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

CTP:phosphocholine cytidylyltransferase (CT) is a rate-limiting and complex ly regulated enzyme in phosphatidylcholine (PC) biosynthesis and is importa nt in the adaptation of macrophages to cholesterol loading. The goal of the present study was to use transgenesis to study the CT reaction in differen tiated macrophages in vivo. We successfully created macrophage-targeted tra nsgenic mice that overexpress a truncated form of CT, called CT-314. Sonica ted homogenates of peritoneal macrophages overexpressing CT-314 protein dem onstrated a two-fold increase in CT activity in vitro compared with homogen ates from nontransgenic macrophages. CT-314 macrophages, however, demonstra ted no increase in CT activity or PC biosynthesis in vivo. This finding cou ld not be explained simply by intracellular mistargeting of CT-314, by the inability of CT-314 to associate with cellular membranes, or by substrate l imitation. To further probe the mechanism, an in vitro assay using intact n uclei was developed in an attempt to preserve interactions between CT, whic h 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 m acrophages. Thus, a sonication-sensitive interaction between excess CT and one or more nuclear molecules may be responsible for the limitation of CT a ctivity in CT-314 macrophages. These data represent the first report of a C T transgenic animal and the first study of a differentiated cell type with excess CT. (C) 1999 Elsevier Science B.V. All rights reserved.