Tang W, Walsh A, Tabas I
The Departments of Medicine, and Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, NY 10032, USA.
Biochim Biophys Acta. 1999 Mar 25;1437(3):301-16. doi: 10.1016/s1388-1981(99)00023-2.
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.
磷酸胆碱胞苷转移酶（CT）是磷脂酰胆碱（PC）生物合成中的一种限速且受复杂调控的酶，在巨噬细胞适应胆固醇负荷过程中起重要作用。本研究的目的是利用转基因技术在体内分化的巨噬细胞中研究CT反应。我们成功创建了巨噬细胞靶向转基因小鼠，其过度表达一种截短形式的CT，称为CT-314。与非转基因巨噬细胞的匀浆相比，过度表达CT-314蛋白的腹膜巨噬细胞的超声破碎匀浆在体外显示CT活性增加了两倍。然而，CT-314巨噬细胞在体内的CT活性或PC生物合成并未增加。这一发现不能简单地用CT-314在细胞内定位错误、CT-314无法与细胞膜结合或底物限制来解释。为了进一步探究其机制，开发了一种使用完整细胞核的体外测定法，试图保留CT（其在巨噬细胞中主要是一种核酶）与其他核分子之间的相互作用。这种完整细胞核测定法忠实地再现了在活巨噬细胞中观察到的情况，即尽管CT-314蛋白增加，但CT活性没有显著增加。相比之下，CT-314巨噬细胞的超声破碎细胞核中的CT活性明显高于非转基因巨噬细胞。因此，过量CT与一种或多种核分子之间的超声敏感相互作用可能是CT-314巨噬细胞中CT活性受限的原因。这些数据代表了CT转基因动物的首次报道以及对具有过量CT的分化细胞类型的首次研究。
