The effects of extracellular nucleotides and parathyroid hormone on bone remodelling.
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
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.