FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2075939587> ?p ?o ?g. }

• W2075939587 endingPage "669" @default.
• W2075939587 startingPage "662" @default.
• W2075939587 abstract "In an attempt to examine the mechanisms by which transcriptional activity of the cyclin D1 promoter is regulated in vascular endothelial cells (EC), we examined the cis-elements in the human cyclin D1 promoter, which are required for transcriptional activation of the gene. The results of luciferase assays showed that transcriptional activity of the cyclin D1 promoter was largely mediated by SP1 sites and a cAMP-responsive element (CRE). DNA binding activity at the SP1 sites, which was analyzed by electrophoretic mobility shift assays, was significantly increased in the early to mid G1 phase, whereas DNA binding activity at CRE did not change significantly. Furthermore, Induction of the cyclin D1 promoter activity in the early to mid G1phase depended largely on the promoter fragment containing the SP1 sites, whereas the proximal fragment containing CRE but not the SP1 sites was constitutively active. Finally, the increase in DNA binding and promoter activities via the SP1 sites was mediated by the Ras-dependent pathway. The results suggested that the activation of the cyclin D1 gene in vascular ECs was regulated by a dual system; one was inducible in the G1phase, and the other was constitutively active. In an attempt to examine the mechanisms by which transcriptional activity of the cyclin D1 promoter is regulated in vascular endothelial cells (EC), we examined the cis-elements in the human cyclin D1 promoter, which are required for transcriptional activation of the gene. The results of luciferase assays showed that transcriptional activity of the cyclin D1 promoter was largely mediated by SP1 sites and a cAMP-responsive element (CRE). DNA binding activity at the SP1 sites, which was analyzed by electrophoretic mobility shift assays, was significantly increased in the early to mid G1 phase, whereas DNA binding activity at CRE did not change significantly. Furthermore, Induction of the cyclin D1 promoter activity in the early to mid G1phase depended largely on the promoter fragment containing the SP1 sites, whereas the proximal fragment containing CRE but not the SP1 sites was constitutively active. Finally, the increase in DNA binding and promoter activities via the SP1 sites was mediated by the Ras-dependent pathway. The results suggested that the activation of the cyclin D1 gene in vascular ECs was regulated by a dual system; one was inducible in the G1phase, and the other was constitutively active. endothelial cells vascular endothelial growth factor cyclin-dependent kinases mitogen-activated protein kinase kinase extracellular signal-regulated kinase cAMP-responsive element binding protein activating transcription factor signal transducers and activators of transcription human umbilical vein endothelial cells bovine aortic endothelial cells hemagglutinin Dulbecco's modified Eagle's medium fetal bovine serum base pair(s) polymerase chain reaction wild type green fluorescence protein adenovirus phosphate-buffered saline electrophoretic mobility shift assay Vascular endothelial cells (EC)1 form a monolayer in the luminal side of the vessels and play pivotal roles, such as prevention of monocyte/macrophage infiltration and provision of a nonthrombogenic surface, which in turn contribute to prevent the development of atherosclerosis (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). When ECs are injured, ECs adjacent to the injured site start to replicate until they come into contact with each other. Prompt and complete repair of defects in the EC monolayer is therefore important to prevent the occurrence and progression of atherosclerosis. Although a variety of mitogens, including endothelin and vascular endothelial growth factor (VEGF) are known to stimulate replication of ECs (2Vigne P. Marsault R. Breittmayer J.P. Frelin C. Biochem. J. 1990; 266: 415-420Crossref PubMed Scopus (135) Google Scholar, 3Plouet J. Schilling J. Gospodarowicz D. EMBO J. 1989; 8: 3801-3806Crossref PubMed Scopus (422) Google Scholar), little is known as to how ECs coordinate signals from multiple mitogens through multiple receptors and re-enter the cell cycle. Progression of the early to mid G1 phase is largely regulated by D-type cyclins, which associate with the cyclin-dependent kinases (cdk) cdk4/6 (4Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar, 5Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2591) Google Scholar). It is well known that expression of cyclin D is regulated, at least partly, at the transcription level and that the p21 ras (Ras)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway is implicated in the expression of the cyclin D1 gene. Several studies have indicated, using a dominant negative Ras mutant, that the Ras signaling pathway is involved in the induction of cyclin D1 (6Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (370) Google Scholar, 7Takuwa N. Takuwa Y. Mol. Cell. Biol. 1997; 17: 5348-5358Crossref PubMed Google Scholar). It has also been reported that the ERK pathway is implicated in VEGF-induced EC proliferation by stimulating cyclin D1 synthesis and cdk4 kinase activity (8Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 1998; 273: 26722-26728Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). However, little is known about the mechanisms by which the Ras-dependent pathway induces expression of the cyclin D1 gene. In other words, it is not clear which cis-elements in the cyclin D1 promoter are involved in the Ras-dependent activation of the cyclin D1 promoter. The promoter region of the cyclin D1 gene contains multiple cis-elements, including binding sites for AP1, for signal transducers and activators of transcription (STAT), for nuclear factor kappa B (NFκB), for activating transcription factor (ATF)/cAMP-responsive element binding protein (CREB), and for SP1. These cis-elements are potentially important for transcriptional activation of the cyclin D1 gene. In fact, it was reported that the AP1 site was implicated in angiotensin II-induced activation of the cyclin D1 promoter (9Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Cytokine-induced transcriptional activation of the cyclin D1 gene was mediated by STAT-binding sites in hematopoietic cells (10Matsumura I. Kitamura T. Wakao H. Tanaka H. Hashimoto K. Albanese C. Downward J. Pestell R.G. Kanakura Y. EMBO J. 1999; 18: 1367-1377Crossref PubMed Scopus (293) Google Scholar). It was also reported that NFκB-binding sites in the cyclin D1 promoter were implicated in transcriptional activation of the gene (11Guttridge D.C. Albanese C. Reuther J.Y. Pestell R.G. Baldwin Jr., A.S. Mol. Cell. Biol. 1999; 19: 5785-5799Crossref PubMed Google Scholar, 12Hinz M. Krappmann D. Eichten A. Heder A. Scheidereit C. Strauss M. Mol. Cell. Biol. 1999; 19: 2690-2698Crossref PubMed Scopus (703) Google Scholar, 13Joyce D. Bouzahzah B. Fu M. Albanese C. D'Amico M. Steer J. Klein J.U. Lee R.J. Segall J.E. Westwick J.K. Der C.J. Pestell R.G. J. Biol. Chem. 1999; 274: 25245-25249Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The ATF/CREB-binding site was also reportedly implicated in the activation of the cyclin D1 gene. pp60 v-src -induced transcriptional activation of the cyclin D1 gene was mediated by the ATF/CREB site (14Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines 3rd., G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Moreover, it was found that estrogen-induced activation of the cyclin D1 gene depended on the ATF/CREB site in which ATF-2 and c-Jun formed heterodimers (15Sabbah M. Courilleau D. Mester J. Redeuilh G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11217-11222Crossref PubMed Scopus (290) Google Scholar). Much less is known about the role of the SP1-binding sites. These results suggest that the transcriptional activation of the cyclin D1 gene may occur in a cell type- and mitogen-specific manner. We therefore sought to determine the cis-elements in the cyclin D1 promoter that are required for the transcriptional activation of the gene in vascular ECs. In the present study, we show that, in vascular ECs, activation of the cyclin D1 promoter is largely mediated by the SP1 and ATF/CREB sites and that DNA binding activity and promoter activity via the SP1 sites increase in the early to mid period of the G1 phase. We also show that the induction of DNA binding activity and transcriptional activity via the SP1 sites is mediated by the Ras-dependent pathway. Human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) were purchased from Sanko Junyaku (Tokyo, Japan). Anti-SP1, -SP2, -SP3, -SP4, -ATF/CREB, -NFκBp50, -NFκBp65, -c-Fos, and -c-Jun antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). HUVECs were cultured in a 1:1 mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 medium containing 10% fetal bovine serum (FBS), 17 ng/ml acidic fibroblast growth factor, and 50 units/ml heparin. To induce cell cycle re-entry, confluent HUVECs were split at a ratio of 1:3. BAECs were cultured in DMEM containing 10% FBS. BAECs were kept in low serum medium (DMEM containing 0.2% FBS) for 48 h to induce quiescence and stimulated with growth medium (DMEM containing 10% FBS) to induce cell cycle reentry. A 1719-bp fragment of the human cyclin D1 promoter was isolated by polymerase chain reaction (PCR) using 1 μg of human genomic DNA (Promega, Madison, WI) as the template. The PCR primers used were as follows: cycD1sense primer, 5′-GCTGATGCTCTGAGGCTTGGCTAT-3′; cycD1antisense primer, 5′-CTCCAGGACTTTGCAACTTCAACAAAACT-3′. The PCR conditions were 1 min at 95 °C, 1 min at 68 °C, and 2 min at 72 °C for 35 cycles, with a final extension for 10 min at 72 °C. To amplify a long fragment of the human cyclin D1 gene accurately, we used an LA Taq DNA polymerase (Takara Shuzou, Osaka, Japan). The PCR-amplified product was digested with SacI and HindIII and subcloned in pGL2-basic vector (pGL2/−1719/wt). The nucleotide sequence of the construct was confirmed by cycle sequencing using an ABI PRISM 310 genetic analyzer (PerkinElmer Life Sciences). The nucleotide sequence of the PCR-amplified product was basically identical with the sequence, which was previously reported (GenBank™ accession number Z29078), except that one extra cytosine was inserted in five consecutive cytosines, which were located at −129 to −125 nucleotides from the transcription start site. To prepare deletion mutants that lacked AP1-, NFκB-, STAT-, SP1-, or ATF/CREB-binding sites, PCR was performed. Constructed plasmids and the sense primers used were as follows: pGL2/−998/AP1: sense primer (5′-GCAGAGGGGACTAATATTTCCAGCA-3′); pGL2/−934/ΔAP1, sense primer (5′-GGAGATCACTGTTTCTCAGCTTTCCA-3′); pGL2/−836/ΔNFκB1, sense primer (5′-GGACCGACTGGTCAAGGTAGGAA-3′); pGL2/−707/ΔNFκB2, sense primer (5′-GAGCGAGCGCATGCTAAGCTGAA-3′); pGL2/−461/ΔSTAT1, sense primer (5′-GCGCCCATTCTGCCGGCTTGGAT-3′); pGL2/−229/ΔSTAT2, sense primer (5′-TTCTATGAAAACCGGACTACAGGGGCAACTC-3′); pGL2/−161/SP1, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTT-3′); pGL2/−95/ΔSP1, sense primer (5′-CGCTCCCATTCTCTGCCGGGCT-3′). The cycD1antisense primer was used as the antisense primer for each PCR reaction. To make a deletion mutant that contained the minimal promoter region (pGL2/−23/ΔNFκB3), a double-stranded oligonucleotide was ligated to the pGL2-basic vector. The sequence of the sense strand was 5′-GTTGAAGTTGCAAAGTCCTGGAG-3′. A double-stranded oligonucleotide, which corresponded to four consecutive SP1 sites of the cyclin D1 promoter, was ligated to the pGL2-basic vector (pGL2/−161/−96). The sense strand was as follows: 5′-CCCCTCGCTGCTCCCGGCGTTTGGCGCCCGCGCCCCCCTCCCCCTGCGCCCGCCCCCGCCCCCCTCCCGCTC-3′. To insert point mutations in the four putative SP1 sites, the ATF/CREB site, or the NFκB site, PCR was performed. The four SP1 sites were designated SP1-1 through -4 in the 5′ to 3′ order. Constructs and primer pairs were as follows: pGL2/−161/SP1-1mut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTTGGCGCaatttCCCCCCTCCCCCTGCGCCCG-3′, antisense primer (cycD1antisense primer); pGL2/−161/SP1-2mut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTTGGCGCCCGCGCCCCCCTaattaTGCGCCCGCCCCCG-3′), antisense primer (cycD1antisense primer); pGL2/−161/SP1-3mut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTTGGCGCCCGCGCCCCCCTCCCCCTGCaatttCCCCCGCCCCCCTCCCGCT-3′), antisense primer (cycD1antisense primer); pGL2/−161/SP1-4mut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTTGGCGCCCGCGCCCCCCTCCCCCTGCGCCCGCCCCCGaattaCTCCCGCTCCCATTCT-3′), antisense primer (cycD1antisense primer); pGL2/−161/ATF/CREBmut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTT-3′), antisense primer (5′-CTCCAGGACTTTGCAACTTCAACAAAACTCCCCTGTAGTCCGTGTGggGgTACTGTTGTTAAGCAAAGA-3′); pGL2/−161/NFκBmut, sense primer (5′-CCCCTCGCTGCTCCCGGCGTTT-3′), antisense primer (5′-CTCCAGGACTTTGCAACTTCAACAAAACTtaCtTGTAGTCCGTGTGACGTTACTGTTGTTAAGCAAAGA-3′. The lowercase, underlined letters indicate the nucleotide substitutions to insert mutations. The nucleotide sequence of each construct was confirmed by cycle sequencing as described above. pRL-TK, which encodes the SeaPansy luciferase gene, was purchased from Toyo Ink (Tokyo, Japan) and used as the internal control for the luciferase assays. BAECs were transiently cotransfected with reporter plasmids encoding the mutants of the cyclin D1 promoter and pRL-TK using LipofectAMINE (Life Technologies, Rockville, MD). To examine the effects of a dominant negative mutant of Ras, the reporter plasmids were transfected in BAECs along with pRL-TK and pcDNA3-HA-mouse RasS17N, which was designed to express an amino-terminally hemagglutinin-epitope tagged RasS17N (16Suzuki E. Nagata D. Kakoki M. Hayakawa H. Goto A. Omata M. Hirata Y. Circ. Res. 1999; 84: 611-619Crossref PubMed Scopus (39) Google Scholar). After serum starvation for 48 h, cells were restimulated with the growth medium for 8 h and harvested. Dual luciferase assay was performed using a luminometer (Lumat LB 9507, Berthold, Bad Wildbad, Germany). Construction of the dominant negative Ras mutant RasS17N has already been described elsewhere (16Suzuki E. Nagata D. Kakoki M. Hayakawa H. Goto A. Omata M. Hirata Y. Circ. Res. 1999; 84: 611-619Crossref PubMed Scopus (39) Google Scholar). The replication-defective adenovirus, which expresses HA-tagged murine RasS17N was constructed according to the COS-TPC method (17Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (787) Google Scholar). RasS17N was excised from the pcDNA3 vector by restriction digestion with BamHI and XhoI. After end-filling, the fragment was ligated to the cosmid vector pAxCAwt at the SwaI site. The cosmid vector, which encoded HA-tagged murine RasS17N, was cotransfected in 293 cells along with DNA-TPC. Recombinant viruses, which expressed HA-tagged murine RasS17N (Ad RasS17N), were generated through homologous recombination. Ad RasS17N was isolated and propagated in HEK293 cells and finally purified by CsCl gradient ultracentrifugation. A recombinant adenovirus, which expresses green fluorescence protein (Ad GFP), was obtained from Quantum Biotechnologies (Montreal, Canada). Subconfluent HUVECs were infected with Ad RasS17N or Ad GFP at a multiplicity of infection varying from 0 to 50. Cells were incubated in the growth medium until they reached complete confluence and then split at a ratio of 1:3 to induce cell cycle re-entry. Proteins were extracted 4 or 8 h after splitting. Whole-cell extracts were prepared from HUVECs and BAECs as described previously (18Suzuki E. Guo K. Kolman M., Yu, Y.-T. Walsh K. Mol. Cell. Biol. 1995; 15: 3415-3423Crossref PubMed Scopus (52) Google Scholar). Briefly, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and collected by centrifugation. The pellet was resuspended in an equal amount of 2× extraction buffer (20 mm HEPES-KOH, pH 7.8, 0.6 m KCl, 1 mm dithiothreitol, 20% glycerol, 2 mm EDTA, 2 μg/ml leupeptin, 2 μg/ml aprotinin) and subjected to three cycles of freezing and thawing. After centrifugation, an aliquot of the protein extract was used for electrophoretic mobility shift assay (EMSA). Protein extraction for Western blot analyses was performed as described previously (19Suzuki E. Nagata D. Yoshizumi M. Kakoki M. Goto A. Omata M. Hirata Y. J. Biol. Chem. 2000; 275: 3637-3644Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In brief, we used Nonidet P-40 cell lysis buffer (50 mmTris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40) containing 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin. Cells were washed twice with ice-cold PBS and collected by centrifugation. Cells were then lysed in Nonidet P-40 cell lysis buffer for 30 min on ice. After centrifugation, the supernatant was stored at −80 °C. Protein concentration was measured according to Bradford's method (Bio-Rad, Melville, NY). Probes and competitor DNAs were double-stranded, synthetic oligonucleotides that were prepared according to the sequence of the cyclin D1 promoter. Two consecutive SP1 sites located upstream (SP1-1/2) and those located downstream (SP1-3/4) were analyzed separately. Nucleotide sequences of the sense strand of the double-stranded oligonucleotides were as follows: SP1/1wt2wt, 5′-GGCGCCCGCGCCCCCCTCCCCCTGC-3′; SP1/1mut2wt, 5′-GGCGCaatttCCCCCCTCCCCCTGC-3′; SP1/1wt2mut, 5′-GGCGCCCGCGCCCCCCTaattaTGC-3′; SP1/1mut2mut, 5′-GGCGCaatttCCCCCTaattaTGC-3′; SP1/3wt4wt, 5′-TGCGCCCGCCCCCGCCCCCCTCC-3′; SP1/3mut4wt, 5′-TGCaatttCCCCCGCCCCCCTCC-3′; SP1/3wt4mut, 5′-TGCGCCCGCCCCCGaattaCTCC-3′; SP1/3mut4mut, 5′-TGCaatttCCCCCGaattaCTCC-3′; ATF/CREBwt, 5′-CTTAACAACAGTAACGTCACACGGACT-3′; ATF/CREBmut, 5′-CTTAACAACAGTAcCccCACACGGACT-3′; NFκBwt, 5′-CGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCT-3′; NFκBmut, 5′-CGGACTACAaGtaAGTTTTGTTGAAGTTGCAAAGTCCT-3′. To confirm the specificity of protein binding, double-stranded oligonucleotides encoding a canonical SP1 binding site or NFκB binding site were used in some experiments. Nucleotide sequences of the sense strand of the double-stranded oligonucleotide were as follows: canonical SP1wt, 5′-ATTCGATCGGGGCGGGGCGAGC-3′; canonical SP1mut, 5′-ATTCGATCaattCGGGGCGAGC-3′; canonical NFκBwt, 5′-AACTGAAAACGGGAAAGTCCCTCTCTCTAACCTG-3′; canonical NFκBmut, 5′-AACTGAAAACGGGAAAGTgggTCTCTCTAACCTG-3′. The lowercase, underlined letters depict nucleotide substitutions to induce point mutations. Electrophoretic mobility shift assays (EMSAs) were performed in reaction mixtures containing 20 μg of the protein extract, 20 fmol of the probe, 1 μg of poly(dI-dC), and 200 ng of a single-stranded oligonucleotide. Electrophoresis was carried out on 5% nondenaturing polyacrylamide gels with 0.5× TBE (0.045 mTris(hydroxymethyl)aminomethane, 0.045 m boric acid, 0.001m EDTA) in a cooled gel box. Protein extracts were separated on 8–10% SDS-polyacrylamide gels and transferred onto nylon membranes (Millipore, Bedford, MA) using a semidry blotting system (Amersham Pharmacia Biotech, Uppsala, Sweden). After blocking in 1× PBS/5% nonfat dry milk/0.2% Tween 20 at 4 °C overnight, the membranes were incubated with the primary antibodies in blocking buffer (1× PBS/2% nonfat dry milk/0.2% Tween 20) for 1 h at room temperature. Antibodies were used at a dilution of 1:200. The membranes were washed three times with the blocking buffer and then incubated with secondary antibodies, which were conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Buckinghamshire, UK) at a final dilution of 1:7000. After final washes with 1× PBS/0.2% Tween 20, the signals were detected using ECL chemiluminescence reagents (Amersham Pharmacia Biotech). The values are the mean ± S.E. Statistical analyses were performed using analysis of variance followed by the Student-Newman-Keul test. Differences with a p value of <0.05 were considered statistically significant. To map cis-elements that were required for transcriptional activity of the cyclin D1 promoter, we transfected a variety of luciferase reporter constructs, which contained various lengths of the human cyclin D1 promoter in BAECs (Fig.1). Surprisingly, deletion of the putative AP1 site did not reduce the promoter activity. Further deletion of two putative NFκB binding sites and two putative STAT binding sites did not reduce the luciferase activity, either. pGL2/−161/SP1, which encoded four consecutive SP1 sites, an ATF/CREB site, and an NFκB binding site, had almost the same promoter activity as pGL2/−1719/wt, which encoded the full-length cyclin D1 promoter. However, when the SP1 sites were deleted (pGL2/−95/ΔSP1), the promoter activity decreased to 30% of that of the full-length cyclin D1 promoter. Further deletion of the putative ATF/CREB site and the NFκB binding site (pGL2/−23/ΔNFκB3) resulted in reduction of the promoter activity almost to the same level as that of the pGL2-basic vector. The results suggested that the activity of the cyclin D1 promoter depended largely on the SP1 sites, the ATF/CREB site, and the NFκB binding site in vascular ECs. To examine the function of each putative DNA-binding site more specifically, a point mutation to each DNA-binding site was introduced in pGL2/−161/SP1, and the luciferase activity of the reporter plasmids was measured (Fig.2). When the SP1 site located at −134 to −126 was mutated (pGL2/−161/SP1-1mut), the promoter activity was rather increased to 147% compared with the wild type construct. However, when the three downstream SP1 sites located at −128 to −120, −115 to −107, or −109 to −101 were mutated (pGL2/−161/SP1-2mut, pGL2/−161/SP1-3mut, and pGL2/−161/SP1-4mut, respectively), the promoter activity was significantly decreased to 47, 65, and 66%, respectively. Insertion of a point mutation in the ATF/CREB site (pGL2/−161/CREB mut) and the NFκB binding site (pGL2/−161/NFκB mut) also significantly reduced the promoter activity to 50 and 52%, respectively, suggesting that these putative DNA-binding sites were functional in these assays. We also tried to transfect the reporter constructs into HUVECs. However, transfection efficiency was too low to map the cis-elements.Figure 2Mutational analysis of the functions of the SP1, ATF/CREB, and NFκB sites in the cyclin D1 promoter. Diagrams show mutant reporter constructs in which each SP1 site, ATF/CREB site, or NFκB site was mutated. The asterisks indicate the positions at which the point mutation was introduced. 2 μg of each luciferase reporter construct was transfected in BAECs along with 0.25 μg of pRL-TK and serum-starved for 48 h. Cells were then restimulated with the growth medium for 8 h and harvested for luciferase assays. The horizontal axis shows the ratio of Photinus pyralis luciferase activity to SeaPansy luciferase activity. #p < 0.01 versus wild type construct (n = 6 per construct).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine whether the SP1 family of transcription factors, ATF/CREB and NFκB actually bound to the putative DNA-binding domains, we performed EMSAs. To analyze the role of four SP1 sites, we designed two double-stranded oligonucleotides. One encoded two consecutive SP1 sites located at −134 to −126 and −128 to −120 (SP1-1/2), and the other encoded another two consecutive SP1 sites located at −115 to −107 and −109 to −101 (SP1-3/4). Two bands were detected in protein extracts prepared from HUVECs when a radiolabeled double-stranded oligonucleotide encoding the SP1-1/2 was used as the probe (A′ and B′ in Fig.3 A, left panel). The DNA binding activity in protein extracts prepared 8 h after splitting of confluent HUVECs was significantly increased compared with that in protein extracts prepared from quiescent HUVECs (SSin Fig. 3 A, 5.3- ± 0.8-fold increase compared with quiescent HUVECs (Q), n = 3,p < 0.05). The DNA-protein complexes were competed away by a 100× molar excess of cold oligonucleotide encoding wild type SP1 sites (SP1-1wt2wt) but not by a 100× molar excess of cold oligonucleotide encoding mutant SP1 sites (SP1-1mut2mut). We also performed a cold competition experiment using a double-stranded oligonucleotide encoding a canonical SP1 site. In this case, the upper band (band A′) was abolished when a molar excess of the oligonucleotide encoding the wild SP1 site was included in the reaction (canon. SP1wt), whereas the band remained when a molar excess of the oligonucleotide encoding the mutant SP1 site was used (canon. SP1mut), suggesting that the upper band represented specific binding of the SP1 family of transcription factors. We next used a radiolabeled double-stranded oligonucleotide encoding SP1-3/4 as the probe (Fig. 3 A, right panel). The DNA binding activity was also induced 8 h after splitting confluent HUVECs (2.5- ± 0.3-fold increase compared with quiescent cells,n = 3, p < 0.01). Two complexes were also detected, both of which were competed away by a molar excess of wild type cold probe (SP1-3wt4wt) but not by a molar excess of mutant cold probe (SP1-3mut4mut). The DNA-protein complex corresponding to the upper band (band X in Fig. 3 A, right panel) was specifically competed away by a molar excess of oligonucleotide encoding the wild type canonical SP1 site but not by that encoding the mutant SP1 site. To examine the role of each SP1 site more precisely and to examine whether the SP1 family of transcription factors specifically bound to the SP1 sites, we performed further competition experiments. The DNA-protein complexes formed at the SP1-1/2 site were competed away by a molar excess of cold double-stranded oligonucleotide encoding one wild type and the other mutant SP1 sites (SP1-1mut2wt and SP1-1wt2mut; Fig. 3 B), suggesting that both SP1 sites were involved in protein binding. The density of the upper band (band A′ in Fig. 3 B) was reduced when anti-SP1 antibody was included in the reaction mixture. The density of the upper band was also reduced when anti-SP2 antibody was included in the reaction mixture, and some of the DNA-protein complex was supershifted. Preincubation with anti-SP3 and -SP4 antibody also reduced the density of the upper band, although the effect appeared to be weaker than that of anti-SP1 and -SP2 antibodies. Preincubation with preimmune serum had no remarkable effect on the DNA-protein complex formation. The DNA-protein complexes formed at the SP1-3/4 site were competed away by a molar excess of cold oligonucleotide encoding one wild type and the other mutant SP1 sites (SP1-3mut4wt and SP1-3wt4mut; Fig. 3 C). However, the complexes were only partially competed away by SP1-3mut4wt, suggesting that the SP1-3 site had higher affinity for the SP1 family of transcription factors in this assay system. Preincubation with anti-SP1 through -SP4 antibodies reduced the density of the upper band (band X in Fig. 3 C), and some of the complex was supershifted with anti-SP2 antibody. Anti-SP3 and -SP4 antibodies tended to have weaker effects for the competition than anti-SP1 and -SP2 antibodies. In contrast, preincubation with preimmune serum had no remarkable effect on the DNA-protein complex formation. We next examined DNA binding activity at the ATF/CREB site. Two bands were detected by EMSAs when a radiolabeled double-stranded oligonucleotide encoding the ATF/CREB was used as the probe (bands X and Y in Fig.4 A). Although the DNA binding activity at the ATF/CREB site tended to increase 8 h after splitting confluent HUVECs, the difference was not statistically significant (0.99- ± 0.04-fold increase compared with quiescent cells,n = 3, not significant). Both bands were abolished by preincubation with a molar excess of cold wild type probe but not by a mutant probe. The lower band (band Y in Fig. 4 A) was supershifted when anti-ATF/CREB antibody was included in the reaction, whereas anti-c-Fos and -c-Jun antibodies did not have remarkable effects on the complex formation. The DNA binding activity at the NFκB site was also examined. When a radiolabeled double-stranded oligonucleotide encoding the cyclin D1 NFκB site located at −33 to −24 was used as the probe, no specific bands were detected (Fig. 4 B, left panel). We therefore used a radiolabeled double-stranded oligonucleotide encoding a canonical NFκB site as the probe. We did observe DNA binding activity in this case. Three bands (bands X, Y, and Zin Fig. 4 B, right panel) were detected. The DNA binding activity at the canonical NFκB site was not increased 8 h after splitting confluent HUVECs, and the DNA-protein complexes were competed away by a molar excess of cold wild type probe but not by a molar excess of cold mutant probe. Bands X and Ywere supershifted by preincubation with anti-p50 antibody. The density of band X was reduced by preincubation with anti-p65 antibody, whereas preimmune serum did not have a remarkable effect on the DNA binding activity. The results suggested that, although HUVECs expressed NFκB proteins, their binding activity at the cyclin D1 NFκB site was under detectable levels in this assay. We also used protein extracts prepared from BAECs. The results obtained were basically the same as those described above (data not shown). The DNA binding activity at the SP1 sites was increased 8 h after quiescent BAECs were stimulated with serum mitogen. ATF/CREB was detected in the DNA-protein complex formed at the ATF/CREB site. No DNA binding activity was detected at the NFκB site. However, preincuba" @default.
• W2075939587 created "2016-06-24" @default.
• W2075939587 creator A5006782913 @default.
• W2075939587 creator A5032355430 @default.
• W2075939587 creator A5032400721 @default.
• W2075939587 creator A5034244372 @default.
• W2075939587 creator A5061663577 @default.
• W2075939587 creator A5085467021 @default.
• W2075939587 creator A5091003441 @default.
• W2075939587 date "2001-01-01" @default.
• W2075939587 modified "2023-09-26" @default.
• W2075939587 title "Transcriptional Activation of the cyclin D1 Gene Is Mediated by Multiple Cis-Elements, Including SP1 Sites and a cAMP-responsive Element in Vascular Endothelial Cells" @default.
• W2075939587 cites W1228798347 @default.
• W2075939587 cites W1565998937 @default.
• W2075939587 cites W1584322138 @default.
• W2075939587 cites W1609315759 @default.
• W2075939587 cites W1807482481 @default.
• W2075939587 cites W1859306058 @default.
• W2075939587 cites W1902352259 @default.
• W2075939587 cites W1922521877 @default.
• W2075939587 cites W193710672 @default.
• W2075939587 cites W1974760224 @default.
• W2075939587 cites W1979721685 @default.
• W2075939587 cites W1980839209 @default.
• W2075939587 cites W1992772703 @default.
• W2075939587 cites W1994953133 @default.
• W2075939587 cites W1996530399 @default.
• W2075939587 cites W2002282120 @default.
• W2075939587 cites W2006705806 @default.
• W2075939587 cites W2018650372 @default.
• W2075939587 cites W2029827117 @default.
• W2075939587 cites W2037230449 @default.
• W2075939587 cites W2041140715 @default.
• W2075939587 cites W2041639550 @default.
• W2075939587 cites W2048721919 @default.
• W2075939587 cites W2052430038 @default.
• W2075939587 cites W2056213387 @default.
• W2075939587 cites W2060203764 @default.
• W2075939587 cites W2066671615 @default.
• W2075939587 cites W2068577315 @default.
• W2075939587 cites W2069104210 @default.
• W2075939587 cites W2070743354 @default.
• W2075939587 cites W2081524299 @default.
• W2075939587 cites W2089126365 @default.
• W2075939587 cites W2091291725 @default.
• W2075939587 cites W2096657295 @default.
• W2075939587 cites W2108954781 @default.
• W2075939587 cites W2110751499 @default.
• W2075939587 cites W2118986158 @default.
• W2075939587 cites W2124608824 @default.
• W2075939587 cites W2132244436 @default.
• W2075939587 cites W2137570167 @default.
• W2075939587 cites W2152105216 @default.
• W2075939587 cites W2152336497 @default.
• W2075939587 cites W2165712565 @default.
• W2075939587 cites W2294953816 @default.
• W2075939587 cites W2318149353 @default.
• W2075939587 cites W2331676402 @default.
• W2075939587 doi "https://doi.org/10.1074/jbc.m005522200" @default.
• W2075939587 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11024050" @default.
• W2075939587 hasPublicationYear "2001" @default.
• W2075939587 type Work @default.
• W2075939587 sameAs 2075939587 @default.
• W2075939587 citedByCount "97" @default.
• W2075939587 countsByYear W20759395872012 @default.
• W2075939587 countsByYear W20759395872013 @default.
• W2075939587 countsByYear W20759395872014 @default.
• W2075939587 countsByYear W20759395872015 @default.
• W2075939587 countsByYear W20759395872016 @default.
• W2075939587 countsByYear W20759395872017 @default.
• W2075939587 countsByYear W20759395872018 @default.
• W2075939587 countsByYear W20759395872019 @default.
• W2075939587 countsByYear W20759395872020 @default.
• W2075939587 countsByYear W20759395872021 @default.
• W2075939587 countsByYear W20759395872022 @default.
• W2075939587 crossrefType "journal-article" @default.
• W2075939587 hasAuthorship W2075939587A5006782913 @default.
• W2075939587 hasAuthorship W2075939587A5032355430 @default.
• W2075939587 hasAuthorship W2075939587A5032400721 @default.
• W2075939587 hasAuthorship W2075939587A5034244372 @default.
• W2075939587 hasAuthorship W2075939587A5061663577 @default.
• W2075939587 hasAuthorship W2075939587A5085467021 @default.
• W2075939587 hasAuthorship W2075939587A5091003441 @default.
• W2075939587 hasBestOaLocation W20759395871 @default.
• W2075939587 hasConcept C104317684 @default.
• W2075939587 hasConcept C1629964 @default.
• W2075939587 hasConcept C185592680 @default.
• W2075939587 hasConcept C199835354 @default.
• W2075939587 hasConcept C29537977 @default.
• W2075939587 hasConcept C54355233 @default.
• W2075939587 hasConcept C86339819 @default.
• W2075939587 hasConcept C86803240 @default.
• W2075939587 hasConcept C95444343 @default.
• W2075939587 hasConceptScore W2075939587C104317684 @default.
• W2075939587 hasConceptScore W2075939587C1629964 @default.
• W2075939587 hasConceptScore W2075939587C185592680 @default.
• W2075939587 hasConceptScore W2075939587C199835354 @default.
• W2075939587 hasConceptScore W2075939587C29537977 @default.

• next