Activation of the P2Y₂ receptor regulates bone cell function by enhancing ATP release

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   Figure 1

   P2Y₂R^−/− mice display age-related increases in trabecular bone. Trabecular bone volume (BV/TV) was increased by ≤46% and ≤48% in the (A) femur and (B) tibiae of P2Y₂R^−/− mice, respectively. Trabecular number (Tb.N) was increased (C) ≤27% in the femur and (D) ≤30% in the tibia. Trabecular thickness (Tb.Th) was ≤17% and ≤10% higher in the (E) femur and (F) tibia, respectively. (G, H) Trabecular BMD was increased ≤12%. (I, J) Cortical bone volume, (K, L) cortical thickness, (M) periosteal diameter and (N) endosteal diameter were unchanged. Values are means ± sem (n = 10), significantly different from controls: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (O) Representative 3D volumetric images of the trabecular and cortical bone of 24-week-old P2Y₂R^−/− and P2Y₂R^+/+ mice.
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   Figure 2

   Increased bone formation by osteoblasts from P2Y₂R^−/− mice. In cultures of (A) calvarial and (B) long-bone osteoblasts from P2Y₂R^−/− mice, the level of mineralised bone nodule formation was increased 3-fold and 5-fold, respectively. Basal TNAP activity was increased by ≤3-fold in P2Y₂R^−/− (C) calvarial and (D) long-bone osteoblasts (n = 6). (F) Serum TNAP activity was increased up to 60% (n = 10). (E) Serum P1NP levels were unchanged in P2Y₂R^−/− mice (n = 10). Values are means ± sem, significantly different from controls: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (G) Representative whole well scans (unstained) and phase-contrast microscopy images (alizarin red stained) showing the increased bone formation in cultures of P2Y₂R^−/− calvarial osteoblasts. Scale bars: whole well = 0.5 cm, microscopy images = 50 µm.
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   Figure 3

   Osteoclasts from P2Y₂R^−/− mice exhibit defective resorption. P2Y₂ receptor deletion (A) had no effect on osteoclast number but (B) decreased resorption per osteoclast by 75% (n = 8). (C) Serum CTX levels were up to 35% lower in P2Y₂R^−/− mice (n = 10). Values are means ± sem, significantly different from controls: * = P < 0.05, *** = P < 0.001. (D) Representative transmitted and reflective light microscopy images showing the decreased resorption seen in P2Y₂R^−/− osteoclast cultures. Scale bar = 50 µm. (E) Qualitative histology suggested that the number of TRAP-positive osteoclasts was reduced on the endocortical and trabecular bone surfaces in 24- but not 8-week-old P2Y₂R^−/− mice. Scale bar = 100 µm.
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   Figure 4

   The role of the P2Y₂ receptor and extracellular ATP in regulating bone resorption. Treatment with (A) UTP (B) 2-thioUTP had no effect on osteoclast formation. The area resorbed per osteoclast was increased up to (C) 80% by UTP and (D) 45% by 2-thioUTP (≥10 nM) in P2Y₂R^+/+ but not P2Y₂R^−/− osteoclasts, (E) P2Y₂R^−/− osteoclasts mice displayed a 53% reduction in basal ATP release. (F) ATP breakdown was unchanged in P2Y₂R^−/− osteoclasts. (G) Culture with apyrase inhibited bone resorption in normal osteoclasts by up to 55%. (H) Addition of exogenous ATP (≥1 µM) returned the level of resorption in P2Y₂R^−/− osteoclast cultures to normal. Values are means ± sem (n = 8), significantly different from controls: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.
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   Figure 5

   The effect of UTP and 2-thioUTP on ATP release from osteoclasts. (A) UTP (≥1 µM) increased extracellular ATP release by ≤2-fold for up to 90 min post treatment. (B, C, D) No effects of UTP on ATP released were seen P2Y₂R^−/− cells. (E) 2-thioUTP (≥0.1 µM) dose-dependently increased extracellular ATP levels by up to 50% (F, G, H) but had no effect in P2Y₂R^−/− osteoclasts. Long-term treatment (7 days) with (I) UTP and (J) 2-thioUTP treatment enhanced ATP release by up to 70% and 65%, respectively, in P2Y₂R^+/+ but not P2Y₂R^−/− osteoclasts. Values are means ± sem (n = 10), significantly different from controls: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Differences between P2Y₂R^+/+ and P2Y₂R^−/−: # = P < 0.05, ## = P < 0.01, ### = P < 0.001. Standard curves used to calculate ATP concentrations in acute (K) UTP and (L) 2-thioUTP experiments.
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   Figure 6

   The role of the P2Y₂ receptor in ATP release from osteoblasts. (A) No differences were observed in the rate of ATP breakdown between P2Y₂R^+/+ and P2Y₂R^−/− osteoblasts. (B) Basal ATP release was up to 60% lower from P2Y₂R^−/− osteoblast. Increased ATP release from P2Y₂R^+/+ but not P2Y₂R^−/− osteoblasts treated for 14 days with (C) UTP (≤4-fold) and (D) 2-thioUTP (≤3-fold). (E) Acute treatment with UTP (≥10 µM) increased ATP release by ≤4-fold for up to 60 min. (F, G, H) No effect of UTP (10 µM) on ATP release from P2Y₂R^−/− osteoblasts. (I) ≥1 µM 2-thioUTP also enhanced ATP release (≤4-fold) from P2Y₂R^+/+osteoblasts but was without effect in P2Y₂R^−/− cells (J, K, L). Values are means ± sem (n = 12), significantly different from controls: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Differences between P2Y₂R^+/+ and P2Y₂R^−/−: # = P < 0.05, ## = P < 0.01, ### = P < 0.001.
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   Figure 7

   Proposed role of the P2Y₂ receptor in osteoclast and osteoblast function. In osteoclasts, UTP acts via the P2Y₂ receptor to promote the release of ATP (via the P2X7 receptor). Once released ATP (and ADP) can act via the P2Y₁ and/or P2Y₁₂ receptors to stimulate bone resorption. UTP can also act via the P2Y₂ receptor to stimulate ATP release from osteoblasts (via vesicular exocytosis). ATP can then act via other P2 receptors (e.g. P2X1 or P2X7) to inhibit bone mineralisation. ATP can also be broken down by NPP1 to produce the mineralisation inhibitor, pyrophosphate (PP_i).

• © 2017 Society for Endocrinology