1,25-dihydroxyvitamin D-3 but not TPA activates PLD in Caco-2 cells via pp60(c-src) and RhoA

In the accompanying paper [Khare et al., Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C-alph a (PKC-alpha) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] and 12-O-tetradecanoy lphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G pr oteins, as well as pp60(c-src) have been implicated in PLD activation. We t herefore determined whether these signal transduction elements were involve d in PLD stimulation by 1,25(OH)(2)D-3 or TPA. Treatment with C3 transferas e, which inhibits members of the Rho family of monomeric G proteins, marked ly diminished the ability of 1,25(OH)(2)D-3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, ho wever, significantly reduce the stimulation of PLD by either of these agent s. Moreover, 1,25(OH)(2)D-3, but not TPA, activated pp60(c-src) and treatme nt with PP1, a specific inhibitor of the pp60(c-src) family, blocked the ab ility of 1,25(OH)(2)D-3 to activate PLD. Pretreatment of cells with pertuss is toxin (PTx) markedly reduced the stimulation of PLD by either agonist. P Tx, moreover, inhibited the stimulation of pp60(c-src) and PKC-alpha by 1,2 5(OH)(2)D-3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)(2)D-3 or inhibit the stimulation of PKC-alpha by TPA. These findings, taken together with those of the accompanying paper, indica te that although 1,25(OH)(2)D-3 and TPA each activate PLD in Caco-2 cells i n part via PKC-alpha, their stimulation of PLD differs in a number of impor tant aspects, including the requirement for pp60(c-src) and RhoA in the act ivation of PLD by 1,25(OH)(2)D-3, but not TPA. Moreover, the requirement fo r different signal transduction elements by 1,25(OH)(2)D-3 and TPA to induc e the stimulation of PLD may potentially underlie differences in the physio logical effects of these agents in Caco-2 cells.