Evaluation of signal transduction mechanisms for the mitogenic effects of prostaglandin E₂ in normal human bone cells in vitro

Prostaglandin E₂ (PGE₂) is one of the most potent stimulators of bone formation in vivo. In these studies, we investigated the mechanism(s) underlying PGE₂ effects on human bone formation by evaluating the effects of PGE₂ on normal human bone cell (HBC) proliferation in vitro. Cell proliferation of normal HBCs was increased by PGE₂ as measured by increased [³H]thymidine incorporation after 18 h and increased cell number after 48 h of treatment. The effect of PGE₂ to stimulate cell proliferation was biphasic, with a maximum stimulation between 0.01 and 1.0 nM PGE₂ in different experiments. At higher concentrations of PGE₂ (0.1 μM), HBC proliferation was inhibited. Signal transduction for PGF₂ has been reported to include both protein kinase A (PKA) and protein kinase C (PKC) pathways. In these studies, concentrations of PGE₂ which stimulated cell proliferation did not increase cyclic adenosine monophosphate (cAMP) production. However, higher concentrations of PGE₂ increased cAMP production (7- to 12-fold at 1- 10 μM) and inhibited cell proliferation. Because stimulators of PKC, such as phorbol esters, have been reported to stimulate cell proliferation, the action of PKC inhibitors were tested. Both staurosporine and sangivamysin (PKC inhibitors) totally abrogated the effect of PGE₂ to stimulate cell proliferation. Additional studies revealed that PGE₂ increased ⁴⁵Ca uptake in a dose-dependent manner with a peak response occuring between 1 and 10 nM PGE₂ concentrations in different experiments. Furthermore, when the calcium channel blocker, verapamil, was added to HBC cultures treated with PGE₂, the stimulation of ⁴⁵Ca uptake and cell proliferation by PGE₂ was completely blocked. These data suggest that PGE₂ increases cell proliferation through activation of a verapamil-sensitive calcium channel. In conclusion, these data are consistent with a model in which stimulation of HBC proliferation by low doses of PGE₂ is mediated by an enhancement of phospholipase C, which results in both an increase in PKC activity and an increase in intracellular calcium influx.