Hoebertz, Astrid; (2001) Purinergic signalling in bone cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is increasing evidence that ATP and other extracellular nucleotides, signalling through P2 receptors, play an important role in bone remodelling. I used immunohistochemistry and in situ hybridisation to study the expression of P2 receptors in rat bone sections and on cultured bone cells: osteoclasts, the multinucleated cells responsible for bone resorption, osteoblasts, the bone-forming cells, and chondrocytes were all shown to express a number of P2 receptors, both of the ionotropic P2X family and the metabotropic P2Y family. ATP has previously been shown to be a potent stimulator of bone resorption, however, it was not known which receptor subtypes mediate this stimulatory effect. Three different models were used to study the effects of a wider range of P2 receptor agonists on osteoclast recruitment and resorption: the 26 hour disaggregated rat osteoclast assay, 72 hour whole organ mouse calvaria cultures, and 10 day mouse marrow cultures. My main discovery is that extracellular ADP, the first degradation product of ATP, is a powerful stimulator, at nanomolar concentrations, of osteoclast activation and recruitment. Furthermore, evidence is provided, using subtype-selective agonists and antagonists, that these ADP effects are probably mediated via the P2Y1 receptor. It is suggested that in vivo, ADP could be involved in the mediation of inflammatory bone loss. The effects of nucleotides on osteoblast function were studied using the bone nodule assay by culturing primary rat calvarial osteoblasts for 3 weeks and by analysing the formation of mineralised bone nodules. Both ATP and UTP, a P2Y2 and P2Y4 receptor agonist, significantly inhibited bone nodule number at concentrations 0.1-10μM, whereas ADP showed no effect. Taken together, these results suggest subtype-specific roles for P2 receptors present in bone cells: ADP is a powerful stimulator of bone resorption signalling via the P2Y1 receptor, whereas UTP, signalling via the P2Y2 receptor, could play a role as an inhibitor of bone formation. In parallel, further functional experiments were carried out to assess the interactions of low pH and of osteolytic stimuli, and to investigate whether human osteoclasts are as acid-sensitive as rodent and avian osteoclasts. Interestingly, both nucleotides (ADP and ATP) and the cytokine RANKL require low pH to show their full stimulatory effect on bone resorption, suggesting that low pH is a key requirement for resorption to occur. In conclusion, this is one of the first studies linking specific P2 receptor subtypes to key functional actions of extracellular nucleotides on bone.

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