The signal transduction mechanism for aluminum (Al
3+)-induced stimulation of bone formation and its crosstalk with the prostaglandin E
2 (PGE
2) signaling pathway were studied in calvarial osteoblasts from 25-week-old rats (MOB) and those from 90-week-old rats (AOB). Alkaline phosphatase activity, the rate of [
3H]proline incorporation into collagenase-digestible proteins, the total area and number of mineralized bone nodules (BN) and the content of calcium in BN, which are the markers for differentiation of osteoblasts, were dose–dependently stimulated by the treatment with Al3+ at a concentration range of 10
-7-10
-5 M in the cultures of both MOB and AOB. The stimulatory effects of Al
3+ on the differentiation markers were abolished by the pretreatment of the cells with pertussis toxin (PTX), an inhibitor of G
i protein, indicating that the effects of Al
3+ are mediated through a receptor coupled with G
i protein. Al
3+ increased inositol-1,4,5-triphosphate (IP
3) production and intracellular concentration of Ca
2+ ([Ca
2+]
i) in the cultures of MOB and AOB: these effects were not observed in the presence of PTX, indicating that the effects of Al
3+ are mediated through the activation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have previously shown that 17-phenyl-
ω-trinor-PGE
2, a selective agonist for an EP
1 subtype of PGE
2 receptor (EP
1), stimulates the differentiation markers in the cultures of MOB through the activation of PI-PLC, but not in those of AOB because of the lack of EP
1. The levels of the differentiation markers obtained in the presence of the EP
1 agonist were increased by the addition of Al
3+ in the cultures of MOB and AOB, while Al
3+ increased the levels of IP
3 production and [Ca
2+]
i in the presence of the EP
1 agonist only in the cultures of AOB. These results indicate a possibility that PI-PLC molecules stimulated by the signal through G
i protein and those stimulated by the signal through EP
1 belong to the same pool and that the Al
3+ signal through G
i protein induces cell differentiation via a pathway(s) independent of PI-PLC in addition to that (those) dependent on the PI-PLC. We have also shown that 11-deoxy-PGE
1, a selective agonist for an EP
2/EP
4 subtype of PGE
2 receptor (EP
2/EP
4), inhibits cell differentiation in the cultures of both MOB and AOB. Al
3+ had no effect on the basal levels of cAMP production, but the levels induced by the EP
2/EP
4 agonist were dose–dependently reduced by the treatment with Al
3+ at a concentration range of 10
-7-10
-5 M. The inhibitory effect of Al
3+ on adenylyl cyclase was abolished by the pretreatment with PTX. These results indicate that Al
3+ suppresses adenylyl cyclase activity induced by the EP
2/EP
4-mediated signal through the G
i protein-coupled receptor.
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