Function of Ca²⁺ influx in phospholipase D activation induced by prostaglandin F_2α in osteoblast-like cells: Involvement of tyrosine kinase

We previously reported that prostaglandin F_2α (PGF_2α) induces Ca²⁺ influx from the extracellular space via protein tyrosine kinase in osteoblast-like MC3T3-E1 cells and that PGF_2α stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells (6, 12). In this study, we examined the relationship between the tyrosine kinase-regulated Ca²⁺ influx by PGF_2α and the activation of phospholipase D in MC3T3-E1 cells. The depletion of extracellular Ca²⁺ by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) markedly reduced the PGF_2α-induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by PGF_2α in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also suppressed the PGF_2α-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the PGF_2α-induced formation of choline. These results strongly suggest that the phospholipase D activation by PGF_2α is dependent on extracellular Ca²⁺ in osteoblast-like cells and that protein tyrosine kinase is involved in the activation of phospholipase D.