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*

Daisuke NagataDagger §, Etsu SuzukiDagger §, Hiroaki Nishimatsu||, Hiroshi SatonakaDagger , Atsuo GotoDagger , Masao OmataDagger , and Yasunobu HirataDagger

From Dagger  The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan and the || Department of Urology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

Received for publication, June 23, 2000, and in revised form, September 28, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 G1 phase 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 G1 phase, and the other was constitutively active.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 (1). 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 (2, 3), 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 (4, 5). It is well known that expression of cyclin D is regulated, at least partly, at the transcription level and that the p21ras (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 (6, 7). 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 (8). 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 (NFkappa B), 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 (9). Cytokine-induced transcriptional activation of the cyclin D1 gene was mediated by STAT-binding sites in hematopoietic cells (10). It was also reported that NFkappa B-binding sites in the cyclin D1 promoter were implicated in transcriptional activation of the gene (11-13). The ATF/CREB-binding site was also reportedly implicated in the activation of the cyclin D1 gene. pp60v-src-induced transcriptional activation of the cyclin D1 gene was mediated by the ATF/CREB site (14). 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 (15). 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents-- 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, -NFkappa Bp50, -NFkappa Bp65, -c-Fos, and -c-Jun antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).

Cell Culture-- 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.

Construction of the Cyclin D1 Promoter Mutants-- 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 (GenBankTM 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-, NFkappa B-, 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/Delta AP1, sense primer (5'-GGAGATCACTGTTTCTCAGCTTTCCA-3'); pGL2/-836/Delta NFkappa B1, sense primer (5'-GGACCGACTGGTCAAGGTAGGAA-3'); pGL2/-707/Delta NFkappa B2, sense primer (5'-GAGCGAGCGCATGCTAAGCTGAA-3'); pGL2/-461/Delta STAT1, sense primer (5'-GCGCCCATTCTGCCGGCTTGGAT-3'); pGL2/-229/Delta STAT2, sense primer (5'-TTCTATGAAAACCGGACTACAGGGGCAACTC-3'); pGL2/-161/SP1, sense primer (5'-CCCCTCGCTGCTCCCGGCGTTT-3'); pGL2/-95/Delta SP1, 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/Delta NFkappa B3), 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 NFkappa B 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/NFkappa Bmut, 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.

Transient Transfection Assays-- 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 (16). 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 a Replication-defective Adenovirus Encoding a Dominant Negative Ras Mutant-- Construction of the dominant negative Ras mutant RasS17N has already been described elsewhere (16). The replication-defective adenovirus, which expresses HA-tagged murine RasS17N was constructed according to the COS-TPC method (17). 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).

Adenoviral Infection-- 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.

Preparation of Protein Extracts-- Whole-cell extracts were prepared from HUVECs and BAECs as described previously (18). 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 (19). In brief, we used Nonidet P-40 cell lysis buffer (50 mM Tris-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).

Electrophoretic Mobility Shift Assay-- 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'; NFkappa Bwt, 5'-CGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCT-3'; NFkappa Bmut, 5'-CGGACTACAaGtaAGTTTTGTTGAAGTTGCAAAGTCCT-3'.

To confirm the specificity of protein binding, double-stranded oligonucleotides encoding a canonical SP1 binding site or NFkappa B 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 NFkappa Bwt, 5'-AACTGAAAACGGGAAAGTCCCTCTCTCTAACCTG-3'; canonical NFkappa Bmut, 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 M Tris(hydroxymethyl)aminomethane, 0.045 M boric acid, 0.001 M EDTA) in a cooled gel box.

Western Blot Analysis-- 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).

Statistical Analyses-- 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Mutational Analysis of the Human Cyclin D1 Promoter Activity-- 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 NFkappa B 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 NFkappa B 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/Delta SP1), 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 NFkappa B binding site (pGL2/-23/Delta NFkappa B3) 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 NFkappa B 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 NFkappa B binding site (pGL2/-161/NFkappa B 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.



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Fig. 1.   Mutational analysis of the cyclin D1 promoter activity in BAECs. Diagrams depict locations of a variety of cis-elements in the cyclin D1 promoter. 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 (n = 6 per construct).



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Fig. 2.   Mutational analysis of the functions of the SP1, ATF/CREB, and NFkappa B sites in the cyclin D1 promoter. Diagrams show mutant reporter constructs in which each SP1 site, ATF/CREB site, or NFkappa B 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).

DNA Binding Activity at the SP1 Sites and ATF/CREB Site in the Cyclin D1 Promoter-- To examine whether the SP1 family of transcription factors, ATF/CREB and NFkappa B 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. 3A, 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 (SS in Fig. 3A, 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. 3A, 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. 3A, 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.



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Fig. 3.   Induction of DNA binding activity at the SP1 sites. A, confluent HUVECs (Q) were split to induce re-entry into the cell cycle, and protein extracts were prepared 8 h after splitting (SS). EMSAs were performed using a radiolabeled double-stranded oligonucleotide probe encoding distal wild type SP1 sites (SP1-1wt2wt, left panel) or proximal wild type SP1 sites (SP1-3wt4wt, right panel). To show the specificity of the bands, a 100× molar excess of cold double-stranded oligonucleotide encoding wild type (canon. SP1wt) or the mutant (canon. SP1mut) canonical SP1 site was used for competition (comp.). B, protein extracts were prepared in the same way as in A. EMSAs were performed using a radiolabeled double-stranded oligonucleotide probe encoding distal wild type SP1 sites (SP1-1wt2wt). Competition experiments were performed using a 100× molar excess of cold double-stranded oligonucleotides encoding one wild type and the other mutant SP1 sites (SP1-1mut2wt and SP1-1wt2mut). Anti-SP1 through -SP4 antibodies were also used to show the specificity of the protein-DNA complexes, and nonimmune serum (NI) was used as the negative control. The relative density of band A' is shown at the bottom of the image. C, experiments were performed basically in the same way as in B. A radiolabeled double-stranded oligonucleotide encoding the proximal wild type SP1 sites (SP1-3wt4wt) was used as the probe. For competition experiments, a 100× molar excess of cold double-stranded oligonucleotides encoding one wild type and the other mutant SP1 sites (SP1-3mut4wt and SP1-3wt4mut) was used. The same anti-sera were used to show the specificity of the bands. The relative density of band X is shown at the bottom.

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. 3B), suggesting that both SP1 sites were involved in protein binding. The density of the upper band (band A' in Fig. 3B) 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. 3C). 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. 3C), 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. 4A). 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. 4A) 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 NFkappa B site was also examined. When a radiolabeled double-stranded oligonucleotide encoding the cyclin D1 NFkappa B site located at -33 to -24 was used as the probe, no specific bands were detected (Fig. 4B, left panel). We therefore used a radiolabeled double-stranded oligonucleotide encoding a canonical NFkappa B site as the probe. We did observe DNA binding activity in this case. Three bands (bands X, Y, and Z in Fig. 4B, right panel) were detected. The DNA binding activity at the canonical NFkappa B 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 Y were 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 NFkappa B proteins, their binding activity at the cyclin D1 NFkappa B site was under detectable levels in this assay.



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Fig. 4.   Characterization of the DNA binding activity at the ATF/CREB and NFkappa B sites. A, protein extracts were prepared in the same way as in Fig. 3. A radiolabeled double-stranded oligonucleotide encoding the wild type ATF/CREB site (ATF/CREBwt) was used as the probe for EMSAs. A 100× molar excess of cold double-stranded oligonucleotide encoding the wild type (ATF/CREBwt) or mutant (ATF/CREBmut) ATF/CREB site was used for competition (comp.) experiments. Anti-ATF/CREB, -c-Fos, and -c-Jun antibodies were also used to show the specificity of the bands. B, experiments were performed in the same way as in A. A radiolabeled double-stranded oligonucleotide encoding the wild type proximal cyclin D1 NFkappa B site (left panel, CycD1NFkappa Bwt) or a canonical NFkappa B site (right panel, canon. NFkappa Bwt) was used as the probe for EMSAs. For competition experiments, a 100× molar excess of cold double-stranded oligonucleotides encoding the wild type (CycD1NFkappa Bwt) or mutant (CycD1NFkappa Bmut) cyclin D1 NFkappa B site, or wild type (canon. NFkappa Bwt) or mutant (canon. NFkappa Bmut) canonical NFkappa B site was used. Anti-p50 and -p65 antibodies were also used for the competition experiment. Nonimmune serum (NI) was used as the negative control. The relative density of bands X and Y is shown at the bottom.

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 NFkappa B site. However, preincubation with anti-SP1 through -SP4 antibodies did not show clear competition, probably because the antibodies used in the experiments did not sufficiently cross-react with the bovine SP1 family of transcription factors.

Effects of a Dominant Negative Ras Mutant on the DNA Binding Activity at the SP1 Sites and Expression of SP1-- To investigate the role of the Ras-dependent pathway in the increase of the DNA binding activity at the SP1 sites, we infected an adenovirus construct encoding RasS17N in HUVECs and examined the effects on the DNA binding activity at the SP1 sites. Ad GFP was used as the control infection. The increase in DNA binding activity at the SP1-1/2 sites (Fig. 5A) and SP1-3/4 sites (Fig. 5B) was significantly inhibited by infection with Ad RasS17N (SP1-1/2: Ad GFP infection versus Ad RasS17N infection, 320 ± 42% versus 167 ± 22% of control level, n = 3, p < 0.01; SP1-3/4: Ad GFP infection versus Ad RasS17N infection, 200 ± 43% versus 93 ± 13% of control level, n = 3, p < 0.05). The specificity of the bands (bands A' and X in Fig. 5) was confirmed by competition with molar excess of cold probes and with anti-SP1 and -SP2 antibodies. We also examined the effects of blocking the Ras-dependent pathway on the expression of SP1. HUVECs were infected with Ad RasS17N and expression of SP1 was detected by Western blot analysis using anti-SP1 antibody. Expression of RasS17N was confirmed by Western blot analysis using anti-hemagglutinin antibody (Fig. 6, lower panel). Two bands corresponding to 95 and 105 kDa of SP1 were detected. The expression level of SP1 in protein extracts prepared 8 h after splitting confluent HUVECs did not change remarkably compared with that in protein extracts prepared from quiescent cells, nor did it change due to infection with Ad RasS17N (Fig. 6, upper panel).



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Fig. 5.   Effects of blocking the Ras-dependent pathway on the DNA binding activity at the SP1 sites. Subconfluent HUVECs were infected with 15 multiplicity of infection of adenovirus constructs expressing GFP (AdGFP) or RasS17N (AdRasS17N) and maintained in the growth medium until they reached confluence. Cells were then split to induce cell cycle re-entry and harvested 8 h after splitting (SS). Confluent HUVECs were used as the control (Q). A, a radiolabeled double-stranded oligonucleotide probe encoding distal wild type SP1 sites (SP1-1wt2wt) was used as the probe for EMSAs. For competition (comp.) experiments, a 100× molar excess of wild type and mutant cold probes, and anti-SP1 and -SP2 antibodies were used. B, experiments were performed in the same way as in A except that a radiolabeled double-stranded oligonucleotide probe encoding proximal wild type SP1 sites (SP1-3wt4wt) was used as the probe for EMSAs, and corresponding cold probes were used for competition experiments. Shown is a representative experiment of three independent ones in which the same results were obtained.



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Fig. 6.   Effects of blocking the Ras-dependent pathway on the expression of SP1. The same protein extracts used in Fig. 5 were used for Western blot analysis to examine the expression level of SP1 (upper panel). The expression of RasS17N was also confirmed by Western blot analysis using anti-HA antibody (lower panel). Shown is a representative experiment of three independent ones in which the same results were obtained.

Induction and Ras Dependence of the Cyclin D1 Promoter Activity in the Early to Mid Period of the G1 Phase Are Largely Mediated by a Fragment Containing the SP1 Sites-- Finally, we examined whether the increase in the cyclin D1 promoter activity in the early to mid period of the G1 phase was mediated by the SP1 sites. As shown in Fig. 7A, when a luciferase reporter construct containing the full-length 161 bp of the promoter region (pGL2/-161/SP1) was used, the promoter activity increased by 2.4-fold (p < 0.01) when quiescent BAECs were stimulated with serum mitogen for 8 h. The promoter activity of a reporter plasmid containing the four SP1 sites, but not the ATF/CREB or NFkappa B site (pGL2-161/-96), increased by 2.7-fold (p < 0.01) after stimulation with serum mitogen for 8 h. In marked contrast, the promoter activity of the reporter containing the ATF/CREB and NFkappa B site (pGL2/-95/Delta SP1) was not induced by stimulation with serum mitogen (1.0-fold induction, not significant). We also examined the effects of RasS17N, and pGL2-161/-96 was cotransfected into BAECs together with increasing amounts of an expression plasmid encoding RasS17N. Expression of RasS17N suppressed the promoter activity of pGL2-161/-96 in a dose-dependent fashion (Fig. 7B).



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Fig. 7.   Induction of the cyclin D1 promoter activity and its dependence upon the Ras-mediated pathway in early to mid G1 phase. A, BAECs cultured in 6-well plates were transfected with 2 µg of several reporter plasmids used in Fig. 1 (-161/SP1 and -95/Delta SP1), along with 0.25 µg of pRL-TK, and serum-starved for 48 h (Q). Half of the wells were then restimulated with growth medium for 8 h (SS), and cells were harvested for the luciferase assay. pGL2-161/-96 (-161/-96), which contained the four consecutive SP1 sites but not the ATF/CREB or NFkappa B site, was also used in these experiments. The vertical axis shows the ratio of Photinus pyralis luciferase activity to SeaPansy luciferase activity (n = 6 per construct). #p < 0.01 versus quiescent (Q). B, BAECs were transfected with 1.5 µg of pGL2-161/-96 (-161/-96) and increasing amounts of pcDNA3-HA-RasS17N (RasS17N), along with 0.25 µg of pRL-TK. After serum starvation for 48 h, the cells were restimulated with growth medium for 8 h and harvested for the luciferase assay. Total amounts of DNA transfected in each well were adjusted by adding pGL2 vector. The vertical axis shows the ratio of Photinus pyralis luciferase activity to SeaPansy luciferase activity (n = 6). #p < 0.01 versus control.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have shown that transcriptional activation of the cyclin D1 gene in vascular ECs was mediated by multiple cis-elements, including SP1 sites and the ATF/CREB site. We have also shown that the DNA binding activity and promoter activity mediated by the SP1 sites were increased in the early to mid period of the G1 phase and that the increase was mediated by the Ras-dependent pathway.

It is well-established that transcriptional activation of the cyclin D1 gene is mediated by the Ras-dependent pathway. Among a variety of cis-elements found in the cyclin D1 promoter regions that are required for the transcriptional activation of the gene, the AP1 site and NFkappa B sites appear to be of particular interest, because AP1 and NFkappa B are potentially activated by the Ras-dependent pathway. It is well known that transcription of the c-fos gene is activated through the Ras/MEK/ERK-dependent pathway (20). It was indeed reported that Ras-dependent activation of the cyclin D1 promoter is mediated by the AP1 site in JEG-3 cells (21). NFkappa B is also a potential target of the Ras-dependent pathway, because p90rsk, a downstream target of Ras/MEK/ERK, phosphorylates and inactivates Ikappa Balpha (22, 23), resulting in nuclear translocation and activation of NFkappa B. It has also been reported that Raf, a downstream target molecule of Ras, activates NFkappa B (24-26). However, these sites do not seem to be involved in the transcriptional activation of the cyclin D1 gene in the early to mid period of the G1 phase in vascular ECs. ATF/CREB is also a molecule that is potentially activated through the Ras-dependent pathway, because the member of the p90rsk family RSK2 is found to phosphorylate and activate CREB (27). However, the ATF/CREB site seemed to be constitutively active in our system.

Several reports have suggested a role for the SP1 sites in the transcriptional activation of D-type cyclins. SP1 sites were found to play critical roles in the transcription of the cyclin D3 gene in megakaryocytes stimulated by thrombopoietin, although the ATF/CREB site was not implicated in the activation of the gene (28). It was also reported that nerve growth factor induces transcription of the cyclin D1 gene in PC12 cells via the SP1 sites, although the role of ATF/CREB site was not examined (29). The SP1 family of transcription factors, which are zinc-finger transcription factors, comprises four structurally related proteins, SP1 through SP4. This family of transcription factors either activates or suppresses the promoter activity of genes, depending on the promoter used and cell types (30, 31). SP3 is known to compete with SP1 and suppress promoter activities (32), although SP3 transactivates the p21cip1 promoter in keratinocytes (33). It has been reported that SP1 sites mediate the transcriptional activation of many genes, including p21cip1, p15INK4B, dihydrofolate reductase, histone H4, VEGF, gastrin, and elastin (29, 34-39). Although SP1 has been considered to be a constitutively active transcription factor (40), it is now known that inducible activation of the SP1 family plays critical roles in the activation of genes. DNA binding activity at the SP1 sites of the p21cip1 promoter increased in differentiating keratinocytes (33). SP1 binding activity in the cyclin D3 gene was significantly increased by the thrombopoietin in megakaryocytes (28). Thus, the SP1 family seems to mediate activation of gene transcription in a constitutive or inducible manner, depending on genes, mitogens, and cell types.

Our data indicated that the increase in DNA binding activity at the SP1 sites in the early to mid period of the G1 phase was mediated by the Ras-dependent pathway. However, we did not detect remarkable changes in the expression of SP1 in vascular ECs. Although the mechanisms by which DNA binding activity at the SP1 sites changed were not clear, it was possible that the phosphorylation status of the SP1 family changed or other nuclear factors associated with the SP1 family were influenced by the Ras-dependent pathway. In fact, it was reported that phosphorylation of SP1 changed its affinity to the SP1 site or its capacity to activate transcription of genes (28, 41). Several reports have shown the Ras-dependent changes in DNA binding activity at SP1 sites (42, 43). It was reported that the Ras-dependent down-regulation of the alpha 2-integrin promoter activity is associated with decreased DNA binding activity at the SP1 site without changes in the expression of SP1 (43). In contrast, a previous report showed that Ras-dependent transcriptional activation of the p21cip1 gene, which is mediated by the SP1 sites, occurs without changes in DNA binding activity at the SP1 sites (44). Therefore, SP1 site-mediated activation of genes appears to depend not only on the SP1 family but also on a variety of combinations of nuclear factors associated with the SP1 family.

Our results indicated that the ATF/CREB site was involved in the maintenance of the basal transcriptional activity but not in the induction of the cyclin D1 promoter activity in the early to mid period of the G1 phase and that ATF/CREB but not c-Fos or c-Jun bound to the site. Several studies have indicated that the ATF/CREB site in the cyclin D1 promoter is implicated in the transcriptional activity of the cyclin D1 gene. The ATF/CREB site was involved in pp60v-src-induced activation of the cyclin D1 promoter and ATF2 and CREB but not c-Jun or c-Fos bound to the site (14). The ATF/CREB site was also implicated in the serum-induced activation of the cyclin D1 promoter in mouse embryonic fibroblasts, and c-Fos, c-Jun, and CREB are found to bind to the site (45). Estrogen-induced activation of the cyclin D1 gene is mediated by the ATF/CREB site and ATF/c-Jun heterodimers bound to the site (15). Activation of the cyclin D1 gene in chondrocytes is mediated by the ATF/CREB site and ATF and CREB but not c-Fos or c-Jun bound to the site (46). Therefore, the role of the ATF/CREB site in the activation of the cyclin D1 gene appears to depend on cell types and mitogens used.

Although the DNA binding activity at the NFkappa B site was under detectable levels in our system, the role of the proximal NFkappa B site could not be excluded, because the site was active in luciferase assays. Possibly, the proximal NFkappa B site was implicated in the stable association of nuclear proteins such as SP1 and ATF/CREB with general transcription factors such as TFIID. Previous studies have suggested that the NFkappa B binding sites in the cyclin D1 promoter were involved in the transcriptional activation of the gene. Forced expression of a super repressor form of Ikappa Balpha to inhibit NFkappa B activity in mouse embryonic fibroblasts retards the expression of cyclin D1 and cell cycle progression (12). In that study, DNA binding activity at the cyclin D1 NFkappa B sites was observed in COS7 cells transfected with expression plasmids encoding the p50 and p65 subunits of NFkappa B. Another report showed that NFkappa B is implicated in the induction of cyclin D1 expression and the inhibition of differentiation of C2C12 myoblasts (11). It was also reported in that study that NFkappa B is involved in the transcriptional activation of the cyclin D1 gene in NIH3T3 cells and that DNA binding activity occurs at multiple NFkappa B sites in the cyclin D1 promoter. Furthermore, Rac-dependent transcriptional activation of the cyclin D1 gene is mediated by the proximal NFkappa B binding site in NIH3T3 cells and DNA binding activity at the proximal NFkappa B site is detected in the cells (13). Thus, the role of NFkappa B in the activation of the cyclin D1 gene also seems to depend on cell types.

In summary, the transcriptional activity of the cyclin D1 promoter was mediated by dual cis-elements in vascular ECs. Induction of the cyclin D1 promoter activity seemed to be largely mediated by SP1 sites, whereas the ATF/CREB site (and the proximal NFkappa B site) appeared to be involved in maintenance of the basal transcriptional activity in the early to mid period of the G1 phase. The increase in DNA binding activity and promoter activity via the SP1 sites was mediated by the Ras-dependent pathway. The roles of each cis-element in the cyclin D1 promoter may be cell type-specific. Further studies are required to elucidate the mechanisms by which Ras induces activation of the cyclin D1 gene via the SP1 sites.


    ACKNOWLEDGEMENTS

We thank Etsuko Taira and Marie Morita for technical assistance.


    FOOTNOTES

* This study was supported by grants-in-aid from the Ministry of Education, Culture and Science of Japan (09281206 and 10218202 awarded to Y. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

To whom correspondence should be addressed: Tel.: 81-3-3815-5411; Fax: 81-3-3814-0021; E-mail: suzuki-2im@h.u-tokyo.ac.jp.

Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M005522200


    ABBREVIATIONS

The abbreviations used are: EC, endothelial cells; VEGF, vascular endothelial growth factor; cdk, cyclin-dependent kinases; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; CREB, cAMP-responsive element binding protein; ATF, activating transcription factor; STAT, signal transducers and activators of transcription; HUVEC, human umbilical vein endothelial cells; BAEC, bovine aortic endothelial cells; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; bp, base pair(s); PCR, polymerase chain reaction; wt, wild type; GFP, green fluorescence protein; Ad, adenovirus; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Ross, R. (1993) Nature 362, 801-809[CrossRef][Medline] [Order article via Infotrieve]
2. Vigne, P., Marsault, R., Breittmayer, J. P., and Frelin, C. (1990) Biochem. J. 266, 415-420[Medline] [Order article via Infotrieve]
3. Plouet, J., Schilling, J., and Gospodarowicz, D. (1989) EMBO J. 8, 3801-3806[Abstract]
4. Pines, J. (1995) Biochem. J. 308, 697-711[Medline] [Order article via Infotrieve]
5. Sherr, C. J. (1994) Cell 79, 551-555[Medline] [Order article via Infotrieve]
6. Aktas, H., Cai, H., and Cooper, G. M. (1997) Mol. Cell. Biol. 17, 3850-3857[Abstract]
7. Takuwa, N., and Takuwa, Y. (1997) Mol. Cell. Biol. 17, 5348-5358[Abstract]
8. Pedram, A., Razandi, M., and Levin, E. R. (1998) J. Biol. Chem. 273, 26722-26728[Abstract/Free Full Text]
9. Watanabe, G., Lee, R. J., Albanese, C., Rainey, W. E., Batlle, D., and Pestell, R. G. (1996) J. Biol. Chem. 271, 22570-22577[Abstract/Free Full Text]
10. Matsumura, I., Kitamura, T., Wakao, H., Tanaka, H., Hashimoto, K., Albanese, C., Downward, J., Pestell, R. G., and Kanakura, Y. (1999) EMBO J. 18, 1367-1377[Abstract/Free Full Text]
11. Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and Baldwin, A. S., Jr. (1999) Mol. Cell. Biol. 19, 5785-5799[Abstract/Free Full Text]
12. Hinz, M., Krappmann, D., Eichten, A., Heder, A., Scheidereit, C., and Strauss, M. (1999) Mol. Cell. Biol. 19, 2690-2698[Abstract/Free Full Text]
13. Joyce, 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., and Pestell, R. G. (1999) J. Biol. Chem. 274, 25245-25249[Abstract/Free Full Text]
14. Lee, R. J., Albanese, C., Stenger, R. J., Watanabe, G., Inghirami, G., Haines, G. K., 3rd., Webster, M., Muller, W. J., Brugge, J. S., Davis, R. J., and Pestell, R. G. (1999) J. Biol. Chem. 274, 7341-7350[Abstract/Free Full Text]
15. Sabbah, M., Courilleau, D., Mester, J., and Redeuilh, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11217-11222[Abstract/Free Full Text]
16. Suzuki, E., Nagata, D., Kakoki, M., Hayakawa, H., Goto, A., Omata, M., and Hirata, Y. (1999) Circ. Res. 84, 611-619[Abstract/Free Full Text]
17. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1320-1324[Abstract/Free Full Text]
18. Suzuki, E., Guo, K., Kolman, M., Yu, Y.-T., and Walsh, K. (1995) Mol. Cell. Biol. 15, 3415-3423[Abstract]
19. Suzuki, E., Nagata, D., Yoshizumi, M., Kakoki, M., Goto, A., Omata, M., and Hirata, Y. (2000) J. Biol. Chem. 275, 3637-3644[Abstract/Free Full Text]
20. Whitmarsh, A. J., Shore, P., Sharrocks, A. D., and Davis, R. J. (1995) Science 269, 403-407[Medline] [Order article via Infotrieve]
21. Albanese, C., Johnson, J., Watanabe, G., Eklund, N., Vu, D., Arnold, A., and Pestell, R. G. (1995) J. Biol. Chem. 270, 23589-23597[Abstract/Free Full Text]
22. Ghoda, L., Lin, X., and Greene, W. C. (1997) J. Biol. Chem. 272, 21281-21288[Abstract/Free Full Text]
23. Schouten, G. J., Vertegaal, A. C., Whiteside, S. T., Israel, A., Toebes, M., Dorsman, J. C., van der Eb, A. J., and Zantema, A. (1997) EMBO J. 16, 3133-3144[Abstract/Free Full Text]
24. Finco, T. S., and Baldwin, A. S., Jr. (1993) J. Biol. Chem. 268, 17676-17679[Abstract/Free Full Text]
25. Kanno, T., and Siebenlist, U. (1996) J. Immunol. 157, 5277-5283[Abstract]
26. Li, S., and Sedivy, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9247-9251[Abstract]
27. Xing, J., Ginty, D. D., and Greenberg, M. E. (1996) Science 273, 959-963[Abstract]
28. Wang, Z., Zhang, Y., Lu, J., Sun, S., and Ravid, K. (1999) Blood 93, 4208-4221[Abstract/Free Full Text]
29. Yan, G. Z., and Ziff, E. B. (1997) J. Neurosci. 17, 6122-6132[Abstract/Free Full Text]
30. Briggs, M. R., Kadonaga, J. T., Bell, S. P., and Tjian, R. (1986) Science 234, 47-52[Medline] [Order article via Infotrieve]
31. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, 1079-1090[Medline] [Order article via Infotrieve]
32. Hagen, G., Muller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843-3851[Abstract]
33. Prowse, D. M., Bolgan, L., Molnar, A., and Dotto, G. P. (1997) J. Biol. Chem. 272, 1308-13014[Abstract/Free Full Text]
34. Li, J. M., Nichols, M. A., Chandrasekharan, S., Xiong, Y., and Wang, X. F. (1995) J. Biol. Chem. 270, 26750-26753[Abstract/Free Full Text]
35. Noe, V., Alemany, C., Chasin, L. A., and Ciudad, C. J. (1998) Oncogene 16, 1931-1938[CrossRef][Medline] [Order article via Infotrieve]
36. Birnbaum, M. J., van Wijnen, A. J., Odgren, P. R., Last, T. J., Suske, G., Stein, G. S., and Stein, J. L. (1995) Biochemistry 34, 16503-16508[Medline] [Order article via Infotrieve]
37. Finkenzeller, G., Sparacio, A., Technau, A., Marme, D., and Siemeister, G. (1997) Oncogene 15, 669-676[CrossRef][Medline] [Order article via Infotrieve]
38. Merchant, J. L., Shiotani, A., Mortensen, E. R., Shumaker, D. K., and Abraczinskas, D. R. (1995) J. Biol. Chem. 270, 6314-6319[Abstract/Free Full Text]
39. Jensen, D. E., Rich, C. B., Terpstra, A. J., Farmer, S. R., and Foster, J. A. (1995) J. Biol. Chem. 270, 6555-6563[Abstract/Free Full Text]
40. Mitchell, P. J., and Tjian, R. (1989) Science 245, 371-378[Medline] [Order article via Infotrieve]
41. Merchant, J. L., Du, M., and Todisco, A. (1999) Biochem. Biophys. Res. Commun. 254, 454-461[CrossRef][Medline] [Order article via Infotrieve]
42. Milanini, J., Vinals, F., Pouyssegur, J., and Pages, G. (1998) J. Biol. Chem. 273, 18165-18172[Abstract/Free Full Text]
43. Ye, J., Xu, R. H., Taylor-Papadimitriou, J., and Pitha, P. M. (1996) Mol. Cell. Biol. 16, 6178-6189[Abstract]
44. Kivinen, L., Tsubari, M., Haapajarvi, T., Datto, M. B., Wang, X. F., and Laiho, M. (1999) Oncogene 18, 6252-6261[CrossRef][Medline] [Order article via Infotrieve]
45. Brown, J. R., Nigh, E., Lee, R. J., Ye, H., Thompson, M. A., Saudou, F., Pestell, R. G., and Greenberg, M. E. (1998) Mol. Cell. Biol. 18, 5609-5619[Abstract/Free Full Text]
46. Beier, F., Lee, R. J., Taylor, A. C., Pestell, R. G., and LuValle, P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1433-1438[Abstract/Free Full Text]


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