Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth

Abstract

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value ( approximately 1,200 microM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

Figure 1

Increases in total and individual phospholipid levels in PC12 cells treated with NGF. PC12 cells were cultured with or without NGF (50 ng/ml) for 2 or 4 days (A) or for 4 days (B). Cell phospholipids were extracted and the amounts of total (A) or individual (B) phospholipids were determined. Results are means ± SEM from three (A) or four (B) independent experiments; duplicate dishes were used for each treatment. ∗ indicate data significantly different from control by unpaired Student’s t test. ∗, P < 0.05; ∗∗, P < 0.01. PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; PS, phosphatidylserine; PI, phosphatidylinositol.

Figure 2

[¹⁴C]choline incorporation into [¹⁴C]PtdCho increases in PC12 cells stimulated with NGF or bFGF. (A) PC12 cells that had been treated with or without NGF (50 ng/ml) or bFGF (50 ng/ml) for 2 or 4 days were labeled with [methyl-¹⁴C]choline for 2 hr, and [¹⁴C]choline incorporation into [¹⁴C]PtdCho was determined. (B) [¹⁴C]choline incorporation was analyzed in PC12 cells treated with the indicated concentrations of NGF for 4 days. Results are means ± SEM from three independent experiments; duplicate dishes were used for each treatment. ∗, P < 0.05; ∗∗, P < 0.01 compared with control. C, control.

Figure 3

Activities of PtdCho biosynthetic enzymes in PC12 cells treated with NGF. PC12 cells were cultured with or without NGF (50 ng/ml) for 2 or 4 days. CK was assayed in soluble fractions, CT in total homogenates, and CPT in membrane fractions. Data are expressed as percentages of control, and represent means ± SEM from at least three experiments. ∗∗, P < 0.01 compared with control. The actual activities in control cells for CK, CT and CPT were 3 ± 0.4, 1.8 ± 0.3, and 7.3 ± 0.6 nmol/min per mg of protein, respectively.

Figure 4

Increases in total DAG levels in PC12 cells treated with NGF. (A) PC12 cells were cultured with or without NGF (50 ng/ml) for 2 or 4 days. DAG masses in cell extracts were determined by enzymatic assay with DAG kinase. (B) DAG levels were measured in PC12 cells treated with the indicated concentrations of NGF for 4 days. Results are expressed as nmol/mg protein, and represent means ± SEM from four (A) or three (B) experiments. ∗∗, P < 0.01 compared with control. C, control.