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.
Quarles
L D
LD
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Hartle
J E
JE
Middleton
J P
JP
Zhang
J
J
Arthur
J M
JM
Raymond
J R
JR
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D016475:3T3 Cells; D000470:Alkaloids / Q000494:pharmacology; D000535:Aluminum / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002999:Clone Cells; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D019852:Diacylglycerol Kinase; D004285:Dogs; D019204:GTP-Binding Proteins / Q000502:physiology*; D005682:Gadolinium / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009355:Neomycin / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000502:physiology*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D006133:Growth Substances; D047908:Intracellular Signaling Peptides and Proteins; D007546:Isoquinolines; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D010879:Piperazines; D011506:Proteins; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004247:DNA; D005682:Gadolinium; D000535:Aluminum; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D019311:Staurosporine; D009355:Neomycin; D013755:Tetradecanoylphorbol Acetate; C038958:gadolinium chloride; D002118:Calcium