD1^CB1−/− animals display an HBM phenotype. We were surprised to find that bone formation and resorption are within normal limits. These findings, at least the consistent set of data obtained in the C57^CB1−/− line, suggest an important role for CB1 signaling in the regulation of bone remodeling and bone mass. Because sympathetic CB1 signaling inhibits norepinephrine (NE) release in peripheral tissues, part of the endocannabinoid activity in bone may be attributed to the regulation of NE release from sympathetic nerve fibers. Several phenotypic discrepancies have been reported between C57^CB1−/− and CD1^CB1−/− mice that could result from genetic differences between the background strains. Unraveling these differences can provide useful information on the physiologic functional milieu of CB1 in bone.

Animals

All animals in the study were 9- to 12-week-old mice. C57^CB1−/− mice were generated as reported previously (Zimmer et al., 1999). We have crossed heterozygous animals of this line for at least 10 generations to WT C57BL/6J mice. Heterozygous animals from the last generation were then intercrossed to obtain congenic C57BL/6J mice that are homozygous for the respective mutation. CD1^CB1−/− mice were generated by homologous recombination as described previously (Ledent et al., 1999). Heterozygous mice were bred for 17 generations on a CD1 background before generating the WT and cb1-null littermates used in this study. To study bone formation, newly formed bone was vitally labeled in mice intended for microcomputed tomographic (μCT)-histomorphometric analysis by the fluorochrome calcein (Sigma, St. Louis, MO), injected i.p. (15 mg/kg) 4 days and 1 day before sacrifice. The use of animals was approved by the Institutional Animal Care Committee of the Hebrew University of Jerusalem.

Immunohistochemistry

Mice were killed by transcardial perfusion of phosphate-buffered saline followed by 4% paraformaldehyde. The femora were dissected and further fixed with paraformaldehyde for 2 h at 4°C. The specimens were decalcified in 0.5 M EDTA, pH 7.4, and embedded in paraffin. For immunohistochemical analysis, serial 5-μm frontal sections were reacted with anti-tyrosine hydroxylase (TH) (Chemicon, Temecula, CA) or anti-CB1 antibodies (Nyíri et al., 2005). Further processing was carried out using the SuperPicture polymer detection Kit (Zymed Laboratories, South San Francisco, CA) according to the manufacturer instructions.

Cell Cultures and mRNA Analysis

Primary bone marrow stromal cell cultures from WT adult femoral and tibial diaphyseal bone marrow were established as described previously. For testing CB1 expression, the cells were grown in osteogenic medium (Ofek et al., 2006). Bone marrow-derived osteoclastogenic cultures were established from Ficoll-separated monocytic precursors and grown for 5 to 6 days in medium containing macrophage colony-stimulating factor (M-CSF) and RANK ligand (RANKL; R&D Systems, Minneapolis, MN) (Ofek et al., 2006). Total RNA was extracted from the cells, purified, and reverse-transcribed using routine procedures. The following primers were used for PCR: CB1, sense: 5′-TGGTGTATGATGTCTTTGGG-3′, antisense: 5′-ATGCTGGCTGTGTTATTGGC-3′; tissue nonspecific alkaline phosphatase, sense: 5′-GACACAAGCATTCCCACTAT-3′, antisense: 5′-ATCAGCAGTAACCACAGTCA-3′; parathyroid hormone receptor I, sense: 5′-CAAGAAGTGGATCATCCAGGT-3′, antisense: 5′-GCTGCTACTCCCACTTCGTGCTTT-3′; and β-actin, sense, 5′-GAGACCTTCAACACCCCAGCC-3′; antisense, 5′-GGCCATCTCTTGCTCGAAGTC-3′.

μCT Analysis

Whole femora were examined by a μCT system (μCT 40; SCANCO Medical, Bassersdorf, Switzerland) as reported recently (Bajayo et al., 2005; Ofek et al., 2006). Scans were performed at a resolution of 20 μm in all three spatial dimensions. Morphometric parameters were determined as reported previously (Kram et al., 2006). Trabecular and cortical bone parameters were measured in metaphyseal and mid-diaphyseal segments, respectively.

Histomorphometry

After μCT image acquisition, the specimens were embedded undecalcified in Technovit 9100 (Heraeus). Longitudinal sections through the midfrontal plane were left unstained for dynamic histomorphometry, based on the vital calcein double labeling. To identify osteoclasts, consecutive sections were stained for tartrate-resistant acid phosphatase. Parameters were determined according to a standardized nomenclature (Parfitt et al., 1987).

Statistical Analysis

Differences between cb1^−/− and WT mice were analyzed with the use of the Student’s t test.

Expression of CB1 in Bone

mRNA analyses were carried out in cells derived from WT C57BL/6J mice. Unlike the expression of CB2, which is absent in undifferentiated bone marrow stromal cells but increases progressively when these cells undergo osteoblastic differentiation (Ofek et al., 2006), we were unable to identify CB1 mRNA transcripts in either undifferentiated or differentiated stromal cells, even after 40 PCR cycles (Fig. 1A). Monocytic osteoclast precursors from these mice also did not show CB1 expression. However, a weak signal was present when these cells underwent osteoclastogenesis with M-CSF and RANKL (Fig. 1B).

Fig. 1

CB1 is expressed in sympathetic nerve fibers in trabecular bone. A and B, RT-PCR analysis. A, bone marrow stromal cells undergoing osteoblastic differentiation in “osteogenic medium”; note absence of CB1-positive bands. PTHRc1, PTH receptor …

Bone, especially trabecular bone, is densely innervated by sympathetic fibers (Serre et al., 1999; Mach et al., 2002). These fibers release norepinephrine, thus potently mediating central signals that restrain bone formation and stimulate bone resorption (Elefteriou et al., 2005). Because CB1 is expressed in such nerve fibers elsewhere (Schlicker and Kathmann, 2001), we further explored its presence in bone sympathetic nerve fibers. Indeed, immunohistochemical analysis using the sympathetic marker TH (Bjurholm et al., 1988) confirmed the occurrence of a network of TH-positive fibers in the intertrabecular spaces of cancellous bone in both C57BL/6J and CD1 mice (Fig. 1, C and G). The fibers were close to the bone trabeculae with terminal nerve processes penetrating the osteoblast palisades, thus being in intimate proximity to these cells (Fig. 1, D and G). Consecutive histological sections show CB1 immunoreactivity of the same nerve fibers (Fig. 1, E, F, and H), indicating the presence of CB1 receptors in sympathetic fibers that innervate the trabecular bone. This CB1 immunoreactivity was missing in the CB1-null mice (data not shown).

Skeletal Phenotype of CB1-Null Mice

Our results demonstrate that the background WT strains, in which the C57^CB1−/− and CD1^CB1−/− mouse lines had been established, display vast differences in both trabecular and cortical bone mass. More importantly, cb1 inactivation in these lines resulted in opposing skeletal effects (Figs. 2–4).

Fig. 2

Bone mass phenotype in CB1-null mice. Tri-dimensional μCT images of distal femoral metaphysial trabecular bone from mice with median BV/TV values. Quantitative data are mean ± S.E.M. obtained in 16 C57^CB1−/− and 16 WT C57BL/6J …

Fig. 4

μCT-based measurements of diaphyseal dimensions in CB1-null mice. A–C, C57^CB1−/− mice and their WT C57BL/6J control mice; D–F, CD1^CB1−/− mice and their WT CD1 control mice. A and D, overall mid-diaphyseal…

Compared with their WT control mice, both male and female C57^CB1−/− mice exhibited low bone mass (LBM) phenotype characterized by a lower density of their trabecular network. The trabecular bone volume density (BV/TV) in female and male null mice was 20 and 15% lower than that of WT C57BL/6J control mice, respectively (Fig. 2). Apparently, the lower BV/TV in the C57^CB1−/− mice resulted from decreases in the trabecular number (Fig. 3A) without changes in the trabecular thickness (Fig. 3B). The trabecular connectivity density, a parameter measuring the structural integrity of the trabecular network (Stampa et al., 2002), was also decreased in these animals (Fig. 3C) but did not reach statistical difference. In addition, both the diaphyseal shaft diameter and medullary cavity diameter were narrower in the C57^CB1−/− mice (Fig. 4, A and B), with unchanged cortical thickness (Fig. 4C).

Fig. 3

μCT-based structural morphometric parameters in secondary spongiosa of distal femoral metaphysis of CB1-null mice. A–C, C57^CB1−/− mice and their WT C57BL/6J control mice; D–F, CD1^CB1−/− mice and …

By contrast, the CD1^CB1−/− skeletal phenotype showed a marked gender bias. The trabecular bone, the main skeletal compartment affected in osteoporosis, appeared normal in female CD1^CB1−/− mice (Figs. 2 and ​and3,3, D–F). Male CD1^CB1−/− mice had a pronounced HBM phenotype demonstrating 27.5% increase in trabecular BV/TV (Fig. 2) accompanied by increased trabecular thickness (Fig. 3E) and slightly decreased connectivity density (Fig. 3F). The female CD1^CB1−/− diaphysis was mildly abnormal, exhibiting cortical expansion portrayed as increases in both diaphyseal shaft diameter and medullary cavity diameter (Fig. 4, D and E). The male CD1^CB1−/− diaphysis appeared normal (Fig. 4, D–F).

To gain further insight into the processes leading to the LBM phenotype in C57^CB1−/−mice, we analyzed their bone remodeling. Consistent with the results of the structural μCT parameters, the histomorphometric analysis demonstrated that the LBM in these mice is associated with unbalanced bone remodeling. The bone formation rate was markedly decreased in both female and male mice (Fig. 5A), mainly because of a decrease in mineral appositional rate, a surrogate of osteoblast activity (Fig. 5B), inasmuch as the mineralizing perimeter, a surrogate of osteoblast number, remains unchanged (Fig. 5C). The osteoclast number was increased, significantly in female mice and insignificantly in male mice (Fig. 5D). We were surprised to find no significant differences in bone remodelling parameters between the CD1^CB1−/− mice and their WT control mice, even not in male mice (Table 1). Together, these results suggest that in the C57BL/6J mice, CB1 signaling positively regulates trabecular bone mass and radial diaphyseal growth by up-regulating bone formation and down-regulating bone resorption. The absence of significant changes in bone remodeling parameters of the male CD1^CB1−/− mice suggests that CB1 in these animals is associated only with the accrual of peak bone mass, which occurs at a younger age than that studied here. Apparently, the LBM phenotype is exhibited in the C57^CB1−/−mice consequent to a decrease in bone formation and increase in bone resorption attributable to the absence of sympathetic CB1, which normally inhibits norepinephrine release (Ishac et al., 1996).

Fig. 5

Histomorphometric bone remodeling parameters in secondary spongiosa of distal femoral metaphysis of C57^CB1−/− mice. A, bone formation rate. B, mineral appositional rate. C, mineralizing perimeter. D, osteoclast number. Data are mean ± …

TABLE 1

Trabecular histomorphometric bone remodeling parameters of male CD1^CB1−/− mice

Although either genetic modification leads to a null mutation missing all CB1 responsiveness to its ligands, the occurrence of phenotypic differences is not entirely surprising, inasmuch as these mouse lines exhibit other substantial discrepancies ranging from nociceptive perception to locomotor activity, life expectancy, and embryo implantation (Lutz, 2002). Furthermore, at least to some extent, skeletal dissimilarity between the C57^CB1−/− and CD1^CB1−/− mice could be expected from the differences in bone mass and structure observed between the WT CD1 and C57BL/6J background strains. More surprising is the gender bias portrayed by the CD1^CB1−/− mice and the absence of changes in bone remodeling in male animals that could explain their HBM. Although a HBM phenotype, unaccompanied by changes in bone remodeling, was reported previously (Idris et al., 2005), it is unclear to us whether it was assigned to male mice, female mice, or both.

Despite the differences between the two mouse lines, the present findings [especially the consistency in C57^CB1−/− mice presented by 1) CB1 expression in bone; 2) LBM; and 3) changes in skeletal turnover parameters] suggest a role for sympathetic CB1 in the control of bone remodeling and bone mass. In fact, unraveling the genetic differences between the C57BL/6J and CD1 strains, as well as the genetic basis for the gender discrimination within the CD1^CB1−/− mouse line, can provide useful information on the physiologic functional milieu of CB1 in bone. Until an explanation for the skeletal (and possibly other) differences between the C57^CB1−/− and CD1^CB1−/− mice is found, it is our approach that only experimental trends shared by both mouse lines should be considered.

We thank Olga Lahat, Malka Attar, Meirav Fogel, and Dr. Ravit Birenboim for expert assistance.

CD1^CB1−/− mice

CB1-null mice generated on a CD1 background

C57^CB1−/− mice

CB1-null mice generated on a C57BL/6J background

M-CSF

macrophage colony-stimulating factor

BV/TV

trabecular bone volume density

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

This work was supported by National Institute on Drug Abuse grants DA9789 (to R.M.) and DA00286, DA11322 (to K.M.), and Israel Science Fondation (ISF) grants 482/01 and 4007/02-Bikura (to I.B. and E.S.). Purchase of the μCT system was supported in part by ISF grant 9007/01 (to I.B.).

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