EFFECT OF PHORBOL-MYRISTATE ACETATE ON RELEASE OF ARACHIDONIC-ACID AND ITS METABOLITES IN THE OSTEOBLASTIC MOB 3-4 CELL-LINE AND ITS SUBCLONE, MOB 3-4-F2

We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4-alpha-phorbol 12, 13-didecanoate (4-alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater-than-or-equal-to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200-mu-M), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca2+-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells. Taken together with Ca2+-dependence of PLA2, these findings suggest that the PMA-induced release of AA and its metabolites in both MOB 3-4 and MOB 3-4-F2 cells is due to activation of PLA2 by both a staurosporine-sensitive mechanism, probably an activation of PKC, and a staurosporine-insensitive mechanism. In addition, the comparison between the MOB 3-4 cell line and its subclone, MOB 3-4-F2, may provide a useful system for studying the physiological or pathological role of PKC in bone-derived cells.