Department of Cell and Developmental Biology, University College London, London, UK.
Bone. 2012 Sep;51(3):389-400. doi: 10.1016/j.bone.2012.06.013. Epub 2012 Jun 26.
Extracellular nucleotides, signalling through P2 receptors, regulate the function of both osteoblasts and osteoclasts. Osteoblasts are known to express multiple P2 receptor subtypes (P2X2,5,7 and P2Y(1),(2,4,6)), levels of which change during differentiation. ATP and UTP potently inhibit bone mineralisation in vitro, an effect mediated, at least in part, via the P2Y(2) receptor. We report here that primary rat osteoblasts express additional, functional P2 receptors (P2X1, P2X3, P2X4, P2X6, P2Y(12), P2Y(13) and P2Y(14)). Receptor expression changed with cellular differentiation: e.g., P2X4 receptor mRNA levels were 5-fold higher in mature, bone-forming osteoblasts, relative to immature, proliferating cells. The rank order of expression of P2 receptor mRNAs in mature osteoblasts was P2X4>>P2Y(1)>P2X2>P2Y(6)>P2X1>P2Y(2)>P2Y(4)>P2X6>P2X5>P2X7>P2X3>P2Y(14)>P2Y(13)>P2Y(12). Increased intracellular Ca(2+) levels following stimulation with P2X-selective agonists indicated the presence of functional receptors. To investigate whether P2X receptors might also regulate bone formation, osteoblasts were cultured for 14days with P2X receptor agonists. The P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP inhibited bone mineralisation by 70% and 90%, respectively at 1μM, with complete abolition at ≥25μM; collagen production was unaffected. Bz-ATP, a P2X7 receptor agonist, reduced bone mineralisation by 70% and 99% at 10μM and 100μM, respectively. Osteoblast alkaline phosphatase activity was similarly inhibited by these agonists, whilst ecto-nucleotide pyrophosphatase/phosphodiesterase activity was increased. The effects of α,β-meATP and Bz-ATP were attenuated by antagonists selective for the P2X1 and P2X7 receptors, respectively. Our results show that normal osteoblasts express functional P2X receptors and that the P2X1 and P2X7 receptors negatively regulate bone mineralisation.
细胞外核苷酸通过 P2 受体信号传导，调节成骨细胞和破骨细胞的功能。已知成骨细胞表达多种 P2 受体亚型（P2X2、5、7 和 P2Y(1)、(2,4,6)），其水平在分化过程中发生变化。ATP 和 UTP 在体外强烈抑制骨矿化，这种作用至少部分通过 P2Y(2)受体介导。我们在这里报告，原代大鼠成骨细胞表达额外的、功能性的 P2 受体（P2X1、P2X3、P2X4、P2X6、P2Y(12)、P2Y(13)和 P2Y(14)）。受体表达随细胞分化而变化：例如，成熟的成骨细胞中 P2X4 受体 mRNA 水平比不成熟的增殖细胞高 5 倍。成熟成骨细胞中 P2 受体 mRNA 的表达顺序为 P2X4>P2Y(1)>P2X2>P2Y(6)>P2X1>P2Y(2)>P2Y(4)>P2X6>P2X5>P2X7>P2X3>P2Y(14)>P2Y(13)>P2Y(12)。用 P2X 选择性激动剂刺激后细胞内 Ca(2+)水平升高表明存在功能性受体。为了研究 P2X 受体是否也可以调节骨形成，用 P2X 受体激动剂培养成骨细胞 14 天。P2X1 和 P2X3 受体激动剂，α,β-meATP 和 β,γ-meATP，在 1μM 时分别抑制骨矿化 70%和 90%，在≥25μM 时完全抑制；胶原生成不受影响。Bz-ATP，一种 P2X7 受体激动剂，在 10μM 和 100μM 时分别减少骨矿化 70%和 99%。这些激动剂也抑制成骨细胞碱性磷酸酶活性，同时增加外核苷酸焦磷酸酶/磷酸二酯酶活性。α,β-meATP 和 Bz-ATP 的作用分别被 P2X1 和 P2X7 受体选择性拮抗剂减弱。我们的结果表明，正常成骨细胞表达功能性 P2X 受体，P2X1 和 P2X7 受体负调节骨矿化。
