INVOLVEMENT OF RHO-FAMILY PROTEINS IN PROSTAGLANDIN-F-2-ALPHA-INDUCEDPHOSPHOLIPASE-D ACTIVATION IN THE OSTEOBLAST-LIKE CELL-LINE-MC3T3-E1

To examine the role of Rho family proteins in prostaglandin F-2 alpha (PGF(2 alpha))-mediated phospholipase D (PLD) activation of osteoblast -like cell line MC3T3-E1 cells, we used Toxin B from Clostridium diffi cile, which inhibits Rho family proteins by monoglucosylation. Pretrea tment of [H-3]myristic acid-labeled MC3T3-E1 cells with Toxin B induce d rounding-up of the cells and inhibited the PGF(2 alpha)-induced PLD activation by 60%, but not the phospholipase C (PLC) activation. Cytoc halasin D also induced rounding the cells, but showed a small inhibiti on in the PLD activation. Brefeldin A (BFA) had marginal inhibitory ef fect on the PGF(2 alpha)-induced PLD activation. In digitonin-permeabi lized MC3T3-E1 cells, [H-3]PBut formation was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or 4 beta-phorbol 12-myristate 13-acetate (PMA) in the presence of Ca2+ (1 mu M) and ATP (1 mM), and phosphatidylinositol 4,5-bisphosphate (PIP2) was also required for it s full PLD activation. Pretreatment of the digitonin-permeabilized MC3 T3-E1 cells with Toxin B reduced the GTP gamma S- and PMA-stimulated P LD activities by 80% and 60%, respectively. On the other hand, C3 toxi n which inhibits Rho by ADP-ribosylation, exerted a partial inhibitory effect on the GTP gamma S-stimulated PLD activity. These results sugg est that Cdc42 as well as RhoA appear to be involved in the PLD activa tion mediated by PGF(2 alpha) and also that the PLD activation may be independent of actin cytoskeIeton in MC3T3-E1 cells.