PROSTAGLANDIN-F2-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 activati on. To elucidate intracellular events elicited by this receptor, we ex amined the effects of PGF(2 alpha) on tyrosine phosphorylation and mit ogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF(2 alpha) rapidly raised the level of phosphotyrosine of cellular protei ns 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-tetrade canoylphorbol 13-acetate pretreatment and by GF 109203X, a potent spec ific PKC inhibitor. The addition of PGF(2 alpha) also transiently acti vated MAPK in the same range of concentrations that stimulated tyrosin e phosphorylation. In addition, PGF(2 alpha) augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellula r signal-regulated protein kinase kinase was less than that of Rat-1 a nd was little affected by PGF(2 alpha). Like the tyrosine phosphorylat ion, these activations of Raf-1 and MAPK activities were reduced by in hibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF(2 alpha), ind icating a tyrosine kinase-independent pathway for Raf-1 activation. Ho wever, the tyrosine kinase inhibitor partially inhibited the MAPK acti vity, suggesting an involvement of another Raf-1-in dependent kinase c ascade for activation of MAPK by PGF(2 alpha). Fluprostenol, a specifi c agonist of PGF(2 alpha) receptor, mimicked the actions of PGF(2 alph a) consistent with a PGF(2 alpha) receptor pathway. Thus, the action o f PGF(2 alpha) on osteoblastic MC3T3-E1 cells appears to involve a sin gle receptor that uses diverse interacting signal transduction systems .