Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

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

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

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 A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 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 A2A, A2B and A3 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

Inhibitors of vesicular ATP release increase bone mineralisation.

<p>(<b>A</b>) Treatment with NEM (100µM), monensin (≥1µM) and brefeldin A (100µM) for 1 hour reduced extracellular ATP levels by up to 90%, 55% and 40%, respectively. (<b>B</b>) Culture with NEM (≥1nM) for 14 days increased bone formation by up to 50%. (<b>C</b>) In osteoblasts treated with brefeldin A, bone formation was increased up to 70%. Values are means ± SEM (n=8-10 replicate wells), *** = p <0.001, ** = p <0.01, * = p <0.05). (<b>D</b>) Phase contrast microscopy images showing the increased mineralised bone nodule formation in osteoblast cultures treated with 100nM NEM and brefeldin A. Scale bar = 50µm.</p

P2X1 and P2X7 receptor antagonists increase bone mineralisation.

<p>(<b>A</b>) Culture with the P2X1 receptor antagonist, ≥0.1µM Ro-0437626 doubled the level of bone mineralisation. Treatment with other P2X1 receptor antagonists (≥1µM) (<b>B</b>) NF279 and (<b>C</b>) PPNDS increased bone mineralisation by ≥ 50% and 70%, respectively. The P2X7 receptor antagonists (<b>D</b>) ≥1µM AZ10606120, (<b>E</b>) ≥0.1µM A740003 and (<b>F</b>) ≥10µM A804598 increased bone mineralisation by ~80%, ~80% and 40%, respectively. Values are means ± SEM (n=6 replicate wells), *** = p <0.001, ** = p <0.01, * = p <0.05.</p

Apyrase treatment removes extracellular ATP.

<p>(<b>A</b>) Within one minute of 0.5U/ml apyrase treatment, a rapid decrease in ATP levels was observed; by 2 minutes and for the remainder of the experiment (10 minutes) ATP levels were negligible. ATP levels in control wells remained relatively constant. (<b>B</b>) ATP levels were measured in osteoblasts cultured with 0.5U/ml apyrase for 4, 7 or 14 days. In the controls wells, ATP levels were between 100–700nM; little or no ATP was detected in apyrase-treated wells. Values are means ± SEM (n=8-10 replicate wells), *** = p <0.001.</p

Apyrase treatment reduces osteoblast number in the early stages of culture.

<p>Osteoblast number was measured 24, 48 and 72 hours and 7 days after seeding with/without apyrase (0.5-1U/ml). Cell number was reduced 30-40% in apyrase-treated cultures at 24, 48 and 72 hours. Values are means ± SEM (n=6 replicate wells), *** = p <0.001, ** = p <0.01, * = p <0.05.</p