title: Purinergic signalling in osteoblasts
Creators: name: Bond, Alistair
Publisher: name: University of Liverpool Repository
Year: 2013

description: ATP is now well established as an extracellular signalling molecule and has been shown to play a role in many different tissues including bone. ATP acting on P2 receptors has been reported to cause an increase in osteoblast proliferation, apoptosis, IL-6 production, AA release and a decrease in bone mineralization. It has also been shown in vivo, by using KO mice, that ATP can cause both an anabolic and catabolic response depending on the P2 receptor involved, with P2Y2 KO mice showing an increased bone formation whilst P2X7 mice have a decrease in the periosteal bone formation. ATP is released from osteoblasts in vitro basally, following trauma to the cell and in response to mechanical strain. This mechanical strain comes in the form of medium displacements, where the medium is taken up in a pipette and then immediately washed over the cells. Work carried out in this thesis looked to refine the experimental procedure of these tests in an attempt to reduce the variation of ATP supernatant measurements following medium displacement experiments. A new experimental procedure has now been implicated that ensures that the luciferase enzyme, used to measure ATP, is kept under constant conditions, the measurements from the wells are taken from the same area and basal levels are measured in each well as it was determined that ATP measurements are not consistent throughout a single well. Further to this a sensitive measure of cell death was developed in order to ensure that the observed ATP release was not due to cell trauma. This used qPCR to measure mtDNA present in the supernatant. Tests were also carried out to investigate the synergy between ATP and PTH as ATP synergises with PTH to increase the expression of c-fos in osteoblasts. c-fos is a master regulator gene that causes an increase in osteoblast proliferation and differentiation. It also causes the secretion of RANKL and decreases secretion of OPG, the RANKL decoy receptor, thus osteoclast fusion is also upregulated with an increase in c-fos production. Given that ATP is a local hormone that is released by mechanical stress and acts in an autocrine/paracrine manner and that PTH is an indiscriminate system wide hormone this synergy could be one mechanism whereby Wolff’s Law is achieved. That is, BMD is increased in areas of higher loading. The mechanism of this synergy is still to be elucidated and is likely to be different between cell types. Experiments on UMR-106 cells has shown that ATP and PTH can cause a potentiation of intracellular calcium release that ultimately leads to increased c-fos transcription. In SaOS-2 cells two separate signalling pathways converge on different areas of the c-fos promoter to cause an increase in c-fos. Work carried out for this project now shows for the first time that ATP sensitises osteoblasts to the action of PTH. Given the short half life of both molecules this increases the likelihood that they do play a significant role in bone remodelling in vivo. The mechanism behind this sensitisation appears to be independent of the known mechanisms described above, as it was shown that the sensitisation was conferred in the medium and was not due to ATP break down products ADP, AMP and adenosine. Thus it is likely another, longer lasting, paracrine factor is involved. BL-1249 is a putative K+ channel opener and inhibits ATP+PTH induced c-fos expression. The inhibition was shown not to be due to BL-1249s effect on K+ channels. It appears more likely that it acts on the cAMP/PKA pathway to inhibit c-fos transcription, as BL-1249 did not decrease FCS induced c-fos expression but did inhibit sp-cAMP induced c-fos expression. Further to this BL-1249 at concentrations of 100 μM caused a massive increase in cell death to the osteosarcoma cell lines SaOS-2, MG-63 and TE-85, however it had no effect on primary human osteoblasts. The apoptotic effect of BL-1249 is very likely due to the activation of K+ channels as it has been reported that opening K+ channels will cause apoptosis through K+ efflux and subsequent cell shrinkage. This was further confirmed by use of TEA, a K+ channel blocker, which acted to reverse the effect of BL-1249. Thus BL-1249 can act to decrease osteosarcoma cell number via inhibition of c-fos and, via K+ xiii channels, cause apoptosis. It is still unclear how apoptosis is targeted to osteosarcoma lines however it is likely through BL-1249s effect on c-fos expression. Occasionally the true effect of a gene or protein can only be elucidated when studying it in the context of a pathology. It has been reported that P2X7 can cause astrocytes to be toxic to motor neurones by secreting factors that contribute towards their death. In situations of increased oxidative stress, such as those found in SOD1 -/- mice, this effect can be increased and cause basal levels of ATP to activate the neurotoxic phenotype of the astrocytes. SOD1 -/- mice have also been examined for the bone and muscle phenotypes. They were shown to have a massive reduction in muscle mass with the peak difference being less than 50% their wild-type counterparts, their bones also had a decrease in BMD, a decreased length of their femur and had a decreased stiffness. Given this link between SOD1, P2 signalling and an altered bone phenotype SOD1-/- mice were examined in more detail. μCT scans were used to analyse the bone structure of these mice, however in this study no differences were observed. P2X4 was increased in SOD1-/- calvarial cells whilst P2Y2 was decreased in these cells suggesting that oxidative stress does cause an altered P2 receptor expression and this may change the BMD of bone.