EFFECTS OF CLOSTRIDIUM-DIFFICILE TOXIN-A AND TOXIN-B ON PHOSPHOLIPASE-D ACTIVATION IN HUMAN PROMYELOCYTIC LEUKEMIC HL60 CELLS

The possible involvement of Rho family GTP-binding proteins in the reg ulation of phospholipase D (PLD) activity has recently been demonstrat ed. In the present study, to further examine the role of Rho family pr oteins in PLD activation of human promyelocytic leukemic HL60 cells, w e used toxin B and toxin B from the anaerobic bacterium Clostridium di fficile, which was shown to glucosylate Rho family proteins and inhibi t their interaction with effecters. Pretreatment of [H-3] oleic acid-l abeled HL60 cell lysates with either one of the toxins resulted in a r emarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). The magnitude of inhibition o f PLD activity was correlated,yell with the extent of toxin A- or B-in duced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more e ffective than toxin A. GTP gamma S-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisph osphate was also inhibited by toxin B. Toxin B had no effect on GTP ga mma S-induced translocation of RhoA from cytosol to membranes. Further more, the toxin B pretreatment also suppressed PLD activation induced by 4 beta-phorbol 12-myristate 13-acetate in HL60 cell lysates. Thus, it was indicated that Rho family proteins play a key role in GTP gamma S- and 4 beta-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells. In addition, the results obtained here indicate that C. d ifficile toxins are a useful tool for researching the regulation of th e Rho family protein-mediated PLD activation and also provide a clue t oward understanding the pathogenic background of pseudomembranous coli tis from the viewpoint of signal transduction.