From 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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 (NF 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.
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,
-NF 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/ 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 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'; NF
To confirm the specificity of protein binding, double-stranded
oligonucleotides encoding a canonical SP1 binding site or NF
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.
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 NF 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 NF
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 NF
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 NF 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).
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/ 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 NF 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
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 NF 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 NF
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 NF
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.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Bp50, -NF
Bp65, -c-Fos, and -c-Jun antibodies were obtained
from Santa Cruz Biotechnologies (Santa Cruz, CA).
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-,
NF
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/
AP1, sense primer
(5'-GGAGATCACTGTTTCTCAGCTTTCCA-3'); pGL2/
836/
NF
B1, sense primer (5'-GGACCGACTGGTCAAGGTAGGAA-3'); pGL2/
707/
NF
B2, sense primer (5'-GAGCGAGCGCATGCTAAGCTGAA-3'); pGL2/
461/
STAT1, sense primer (5'-GCGCCCATTCTGCCGGCTTGGAT-3'); pGL2/
229/
STAT2, sense primer (5'-TTCTATGAAAACCGGACTACAGGGGCAACTC-3'); pGL2/
161/SP1, sense
primer (5'-CCCCTCGCTGCTCCCGGCGTTT-3'); pGL2/
95/
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/
NF
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 NF
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/NF
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.
80 °C. Protein concentration was measured according
to Bradford's method (Bio-Rad, Melville, NY).
Bwt,
5'-CGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCT-3'; NF
Bmut,
5'-CGGACTACAaGtaAGTTTTGTTGAAGTTGCAAAGTCCT-3'.
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 NF
Bwt,
5'-AACTGAAAACGGGAAAGTCCCTCTCTCTAACCTG-3'; canonical NF
Bmut,
5'-AACTGAAAACGGGAAAGTgggTCTCTCTAACCTG-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 NF
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/
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
NF
B binding site (pGL2/
23/
NF
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
NF
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 NF
B binding site (pGL2/
161/NF
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.
View larger version (25K):
[in a new window]
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).
View larger version (21K):
[in a new window]
Fig. 2.
Mutational analysis of the functions of the
SP1, ATF/CREB, and NF B sites in the cyclin D1
promoter. Diagrams show mutant reporter constructs in which each
SP1 site, ATF/CREB site, or NF
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).
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.
View larger version (62K):
[in a new window]
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.
B site was also examined. When a radiolabeled
double-stranded oligonucleotide encoding the cyclin D1 NF
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
NF
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 NF
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 NF
B proteins, their binding activity at the cyclin D1
NF
B site was under detectable levels in this assay.
View larger version (71K):
[in a new window]
Fig. 4.
Characterization of the DNA binding activity
at the ATF/CREB and NF 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 NF
B site (left panel,
CycD1NF
Bwt) or a canonical NF
B site
(right panel, canon. NF
Bwt) was
used as the probe for EMSAs. For competition experiments, a 100× molar
excess of cold double-stranded oligonucleotides encoding the wild type
(CycD1NF
Bwt) or mutant
(CycD1NF
Bmut) cyclin D1 NF
B site, or wild
type (canon. NF
Bwt) or mutant (canon.
NF
Bmut) canonical NF
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.
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.
View larger version (42K):
[in a new window]
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.
View larger version (56K):
[in a new window]
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.
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 NF
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 NF
B site (pGL2/
95/
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).
View larger version (12K):
[in a new window]
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/
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 NF
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
B sites appear to be of
particular interest, because AP1 and NF
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). NF
B is also a
potential target of the Ras-dependent pathway, because
p90rsk, a downstream target of Ras/MEK/ERK, phosphorylates and
inactivates I
B
(22, 23), resulting in nuclear translocation and
activation of NF
B. It has also been reported that Raf, a downstream
target molecule of Ras, activates NF
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.
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.
B site was under
detectable levels in our system, the role of the proximal NF
B site
could not be excluded, because the site was active in luciferase assays. Possibly, the proximal NF
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 NF
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 I
B
to inhibit NF
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 NF
B sites was observed in COS7 cells transfected with
expression plasmids encoding the p50 and p65 subunits of NF
B.
Another report showed that NF
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 NF
B is
involved in the transcriptional activation of the cyclin D1
gene in NIH3T3 cells and that DNA binding activity occurs at multiple
NF
B sites in the cyclin D1 promoter. Furthermore, Rac-dependent transcriptional activation of the
cyclin D1 gene is mediated by the proximal NF
B binding
site in NIH3T3 cells and DNA binding activity at the proximal NF
B
site is detected in the cells (13). Thus, the role of NF
B in the
activation of the cyclin D1 gene also seems to depend on
cell types.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
9. |
Watanabe, G.,
Lee, R. J.,
Albanese, C.,
Rainey, W. E.,
Batlle, D.,
and Pestell, R. G.
(1996)
J. Biol. Chem.
271,
22570-22577 |
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 |
11. |
Guttridge, D. C.,
Albanese, C.,
Reuther, J. Y.,
Pestell, R. G.,
and Baldwin, A. S., Jr.
(1999)
Mol. Cell. Biol.
19,
5785-5799 |
12. |
Hinz, M.,
Krappmann, D.,
Eichten, A.,
Heder, A.,
Scheidereit, C.,
and Strauss, M.
(1999)
Mol. Cell. Biol.
19,
2690-2698 |
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 |
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 |
15. |
Sabbah, M.,
Courilleau, D.,
Mester, J.,
and Redeuilh, G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11217-11222 |
16. |
Suzuki, E.,
Nagata, D.,
Kakoki, M.,
Hayakawa, H.,
Goto, A.,
Omata, M.,
and Hirata, Y.
(1999)
Circ. Res.
84,
611-619 |
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 |
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 |
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 |
22. |
Ghoda, L.,
Lin, X.,
and Greene, W. C.
(1997)
J. Biol. Chem.
272,
21281-21288 |
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 |
24. |
Finco, T. S.,
and Baldwin, A. S., Jr.
(1993)
J. Biol. Chem.
268,
17676-17679 |
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 |
29. |
Yan, G. Z.,
and Ziff, E. B.
(1997)
J. Neurosci.
17,
6122-6132 |
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 |
34. |
Li, J. M.,
Nichols, M. A.,
Chandrasekharan, S.,
Xiong, Y.,
and Wang, X. F.
(1995)
J. Biol. Chem.
270,
26750-26753 |
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 |
39. |
Jensen, D. E.,
Rich, C. B.,
Terpstra, A. J.,
Farmer, S. R.,
and Foster, J. A.
(1995)
J. Biol. Chem.
270,
6555-6563 |
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 |
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 |
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 |