Free cholesterol loading of macrophages stimulates phosphatidylcholine biosynthesis and up-regulation of CTP: phosphocholine cytidylyltransferase

Atheroma macrophages accumulate large amounts of free cholesterol (FC) as well as cholesteryl ester (CE). An important adaptive response to FC loading might be increased cellular phospholipid to accommodate the excess FC. To explore this idea, J774 macrophages were incubated for 48 h without lipid, with acetyl-low density lipoprotein to induce mostly CE loading, or with acetyl-low density lipoprotein plus an acyl-CoA:cholesterol O-acyltransferase inhibitor (58035) to induce marked FC loading. The total phospholipid content approximately doubled in FC-loaded versus control or CE-loaded macrophages, with phosphatidylcholine showing the largest increase (approximately 2.5-fold versus control). Electron micrographs revealed the presence of multiple intracellular membrane whorls in the FC-loaded macrophages but not in the control or CE-loaded macrophages. [3H]Choline incorporation into phosphatidylcholine was also greater in FC-loaded macrophages versus control or CE-loaded macrophages, whereas [3H]phosphatidylcholine degradation was similar in all of the macrophages. In these experiments and in others that used non-lipoprotein cholesterol, there was a very close correlation between cellular FC content and phosphatidylcholine biosynthesis. To determine the mechanism of increased phosphatidylcholine synthesis, FC-loaded and CE-loaded macrophages were pulsed with [3H]choline, then chased and assayed for labeled phosphatidylcholine biosynthetic precursors. The only major differences were a 2-fold greater disappearance of label from [H]choline phosphate and a 5-fold greater appearance of label in CDP-[3H]choline in the FC-loaded macrophages. These data suggest a stimulation of CTP-phosphocholine cytidylyltransferase (CT), which was confirmed by microsomal CT assays. Further studies revealed that the increase in phosphatidylcholine biosynthesis in FC-loaded macrophages was: (a) reversible under conditions of high density lipoprotein3-mediated cellular cholesterol efflux; (b) not blocked by cycloheximide-induced protein synthesis inhibition; and (c) not associated with increased CT mRNA levels. Thus, FC loading of macrophages leads to an increase in phosphatidylcholine mass which is caused by increased phosphatidylcholine biosynthesis. The mechanism appears to be FC-mediated post-translational activation of CT. This adaptive response may be important for atheroma macrophage survival, and disruption of the response may lead to macrophage necrosis and lesion progression.