Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

CB2 cannabinoid receptor targets mitogenic Gi protein-cyclin D1 axis in osteoblasts.

CB2 is a Gi protein-coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase-activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein-cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis.

Peripheral cannabinoid receptor, CB2, regulates bone mass.

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.

Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling.

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 in cb1-/- 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.