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 pot ent stimulus for DNA synthesis in non-transformed osteoblasts in vitro . The recent identification of a G-protein coupled cation-sensing rece ptor (BoPCaR), which is activated by polyvalent agonists [e.g., gadoli nium (Gd3+) > neomycin > calcium (Ca2+)], suggests that a similar phys iologically important cation sensing receptor may be present in osteob lasts and pharmacologically activated by Al3+. To evaluate that possib ility, we assessed whether known BoPCaR agonists stimulate DNA synthes is in MC3T3-E1 osteoblasts and examined the additive effects of Al3+ a nd 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 co ncentrations (EC(50)) Of 10 mu M, 30 mu M, 60 mu M, and 2.5 mM, respec tively. Al3+ displayed non-additive effects on DNA synthesis with the BoPCaR agonists as well as an unrelated G-protein coupled receptor ago nist, PGF(2 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 d istinct signalling pathways. AlCl3 (25 mu M) induced DAG levels 2-fold and the phosphorylation of the myristoylated alanine-rich C kinase (M ARCKS) substrate 4-fold, but did not increase intracellular calcium co ncentrations. Down-regulation of PKC by pre-treatment with phorbol 12- myristate 13-acetate as well as PKC inhibition by H-7 and staurosporin e blocked Al3+-induced DNA synthesis. Finally, Al3+, Gd3+, neomycin, a nd Ca2+ activated G-proteins in osteoblast membranes as evidenced by i ncreased covalent binding of [P-32]-CTP-azidoanilide to putative Got s ubunits. Our findings suggest that Al3+ stimulates DNA synthesis in os teoblasts through a cation sensing mechanism coupled to G-protein acti vation and signalling cascades involving DAG and PKC-dependent pathway s, (C) 1994 Wiley-Liss, Inc.