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In Chapter 4, I investigated the roles of the RANK signalling partners RANK ligand (RANKL) and osteoprotegerin (OPG) in the pathogenesis of sporadic and familial PDB. One polymorphism in the RANK gene and five polymorphisms in the OPG gene were examined in sporadic PDB cases and in sex- and age-matched controls. No allele-disease or genotype-disease association was observed for the <i>RANKL </i>polymorphism, suggesting RANKL is not directly involved in susceptibility to sporadic PDB. Genotypes at two <i>OPG</i> polymorphisms did significantly predict disease status in individuals affected with sporadic PDB, suggesting a role for OPG in the pathogenesis of sporadic PDB. The five <i>OPG</i> polymorphisms were also examined in families affected with PDB. No evidence was found to either suggest or exclude the involvement of any of the <i>OPG</i> polymorphisms in familial PDB. In Chapter 5, I performed a genome-wide search for PDB susceptibility loci in families with inherited PDB. Three regions of potential linkage were identified at 2q36, 5q35 and 10p11. Fine mapping was performed for the candidate region on chromosome 5q35, and eight families with a high probability of linkage to 5q35 were identified. In seven of the families, a shared haplotype transmitted only with affected family members was present. The shared haplotype varied between families and no common allele existed in the seven families for any of the nine markers studied. However, one area of shared haplotype occurred in all seven families across three of the markers, supporting evidence for a susceptibility gene for PDB on 5q35 in these families and narrowing the candidate region. In summary, this study has further highlighted the importance of genetic heterogeneity in the pathogenesis of PDB, excluding the previously identified PDB2 susceptibility locus and identifying three novel regions potentially harbouring susceptibility loci in the families studied. This study has also further defined the role of members of the RANK signalling pathway in the pathogenesis of familial and sporadic PDB.

Bone and skeletal joint disorders affect millions of people with significant morbidity and mortality. Over the last few decades it has become apparent that the physiological structure of bone is maintained by the balanced functioning of bone-forming osteoblast and bone-resorbing osteoclast cells. Thus study of the regulation and function of osteoclasts became the focus of scientific research to find therapeutic targets for bone related disorders. The aim of this study was to elucidate the roles of two pathways, namely Receptor Activator of NFB (RANK) signaling pathway and mevalonate pathway in relation to the pathology and pharmacology of osteoclasts. RANK mutations associated with osteopetrosis were studied to elucidate the molecular mechanism of activation of the RANK signaling pathway. The data demonstrate that, unlike other TNF receptors, the C-terminal PreLigand Assembly Domain (PLAD or ‘IVVY' motif) is not essential for ligand-induced activation of RANK signaling. The study of the extracellular RANK mutations provided the opportunity to examine role of three cysteine rich domains (CRD) within extracellular RANK in its interaction with RANK ligand. The binding affinities of RANK ligand to wild type and six extracellular mutant RANK proteins were studied by surface plasmon resonance. It showed that CRD 1, 3 and 4 played crucial roles, despite previous crystallography studies predicting roles for only CRD 2 and CRD 3 in RANK ligand binding. In the second part of the thesis the mevalonate pathway was studied in relation to mevalonate kinase deficiency and osteoporosis. Mevalonate kinase deficiency is a hereditary disorder caused by mutations within the mevalonate kinase gene and manifests as autoimmune disorder due to deficiency of end products in mevalonate pathway. Due to rarity of the disorder, access to patient samples is extremely limited and we aimed to develop an in vitro model of mevalonate kinase deficiency to develop a better understanding of regulation of the mevalonate pathway. In osteoporosis, nitrogen-containing bisphosphonate drugs target farnesyl pyrophosphate synthase (FPPS) within the mevalonate pathway. A small number of patients develop resistance to bisphosphonate therapy, but the molecular mechanism is unknown. It was hypothesized that upregulation of FPPS would confer resistance to bisphosphonates and this study showed that upregulated endogenous FPPS introduced bisphosphonate resistance at therapeutic concentrations in vitro. Furthermore, it was observed that mitochondrial isoforms of FPPS were unlikely to play any role in bisphosphonate resistance. In conclusion, these data suggest that therapeutic targeting of the PLAD motif in RANK may not be as effective as previously proposed, but the extracellular domain of RANK may be a potential target for the development of novel therapies in the prevention and treatment of osteoporosis. In addition, prior screening for expression levels of FPPS patients with osteoporosis for may identify those at risk of resistance to nitrogen-containing bisphosphonate therapy.

This thesis focuses on evaluating the osteogenic potential of two synthetic bone graft materials either in vitro and/or in established rodent bone defect models.

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The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated.  At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB₁, CB₂ or TRPV1 receptors, indicating a non-receptor mediated effect.  Conversely, anandamide and 2-AG increased mouse osteoclast formation.  The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB_1/2 knockout mice.  The effect of 2-AG was not eliminated in CB_1/2 knockout cells, indicating a non-CB₁/CB₂ action.  The CB₁ antagonist, AM251, and the CB₂ antagonist, AM630, both inhibited mouse osteoclast formation.  These effects were not rescued in the CB_1/2-knockout mouse cells.  Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast.  This was prevented in the presence of AM630 but not AM251, indicating a CB₂-mediated response.  The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E₂-glycerol ester (PGE₂-G), on human osteoclasts were examined.  Treatment with PGE₂-G inhibited formation and ERK phosphorylation of human osteoclasts.  These effects were attenuated by a selective EP₄ antagonist and mimicked by PGE₂ alone, indicating that PGF₂-G is rapidly metabolised into PGE₂ in human osteoclast cultures.  However, PGE₂-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP₃- dependent mechanism, indicative of a G-protein coupled receptor effect.  This was not mimicked by PGE₂, or prevented by the EP₄ antagonist, but blocked by a putative PGE₂-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE₂-G receptor antagonist.

Osteoclasts are the cells that resorb bone to maintain a healthy skeleton. Receptor activator of NFkB (RANK) is a receptor that is critical for the formation, activity and survival of osteoclasts. A number of mutations have been identified within RANK that cause bone diseases with opposite osteoclast phenotypes. The aim of this thesis was to study the downstream consequences of these disease-associated mutations for RANK protein processing and activation of the RANK signalling pathway. Early onset Paget’s disease of bone (ePDB), Familial Expansile Osteolysis (FEO) and Expansile Skeletal Hyperphosphatasia (ESH) are conditions featuring focal areas of increased bone resorption driven by overactive osteoclasts. These conditions are caused by heterozygous insertion mutations in the signal peptide region of the RANK gene, but the mechanisms underlying the development of overactive osteoclasts are not known. In this thesis, in vitro study of the mutant RANK proteins demonstrated that homozygous overexpression caused inactivation of RANK signalling due to intracellular accumulation of RANK within an extended form of the endoplasmic reticulum. By contrast, when expressed in a heterozygous manner, the mutant proteins were found at the plasma membrane and caused prolonged ligand-dependent signalling. Taken together and as predicted by the clinical situation, these data strongly suggest that heterozygous expression of the mutant RANK proteins hold the key to the hyperactive osteoclast phenotype associated with these diseases. Osteoclast-poor osteopetrosis is a disease in which osteoclasts do not form leading to a high bone mass phenotype. Single base pair mutations within RANK have been identified in some patients with this condition. These mutant RANK proteins were studied and the findings related to regions within RANK in which the mutations occur that have been shown to be critical for its function. In addition, osteoclast formation was assessed in cultures of peripheral blood mononuclear cells isolated from patients with osteopetrosis with unidentified genetic background in order to further characterise the osteoclast phenotype for each patient. In summary, the findings presented in this thesis begin to elucidate the molecular mechanisms leading to diseases of bone with opposite osteoclast phenotypes that, paradoxically, are all caused by inactivating mutations in the RANK gene.

Osteoclasts polarise upon activation and form four distinct membrane domains; the basolateral domain, the sealing zone, the functional secretory domain and the ruffled border. The ruffled border is the resorptive organelle of the cell and provides a large surface area for the release of protons and enzymes into the space beneath the osteoclast. Defects in osteoclast formation or function can lead to diseases such as osteopetrosis. Ruffled border formation is a critical event in osteoclast function but the process by which it and other membrane domains form is only partially understood. Vesicular trafficking is essential for the tight regulation of the osteoclast membrane domains and it has been shown previously that treatment with pharmacological inhibitors causes disruption of trafficking. The aims of this PhD were to increase our understanding of vesicular trafficking in osteoclasts and to optimise ways of visualising osteoclast membrane domains. My studies of patients with osteoclast-poor osteopetrosis identified defects in RANKL as a cause of the defect. This in turn has identified a potential therapy of recombinant RANKL for patients with this form of the disease. Although purification of wild type or mutant RANKL was not completely successful, it did suggest that the mutant forms of RANKL were not functional. I have used pharmacological inhibitors to study osteoclast membrane domains, and found that transmission electron microscopy is an essential tool for studying membrane changes following pharmacological inhibition at the ultrastructural level. I also established that the study of vesicular trafficking to analyse formation of membrane domains can make excellent use of immuno-electron methods. Furthermore, genetic diseases associated with defective ruffled border formation such as XLA and osteopetrosis provide useful tools to further analyse the dynamics involved in the formation and maintenance of the ruffled border, as well as revealing more about the diseases themselves.