Macrophage-targeted CTP:phosphocholine cytidylyltransferase (1-314) transgenic mice

CTP:phosphocholine cytidylyltransferase (CT) is a rate-limiting and complexly regulated enzyme in phosphatidylcholine (PC) biosynthesis and is important in the adaptation of macrophages to cholesterol loading. The goal of the present study was to use transgenesis to study the CT reaction in differentiated macrophages in vivo. We successfully created macrophage-targeted transgenic mice that overexpress a truncated form of CT, called CT-314. Sonicated homogenates of peritoneal macrophages overexpressing CT-314 protein demonstrated a two-fold increase in CT activity in vitro compared with homogenates from nontransgenic macrophages. CT-314 macrophages, however, demonstrated no increase in CT activity or PC biosynthesis in vivo. This finding could not be explained simply by intracellular mistargeting of CT-314, by the inability of CT-314 to associate with cellular membranes, or by substrate limitation. To further probe the mechanism, an in vitro assay using intact nuclei was developed in an attempt to preserve interactions between CT, which is primarily a nuclear enzyme in macrophages, and other nuclear molecules. This intact-nuclei assay faithfully reproduced the situation observed in living macrophages, namely, no significant increase in CT activity despite increased CT-314 protein. In contrast, CT activity in sonicated nuclei from CT-314 macrophages was substantially higher than that from nontransgenic macrophages. Thus, a sonication-sensitive interaction between excess CT and one or more nuclear molecules may be responsible for the limitation of CT activity in CT-314 macrophages. These data represent the first report of a CT transgenic animal and the first study of a differentiated cell type with excess CT. (C) 1999 Elsevier Science B.V. All rights reserved.