Aluminum-induced DNA synthesis in osteoblasts: mediation by a G-protein coupled cation sensing mechanism

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.