Prostaglandin E-2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E-2 (PGE(2)) on the proliferation and differen tiation of osteoblastic cells were studied in osteoblast-like cells isolate d from adult rat calvaria. Treatment of the cells with PGE(2) within the co ncentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [H-3]proline incorporation into collag enase-digestible protein, and mineralized bone nodule (BN) formation, as we ll as a dose-dependent decrease in [H-3]thymidine incorporation into the ce lls. PGE(2) also caused a dose-dependent increase in the intracellular cycl ic adenosine monophosphate (cAMP) content, with a maximal effective concent ration of 10(-5) M; this effect of PGE(2) was mimicked by forskolin, an ade nylate cyclase activator. The treatment of adult calvarial cells with forsk olin decreased BN formation, ALP activity, and collagen synthesis. These re sults suggested that cAMP does not have a stimulatory, but rather a suppres sive, effect on the differentiation of adult rat calvarial cells. A time-co urse study of cAMP accumulation showed that both PGE(2)- and forskolin-indu ced cAMP reached a maximum at 5 min after the treatment, but the former rap idly returned to the basal level by 40 min, while the latter declined slowl y and was still at 70% of the maximal level at 60 min, suggesting that PGE( 2) activates phosphodiesterase as well as adenylate cyclase. The presence o f N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin an tagonist, reduced the rate of degradation of cAMP formed after PGE(2) treat ment, suggesting the involvement of calmodulin in the activation of phospho diesterase. However, PGE(2) also caused the production of inositol 1,4,5-tr iphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ( [Ca2+](i)), both of which peaked at 15 s and returned to the basal level wi thin 1 min. Submaximal responses of the IP3 production and the [Ca2+](i) el evation to PGE(2) were obtained at 10(-5) M. W-7 decreased both basal and P GE(2)-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE(2)- induced differentiation of calvarial cells. From these results, we conclude d that PGE(2) inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+](i) through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyc lase. We also found that BN formation varies, depending on the lime of PGE( 2) addition, suggesting that responsiveness of the cells to PGE(2) may chan ge during the culture period. J. Cell. Biochem. 73:36-48, 1999. (C) 1999 Wi ley-Liss, Inc.