Phospholipase D-derived products in the regulation of 12-O-tetradecanoylphorbol-13-acetate-stimulated prostaglandin synthesis in madin-darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of ethanol synthesize phosphatidylethanol (PEt) instead of phosphatidic acid (PA) and diglyceride (DG). We have used ethanol to block the production of phospholipase D (PLD)-derived PA and DG (from PA hydrolysis) to study their role in signal transduction. In MDCK cells, TPA-stimulated prostaglandin E2 (PGE2) synthesis was inhibited by ethanol at concentrations which inhibit PA and DG formation. In addition, TPA elicited a prolonged increase in PGE2 synthesis that is dependent upon continuous activation of PLD. The TPA-stimulated translocation of protein kinase Calpha (PKCalpha) from cytosol to membrane was unaffected by ethanol. This suggests that PLD-derived products act downstream of PKC in TPA-stimulated prostaglandin synthesis. The calcium ionophore, A23187, did not activate PLD, and PGE2 synthesis in response to A23187 was unaffected by ethanol. TPA increased prostaglandin endoperoxide H synthase (PGHS) activity and increased the amount of immunodetectable prostaglandin endoperoxide H synthase 2 (PGHS-2). A23187 did not induce PGHS-2 and A23187-stimulated PGE2 synthesis appears to be due to the constitutively expressed PGHS-1. Blocking the formation of PLD-derived products, PA and DG, inhibited the induction of PGHS-2 by TPA. These results indicate that prolonged PGE2 synthesis in response to TPA is due to the continuous induction of PGHS-2, which is dependent upon PLD activation. In contrast, induction of PGHS-2 by epidermal growth factor was not affected by ethanol. Epidermal growth factor did not induce PKCalpha translocation nor activate PLD. Taken together, these data suggest that PLD-derived PA or DG act as second messengers in the induction of PGHS-2 by PKC-dependent pathways. The demonstration that inhibition of TPA-induced PA formation inhibits Raf-1 translocation in MDCK cells (Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L. W. , and Bell, R. M. (1996) J. Biol. Chem. 271, 8472-8480) suggests that PA is the active PLD metabolite in TPA-stimulated signaling.