Involvement of Rho Family Proteins in Prostaglandin F2α-Induced Phospholipase D Activation in the Osteoblast-like Cell Line MC3T3-E1

Abstract

To examine the role of Rho family proteins in prostaglandin F_2α (PGF_2α)-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 [³H]myristic acid-labeled MC3T3-E1 cells with Toxin B induced rounding-up of the cells and inhibited the PGF_2α-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_2α-induced PLD activation. In digitonin-permeabilized MC3T3-E1 cells, [³H]PBut formation was stimulated by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or 4β-phorbol 12-myristate 13-acetate (PMA) in the presence of Ca²⁺ (1 μM) 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 GTPγS- 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 GTPγS-stimulated PLD activity. These results suggest that Cdc42 as well as RhoA appear to be involved in the PLD activation mediated by PGF_2α and also that the PLD activation may be independent of actin cytoskeleton in MC3T3-E1 cells.