Extracellular Nucleotides Block Bone Mineralization in Vitro: Evidence for Dual Inhibitory Mechanisms Involving Both P2Y2 Receptors and Pyrophosphate

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114

It is now widely recognised that extracellular nucleotides, signalling via purinergic receptors, participate in numerous biological processes in most tissues. It has become evident that extracellular nucleotides have significant regulatory effects in the musculoskeletal system. In early development, ATP released from motor nerves along with acetylcholine acts as a cotransmitter in neuromuscular transmission; in mature animals, ATP functions as a neuromodulator. Purinergic receptors expressed by skeletal muscle and satellite cells play important pathophysiological roles in their development or repair. In many cell types, expression of purinergic receptors is often dependent on differentiation. For example, sequential expression of P2X5, P2Y 1 and P2X2 receptors occurs during muscle regeneration in the mdx model of muscular dystrophy. In bone and cartilage cells, the functional effects of purinergic signalling appear to be largely negative. ATP stimulates the formation and activation of osteoclasts, the bone-destroying cells. Another role appears to be as a potent local inhibitor of mineralisation. In osteoblasts, the bone-forming cells, ATP acts via P2 receptors to limit bone mineralisation by inhibiting alkaline phosphatase expression and activity. Extracellular ATP additionally exerts significant effects on mineralisation via its hydrolysis product, pyrophosphate. Evidence now suggests that purinergic signalling is potentially important in several bone and joint disorders including osteoporosis, rheumatoid arthritis and cancers. Strategies for future musculoskeletal therapies might involve modulation of purinergic receptor function or of the ecto-nucleotidases responsible for ATP breakdown or ATP transport inhibitors.

Section: Functional Effects Of P2 Receptor-mediated Signalling In Ostmentioning

confidence: 99%

“…Expression of multiple NTPdases and NPPs by osteoblasts has now been reported [203,204]. Of these, NPP1 has emerged as having a particularly important role in bone.…”

Section: Atp Hydrolysis and The Effects On Bone Mineralisationmentioning

confidence: 99%

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Purinergic signalling in the musculoskeletal system

Burnstock

Arnett

Orriss

2013

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111

114

“…This release has been reported to be increased by mechanical stress and is known to have a paracrine or autocrine effect on osteoblasts and osteoclasts partly via activation of specific P2 receptors. However, this ATP is also known to be rapidly metabolised to adenosine (which can then act via its own receptors) by the sequential actions of a number of enzymes, including ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), CD73 and alkaline phosphatases [4,17]. Interestingly, a mouse model lacking ENPP1 has recently been shown to have a severe disruption to the architecture and mineralization of longbones [18].…”

Section: Commentarymentioning

confidence: 99%

Does adenosine play a role in bone formation, resorption and repair?

Evans

2012

There has been increasing interest recently in the role purinergic signalling in the physiology and pathophysiology of musculoskeletal tissues, especially bone. Whereas the role of ATP in bone metabolism [1,2] has been revealed to some extent and quite a few papers have been published in this respect, functions of its metabolite adenosine are not understood. Bone remodelling is a continuous, life-long process that maintains skeletal integrity. It is orchestrated by the three bone cell types (osteoblasts, osteoclasts and osteocytes), and impairments eventually result in diseases such as osteoporosis. Although there is much insight into the systems that maintain healthy bone, there is still a need to develop new therapies. The hypothesis that the adenosine signalling pathways might provide new targets for bone disease has gained further momentum recently with the publication of the following two papers that are commented on by Bronwen A. J. Evans of Cardiff University. Interestingly enough, the first article deals with findings obtained using CD73 null mice, that have also been the subject of another recently published Highlight [3] that focused on the role of local and systemic adenosine in modulation of antitumour responses in vivo. Here, CD73 null mice have been exploited to unveil a potential role of adenosine in osteoblast differentiation. In the second commented article, by using adenosine A2B receptor knockout mice, this receptor is identified as a main target for adenosine in promoting the differentiation of mesenchymal stem cell to osteoblasts. Article summaryM. Takedachi and colleagues hypothesized that CD73 may be involved in regulating function of the bone forming cell (osteoblast) through modulating nucleotide metabolism and generating extracellular adenosine that can activate adenosine receptors (ARs). To address this hypothesis, they asked whether CD73 functionally regulates bone metabolism in vivo by characterizing the bone phenotype of cd73−/− mice. In addition, they investigated the involvement of CD73 and AR signalling in osteoblast differentiation in vitro.Micro-computed tomography (μCT) showed that 13 week old male mice had osteopenia, and that this was due to reduced trabecular bone volume, decreased trabecular number and thickness, and increased trabecular separation in cd73−/− mice when compared to wild-type controls. Subsequent work measuring biochemical markers of bone formation showed that osteocalcin (bone formation marker) was decreased in cd73−/− mice whereas bone resorption markers (TRAP5b and fragments of type I collagen) were comparable to wild-type animals. The association of the osteoblast to the impared bone phenotype seen in cd73−/− mice was further substantiated using quantitative RT-PCR. These

“…Osteoblasts, the bone-forming cells, express multiple P2 receptors [5,6], in a differentiationdependent manner [7,8], and respond to extracellular nucleotides with a prompt increase in intracellular calcium [7][8][9][10]. Importantly, exogenous ATP, UTP and other nucleotide analogues also potently inhibit mineralisation of bone formed by osteoblasts in culture [8,11,12]. Moreover, endogenous ATP released by osteoblasts also appears to act as a significant local inhibitor of mineralisation [13].…”

Section: Introductionmentioning

confidence: 99%

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

Hajjawi

Patel

Corcelli

et al. 2016

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Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A 1 and A 2B receptors was expressed by all primary osteoblasts, A 2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A 3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30-50 % at a concentration of 1 μM. Osteoclasts were found to express the A 2A , A 2B and A 3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone.