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Correspondence to:
Peter A. Edwards, at the Department of Biological Chemistry, CHS, 33-257 UCLA School of Medicine, Los Angeles, CA 90095-1769., pedwards{at}mednet.ucla.edu (E-mail)
The CTP:phosphocholine cytidylyltransferase (CT) gene encodes the rate-controlling enzyme in the phosphatidylcholine biosynthesis pathway. CT
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. J. Lipid Res. 2001. 42: 1266;–1272.
Supplementary key words:
phosphatidylcholine, sterol response element, CT
CTP:phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the synthesis of the major membrane phospholipid phosphatidylcholine (1). Two genes, CT
In addition to these posttranslational regulatory events, CT
Studies have identified both positive and negative cis elements within the proximal promoter of the murine CT
Sterol response element-binding proteins (SREBP-1a, -1c, and -2) are positive transcription factors that bind to SRE in the promoters of target genes that are involved in the synthesis of cholesterol, triglycerides, and saturated and unsaturated fatty acids, as well as the endocytosis of LDL and the production of NADPH (14) (15) (16) (17). SREBPs are synthesized as 125-kDa proteins that are bound to the endoplasmic reticulum (ER). Cellular sterol depletion results in formation of an SREBP cleavage-activating protein/SREBP complex that is translocated from the ER to the Golgi, whereupon SREBP is cleaved, resulting in release from the Golgi of the mature 68-kDa SREBP (15) (18). Mature SREBPs enter the nucleus, bind to SRE in the promoters of specific target genes, and activate transcription (16) (17). Consistent with these observations, excess cellular sterols/oxysterols were reported to prevent the cleavage and maturation of SREBP and to inhibit transcription of SREBP-dependent target genes [reviewed in refs. (14) and (17)]. However, studies have demonstrated that specific oxysterols or other ligands that activate the nuclear receptor LXR increase the mRNA levels of SREBP-1c, but have no effect on the mRNA levels of SREBP-1a or SREBP-2 (19) (20). The SREBP-1 gene has two promoters (15). The increase in SREBP-1c mRNA levels is dependent on the LXR response element in the promoter that controls the expression of SREBP-1c (19). Thus, specific oxysterols can have diverse roles that can lead to enhanced transcription of SREBP-1c and to inhibition of the cleavage and maturation of SREBP. The relative effects of specific oxysterols on these two processes are likely to affect the expression of target genes that are preferentially activated by either SREBP-1a, SREBP-1c, or SREBP-2.
The nucleotide sequences of the known SREs are divergent, but often consist of two half-sites (C/TCAC) with 1-bp spacing between the half-sites (17). In previous studies, we identified SREBP target genes by a variety of approaches that included visual scanning of published promoter sequences to identify SREs (21) and mRNA differential display (22). The current report was based on our visual inspection of the published sequence of the proximal promoter of the murine CT
Materials
Cell culture
RNA isolation, Northern blot analyses
RT-PCR
CT
Transient transfections and reporter gene assays
Electrophoretic mobility shift assays
Exogenous sterols regulate CT
We further examined the sterol responsiveness of the CT
Proximal promoter of the CT
SREBP binds the SRE within the CT
SREBP trans-activates CT
In other studies, HepG2 cells, cultured in MEM + 10% FBS in 48-well dishes, were transfected with the CT
Coexpression of a dominant-negative SREBP-1a attenuates induction of the CT
Induction of CT
Herein, the data of Fig 1 and Fig 2 demonstrate that transcription of the CT
The current studies extend those of Ryan and colleagues and demonstrate that the increase in CT
In contrast to the data in the present report and to those of Ryan et al. (9), other investigators have reported that CT
SREBP-mediated induction of target genes occurs in concert with the binding of additional coregulatory transcription factors Sp1 and/or NF-Y to the promoter, resulting in synergistic levels of transcriptional activation (21) (24) (36) (37) (38). The CT
We have previously shown that SREBP directly binds to the promoter of GPAT and is able to trans-activate the gene (21). The enzyme GPAT is involved in the initial step in the synthesis of both triglycerides and phospholipids (39). As in the cholesterol biosynthetic pathway, in which multiple steps are regulated by SREBP (HMG-CoA reductase, FPPS, etc.), it appears that there is coordinate regulation of genes within the phosphatidylcholine biosynthetic pathway by SREBP (GPAT and CT
The current studies extend the list of enzymes and lipid pathways that are directly regulated by SREBP; these include the activation of acetate to acetyl-CoA and its conversion to cholesterol, unsaturated and saturated fatty acids, triglycerides, and, with the current report, phospholipids. The characterization of CT
Abbreviations: CT, CTP:phosphocholine cytidylyltransferase; FPPS, farnesyl diphosphate synthase; LDLr, LDL receptor; LPDS, lipoprotein-deficient serum; SRE, sterol response element; SREBP, sterol response element-binding protein.
This work was supported by grants from the National Institutes of Health (HL30568 to P.A.E.), the Laubisch Fund (to P.A.E.), and predoctoral training grants (U.S. Department of Education #P200A80113) (to H.R.K. and A.M.A).
Manuscript received February 13, 2001; and in revised form April 6, 2001
Copyright © 2001 by Lipid Research, Inc.
Original Article
CTP:phosphocholine cytidylyltransferase, a new sterol- and SREBP-responsive gene
Heidi Rachelle Kasta,
Catherine M. Nguyena,
Andrew M. Anisfelda,
Johan Ericssonb, and
Peter A. Edwardsa,c
a Departments of Biological Chemistry and Medicine, University of California, Los Angeles, CA 90024
b Ludwig Institute for Cancer Research, S-751 24 Uppsala, Sweden
c Molecular Biology Institute, University of California, Los Angeles, CA 90095
ABSTRACT
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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 CT gene. Electrophoretic mobility shift assays demonstrate that recombinant SREBP-1a binds to the wild-type SRE identified in the CT promoter but not to oligonucleotides containing two mutations in the SRE. In other studies, a luciferase reporter construct under the control of the murine CT 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 CT-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 CT promoter-reporter gene contained two mutations in the SRE. In addition, 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. gene
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and CTß, encode three isoforms, namely CT, CTß1, and CTß2 (2) (3). CT contains four functional domains that have been characterized as a nuclear localization signal at the amino terminus, a catalytic domain, a hydrophobic lipid-binding domain, and a kinase domain at the carboxy terminus (1) (3) (4). The activity of the enzyme is controlled by multiple mechanisms, including its phosphorylation state, and its association with lipids and membranes (1). CT interconverts between an inactive phosphorylated cytosolic form, and an enzymatically active nonphosphorylated membrane-bound form (1) (5). The association of CT with membranes, and thus its activity, is stimulated by unsaturated fatty acids, diacylglycerol, and anionic phospholipids [reviewed in ref. (6)]. These posttranscriptional events appear to be particularly important in the regulation of CT activity (1). mRNA levels have also 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). Interestingly, CTß2 mRNA levels are increased in CT-null peritoneal macrophages (10). However, the mechanism for this increase has yet to be determined. gene (11) (12). These include several consensus transcription factor-binding sites, including those recognized by the transcriptional enhancer factor 4, Sp1, Sp2, Sp3, and cAMP response element-binding protein (11) (12) (13). In addition, transcription of the CT gene has been shown to be dependent in part on the relative concentrations of Sp1, Sp2, and Sp3 (13). A putative sterol response element (SRE) was also identified in the proximal promoter of the murine CT gene (13), although no reports as to the significance of this element in controlling CT transcription have been reported to date. gene; this approach identified a putative SRE. Bakovic and colleagues (12) independently identified the same potential SRE. We now demonstrate that this putative SRE is functionally important for the transcriptional regulation of the CT gene in response to alterations in cellular sterols. These studies identify CT as a novel SREBP target gene, and we propose that nuclear SREBP regulates phospholipid biosynthesis, in addition to the well-established role of SREBP in the synthesis of cholesterol, fatty acids, triglycerides, and NADPH and in the endocytosis of LDL (14) (17).
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The human CT cDNA was a kind gift of C. Kent (University of Michigan, Ann Arbor, MI) (23). The LDL receptor promoter-luciferase reporter construct (LDLr-SRE) was described previously (24). The cDNA for rat 18S ribosomal protein, used as a control for RNA loading, was generously given by B. Quednan (University of California, Los Angeles, Los Angeles, CA). All other materials have been described (21) (25).
HepG2 cells were cultured in MEM supplemented with 10% FBS (25). Chinese hamster ovary (CHO) cells were cultured in Ham's F-12 medium supplemented with 10% FBS (22). 3T3L1 cells (preadipocytes) were cultured in DMEM supplemented with 10% FBS. To differentiate the preadipocytes into adipocytes, cells were allowed to reach confluence and then were treated for 2 days with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO), 0.25 µM dexamethasone (Sigma), and insulin (1 µg/ml; Sigma) (21). Fresh medium (DMEM-10% FBS) was added and changed every 2 days thereafter.
CHO cells were cultured in Ham's F-12 medium supplemented with 10% FBS. To initiate experiments the medium was replaced with Ham's F-12 medium supplemented with 10% lipoprotein-deficient serum (LPDS) and either 5 µM compactin and 100 µM mevalonic acid (sterol depleted) or sterols (cholesterol at 10 µg/ml and 25-hydroxycholesterol at 1 µg/ml) and 5 µM compactin and 100 µM mevalonic acid (sterol loading). After 36 h total RNA was isolated with TRIzol reagent (Life Technologies, Rockville, MD) and poly(A)+ RNA was purified with an oligo(dT) column (Ambion, Austin, TX). The RNA was resolved (3 µg/per lane) on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and cross-linked to the membrane with UV light. cDNA probes were radiolabeled with [-32P]dCTP, using the random priming labeling method (Amersham Pharmacia Biotech, Piscataway, NJ), and incubated with the membrane as described (25). Variations in RNA loading in each lane were normalized by hybridization of a control cDNA probe to the 18S ribosomal RNA. The RNA levels were quantitated with a PhosphorImager (with Image-Quant software; Molecular Dynamics, Sunnyvale, CA).
The human monocyte cell line THP-1 was treated with phorbol esters (26) for 24 h to induce differentiation into macrophages. The latter cells were incubated for 24 h in the presence of DMEM and 10% LPDS supplemented with either acetylated LDL (100 µg/ml), cholesterol (10 µg/ml; Sigma), 25-hydroxycholesterol (1 µg/ml; Sigma), and 100 µM mevalonic acid (Sigma) (sterol loaded), or ethanol (1 µl/ml), 5 µM mevinolin, and 100 µM mevalonic acid (sterol depleted). Total RNA was isolated with TRIzol reagent (Life Technologies). Reverse transcription was carried out with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) according to the manufacturer's protocol, followed by 35 cycles of PCR amplification (which was within the linear range of amplification for the target genes) with human CT cDNA-specific primers 5'-agcgaagaaccttttccctaat-3' and 5'-gtttagtgttggggtcacaattcg-3' with Taq polymerase (GIBCO-BRL, Gaithersburg, MD) in the presence of [-32P]dCTP. The linear range of amplification had been determined by running 10-µl aliquots of PCR product, removed at intervals of five cycles from 10 to 45 cycles, on an 8% polyacrylamide gel and exposing the dried gel to an autoradiogram. The intensities of the specific PCR products were quantitated with a PhosphorImager (with ImageQuant software; Molecular Dynamics). promoter-reporter constructs
The proximal promoter of CT (– 160 to +44 bp) was amplified from SVJ129 mouse genomic DNA, using primers 5'-taccctc gagctcagtcaccccacgcgccc-3' and 5'-acttaagcttcggatcccttccccgcgccg-3', and cloned into XhoI-HindIII sites of the PGL2 vector (Promega). The mutant CT promoter reporter was created in a similar manner, using 5'-taccctcgagctcagtaaacccacgcgccc-3' and the common downstream primer located at position +44 bp (mutations are italicized and underlined). The wild-type and mutant CT promoter sequences were confirmed with a T7 Sequenase 7-deaza-dGTP sequencing kit (U.S. Biochemical, Cleveland, OH).
HepG2 cells were transiently transfected, using an MBS mammalian transfection kit (Stratagene, La Jolla, CA) with minor modifications, as described (22). pCI-SREBP-1a, which encodes amino acids 1;–490 of SREBP-1a, and pCI-SREBP-2, which encodes amino acids 1;–485 of SREBP-2, were cloned into the pCI-neo expression vector. Reporter plasmid pCI-SREBP1a or pCI-SREBP2, and pCMV-ß-galactosidase, were transfected into HepG2 cells in 60-mm dishes or (where indicated) 48-well plates. After 3.5 h the cells were incubated in medium supplemented with 10% LPDS and either ethanol or sterols (cholesterol at 10 µg/ml and 25-hydroxycholesterol at 1 µg/ml). After 24 h, the cells were lysed and assayed for luciferase and ß-galactosidase activity and values were normalized to account for variations in transfection efficiency as described (22).
Complementary single-stranded oligonucleotides containing the wild-type or mutant nucleotide sequence were annealed, and subsequently end labeled with [
-32P]dCTP. Purified recombinant SREBP-1a (1 µl) (21) (24) was incubated in binding buffer [10 mM HEPES (pH 7.9), 0.5 mM DTT, 2.5 mM MgCl2, 0.05% (v/v) glycerol, nonfat milk (50 mg/ml), and 50 mM NaCl] at room temperature for 15 min. Radiolabeled probe (30,000 cpm, 1.5 fmol) was added and the incubation was continued at room temperature for 1 h. DNA-protein complexes were resolved by 4% nondenaturing polyacrylamide gel electrophoresis at 4°C for 2.5 h at 250 V. The gels were dried and analyzed by autoradiography and with a PhosphorImager (25).
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mRNA levels
To determine whether the CT gene is regulated by sterols, poly(A)+ RNA was isolated from CHO cells that had been cultured either in the presence of excess sterols (cholesterol and 25-hydroxycholesterol) or in the presence of medium supplemented with 10% LPDS and compactin, an inhibitor of HMG-CoA reductase and cholesterol synthesis. Northern blot assays demonstrated that the CT mRNA is expressed at low levels in sterol-treated cells and is induced 4.7-fold in cells incubated in lipid-depleted medium ( Fig 1A). Farnesyl diphosphate synthase (FPPS) mRNA levels were induced 3.6-fold when cells were incubated in the lipid-depleted medium (Fig 1), consistent with previous reports (27). Because FPPS is transcriptionally activated in response to increasing nuclear levels of one or more forms of SREBP (27) (28), the results of Fig 1A suggest that the CT gene might be activated by a similar mechanism.
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Figure 1.
Regulation of CT mRNA levels by exogenous sterols. A: CHO cells were incubated for 36 h in medium supplemented with 10% LPDS and compactin (LPDS) or LPDS and sterols (cholesterol at 10 µg/ml and 25-hydroxycholesterol at 1 µg/ml) (sterols). Poly(A)+ RNA (3 µg/lane) was separated on a 1% agarose-formaldehyde gel, transferred to nylon membranes, and hybridized to the indicated radiolabeled probes. Quantitation was performed with a PhosphorImager (Molecular Dynamics). The relative levels of CT mRNA are shown graphically and were determined after normalization to the control probe (18S). B: THP-1-derived macrophages were cultured for 24 h in medium containing 10% LPDS and compactin or LPDS and sterols, as described in Materials and Methods. RNA was isolated and reverse transcribed, and DNA fragments of the CT mRNA (900 bp) or GAPDH (301 bp) were generated by 35 cycles of PCR and human-specific primers in the presence of [ -32P]dCTP. A similar induction of the CT mRNA was observed after 40 cycles of PCR (data not shown). The blots are representative of at least two experiments. 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 then incubated for 24 h in medium supplemented with 10% LPDS in the presence or absence of sterols. RNA was isolated, and the CT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were determined by RT-PCR. CT mRNA levels were induced 7.4-fold in cells cultured in lipid-depleted medium (Fig 1B). We conclude that CT mRNA levels are regulated in both human and hamster cells as a result of changes in the levels of exogenous sterols. gene contains a functional SRE
We analyzed the published sequence (29) of the proximal promoter of the murine CT gene and identified a putative SRE (5'-GTCACCCCAC-3') 156 bp upstream of the transcription start site. To determine whether this SRE was necessary for sterol-regulated transcription of the CT gene in vivo, we fused the wild-type murine CT promoter (-160 to +44) or a mutant CT promoter, containing two 1-bp mutations in the putative SRE, to a luciferase reporter gene. The constructs were transiently transfected into HepG2 cells together with a cytomegalovirus-driven plasmid expressing ß-galactosidase, and the cells were incubated in medium supplemented with 10% LPDS and either cholesterol and 25-hydroxycholesterol or ethanol ( Fig 2). There was a modest, but reproducible, regulation of the wild-type CT promoter-reporter gene; the activity was approximately 1.7-fold higher in cells incubated in 10% LPDS (sterol depleted). In contrast, the activity of the mutant CT promoter-reporter gene remained at basal levels and was not induced when cells were incubated in 10% LPDS in the absence of sterols (Fig 2). As expected, the LDLr promoter-reporter construct was expressed at low levels in the cells incubated in the presence of 10% LPDS and exogenous sterols and was induced significantly (8.8-fold) when sterols were absent from the medium (Fig 2).
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Figure 2.
Exogenous sterols regulate CT reporter genes. A: Duplicate dishes of HepG2 cells were transiently transfected with ß-galactosidase and luciferase reporter constructs under the control of the sequences derived from either the proximal promoter of the LDLr, wild-type CT, or mutant CT (mut CT) gene. The latter contains two nucleotide mutations in the putative SRE. Cells were cultured in media supplemented with 10% LPDS [(–) sterols] or LPDS plus cholesterol and 25-hydroxycholesterol [(+) sterols]. After 48 h, the cells were lysed and the normalized luciferase activity was determined. Relative levels of the luciferase reporter genes are shown. The bars represent the range between duplicate samples. The results are representative of five separate experiments. proximal promoter
The levels of nuclear, transcriptionally active SREBP-1a and -2 and the mRNAs of their target genes are repressed in cholesterol-loaded cells and elevated when cells are incubated in cholesterol-poor medium (15) (17). The results of Fig 1 and Fig 2 suggest that transcription of the CT gene is also controlled by nuclear SREBP. Analysis of the murine CT proximal promoter identified a putative SRE that differs by one nucleotide from the LDLr SRE in the promoter of the human LDLr gene ( Fig 3). We utilized recombinant SREBP-1a protein and electrophoretic mobility shift assays to determine whether this putative SRE in the murine CT promoter could form a complex with SREBP1a. Fig 4 demonstrates both that a DNA-protein complex was formed when SREBP-1a was incubated with double-stranded DNA containing a wild-type SRE, and that the formation of the complex was greatly attenuated when two mutations were introduced into the SRE. These results demonstrate that SREBP-1a binds in vitro to nucleotides -156 to -147 in the proximal promoter of the CT gene.
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Figure 3.
Schematic illustration of the LDLr and CT proximal promoters. The SRE- and Sp1-binding sites in the proximal promoters of the human LDLr, murine LDLr, and murine CT genes are shown. The mutant murine CT SRE is shown for comparison.
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Figure 4.
SREBP binds the SRE in the proximal promoter of CT . Purified recombinant SREBP1a (1 µl) was incubated with radiolabeled probes of similar specific activity corresponding to either the wild-type CT promoter or the mutant CT promoter containing two mutations in the putative SRE, as described in Materials and Methods. The shifted SREBP-1a-DNA complex is indicated. The free probe is not shown. No other complexes were observed (data not shown). WT CTPCT, Wild-type CTP:phosphocholine cytidylyltransferase. promoter-luciferase reporter genes
The results of Fig 1 Fig 2 Fig 3 Fig 4 suggest that SREBP will trans-activate a CT promoter-reporter construct. To test this proposal HepG2 cells, maintained in MEM + 10% FBS to prevent the proteolytic cleavage and maturation of endogenous SREBP, were transiently transfected with CT-luciferase reporter constructs in the presence or absence of plasmids that express either SREBP-1a or SREBP-2. The data of Fig 5A show that, at equivalent plasmid concentrations, the wild-type CT promoter was more potently induced by SREBP-1a than SREBP-2. In contrast, the activity of the mutant CT promoter was low and was unaffected by cotransfection of either SREBP-1a or SREBP-2 (Fig 5A)
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Figure 5.
SREBP trans-activation of CT reporter genes requires an intact SRE. A: Duplicate dishes of HepG2 cells were transiently transfected with ß-galactosidase and wild-type or mutant CT promoter-reporter constructs and, where indicated, SREBP-1a or SREBP-2. Cells were harvested 24 h after the transfection. Fold induction and the range for duplicate samples are shown after normalization with ß-galactosidase. B: HepG2 cells in 48-well dishes were transiently transfected with the wild-type CT promoter-reporter construct (100 ng) and increasing amounts of SREBP-1a or SREBP-2. Cells were harvested 24 h after the transfection. The data are representative of six independent experiments. RLU, Relative light units. wild-type reporter constructs along with increasing amounts of plasmids encoding either SREBP-1a or SREBP-2 (Fig 5B). The results demonstrate that the wild-type CT promoter-reporter gene is induced by SREBP in a dose-dependent manner, that induction is linear, and that at each concentration of expression plasmid, SREBP-1a was a more potent activator of the reporter gene than SREBP-2 (Fig 5B). This difference is not due to differential expression of SREBP-1a and SREBP-2, because in other studies we have shown that the FPPS promoter-luciferase reporter gene is induced by SREBP-2 to a greater extent than by SREBP-1a (21). promoter-reporter construct
To further define the role of SREBP in the regulated transcription of the CT gene we transiently transfected cells with a CT promoter-reporter gene and dominant-negative forms of SREBP-1a. The latter protein binds the SRE but is unable to trans-activate genes as a result of deletion of the amino-terminal 90 amino acids (30). Fig 6 demonstrates that the induction of both the CT- and LDLr-reporter genes that normally occurs in cells incubated in lipid-depleted medium is attenuated when the dominant-negative (DN) SREBP-1a was coexpressed.
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Figure 6.
Dominant-negative SREBP-1a attenuates the induction of the CT reporter gene. Duplicate dishes (60 mm) containing HepG2 cells were transiently transfected with ß-galactosidase and luciferase reporter constructs fused to the promoter of either the LDL receptor (LDLr) or wild-type CT (CT) along with dominant-negative SREBP-1a, as indicated. Cells were cultured in media supplemented with 10% LPDS or LPDS, cholesterol, and 25-hydroxycholesterol. After 48 h, the cells were lysed and the normalized luciferase activity was determined. Relative levels of the luciferase reporter genes in the absence of sterols versus in the presence of sterols are shown. These results are representative of three separate experiments, each performed in duplicate. DN, Dominant negative. mRNA during adipocyte differentiation
Previous studies have shown that as preadipocytes differentiate into adipocytes, the nuclear levels of SREBP-1c/adipocyte determination and differentiation-dependent factor 1 and SREBP target genes increase (21) (31) (32). Fig 7 shows that CT mRNA levels are induced between days 16 and 32 of cell differentiation. In contrast, the mRNAs encoding two other SREBP target genes, namely HMG-CoA synthase and glycerol 3-phosphate acyltransferase (GPAT), are maximally induced at days 3 and 8, respectively (Fig 7) (21). We conclude that different SREBP target genes are transcriptionally activated at different times after the initiation of differentiation of preadipocytes into adipocytes. Such differences in induction likely depend on the presence of additional factors that are required for maximal induction of specific SREBP target genes.
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Figure 7.
Increased expression of CT mRNA during adipocyte differentiation. 3T3L1 preadipocytes were grown to confluence to induce differentiation into adipocytes. On day 0 (when the cells reached confluence) the cells were treated with a differentiation cocktail as described in Methods and Materials. Total RNA was isolated from the 3T3L1 preadipocytes on the day indicated. Ten micrograms of RNA was separated on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to the indicated radiolabeled probes. Quantitation was performed with a PhosphorImager (Molecular Dynamics). The fold induction was determined after normalization to the control probe (36B4). Similar results were obtained when cells were differentiated on three separate occasions.
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gene is regulated in response to changes in the concentration of sterols in the medium; the data demonstrate that CT mRNA levels are induced 4.7-fold to 7.4-fold, respectively, when CHO cells or THP-1-derived macrophages are incubated in lipid-depleted medium and that these same conditions result in increased activity of a CT promoter-luciferase reporter gene (1.7-fold). Ryan et al. (9) reported that CT mRNA levels increased 2-fold when an alveolar type II epithelial cell line (MLE-12) was switched from a growth medium supplemented with 10% FBS (lipid-cholesterol rich) to one containing 10% LPDS (lipid-cholesterol depleted). On the basis of the results of nuclear runon assays, Ryan et al. (9) proposed that the increase in CT mRNA levels resulted from increased transcription of the gene. mRNA levels, which occurs when cells are incubated in lipid-depleted medium, is the result of SREBP-dependent trans-activation of the CT gene. We demonstrate that this activation depends on the interaction of SREBP with the SRE in the proximal promoter of the CT gene. We also note that at equivalent plasmid concentrations, SREBP-1a is a more potent trans-activator of CT transcription than SREBP-2. This latter result is consistent with previous studies that have reported that genes involved in fatty acid and triglyceride synthesis are more responsive to SREBP-1a or SREBP-1c than SREBP-2, whereas genes involved in cholesterol biosynthesis are more responsive to SREBP-2 than SREBP-1 (17). mRNA levels are not regulated significantly after the addition to cells of cholesterol/sterols; CT mRNA levels were reported to be unchanged when macrophages were loaded with unesterified cholesterol, as a result of treatment with inhibitors of ACAT (33). CT mRNA levels were also reported to be unchanged in SRD4 cells that express a mutant inactive form of ACAT and constitutively process SREBP (34) or in SRD6 cells that fail to process SREBP (35). Despite the lack of effect on CT mRNA levels these mutants exhibit altered enzyme activity levels of CT and changes in the rate of phospholipid biosynthesis. These different results noted in these various studies regarding the regulation of CT mRNA levels are difficult to reconcile, but may depend on a number of factors including the relative expression of SREBP-1c in the various cell types under the different conditions. promoter has several Sp1-binding sites, one of which is located within 2 bp of the SRE (Fig 3) (12). Expression of a CT promoter-reporter gene has been shown to be dependent on binding of Sp1, or related proteins, to the proximal promoter (12) (13). We hypothesize that high rates of transcription of the CT gene require functional cis elements that bind SREBP and Sp1. ). as an SREBP target gene identifies a mechanism by which cholesterol, fatty acid, and triglyceride syntheses are coordinately regulated with phosphatidylcholine biosynthesis.
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 J. Zhou, Y. You, J. Zabner, A. J. Ryan, and R. K. Mallampalli The CCT Promoter Directs High-Level Transgene Expression in Distal Lung Epithelial Cell Lines Am. J. Respir. Cell Mol. Biol., January 1, 2004; 30(1): 61 - 68. [Abstract] [Full Text] [PDF]
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K. N. Maxwell, R. E. Soccio, E. M. Duncan, E. Sehayek, and J. L. Breslow Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice J. Lipid Res., November 1, 2003; 44(11): 2109 - 2119. [Abstract] [Full Text] [PDF]
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C. Banchio, L. M. Schang, and D. E. Vance Activation of CTP:Phosphocholine Cytidylyltransferase {alpha} Expression during the S Phase of the Cell Cycle Is Mediated by the Transcription Factor Sp1 J. Biol. Chem., August 22, 2003; 278(34): 32457 - 32464. [Abstract] [Full Text] [PDF]
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H. Sugimoto, S. Sugimoto, K. Tatei, H. Obinata, M. Bakovic, T. Izumi, and D. E. Vance Identification of Ets-1 as an Important Transcriptional Activator of CTP:Phosphocholine Cytidylyltransferase {alpha} in COS-7 Cells and Co-activation with Transcriptional Enhancer Factor-4 J. Biol. Chem., May 23, 2003; 278(22): 19716 - 19722. [Abstract] [Full Text] [PDF]
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M. Bakovic, K. Waite, and D. E. Vance Oncogenic Ha-Ras Transformation Modulates the Transcription of the CTP:Phosphocholine Cytidylyltransferase alpha Gene via p42/44MAPK and Transcription Factor Sp3 J. Biol. Chem., April 18, 2003; 278(17): 14753 - 14761. [Abstract] [Full Text] [PDF]
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 A. C.M. Bonne, M. G. den Bieman, G. F. Gillissen, A. Lankhorst, C. J. Kenyon, B. F.M. van Zutphen, and H. A. van Lith Quantitative Trait Loci Influencing Blood and Liver Cholesterol Concentration in Rats Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 2072 - 2079. [Abstract] [Full Text] [PDF]
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P. A. Edwards, H. R. Kast, and A. M. Anisfeld BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis J. Lipid Res., January 1, 2002; 43(1): 2 - 12. [Abstract] [Full Text] [PDF]
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L. S. Golfman, M. Bakovic, and D. E. Vance Transcription of the CTP:Phosphocholine Cytidylyltransferase alpha Gene Is Enhanced during the S Phase of the Cell Cycle J. Biol. Chem., November 16, 2001; 276(47): 43688 - 43692. [Abstract] [Full Text] [PDF]
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