Abstract
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.
Highlights
The tumor promoter, TPA,1 stimulates PC turnover in Madin-Darby canine kidney (MDCK) cells, resulting in the generation of DG, the major endogenous activator of PKC [1]
When we used ethanol to block phosphatidic acid (PA) and DG formation by phospholipase D (PLD), TPA (10 nM)-induced PEt formation increased with increasing concentrations of ethanol (0 –2%, v/v), while the formation of TPA-induced prostaglandin E2 (PGE2) was inversely proportional to the concentration of ethanol (Fig. 1)
It is possible that PA generated directly from PLD serves as a second messenger in cellular signaling
Summary
The tumor promoter, TPA,1 stimulates PC turnover in MDCK cells, resulting in the generation of DG, the major endogenous activator of PKC [1]. We report that the inhibition of PA and DG synthesis by ethanol blocks TPA-induced PGE2 production in MDCK cells. MDCK cells were stimulated with TPA (10 nM), and the media was removed and replaced with fresh completed-DMEM containing ethanol (1%) for a 2-h pulse prior to harvesting at the indicated times (Fig. 3). In order to determine if ethanol is acting on a specific signaling pathway, we compared ethanol’s effect on 20:4 release and PGE2 production induced by the calcium ionophore, A23187, or TPA (Fig. 5).
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