Phorbol ester stimulation of phosphatidylcholine synthesis requires expression of both protein kinase C-α and phospholipase D

Abstract

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 suspended NIH 3T3 fibroblasts as well as variants of the MCF-7 human breast carcinoma cell line expressing PKC-α and a PtdCho-specific PLD activity at widely different levels were used to determine the possible role of PKC-α, PtdCho hydrolysis, and choline uptake in the mediation of PMA effect on PtdCho synthesis. In wild-type MCF-7 cells, which express both PKC-α and PLD activities at very low levels, PMA had little effects on the uptake or incorporation [${}^{14}\text{C}$]choline into PtdCho. In multidrug resistant MCF-7/MDR1 cells, which highly express PKC-α but lack the PtdCho-specific PLD activity, 100-nM PMA had relatively small stimulatory effects on the uptake of [${}^{14}\text{C}$]choline (∼1.5-fold) and [${}^{14}\text{C}$]PtdCho synthesis (1.5- to 2-fold). In NIH 3T3 fibroblasts and MCF-7/PKC-α cells, both expressing PKC-α and PLD activities at high levels, 10–100-nM PMA enhanced [${}^{14}\text{C}$]choline uptake only slightly (1.7- to 2.2-fold), while it had much greater (∼4–9-fold) stimulatory effects on PtdCho synthesis. PMA significantly enhanced the formation of phosphatidic acid (PtdOH) in MCF-7/PKC-α cells (2.8-fold increase), but not in MCF-7/MDR1 cells (1.4-fold increase), while in both cell lines it had only small (1.3–1.5-fold) stimulatory effects on 1,2-diacylglycerol (1,2-DAG) formation. In suspended 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 effect on PtdCho synthesis. In attached NIH 3T3 cells, dimethylbenz[a]anthracene enhanced phosphocholine formation and, thus, choline uptake without increasing PtdCho synthesis or modifying the effect of PMA. While the results indicate that the stimulatory effect of PMA on PtdCho synthesis requires the expression of both PKC-α and a PtdCho-specific PLD, they do not support a role for 1,2-DAG, PtdOH or choline in the mediation of PMA effect.