Prostaglandin F-2 alpha stimulates tyrosine phosphorylation and mitogen-activated protein kinase in osteoblastic MC3T3-E1 cells via protein kinase C activation

PGF(2 alpha) stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF(2 alpha) receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF(2 alpha) on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF(2 alpha) rapidly raised the level of phosphotyrosine of cellular proteins with M-r values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF(2 alpha)-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF(2 alpha) also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF(2 alpha) augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Rat-1 and was little affected by PGF(2 alpha). Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF(2 alpha), indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-in dependent kinase cascade for activation of MAPK by PGF(2 alpha). Fluprostenol, a specific agonist of PGF(2 alpha) receptor, mimicked the actions of PGF(2 alpha) consistent with a PGF(2 alpha) receptor pathway. Thus, the action of PGF(2 alpha) on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.