Quarles L D, Hartle J E, Middleton J P, Zhang J, Arthur J M, Raymond J R
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Cell Biochem. 1994 Sep;56(1):106-17. doi: 10.1002/jcb.240560115.
Aluminum (Al3+) stimulates de novo bone formation in dogs and is a potent stimulus for DNA synthesis in non-transformed osteoblasts in vitro. The recent identification of a G-protein coupled cation-sensing receptor (BoPCaR), which is activated by polyvalent agonists [e.g., gadolinium (Gd3+) > neomycin > calcium (Ca2+)], suggests that a similar physiologically important cation sensing receptor may be present in osteoblasts and pharmacologically activated by Al3+. To evaluate that possibility, we assessed whether known BoPCaR agonists stimulate DNA synthesis in MC3T3-E1 osteoblasts and examined the additive effects of Al3+ and BoPCaR agonists on DNA synthesis in MC3T3-E1 osteoblast-like cells. We found that Al3+, Gd3+, neomycin, and Ca2+ stimulated DNA synthesis in a dose-dependent fashion, achieving 50% effective extracellular concentrations (EC50) of 10 microM, 30 microM, 60 microM, and 2.5 mM, respectively. Al3+ displayed non-additive effects on DNA synthesis with the BoPCaR agonists as well as an unrelated G-protein coupled receptor agonist, PGF2 alpha, suggesting shared mechanisms of action. In contrast, the receptor tyrosine kinase agonist, IGF-I (10 eta g/ml), displayed additive proliferative effects when combined with AlCl3, indicating distinct signalling pathways. AlCl3 (25 microM) induced DAG levels 2-fold and the phosphorylation of the myristoylated alanine-rich C kinase (MARCKS) substrate 4-fold, but did not increase intracellular calcium concentrations. Down-regulation of PKC by pre-treatment with phorbol 12-myristate 13-acetate as well as PKC inhibition by H-7 and staurosporine blocked Al(3+)-induced DNA synthesis. Finally, Al3+, Gd3+, neomycin, and Ca2+ activated G-proteins in osteoblast membranes as evidenced by increased covalent binding of [32P]-GTP-azidoanilide to putative G alpha subunits. Our findings suggest that Al3+ stimulates DNA synthesis in osteoblasts through a cation sensing mechanism coupled to G-protein activation and signalling cascades involving DAG and PKC-dependent pathways.
铝离子（Al3+）可刺激犬类的骨新生，并且在体外对未转化的成骨细胞中的DNA合成是一种强效刺激物。最近鉴定出一种G蛋白偶联阳离子传感受体（BoPCaR），它可被多价激动剂激活（例如，钆离子（Gd3+）>新霉素>钙离子（Ca2+）），这表明成骨细胞中可能存在类似的具有重要生理意义的阳离子传感受体，并且Al3+可通过药理学方式激活该受体。为了评估这种可能性，我们检测了已知的BoPCaR激动剂是否能刺激MC3T3-E1成骨细胞中的DNA合成，并研究了Al3+和BoPCaR激动剂对MC3T3-E1成骨样细胞中DNA合成的叠加效应。我们发现Al3+、Gd3+、新霉素和Ca2+均以剂量依赖性方式刺激DNA合成，其50%有效细胞外浓度（EC50）分别为10微摩尔、30微摩尔、60微摩尔和2.5毫摩尔。Al3+与BoPCaR激动剂以及一种不相关的G蛋白偶联受体激动剂前列腺素F2α（PGF2α）对DNA合成均无叠加效应，这表明它们具有共同的作用机制。相比之下，受体酪氨酸激酶激动剂胰岛素样生长因子-I（IGF-I，10微克/毫升）与AlCl3联合使用时表现出叠加的增殖效应，表明存在不同的信号通路。AlCl3（25微摩尔）使二酰基甘油（DAG）水平升高2倍，使富含豆蔻酰化丙氨酸的蛋白激酶C（PKC）底物的磷酸化水平升高4倍，但并未增加细胞内钙离子浓度。用佛波醇12-肉豆蔻酸酯13-乙酸酯预处理下调PKC以及用H-7和星形孢菌素抑制PKC均可阻断Al(III)诱导的DNA合成。最后，Al3+、Gd3+、新霉素和Ca2+激活了成骨细胞膜中的G蛋白，这可通过[32P]-GTP-叠氮苯胺与假定的Gα亚基的共价结合增加来证明。我们的研究结果表明，Al3+通过与G蛋白激活以及涉及DAG和PKC依赖性途径的信号级联相关的阳离子传感机制刺激成骨细胞中的DNA合成。
