Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Abstract

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.

The role of purinergic signalling in the musculoskeletal system

“…There is now significant evidence suggesting that extracellular ATP is a key source of PPi in bone Orriss et al, 2013;Orriss et al, 2007). Thus, it is likely that nucleotide triphosphates exert a dual inhibitory action on bone mineralisation via both P2 receptor mediated signalling and direct hydrolysis to PPi (Fig.…”

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

“…Osteoblast cell number was measured at regular intervals throughout the culture period using a commercially available kit (CytoTox 96, Promega UK, Southampton, UK), as previously described [13]. This assay measures the activity of lactate dehydrogenase (LDH), a cytosolic enzyme which is released on cell lysis.…”

“…Moreover, endogenous ATP released by osteoblasts also appears to act as a significant local inhibitor of mineralisation [13]. The action of UTP, together with data from pharmacological studies using other selective P2 receptor agonists and antagonists indicated that P2Y 2 , P2X1 and P2X7 receptors could be involved in mediating the inhibition of mineralisation by ATP [8,12,13].…”

Purinergic signalling in the musculoskeletal system

“…This study used selective P2 receptor antagonists to suggest that the effects were mediated via the P2X1 and P2X7 receptor subtypes [167]. Furthermore, a recent investigation demonstrated increased bone mineralisation in osteoblast cultures treated with apyrase, suggesting that endogenous ATP released by osteoblasts acts as an important local brake on mineralisation [205]. Combined these data suggests that ATP acts as a key endogenous inhibitor of bone mineralisation.…”

“…A recent detailed study of the mouse model lacking NPP1 showed that this enzyme is essential for normal bone development and control of physiological bone mineralisation [242]. It is now becoming apparent that extracellular ATP is a key source of PP i in bone [205]. Thus, it is likely that nucleotide triphosphates exert a dual inhibitory action on bone mineralisation via both P2 receptor-mediated signalling and direct hydrolysis to PP i [111,203,205] (Fig.…”