Effect of prostaglandin E₂ on phospholipase D activity in osteoblast‐like MC3T3‐E1 cells

Recent evidence indicates that phosphatidylcholine breakdown by phospholipase D (PLD) is an important cellular control mechanism. We investigated the signaling pathway participating in prostaglandin E₂ (PGE₂)–induced PLD activation in osteoblast‐like MC3T3‐E1 cells. PGE₂ stimulated PLD activity, as measured by choline generated from phosphatidylcholine, just after the stimulation. The reaction reached a plateau 15 minutes later. PGE₂ stimulated PLD activity in a dose‐related manner and also increased inositol phosphate (IP) formation. However, the EC₅₀ value for PGE₂‐induced IP formation is lower than that for PLD activation. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator, stimulated PLD activity, and a combination of PGE₂ and TPA potentiated it in an additive manner. Although NaF, a heterotrimeric GTP‐binding protein activator, significantly stimulated PLD activity, this effect was not augmented by combination with PGE₂. PGE₂‐induced PLD activity was markedly suppressed by either chelating extracellular Ca²⁺ by EGTA or pertussis toxin. These findings suggest that osteoblasts might have at least two PLD activation mechanisms which involve PKC‐dependent or ‐independent pathways. However, present results indicate that PKC is unlikely to be essential to PGE₂‐induced PLD activation. On the contrary, pertussis toxin‐sensitive GTP‐binding protein and extracellular Ca²⁺ might play important roles in the pathway of PGE₂‐induced PLD activation.