Abstract
The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway. CTα mRNA levels, like farnesyl diphosphate synthase and the LDL receptor, are repressed when human or rodent cells are incubated with exogenous sterols and induced when cells are incubated in lipid-depleted medium. A putative sterol response element (SRE) was identified 156 bp upstream of the transcription start site of the CTα gene. Electrophoretic mobility shift assays demonstrate that recombinant SREBP-1a binds to the wild-type SRE identified in the CTα promoter but not to oligonucleotides containing two mutations in the SRE. In other studies, a luciferase reporter construct under the control of the murine CTα proximal promoter was transiently transfected into cells. The activity of the reporter was repressed after addition of sterols to the medium and induced when the cells were incubated in lipid-depleted medium. The activity of the CTα-luciferase reporter was also induced when cells were cotransfected with plasmids encoding either SREBP-1a or SREBP-2. In contrast, no induction was observed under the same conditions when the CTα promoter-reporter gene contained two mutations in the SRE. In addition, the induction of the wild-type CTα promoter-reporter gene that occurs in cells incubated in lipid-depleted medium is attenuated when dominant-negative SREBP is cotransfected into the cells. These studies demonstrate that transcription of the CTα gene is inhibited by sterols and activated by mature forms of SREBP. We conclude that SREBP-regulated genes are involved not only in the synthesis of cholesterol, fatty acids, triglycerides, and NADPH, but also, as shown here, in the synthesis of phospholipids. —Kast, H. R., C. M. Nguyen, A. M. Anisfeld, J. Ericsson, and P. A. Edwards. CTP:phosphocholine cytidylyltransferase, a new sterol- and SREBP-responsive gene.
Highlights
The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway
Exogenous sterols regulate CT␣ mRNA levels To determine whether the CT␣ gene is regulated by sterols, poly(A)ϩ RNA was isolated from Chinese hamster ovary (CHO) cells that had been cultured either in the presence of excess sterols or in the presence of medium supplemented with 10% lipoproteindeficient serum (LPDS) and compactin, an inhibitor of HMG-CoA reductase and cholesterol synthesis
We further examined the sterol responsiveness of the CT␣ gene in the human monocyte cell line THP-1, using semiquantitative RT-PCR; THP-1 cells were converted to macrophages by treatment with phorbol esters and incubated for 24 h in medium supplemented with 10% LPDS in the presence or absence of sterols
Summary
The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway. The induction of the wild-type CT␣ promoter-reporter gene that occurs in cells incubated in lipid-depleted medium is attenuated when dominant-negative SREBP is cotransfected into the cells. These studies demonstrate that transcription of the CT␣ gene is inhibited by sterols and activated by mature forms of SREBP. These posttranscriptional events appear to be important in the regulation of CT␣ activity [1] In addition to these posttranslational regulatory events, CT␣ mRNA levels have been shown to be regulated; they are induced in hepatic tissue after partial hepatectomy [7], in macrophages treated with colony-stimulating factor 1 [8], and in an alveolar type II epithelial cell line incubated in lipid-depleted medium [9]. CT contains four functional domains that have been characterized as a nuclear
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