Peripheral cannabinoid receptor, CB2, regulates bone mass

Abstract

The endogenous cannabinoids bind to and activate two G protein-coupled receptors, the predominantly central cannabinoid receptor type 1 (CB1) and peripheral cannabinoid receptor type 2 (CB2). Whereas CB1 mediates the cannabinoid psychotropic, analgesic, and orectic effects, CB2 has been implicated recently in the regulation of liver fibrosis and atherosclerosis. Here we show that CB2-deficient mice have a markedly accelerated age-related trabecular bone loss and cortical expansion, although cortical thickness remains unaltered. These changes are reminiscent of human osteoporosis and may result from differential regulation of trabecular and cortical bone remodeling. The CB2(-/-) phenotype is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors. CB2 is expressed in osteoblasts, osteocytes, and osteoclasts. A CB2-specific agonist that does not have any psychotropic effects enhances endocortical osteoblast number and activity and restrains trabecular osteoclastogenesis, apparently by inhibiting proliferation of osteoclast precursors and receptor activator of NF-kappaB ligand expression in bone marrow-derived osteoblasts/stromal cells. The same agonist attenuates ovariectomy-induced bone loss and markedly stimulates cortical thickness through the respective suppression of osteoclast number and stimulation of endocortical bone formation. These results demonstrate that the endocannabinoid system is essential for the maintenance of normal bone mass by osteoblastic and osteoclastic CB2 signaling. Hence, CB2 offers a molecular target for the diagnosis and treatment of osteoporosis, the most prevalent degenerative disease in developed countries.

Fig. 1.

Low trabecular bone mass and cortical expansion in CB2^–/– mice. (A–C) Distal femoral metaphysis. (A) 3D trabecular bone structure in 51-week-old mice. (B) Trabecular bone volume density as percent trabecular network of total metaphyseal volume (BV/TV). (C) Trabecular number per millimeter metaphyseal line (Tb.N). Open circles, CB2^–/– mice; filled circles, WT mice. (D) Femoral mid-diaphysis of 8-week-old mice. Microcomputed tomographic (μCT) analysis in male mice. Quantitative data are mean ± SE. *, P < 0.05.

Fig. 2.

CB2 expression in normal bone. (A) Real-time RT-PCR of CB2 and osteoblast differentiation markers in bone marrow-derived primary stromal cell cultures undergoing osteoblastic differentiation in osteogenic medium. NOM, cells grown for 20 days in nonosteogenic medium; RUNX2, runt-related transcription factor 2; TNSALP, tissue nonspecific alkaline phosphatase. (B) RT-PCR analysis in MC3T3 E1 osteoblastic cell line grown in osteogenic medium. PTHRc1, parathyroid hormone (PTH)/PTH-related protein receptor 1. (C) RT-PCR analysis in primary culture of monocytic cells undergoing osteoclastogenesis. Upper lanes, bone marrow-derived culture grown in the presence of M-CSF and RANKL; lower lanes, RAW 264.7 cells grown with RANKL; Mono, undifferentiated monocytes; Ocl, osteoclast-like, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. (D and E) Immunohistochemical localization of CB2-positive osteoblasts (arrowheads), osteocytes (double arrowhead), and osteoclasts (arrows) in distal femoral metaphysis of WT mice (D) but not of CB2^–/– mice (E).

Fig. 3.

CB2 signaling regulates supply of diaphyseal osteoblasts. (A) CB2-specific agonist HU-308 stimulates the number of diaphyseal-derived bone marrow stromal cells. Cells were grown for 10 days in osteogenic medium followed by a 48-h challenge with HU-308 (see further details in Methods). Filled circles, cells derived from WT mice; open circles, cells derived from CB2^–/– mice. Data are mean ± SE obtained in triplicate culture wells per condition. (B) Ex vivo regulation of bone marrow-derived CFU-OB by CB2^–/– signaling. Data are mean ± SE obtained in five CB2^–/– and four WT mice per condition (10 wells per mouse). *, P < 0.05.

Fig. 4.

CB2-specific agonist stimulates growth and differentiation of newborn calvarial osteoblasts derived from WT but not from CB2^–/– mice. Cells were grown for 12 days in osteogenic medium before challenging with 10^–8 M HU-308. (A) DNA synthesis. (B) TNSALP activity. pNPP, p-nitrophenyl phosphate. (C) Mineral staining with alizarin red S. Cells were treated with HU-308 for 48 h in A and 10 days in B and C. Data in A and B are mean ± SE obtained in 12 and 3 culture wells per condition, respectively. Images in C are representative of 3–6 wells per condition. *, P < 0.05.

Fig. 5.

CB2-specific agonist, HU-308, restrains osteoclastogenesis. (A) Number of TRAP-positive multinucleated cells in primary bone marrow-derived monocyte culture from WT (filled squares) and CB2^–/– (open squares) mice maintained for 6 days in osteoclastogenic conditions (M-CSF and RANKL) with the indicated HU-308 concentrations. Cntl, control without HU-308. Data are mean ± SE obtained in 24 culture wells per condition. (B) Number of TRAP-positive multinucleated cells in RAW 264.7 cell cultures incubated for 7 days with RANKL and the indicated HU-308 concentrations. (C) DNA synthesis in RAW 264.7 cultured for 3 days with (open circles) or without (filled circles) RANKL and HU-308 as indicated. Data in B and C are mean ± SE obtained in triplicate culture wells per condition. (D) RT-PCR analysis of RANKL and osteoprotegerin (OPG) expression in bone marrow-derived primary stromal cell cultures. Cells from WT mice were incubated for 10 days in osteogenic medium and then challenged for 8 h with HU-308 or control (Cntl) medium. *, P < 0.05.

Fig. 6.

CB2-specific agonist, HU-308, attenuates OVX-induced femoral bone loss in sexually mature C3H mice. A 4-week treatment with HU-308 at 10 mg/kg per day commenced at the time of OVX. (A) μCT analysis of trabecular bone volume density. (B) Histomorphometric analysis of osteoclast number per bone surface area (Oc.N/BS). (C) Histomorphometric analysis of bone formation rate (BFR). A–C were analyzed in the distal femoral metaphysis. (D) Mid-diaphyseal μCT (Upper) and histomorphometric (Lower) analyses. Quantitative microtomograpic and histomorphometric parameters are as defined in Fig. 1. Data are mean ± SE. *, P < 0.05.