EVALUATION OF SIGNAL-TRANSDUCTION MECHANISMS FOR THE MITOGENIC EFFECTS OF PROSTAGLANDIN E(2) IN NORMAL HUMAN BONE-CELLS IN-VITRO

Prostaglandin E(2) (PGE(2)) is one of the most potent stimulators of b one formation in vivo. In these studies, we investigated the mechanism (s) underlying PGE(2) effects on human bone formation by evaluating th e effects of PGE(2) on normal human bone cell (HBC) proliferation in v itro. Cell proliferation of normal HBCs was increased by PGE(2) as mea sured by increased [H-3]thymidine incorporation after 18 h and increas ed cell number after 48 h of treatment. The effect of PGE(2) to stimul ate cell proliferation was biphasic, with a maximum stimulation betwee n 0.01 and 1.0 nM PGE(2) in different experiments. At higher concentra tions of PGE(2) (0.1 mu M), HBC proliferation was inhibited. Signal tr ansduction for PGE(2) has been reported to include both protein kinase A (PKA) and protein kinase C (PKC) pathways. In these studies, concen trations of PGE(2) which stimulated cell proliferation did not increas e cyclic adenosine monophosphate (cAMP) production. However, higher co ncentrations of PGE(2) increased cAMP production (7- to 12-fold at 1-1 0 mu M) and inhibited cell proliferation. Because stimulators of PKC, such as phorbol esters, have been reported to stimulate cell prolifera tion, the action of PKC inhibitors were tested. Both staurosporine and sangivamysin (PKC inhibitors) totally abrogated the effect of PGE(2) to stimulate cell proliferation. Additional studies revealed that PGE( 2) increased Ca-45 uptake in a dose-dependent manner with a peak respo nse occuring between 1 and 10 nM PGE(2) concentrations in different ex periments. Furthermore, when the calcium channel blocker, verapamil, w as added to HBC cultures treated with PGE(2), the stimulation of Ca-45 uptake and cell proliferation by PGE(2) was completely blocked. These data suggest that PGE(2) increases cell proliferation through activat ion of a verapamil-sensitive calcium channel. In conclusion, these dat a are consistent with a model in which stimulation of HBC proliferatio n by low doses of PGE(2) is mediated by an enhancement of phospholipas e C, which results in both an increase in PKC activity and an increase in intracellular calcium influx.