Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo.

Bone is a dynamic living organ that differentiates vertebrates from other animals. Calcification makes the tissue dense, stiff, strong and careful control of the material and geometrical properties of bone attempts to match their properties to the prevailing mechanical environment. Bone homeostasis involves an array of factors, only a few of which are known. The cells that produce bone, osteoblasts, share a common origin with adipocytes in mesenchymal stem cells (MSC) and there is mounting evidence of dysregulation of MSC differentiation in bone disorders such as osteoarthritis and osteoporosis. The cannabinoid system has recently been shown to be involved in bone homeostasis and has been proposed as a potential therapeutic target for treatment of bone diseases. However studies published to date look almost exclusively at its effect on trabecular bone. A full understanding of any regulatory factor can come only by considering both cortical and trabecular bone. This thesis describes experiments to characterize the mechanical, material and geometrical properties of cortical and trabecular bone from mice in which cannabinoid, CB1, CB2, or putative cannabinoid, GPR55, receptors have been deleted. Further studies started to explore the biological basis underlying the role of GPR55 and one of its antagonists, cannabidiol (CBD). The hypothesis was that endogenous cannabinoids can regulate bone properties and the aim Abstract ii was to resolve discrepancies found in previous studies and begin to explore the effect of cannabidiol acting through the GPR55 receptor on MSC differentiation. CB1 -/- mice had inferior mechanical properties to wild type mice due to reduced geometrical properties while the material properties remained unchanged. No change was found in trabecular bone volume in males but female CB1 -/- mice showed a high bone mass phenotype. CB2 -/- mice had mechanically superior bones to wild type animals due to larger geometrical properties. These superior characteristics were also seen in trabecular bone, where the female KOs had a high bone mass phenotype. The effect of knocking out GPR55 showed an increase in the amount of both cortical and trabecular bone. The knockout mice were fatter and were protected against age related bone loss. In addition, CBD enhanced adipogenesis in both humans and mice, while no effects were seen on osteogenesis. Enhanced adipogenesis was also found in GPR55-/- cell cultures. These results suggest that blocking GPR55 with small molecules such as CBD may be beneficial in bone diseases such as osteoporosis but not osteoarthritis.

Microarray analysis of gene expression in osteoblasts from patients with osteoporosis (OP) and osteoarthritis (OA) showed that 115 genes were robustly differentially expressed (P<0.05). Functional annotation clustering revealed cell adhesion to be the gene ontology classification most likely to be associated with this gene list. In addition scrutiny of the list revealed several genes with strong biological support for the involvement in bone homeostasis (FOSL1, BMPR2 and TGFBR1). Real -time PCR validated the trends seen in the microarray analysis, but failed to reach statistical significance for any of the genes examined. This analysis supports the value and potential of larger scale comparison of gene expression in OA and OP osteoblasts as a method for identifying novel factors involved in bone homeostasis. The cannabinoid system has recently been identified as involved in the regulation of bone homeostasis. In vitro investigation revealed that although cannnabinoid receptor agonists N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG) and JWH015 had no effect on metabolic activity, cell number, or alkaline phosphatase activity of calvarial mouse osteoblasts there were changes in gene expression. RankL expression was reduced relative to Opg expression by both JWH015 and AEA. Preliminary results indicate that JWH015 was also capable of increasing PPARγ expression which could alter the balance of osteoblastic and adipocytic differentiation of mesenchymal stem cells (MSC). This could have implications for use of these drugs in vivo. Using the 3T3-F442A cell line to develop a model of MSC differentiation highlighted difficulties associated with using cell models. Necessary additional factors required to induce differentiation of a cell line compared to a primary cell make interpretation of results more complicated. This model also highlighted that alkaline phosphatase and osteocalcin (markers usually used to identify osteogenic differentiation) were expressed during adipocytic differentiation. Future use of such markers in MSC models should be closely scrutinized.