FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068389722> ?p ?o ?g. }

• W2068389722 endingPage "19722" @default.
• W2068389722 startingPage "19716" @default.
• W2068389722 abstract "Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTα in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338–12344). To identify the most important transcription factor involved in basal CTα transcription, we made CTα promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CTα transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CTα promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTα mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTα gene and that Ets-1 activity is enhanced by TEF-4. Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTα in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338–12344). To identify the most important transcription factor involved in basal CTα transcription, we made CTα promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CTα transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CTα promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTα mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTα gene and that Ets-1 activity is enhanced by TEF-4. Phosphatidylcholine (PC) 1The abbreviations used are: PC, phosphatidylcholine; CREBP, cAMP responsive element-binding protein; CTα, CTP:phosphocholine cytidylyltransferase α; Ctpct, CTP:phosphocholine cytidylyltransferase α gene; EBS, Ets-binding site; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; TEF-4, transcriptional enhancer factor-4; RT, reverse transcription; CMV, cytomegalovirus; MAPK, mitogen-activated protein kinase; LUC, luciferase. is the major membrane phospholipid in mammalian cells and tissues. PC is made in all nucleated cells via the CDP-choline pathway in which CTP:phosphocholine cytidylyltransferase (CT) is recognized as an important rate-limiting and regulated enzyme (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 2Lykidis A. Jackowski S. Prog. Nuc. Acid Res. & Mol. Biol. 2000; 65: 361-393Crossref Google Scholar, 3Johnson J.E. Cornell R.B. Mol. Mem. Biol. 1999; 16: 217-235Crossref PubMed Scopus (242) Google Scholar, 4Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam2002: 205-232Google Scholar). The first mammalian CT was purified from rat liver (5Weinhold P.A. Rounsifer M.E. Feldman D.A. J. Biol. Chem. 1986; 261: 5104-5110Abstract Full Text PDF PubMed Google Scholar, 6Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar), and the corresponding CTα cDNA was cloned and expressed (7Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (130) Google Scholar). CTα is ubiquitous and the most active form of CT (8Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (114) Google Scholar, 9Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) although cDNAs for two other isoenzymes, CTβ1 (8Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (114) Google Scholar) and its splice variant CTβ2 (9Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), were recently cloned, and are encoded by a second gene. All isoforms contain a highly conserved catalytic domain and a helical lipid-binding domain (8Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (114) Google Scholar). CTα and CTβ2 contain a highly phosphorylated domain at their carboxyl termini (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 2Lykidis A. Jackowski S. Prog. Nuc. Acid Res. & Mol. Biol. 2000; 65: 361-393Crossref Google Scholar, 3Johnson J.E. Cornell R.B. Mol. Mem. Biol. 1999; 16: 217-235Crossref PubMed Scopus (242) Google Scholar, 4Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam2002: 205-232Google Scholar, 9Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Pelech S.L. Vance D E. J. Biol. Chem. 1982; 257: 14198-14202Abstract Full Text PDF PubMed Google Scholar, 11Yang W. Jackowski S. J. Biol. Chem. 1995; 270: 16503-16506Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), whereas CTβ1 lacks this domain (8Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (114) Google Scholar). The functions of the individual domains for regulation of CTα activity have been extensively studied. The helical domain for the binding of specific lipids (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 2Lykidis A. Jackowski S. Prog. Nuc. Acid Res. & Mol. Biol. 2000; 65: 361-393Crossref Google Scholar, 3Johnson J.E. Cornell R.B. Mol. Mem. Biol. 1999; 16: 217-235Crossref PubMed Scopus (242) Google Scholar, 4Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam2002: 205-232Google Scholar, 12Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Sohal P.S. Cornell R.B. J. Biol. Chem. 1990; 265: 11746-11750Abstract Full Text PDF PubMed Google Scholar), and a highly phosphorylated domain at the carboxyl terminus (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 2Lykidis A. Jackowski S. Prog. Nuc. Acid Res. & Mol. Biol. 2000; 65: 361-393Crossref Google Scholar, 3Johnson J.E. Cornell R.B. Mol. Mem. Biol. 1999; 16: 217-235Crossref PubMed Scopus (242) Google Scholar, 4Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam2002: 205-232Google Scholar, 11Yang W. Jackowski S. J. Biol. Chem. 1995; 270: 16503-16506Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14MacDonald J.I.S. Kent C. J. Biol. Chem. 1994; 269: 10529-10537Abstract Full Text PDF PubMed Google Scholar, 15Wang Y. Kent C. J. Biol. Chem. 1995; 270: 17843-17849Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Arnold R.S. DePaoli-Roach A.A. Cornell R.B. Biochemistry. 1997; 36: 6149-6156Crossref PubMed Scopus (61) Google Scholar, 17Houweling M. Jamil H. Hatch G.M. Vance D.E. J. Biol. Chem. 1994; 269: 7544-7551Abstract Full Text PDF PubMed Google Scholar), are important for the modulation of CTα activity. However, less is known about the regulation or function of CTβ1 and CTβ2. Zhang et al. (18Zhang D. Tang W. Yao P.M. Yang C. Xie B. Jackowski S. Tabas I. J. Biol. Chem. 2000; 275: 35368-35376Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) showed that CTβ2 is induced in macrophages from CTα-deficient mice, and a low level of CTβ2 activity is apparently enough to keep the cells viable. In addition to post-translational regulation, CTα mRNA levels have been reported to be regulated transcriptionally and post-transcriptionally. Tessner et al. (19Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar) provided the first evidence for increased CT mRNA in colony stimulating factor 1-stimulated macrophages. Houweling et al. (20Houweling M. Tijburg L.B.M. Vaartjes W.J. Batenburg J.J. Kalmar G.B. Cornell R.B. van Golde L.M.G. Eur. J. Biochem. 1993; 214: 927-933Crossref PubMed Scopus (29) Google Scholar) demonstrated that CTα is regulated at the level of its mRNA in rat liver after partial hepatectomy. The maximal increase in the CTα mRNA coincided with maximal DNA synthesis 24 h after partial hepatectomy (21Houweling M. Cui Z. Tessitore L. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar). CTα is highly expressed during the perinatal period, and the expression of CTα is positively associated with hepatic cell division (22Sesca E. Perletti G.P. Binasco V. Chiara M. Tessitore L. Biochem. Biophys. Res. Comm. 1996; 229: 158-162Crossref PubMed Scopus (29) Google Scholar, 23Cui Z. Shen Y.J. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 10-16Crossref PubMed Scopus (44) Google Scholar). Golfman et al. (24Golfman L.S. Bakovic M. Vance D.E. J. Biol. Chem. 2001; 276: 43688-43692Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) recently showed that CTα mRNA increased during the S phase of the cell cycle in C3H10T1/2 cells. Several reports indicate that CTα can be regulated post-transcriptionally, apparently by reduction in the rate of mRNA degradation (19Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar, 25Hogan M. Kuliszewski M. Lee W. Post M. Biochem. J. 1996; 314: 799-803Crossref PubMed Scopus (28) Google Scholar). However, the precise mechanisms involved in regulating CT mRNA stability still have to be elucidated. Tang et al. (26Tang W. Keesler G.A. Tabas I. J. Biol. Chem. 1997; 272: 13146-13151Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) isolated the murine CTα gene (Ctpct) and showed that the exon/intron organization of the gene closely resembles the functional domains of the enzyme. The gene is transcribed from two transcriptional start sites, lacks a TATA box, but contains GC-rich regions. The 5′-terminal, ∼200 bp of the proximal promoter, appears to contain Sp1, NF-κB, Ets, and cAMP responsive element-binding protein (CREBP)-binding sites as shown in Fig. 1. Bakovic et al. (27Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar) characterized its regulatory elements and associated factors. Three Sp1-binding regions (-31/-9, -88/-50, and -148/-128) have basal, activator, and suppressor promoter activities, respectively. Sp1, Sp2, and Sp3 can competitively bind to these regions, and the relative abundance of these factors regulates promoter activity of the CTα gene (28Bakovic M. Waite K. Vance D.E. J. Lipid Res. 2000; 41: 583-594Abstract Full Text Full Text PDF PubMed Google Scholar). More recently we have shown that transcriptional enhancer factor-4 (TEF-4) binds to the promoter region between -97 and -89 as shown in Fig. 1 and stimulates the expression of the CTα gene by association with the basal transcription machinery (29Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). TEF-4 is closely related to TEF-1 (30Yasunami M. Suzuki K. Houtani T. Sugimoto T. Ohkubo H. J. Biol. Chem. 1995; 270: 18649-18654Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Jaquemin P. Hwang J.-J. Martial J.A. Dolle P. Davidson I. J. Biol. Chem. 1996; 271: 21775-21785Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) that was previously isolated as a regulatory protein of the SV40 enhancer (32Xiao J.H. Davidson I. Matthes H. Garnier J.-M. Chambon P. Cell. 1991; 65: 551-568Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Kast et al. (33Kast H.R. Nguyen C.M. Anisfeld A.M. Ericsson J. Edwards P.A. J. Lipid Res. 2001; 42: 1266-1272Abstract Full Text Full Text PDF PubMed Google Scholar) and Mallampalli et al. (34Mallampalli R.K. Ryan A.J. Carroll J.L. Osborne T.F. Thomas C.P. Biochem. J. 2002; 362: 81-88Crossref PubMed Google Scholar) reported a functional sterol response element 156 bp upstream of the transcriptional start site. Induction of CT mRNA was observed when Chinese hamster ovary cells and alveolar type II epithelial cells were cultured in lipoprotein-deficient serum, respectively. However, the physiological importance of this sterol response site in transcriptional regulation of the CTα gene is not clear (35Lagace T.A. Storey M.K. Ridgway N.D. J. Biol. Chem. 2000; 275: 14367-14374Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In this study, we provide evidence that Ets-1 is a major transcription factor involved in the basal transcription of the CTα gene. Ets-1 was first reported as a proto-oncogene for avian erythroblastosis (36Nun M.F. Seeburg P.H. Moscovici C. Duesberg P.H. Nature. 1983; 306: 391-395Crossref PubMed Scopus (289) Google Scholar). Materials—The luciferase vector, pGL3-basic, that contains the cDNA for Photinus pyraris luciferase, the control pRL-CMV vector that contains the cDNA for Renilla reniformis luciferase, and the Dual-luciferase Reporter Assay system were obtained from Promega (Madison, WI). FuGENE 6TM transfection reagents, Dulbecco's modified Eagle's medium (high glucose), and fetal bovine serum were from Roche Applied Science, Sigma, and Invitrogen, respectively. COS-7 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Preparation of Deleted and Mutated CTα Promoter-Luciferase Reporters—Various 5′-deleted CTα promoter regions, LUC.C7 (-1268/+38), LUC.C8 (-201/+38), LUC.D1 (-90/+38), LUC.D2 (-130/+38), LUC.D3 (-52/+38), LUC.D4 (-10/+38), and LUC.D5 (+10/+38), inserted into the promoter-less luciferase vector pGL3-basic (Promega) were prepared as described previously (27Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar). LUC.D3.25 (-43/+38), LUC.D3.5 (-31/+38), and LUC.D1.5 (-71/+38) clones were obtained from LUC.C8 by PCR using the corresponding forward primers, 5′-CAGGTACCCAGTCCGGTCAGATGTTTCCCG-3′, 5′-CAGGTACCCAGATGTTTCCCGGGCGTCTCC-3′, and 5′-CAGGTACCAGGGCGGGCGGGAGGCGGGACT-3′, respectively, and GL Primer 2 (Promega) as a universal reverse primer from the luciferase vector. The amplified fragments were purified, cut with KpnI/HindIII, and cloned into the corresponding site of the pGL3-basic vector. Fig. 1 shows the partial structure of the mouse Ctpct promoter from -212 to +38 and indicates the start positions of the deletion mutants. When we searched the CTα promoter region by TRANSFAC transcription factor data base, several important consensus elements for the binding of transcription factors were identified (Fig. 1). To prepare mutated promoters, GCCC (-139/-136) was mutated to AGCT and named LUC.mSp1 (1)/C7 or D1.5, CGGGCG (-67/-62) was changed to AATTCA and named LUC.mSp1 (2)/C7 or D1.5, GGCGG (-58/-54) was mutated to AACAA and named LUC.mNF-κB/C7 or D1.5, GACTTC (-53/-48) was converted to ACCAAA and named LUC.mEts-a/C7 or D1.5, TCC (-49/-47) was changed to AAA and named LUC.mEts-b/C7 or D1.5, and TGAC (+20/+23) was mutated to AAAA and named LUC.mCREB/C7 or D1.5. These mutations were created from LUC.C7 or LUC.D1.5 by PCR using QuikChangeTM site-directed mutagenesis kits (Stratagene, La Jolla, CA). The mutated complementary primers were used for making each mutated construct, and the resulting plasmids were used for transformation of JM109 cells. The amplified plasmids were cut with KpnI/HindIII and cloned into the corresponding site of the pGL3-basic vector. All deleted and mutated constructs were sequenced and correct ones were selected for further experiments. Construction of TEF-4 and Ets-1 Expression Vectors—The mouse TEF-4 expression vector carrying the cDNA of TEF-4 was obtained by the yeast one-hybrid system as reported (29Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The mouse full-length cDNA coding murine Ets-1 was obtained through PCR of a reverse-transcribed product of mRNA from NIH cells using Superscript II (Invitrogen). PCR was performed with the complementary primers, 5′-GGCACCATGAAGGCGGCCGTCGATC-3′, and 5′-GTCAGCATCCGGCTTTACATCCAGC-3′ with Takara Ex TaqTM DNA polymerase (Takara-Bio. Co., Tokyo, Japan). The 1.4-kb PCR product was cloned into pcDNA3.1/V5 (Invitrogen) in frame with V5-tag, and the sequence was confirmed. Tissue Culture, Transfection, and Luciferase Assays—COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells (1 × 105) were plated on 35-mm plates (Falcon-Becton Dickson Labware, Franklin Lakes, NJ) and grown overnight. Three μl of FuGENE 6TM was suspended in 100 μl of serum-free medium and mixed with 0.5 μg of the CTα promoter-luciferase constructs (see above), 0.001 μg of pRL-CMV Renilla vector as a transfection control, and 0.5 μg of either pcTEF-4 or pcDNA (Invitrogen). Transfection was initiated by dropwise addition of DNA suspension to the cell culture. Forty-eight hours later cells were harvested, lysed in 200 μl of Passive lysis buffer (Promega), and 10 μl of the cell lysate was used for the dual-luciferase assay according to the manufacturer's instructions. Luciferase activity was normalized for transfection efficiency by using the ratio of the activities obtained with the CTα promoter deletion constructs (see above) and the pRL-CMV construct carrying the cytomegalovirus promoter-luciferase fusion. When we assayed luciferase activities with Ets-1, pcDNA was used so that the plasmid content in each experiment was equal, and luciferase values were normalized against total protein concentrations determined by protein assay (Bio-Rad). Preparation of Nuclear Extracts and Electromobility Gel-shift Assays—Nuclear extracts from COS-7 cells were prepared according to Andrews and Faller (37Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2214) Google Scholar) with minor modifications (27Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar). Five hundred pmol of the opposite strands of pEts (5′-GGCGGGAGGCGGGACTTCCGGTCCGCAGTC-3′), pmEts-a (5′-GGCGGGAGGCGGaccaaaCGGTCCGCAGTC-3′), and pmEts-b (5′-GGCGGGAGGCGGGACTaaaGGTCCGCAGTC-3′) were annealed (70 °C, 10 min) in 100 μl of 25 mm Tris-HCl, pH 8.0, 0.5 mm MgCl2, and 25 mm NaCl and then cooled to room temperature. An aliquot (10 pmol) of the double-stranded oligonucleotides was 5′-end-labeled with [32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase and purified on a Sephadex G-25 column (Amersham Biosciences). A DNA protein-binding reaction was performed for 30 min at room temperature in 40 μlof1× binding buffer (40 mm Tris-HCl, pH 7.9, 4 mm MgCl2, 2 mm EDTA, 100 mm NaCl, 2 mm dithiothreitol, 200 μg/ml bovine serum albumin, 20% glycerol, and 0.2% Nonidet P-40) containing 1 μg of poly(dI-dC) (Amersham Biosciences), 1 μl of the radiolabeled probe (50,000–80,000 cpm), and nuclear extracts of COS-7 cells. In some cases, unlabeled double-stranded pEts (100-fold molar excess), anti-Ets-1 or anti-Ets-1/Ets-2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti-V5 antibody (Invitrogen) was included in the incubation mixture. The labeled probe was separated from DNA-protein complexes by electrophoresis on 6% non-denaturing polyacrylamide gels in Tris borate/EDTA buffer (44.5 mm Tris-HCl, pH8.3, 44.5 mm boric acid, and 1 mm EDTA) at 4 °C until the xylene cyanol dye reached 5 cm from the bottom of the gel. Autoradiography was performed by exposure of the gel to an imaging plate for 15–30 min, and images were analyzed by Fuji BAS-2000 (Fiji Photo Film Co., Ltd., Tokyo, Japan). The intensity of the gel-shift band was calculated using the Quantity One software (PDI, Huntington Station, NY). SDS-PAGE and Immunoblot Analysis—The proteins were separated by SDS-PAGE according to the method of Laemmli (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) with 10% (w/v) gels, and transferred to a nitrocellulose membrane (Hybond-C, Amersham Biosciences) with a semi-dry electroblotter (Sartorius, Goetingen, Germany). The membrane was treated with 5% (w/v) dried skim milk in 20 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl at 4 °C overnight, washed, and then treated with anti-Ets-1/Ets-2, anti-Ets-1, or anti-actin (Santa Cruz Biotechnology, Inc.) antiserum diluted 1:200 for 2 h, followed by washing with 20 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl. Immunoreactive proteins were visualized by treatment for 30 min with protein A-peroxidase complex (Zymed Laboratories, San Francisco, CA), diluted 1:2,500, and the peroxidase immunostaining kit (Wako, Osaka, Japan). RT-PCR—COS-7 cells (2.5 × 105) were transfected with pcDNA (1.25 μg), pcEts-1 (1.25 μg), pcTEF-4 (1.25 μg), or pcEts-1 and pcTEF-4 (1.25 μg each) in 60-mm plates as described above. pcDNA was used so that the plasmid content in each experiment was equal. After cells were cultured for 24 h, total mRNA was obtained with RNA extraction kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. One μg of total RNA was reverse-transcribed at 50 °C for 30 min, then subjected to 25 cycles of amplification (94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min) using the one-step RT-PCR kit (Qiagen). The primers used for CTα were 5′-ATGCACAGTGTTCAGCCAA-3′ (sense) and 5′-GGGCTTACTAAAGTCAACTTCAA-3′ (antisense), and they produce an ∼200-bp CTα fragment. Primers for glycero-3-phosphate dehydrogenase (G3PDH) were 5′-TCCACCACCCTGTTGCTGTA-3′ (sense) and 5′-ACCACAGTCCATGCCATCAC-3′ (antisense). The intensities of the CTα bands were normalized to those of the G3PDH bands using the Quantity One software. Statistical Analysis—All values are expressed as means ± S.D. Group means were compared by Student's t test or Cochran-Cox test after analysis of variance to determine the significance of difference between the individual means. Statistical significance was assumed at p < 0.05. Deletion and Mutation Analysis of Chimeric CTα Promoter-Luciferase Reporters and Their Activation by TEF-4 in COS-7 Cells—To examine the promoter region for CTα basal transcription, we prepared various CTα promoter deletion constructs linked to the luciferase reporter and transfected into COS-7 cells. The expression of luciferase activity was determined by dual-luciferase assays and normalized for transfection efficiency after co-transfection with pRL-CMV Renilla vector. The Renilla luciferase activity of the CMV promoter-driven controls was constant and was not affected by TEF-4 throughout the experiments. As shown in Fig. 2A, when cells were transfected with CTα promoter-luciferase constructs, luciferase activity was increased dependent on the length of the promoter region. The deletion analysis clearly demonstrated that constructs D3 (-52/+38) or shorter had minimal luciferase activity compared with D1.5 (-71/+38). These results suggested that important positive regulatory regions for basal CTα transcription in COS-7 cells resided between positions -52 and -71. To substantiate this proposal we made mutated constructs of LUC.C7 and LUC.D1.5 at each putative transcription factor binding site shown in Fig. 1. After transfection, the luciferase activities of the mutated LUC.C7 and LUC.D1.5 constructs were assayed (Fig. 2, B and C, respectively). When the Sp1 site at -139 was mutated, the luciferase activity of the LUC.mSp1 (1)/C7 was decreased significantly, but only 25% compared with LUC.C7. When the Sp1 site at -67 was mutated, the luciferase activities of LUC.mSp1 (2)/C7 did not decrease compared with LUC.C7. The luciferase activity of the NF-κB site mutant (LUC.mNF-κB/C7) decreased by 25% compared with LUC.C7 (Fig. 2B). Mutation in the CREBP-binding site at +20 slightly decreased the luciferase activity (Fig. 2B). However, the luciferase activities of mutated constructs in the putative Ets-binding site (EBS), LUC.mEts-a/C7 and LUC.mEts-b/C7, were decreased by ∼80% compared with LUC.C7 activity as shown in Fig. 2B. The expression of the shorter constructs, LUC.mEts-a/D1.5 and LUC.mEts-b/D1.5, were very low, only ∼5% compared with LUC.D1.5 (Fig. 2C). These results strongly suggest that the promoter region between -53 and -47 is an important site for basal CTα transcription. We reported TEF-4 enhanced the luciferase activity of LUC.C7 and LUC.D1.5 about twice (Ref. 29Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar and Fig. 2). When the mutations in EBS were created, co-transfection with TEF-4 cDNA also stimulated the activity of LUC.mEts-a/C7 or D1.5 and LUC.mEts-b/C7 or D1.5 about 2-fold. However, the TEF-4-enhanced luciferase activities with LUC.mEts-a/C7 or D1.5 and LUC.mEts-b/C7 or D1.5 were much smaller than LUC.C7 or LUC.C1.5. Thus, mutation of the Ets binding of the CTα promoter decreased luciferase activity to a low level (Fig. 2, A–C). DNA Binding Properties of the Nuclear Extracts of COS-7 Cells—To test whether or not there is a protein in the nuclear extracts from COS-7 cells that binds to the putative EBS (-49/-47), we prepared probes with or without a mutation in the EBS. As shown in Fig. 3A, lanes 2 and 3, DNA-protein complexes with the pEts (-65/-36) probe were clearly identified. When the EBS in pEts was mutated from GACTTC to ACCAAA (-53/-48) (pmEts-a) (Fig. 3A, lanes 7 and 8) or from TCC to AAA (-49/-47) (pmEts-b) (Fig. 3A, lanes 10 and 11), the bands were attenuated or disappeared. These results strongly suggested that there was a protein(s) in the extracts of COS-7 cells that bound to EBS in the pEts probe. After transfection with pcTEF-4, the band intensities of the specific DNA-protein complexes were significantly increased (Fig. 3A, lanes 2 and 3, and Fig. 3B). This result indicated that TEF-4 enhanced the binding of pEts probe and protein complexes to EBS. Identification of Ets-1 as the Protein That Binds to the pEts Probe—To identify the protein(s) binding to pEts, we used an antibody, anti-Ets-1/Ets-2, with specificity for the DNA-binding site in the C terminus of Ets family proteins. Immunoblot analysis indicated the existence of a 54-kDa protein that cross-reacted with anti-Ets-1/Ets-2 antibody in the nuclear extracts from COS-7 cells (Fig. 4A, upper panel). When various amounts of the anti-Ets-1/Ets-2 antibody were added to incubations of nuclear extracts with the pEts probe, the intensity of the immunoreactive band decreased (Fig. 4B, left). This result indicated that the bands revealed by the gel-shift analysis resulted from a complex of Ets-1 or Ets-2 with the pEts probe. To determine which member of the Ets family was binding to pEts, we used an Ets-1-specific antibody. Immunoblot analysis showed a 54-kDa protein that cross-reacted with anti-Ets-1 antibody in the nuclear extracts from COS-7 cells (Fig. 4A, middle panel). When we used the anti-Ets-1 antibody with the probe in gel shift analyses, the decrease in the intensity of the band was similar to anti-Ets-1/-2 (Fig. 4B, right). Thus, it appears that Ets-1 is the protein that binds to the EBS of pEts. When COS-7 cells were stimulated with TEF-4, the basal luciferase activities of LUC.C7 and LUC.D1.5 were increased (Fig. 2), and the binding of endogenous Ets-1 to pEts was significantly enhanced (Fig. 3, lane 3). However, immunoblot analysis showed that the amount of Ets-1, or actin as the internal standard, was not changed by TEF-4 stimulation (Fig. 4A). Thus, TEF-4 might modify the binding of Ets-1 to the promoter, and enhance Ets-1-stimulated CTα transcription. Over-expression of Ets-1 Increases the Binding of Ets to the CTα Promoter—In another approach to confirm that Ets does indeed bind to the CTα promoter, we transfected COS-7 cells with a vector that contained the cDNA encoding Ets-1 (pcEts-1) or the vector alone (pcDNA). Nuclear extracts were prepared after 48 h and i" @default.
• W2068389722 created "2016-06-24" @default.
• W2068389722 creator A5008956250 @default.
• W2068389722 creator A5014399804 @default.
• W2068389722 creator A5024316557 @default.
• W2068389722 creator A5039896704 @default.
• W2068389722 creator A5063670502 @default.
• W2068389722 creator A5073998071 @default.
• W2068389722 creator A5078870288 @default.
• W2068389722 date "2003-05-01" @default.
• W2068389722 modified "2023-10-16" @default.
• W2068389722 title "Identification of Ets-1 as an Important Transcriptional Activator of CTP: Phosphocholine Cytidylyltransferase α in COS-7 Cells and Co-activation with Transcriptional Enhancer Factor-4" @default.
• W2068389722 cites W1490431590 @default.
• W2068389722 cites W1524917676 @default.
• W2068389722 cites W1531352737 @default.
• W2068389722 cites W1542145035 @default.
• W2068389722 cites W155985007 @default.
• W2068389722 cites W1565685625 @default.
• W2068389722 cites W1576342917 @default.
• W2068389722 cites W1592254226 @default.
• W2068389722 cites W1923711010 @default.
• W2068389722 cites W1963489817 @default.
• W2068389722 cites W1967012124 @default.
• W2068389722 cites W1967871528 @default.
• W2068389722 cites W1969198017 @default.
• W2068389722 cites W1969499006 @default.
• W2068389722 cites W1974917376 @default.
• W2068389722 cites W1976625108 @default.
• W2068389722 cites W1977385412 @default.
• W2068389722 cites W1986030520 @default.
• W2068389722 cites W1996353071 @default.
• W2068389722 cites W2006308545 @default.
• W2068389722 cites W2007487901 @default.
• W2068389722 cites W2011007912 @default.
• W2068389722 cites W2011487859 @default.
• W2068389722 cites W2016973558 @default.
• W2068389722 cites W2017387476 @default.
• W2068389722 cites W2017900917 @default.
• W2068389722 cites W2018095558 @default.
• W2068389722 cites W2018950363 @default.
• W2068389722 cites W2023944219 @default.
• W2068389722 cites W2024931988 @default.
• W2068389722 cites W2027147108 @default.
• W2068389722 cites W2038113019 @default.
• W2068389722 cites W2041798139 @default.
• W2068389722 cites W2042001534 @default.
• W2068389722 cites W2045893838 @default.
• W2068389722 cites W2049792868 @default.
• W2068389722 cites W2050477788 @default.
• W2068389722 cites W2051703449 @default.
• W2068389722 cites W2052411313 @default.
• W2068389722 cites W2055115648 @default.
• W2068389722 cites W2057885570 @default.
• W2068389722 cites W2068560412 @default.
• W2068389722 cites W2073905990 @default.
• W2068389722 cites W2078028419 @default.
• W2068389722 cites W2079045007 @default.
• W2068389722 cites W2081855564 @default.
• W2068389722 cites W2082214219 @default.
• W2068389722 cites W2083757724 @default.
• W2068389722 cites W2088884715 @default.
• W2068389722 cites W2094646101 @default.
• W2068389722 cites W2097513054 @default.
• W2068389722 cites W2100837269 @default.
• W2068389722 cites W2106139478 @default.
• W2068389722 cites W2109906218 @default.
• W2068389722 cites W2117671999 @default.
• W2068389722 cites W2119973182 @default.
• W2068389722 cites W2125263842 @default.
• W2068389722 cites W2126469537 @default.
• W2068389722 cites W2138292652 @default.
• W2068389722 cites W2151473045 @default.
• W2068389722 cites W2153892426 @default.
• W2068389722 cites W2155776330 @default.
• W2068389722 cites W2160659225 @default.
• W2068389722 cites W2235552068 @default.
• W2068389722 cites W2318613091 @default.
• W2068389722 cites W2319829720 @default.
• W2068389722 cites W4244518691 @default.
• W2068389722 cites W4562572 @default.
• W2068389722 doi "https://doi.org/10.1074/jbc.m301590200" @default.
• W2068389722 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12642588" @default.
• W2068389722 hasPublicationYear "2003" @default.
• W2068389722 type Work @default.
• W2068389722 sameAs 2068389722 @default.
• W2068389722 citedByCount "15" @default.
• W2068389722 countsByYear W20683897222014 @default.
• W2068389722 countsByYear W20683897222019 @default.
• W2068389722 crossrefType "journal-article" @default.
• W2068389722 hasAuthorship W2068389722A5008956250 @default.
• W2068389722 hasAuthorship W2068389722A5014399804 @default.
• W2068389722 hasAuthorship W2068389722A5024316557 @default.
• W2068389722 hasAuthorship W2068389722A5039896704 @default.
• W2068389722 hasAuthorship W2068389722A5063670502 @default.
• W2068389722 hasAuthorship W2068389722A5073998071 @default.
• W2068389722 hasAuthorship W2068389722A5078870288 @default.
• W2068389722 hasBestOaLocation W20683897221 @default.
• W2068389722 hasConcept C104317684 @default.

• next