Involvement of Rho family proteins in prostaglandin F-2 alpha-induced phospholipase 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 difficile, which inhibits Rho family proteins by monoglucosylation. Pretreatment of [H-3]myristic acid-labeled MC3T3-E1 cells with Toxin B induced rounding-up of the cells and inhibited the PGF(2 alpha)-induced PLD activation by 60%, but not the phospholipase C (PLC) activation. Cytochalasin D also induced rounding the cells, but showed a small inhibition in the PLD activation. Brefeldin A (BFA) had marginal inhibitory effect on the PGF(2 alpha)-induced PLD activation. In digitonin-permeabilized 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 its full PLD activation. Pretreatment of the digitonin-permeabilized MC3T3-E1 cells with Toxin B reduced the GTP gamma S- and PMA-stimulated PLD activities by 80% and 60%, respectively. On the other hand, C3 toxin which inhibits Rho by ADP-ribosylation, exerted a partial inhibitory effect on the GTP gamma S-stimulated PLD activity. These results suggest that Cdc42 as well as RhoA appear to be involved in the PLD activation mediated by PGF(2 alpha) and also that the PLD activation may be independent of actin cytoskeIeton in MC3T3-E1 cells.