PHORBOL ESTER STIMULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS REQUIRES EXPRESSION OF BOTH PROTEIN-KINASE-C-ALPHA AND PHOSPHOLIPASE-D

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate ( PMA) stimulates both the synthesis and phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho). Here, attached and suspend ed NIH 3T3 fibroblasts as well as variants of the MCF-7 human breast c arcinoma cell line expressing PKC-alpha and a PtdCho-specific PLD acti vity at widely different levels were used to determine the possible ro le of PKC-alpha, PtdCho hydrolysis, and choline uptake in the mediatio n of PMA effect on PtdCho synthesis. In wild-type MCF-7 cells, which e xpress both PKC-alpha and PLD activities at very low levels, PMA had l ittle effects on the uptake or incorporation [C-14]choline into PtdCho . In multidrug resistant MCF-7/MDR1 cells, which highly express PKC-al pha but lack the PtdCho-specific PLD activity, 100-nM PMA had relative ly small stimulatory effects on the uptake of [C-14]choline(similar to 1.5-fold) and [C-14]PtdCho synthesis (1.5- to 2-fold). In NIH 3T3 fib roblasts and MCF-7/PKC-alpha cells, both expressing PKC-alpha and PLD activities at high levels, 10-100-nM PMA enhanced [C-14]choline uptake only slightly (1.7- to 2.2-fold), while it had much greater (similar to 4-9-fold) stimulatory effects on PtdCho synthesis. PMA significantl y enhanced the formation of phosphatidic acid (PtdOH) in MCF-7/PKC-alp ha cells (2.8-fold increase), but not in MCF-7/MDR1 cells (1.4-fold in crease), while in both cell lines it had only small (1.3-1.5-fold) sti mulatory effects on 1,2-diacylglycerol (1,2-DAG) formation. In suspend ed NIH 3T3 cells, 200-300-mM ethanol blocked the stimulatory effect of PMA on PtdOH formation without affecting PtdCho synthesis indicating that neither PtdOH nor 1,2-DAG derived from it is a mediator of PMA ef fect on PtdCho synthesis. In attached NIH 3T3 cells, dimethylbenz[a]an thracene enhanced phosphocholine formation and, thus, choline uptake w ithout increasing PtdCho synthesis or modifying the effect of PMA. Whi le the results indicate that the stimulatory effect of PMA on PtdCho s ynthesis requires the expression of both PKC-alpha and a PtdCho-specif ic PLD, they do not support a role for 1,2-DAG,PtdOH or choline in the mediation of PMA effect. (C) 1998 Elsevier Science B.V. All rights re served.