title: The effects of extracellular nucleotides and parathyroid hormone on bone remodelling.
Creators: name: Anne. Buckley, Katherine
Publisher: name: University of Liverpool Repository
Year: 2023

description: Osteoclasts are multinucleated, terminally differentiated cells found in bone tissue, which have the unique ability to resorb calcified substrates. The study of human osteoclasts has been restricted in the past due to difficulties in obtaining these cells. Recently, however, cell culture techniques have been devised that allow human osteoclasts to be grown in culture from their mononuclear precursors found in peripheral blood, therefore providing a constant supply of these cells. These cultures are based on the discovery of RANKL (receptor activator for NFJ(B ligand), which has recently been shown to playa central role in osteoclast formation and activation. This thesis has initially characterised cells grown in such cultures to confirm that they are authentic human osteoclasts, possessing osteoclast markers and with the ability to resorb calcified substrates. Once these cultures were established, and the authenticity of human osteoclasts grown in these cultures was confirmed, these cells were used to study the effects of extracellular nucleotides on human osteoclast acti vity. Adenosine-5' -triphosphate (ATP) is well known as an intracellular energy source. This and other nucleotides, however, also exist extracellularly, where they are agonists at a group of receptors termed P2 receptors. This receptor family is subdivided into P2X ligand gated ion channels, and P2Y G-protein coupled receptors. Bone-forming osteoblast cells, and boneresorbing osteoclast cells both express multiple SUbtypes of these receptors. Studies examining the effects of extracellular nucleotides on osteoclasts have been largely limited to non-human cells in the past due to the difficulty in obtaining human osteoclasts. This thesis, therefore, has examined the effects of extracellular nucleotides on human osteoclast activity. Human osteoclasts and their precursors were shown to express mRNA for nearly all of the P2Y and P2X receptor subtypes. ATP was found to both activate human osteoclast formation, by acting directly on P2X receptors expressed by osteoclast precursors, and to stimulate osteoclast resorption indirectly by acting at osteoblast-expressed P2Y) receptors to induce elevated RANKL expression by these cells. Activation of P2Y receptors expressed by osteoclasts was shown to result in downstream activation of MAPKinase pathways. Parathyroid hormone (PTH) is considered to be one of, if not the most important systemic factor in the regulation of bone. Co-stimulation of UMR-I 06 osteoblast-like cells with this hormone and P2Y) agonists resulted in potentiation of P2Y) agonist-induced [Ca2+Ji release by PTH, while PTH alone produced no [Ca2+]j elevation at all. The mechanism of this potentiation was attributed to Gs activation following PTH receptor stimulation, Gq having no involvement. Co-stimulation of these cells by PTH and P2Y) agonists also resulted in synergistic immediate early gene expression. These findings suggested that extracellular nucleotides are able to sensitize osteoblasts to the actions of PTH, providing a mechanism for localizing the response to this systemic hormone in bone, consistent with the discrete pattern of remodelling observed in the skeleton. Finally, the involvement of PTH on osteoclast formation was investigated. PTH was found to inhibit this process via activation of contaminating T lymphocytes present in osteoclast cultures. In conclusion this thesis presents evidence to suggest that extracellular nucleotides are important local signaling molecules in bone, affecting both osteoclast and osteoblast activity, alone and in combination with systemic factors such as PTH. Additionally, a novel action of PTH acting via lymphocytes to affect osteoclast formation is described.