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J.Health Sci., 50(1), 47-57, 2004
Mechanism Underlying the Aluminum-Induced Stimulation of Bone Nodule Formation by Rat
Calvarial Osteoblasts
Hiroyuki Kaneki,*, a Keiko
Ishibashi,a Minoru
Kurokawa,b Masaki
Fujieda,a Michiaki
Kiriu,a Shigeki
Mizuochi,a and Hayao Idea
aDepartment of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba
274-8510, Japan and bDepartment of Pharmacy, Omori Hospital, Faculty of Medicine, Toho University, 5-21-16 Omorinishi, Ota-ku,
Tokyo 143-8540, Japan
The signal transduction mechanism for aluminum
(Al3+)-induced stimulation of bone formation and its
crosstalk with the prostaglandin E2
(PGE2) 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
Al3+ on the differentiation markers were abolished by the pretreatment of
the cells with pertussis toxin (PTX), an inhibitor of
Gi protein, indicating that the effects of
Al3+ are mediated through a receptor coupled with
Gi protein. Al3+ increased inositol-1,4,5-triphosphate
(IP3) production and intracellular concentration of
Ca2+
([Ca2+]i) in the cultures of MOB and AOB: these effects were not observed in the
presence of PTX, indicating that the effects of
Al3+ are mediated through the activation of
phosphatidylinositol-specific phospholipase C (PI-PLC). We have previously shown that
17-phenyl-omega-trinor-PGE2, a selective
agonist for an EP1 subtype of
PGE2 receptor (EP1), 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
EP1. The levels of the differentiation markers obtained in the presence of the
EP1 agonist were increased by the addition of
Al3+ in the cultures of MOB and AOB, while
Al3+ increased the levels of
IP3 production and
[Ca2+]i in the presence of the
EP1 agonist only in the cultures of AOB. These results indicate a possibility that PI-PLC molecules stimulated by the signal
through Gi protein and those stimulated by the signal through
EP1 belong to the same pool and that the
Al3+ signal through Gi 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-PGE1, a selective agonist for an
EP2/EP4 subtype of
PGE2 receptor
(EP2/EP4), inhibits cell differentiation in the cultures of both MOB and AOB.
Al3+ had no effect on the basal levels of cAMP production, but the levels induced by the
EP2/EP4 agonist were dose-dependently
reduced by the treatment with Al3+ at a concentration range of
10-7-10-5 M. The inhibitory effect of
Al3+ on adenylyl cyclase was abolished by the pretreatment with PTX. These results indicate that
Al3+ suppresses adenylyl cyclase activity induced by the
EP2/EP4-mediated signal through the
Gi protein-coupled receptor.
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