1,25-dihydroxyvitamin D3 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-alpha (PKC-alpha) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3] and 12-O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60(c-src) have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3 or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60(c-src) and treatment with PP1, a specific inhibitor of the pp60(c-src) family, blocked the ability of 1,25(OH)2D3 to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60(c-src) and PKC-alpha by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3 or inhibit the stimulation of PKC-alpha by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3 and TPA each activate PLD in Caco-2 cells in part via PKC-alpha, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60(c-src) and RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3 and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.