Phosphatidylethanolamine N-Methyltransferase 2 and CTP-Phosphocholine Cytidylyltransferase Expressions Are Related with Protein Kinase C Isozymes in Developmental Liver Growth

2010

Phosphatidylcholine is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for phosphatidylcholine biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have now identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. We have found that PEMT mRNA and protein levels increased dramatically in 3T3-L1 cells upon differentiation to adipocytes. 5-Deletion analysis of the PEMT promoter-luciferase constructs stably expressed in 3T3-L1 adipocytes identified a regulatory region between ؊471 and ؊371 bp (relative to the transcriptional start site). Competitive and supershift assays demonstrated binding sites for transcription factors Sp1, Sp3 (؊408 to ؊413), and YY1 (؊417 to ؊420). During differentiation of 3T3-L1 cells to adipocytes, the amount of Sp1 protein decreased by ϳ50% just prior to activation of PEMT. Transduction of 3T3-L1 adipocytes with retrovirus containing Sp1 cDNA demonstrated that Sp1 inhibited PEMT transcriptional activity. Similarly, short hairpin RNA directed against Sp1 in 3T3-L1 adipocytes enhanced PEMT transcriptional activation. Chromatin immunoprecipitation assays confirmed that Sp1 binds to the PEMT promoter, and this interaction decreases upon differentiation to adipocytes. These experiments directly link increased PEMT expression in adipocytes to decreased transcriptional expression of Sp1. In addition, our data established that Sp1 binding was required for tamoxifen-mediated inhibition of Pemt promoter activity.

Section: Implications For Transcriptional Regulation Of the Pemt Genementioning

confidence: 99%

A Role for Sp1 in Transcriptional Regulation of Phosphatidylethanolamine N-Methyltransferase in Liver and 3T3-L1 Adipocytes

Cole¹,

2010

“…CT mRNA is increased after partial hepatectomy in rats (13), after stimulation with colony-stimulating factor 1 in macrophages (24), and during development and growth (25)(26)(27)(28). It remains to be determined whether these increases in the message levels are the result of CT␣ and/or CT␤1/2 mRNA stability, an increase in gene transcription, or a combination of both.…”

Section: Phosphatidylcholine (Pc)mentioning

confidence: 99%

Oncogenic Ha-Ras Transformation Modulates the Transcription of the CTP:Phosphocholine Cytidylyltransferase α Gene via p42/44MAPK and Transcription Factor Sp3

Bakovic¹,

Waite²,

2003

Phosphatidylcholine (PC)1 is the most abundant phospholipid in mammalian cellular membranes. Besides having a structural role in membranes and lipoproteins (1-3), PC plays an important role in signal transduction as a source of lipid second messengers (1-3). In all nucleated cells, PC is made primarily through the CDP-choline pathway in which the key enzyme is CTP:phosphocholine cytidylyltransferase (CT) (2-6).Two genes that encode CT activity have been identified and characterized. CT␣ is expressed in many cells and tissues (7-13) and has a predicted structure that contains catalytic, phosphorylation, and lipid binding domains as well as a nuclear localization sequence (8, 14 -21). Recently, CT␤ has been identified in human tissues and appears to exist as two splice variants, CT␤1 and CT␤2, differing at their C termini (22, 23). Like CT␣, CT␤1/2 contains catalytic and lipid binding domains. However, CT␤1 lacks the phosphorylation domain, and both CT␤1 and CT␤2 lack the nuclear localization sequence (23).In the last several years, studies have shown that CT can be regulated at both the transcriptional level and post-transcriptionally. CT mRNA is increased after partial hepatectomy in rats (13), after stimulation with colony-stimulating factor 1 in macrophages (24), and during development and growth (25)(26)(27)(28). It remains to be determined whether these increases in the message levels are the result of CT␣ and/or CT␤1/2 mRNA stability, an increase in gene transcription, or a combination of both.The murine CT␣ gene (Ctpct) was cloned and characterized by Tang and co-workers (29). The Ctpct promoter contains several putative elements for binding transcription factors, including Ap1, an overlapping site for nuclear factor-B, E2F, and Elk1, one sterol response element, as well as three elements for Sp-related factors (30). We have shown that Sp1, Sp2, and Sp3 bind competitively to three GC-rich elements and that relative promoter activity depends upon the abundance of these factors (31). We further established that transcription enhancer factor-4 can bind to an upstream regulatory element (Ϫ103/Ϫ82) and enhance the CT␣ gene expression through its interactions with the basal transcriptional machinery (32). In agreement with the finding that lipoprotein deficiency induces the expression of CT␣ mRNA and protein in alveolar type II epithelial cells (33), and our observation that the CT␣ promoter contains a putative sterol response element (30), studies by Kast et al. (34) indicate a role for cholesterol/sterol response element-binding protein and the functionality of the sterol response element in the regulation of CT␣ gene expression in Chinese hamster ovary cells and THP-1 cells. On the other hand, Lagace et al. (35) have shown that cholesterol/sterol response element-binding protein can stimulate PC biosynthe-

Section: Phosphatidylcholine (Pc)mentioning

confidence: 99%

“…The maximal increase in the CT mRNA coincided with maximal DNA synthesis 24 h after partial hepatectomy (19). CT is highly expressed during the perinatal period and the expression of CT is positively associated with hepatic cell division (20,21). Tang et al (22) isolated and characterized the murine CT␣ gene and demonstrated that the exon/intron organization of the gene corresponded to enzyme functional domains and the gene possessed two transcriptional start sites.…”

mentioning

confidence: 99%

Transcription of the CTP:Phosphocholine Cytidylyltransferase α Gene Is Enhanced during the S Phase of the Cell Cycle

Golfman

Bakovic²,

2001

We have studied the transcription of the CTP:phosphocholine cytidylyltransferase ␣ (CT␣) gene in C3H10T1/2 fibroblasts as a function of the cell cycle. The cells were incubated for 48 h with 0.5% fetal bovine serum. The cells were induced into the G 1 phase of the cell cycle by the addition of medium with 10% fetal bovine serum. The cells began the synthesis of DNA after 12 h. At 16 and 20 h there was an increased amount of CT␣ mRNA that coincided with an increase in the expression of CT␣ proximal promoter-luciferase constructs (؊201/؉38 and ؊130/؉38). Luciferase constructs with the basal promoter (؊52/؉38) showed no change in activity during the cell cycle. Incorporation of [ 3 H]choline into phosphatidylcholine began to increase by 8 h after the addition of serum and peaked at 18 h. The mass of phosphatidylcholine nearly doubled between 8 and 26 h after addition of serum. CT activity increased by 6 h after serum addition and was maintained until 22 h. Thus, the increase of phosphatidylcholine biosynthesis in the G 1 phase of the cell cycle is not due to enhanced transcription of the CT␣ gene. Instead increased transcription of the CT␣ gene occurred during the S phase of the cell cycle in preparation for mitosis. Phosphatidylcholine (PC)1 represents ϳ50% of the membrane phospholipids in animal cells (1). PC is a substrate for the generation of bioactive lipid messengers (2), a precursor in the synthesis of sphingomyelin, phosphatidylethanolamine, and phosphatidylserine (3, 4) and is an important component of serum lipoproteins (5) and bile (6). PC biosynthesis occurs in all nucleated cells mainly via the CDP-choline/Kennedy pathway in which CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) is considered to be the rate-limiting and regulated enzyme of this pathway (1,7,8). The first mammalian CT was purified from rat liver (9, 10) as a homodimer with a subunit molecular mass of 42 kDa and was the basis for the cloning of the corresponding cDNA encoding a 367-amino acid (CT␣) protein (11). To date, two additional isoforms of CT, CT␤1 and CT␤2, have been identified that are encoded by a second gene (12,13).While the primary mechanism by which CT activity is regulated in cells is activation by lipids after translocation to cellular membranes (1,8,14,15), it is now recognized that PC synthesis can also be regulated by changes in CT mRNA turnover and by CT gene transcription. Several reports indicate that CT can be regulated post-transcriptionally, apparently by reducing the rate of mRNA degradation (16,17). However, the precise mechanisms involved in regulating CT mRNA stability still have to be elucidated. Tessner et al. (16) provided the first evidence for increased CT mRNA during growth factor-stimulated PC synthesis in a murine macrophage cell line. Houweling et al. (18) demonstrated that CT is regulated at the level of its mRNA in rat liver after partial hepatectomy. The maximal increase in the CT mRNA coincided with maximal DNA synthesis 24 h after partial hepatectomy (19). CT is highly expressed dur...