From the Cardiovascular Division, Departments of Medicine and
Pharmacology, Vanderbilt University Medical Center, Nashville Veterans
Affairs Medical Center, Nashville, Tennessee 37232
This study was designed to characterize the
direct effects of hyperglycemia on plasminogen activator inhibitor-1
(PAI-1) expression in cultured vascular smooth muscle cells. Glucose
induced dose- and time-dependent increases of PAI-1
mRNA expression in rat aortic smooth muscle (RASM) cells in
vitro. Using a series of luciferase reporter gene constructs
containing PAI-1 5'-flanking sequence (from
6.4 kilobase to
42 base
pairs (bp)) transfected into RASM, we found that glucose (25 mM) consistently induced a 4-fold increase in luciferase
activity, with the response localized to sequence between
85 and
42
bp. Mutagenesis of two putative Sp1-binding sites located in the region
of interest essentially obliterated the glucose-response.
Electrophoretic mobility shift assays with radiolabeled
oligonucleotides containing the two putative Sp1-binding sites from
PAI-1 promoter and nuclear extracts from RASM cells revealed that
glucose treatment markedly changed the mobility pattern of the major
protein-DNA complexes. Supershift assay showed that transcription
factor Sp1 was present in the complexes under control and hyperglycemic
conditions. These results suggest that glucose regulates PAI-1 gene
expression in RASM cells through an effect on two adjacent Sp1 sites
located between
85 and
42 bp of the PAI-1 5'-flanking region and
that the release of a transcriptional repressor from the Sp1 complexes
may explain the activation of the PAI-1 gene under high glucose
conditions in RASM cells.
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INTRODUCTION |
A number of different factors contribute to the development of
atherosclerosis in patients with diabetes mellitus (1). Hyperglycemia
has direct toxic effects on vascular tissue through the glycosylation
of structural proteins (2). Hyperglycemia also appears to facilitate
free radical formation and oxidative damage to the vascular wall (3).
Conversely, the aggressive control of plasma glucose levels appears to
delay the development of the common complications of diabetes in
insulin-dependent diabetic patients (4). Many common risk
factors also contribute to the atherogenic tendency in diabetes. These
factors include abnormal lipoprotein metabolism, hypertension, and
endothelial dysfunction. Recent studies have identified elevated levels
of the fibrinolytic inhibitor, plasminogen activator inhibitor-1
(PAI-1),1 in diabetic
patients (5, 6). There is a developing appreciation for the role of
PAI-1 plays in the pathogenesis in atherothrombotic disorders (7), and
recent evidence suggests that increased PAI-1 production may be an
important contributor to the development of vascular disease in
diabetics (8). The increased production of PAI-1 seen in diabetic
patients has been attributed directly to glucose, which increases PAI-1
production in cultured endothelium (9, 10), and by insulin and
pro-insulin peptides (11), which induce PAI-1 production in cultured
hepatocytes.
Recent studies have identified a cis-acting carbohydrate responsive
element in the promoter region of the rat S14 gene (12). The
carbohydrate responsive element consensus sequence has been deduced as
5'-CACGTGNNNGCC-3' (13). This DNA motif has been shown to mediate
glucose responsiveness when placed 5' of genes that do not normally
respond to glucose. Similar or identical DNA motifs have also
been identified in other carbohydrate-responsive genes, including the
genes for pyruvate kinase and the fatty acid synthase gene (13).
Although it is clear that glucose can directly induce gene expression
through a motif CACGTG that is related to the consensus binding site
for the c-myc family of transcription factors, other glucose
responsive elements have recently been identified, including Sp1 sites
in the promoter II of the gene for acetyl-CoA carboxylase (14).
This study was designed to characterize the mechanism through which
glucose regulates PAI-1 expression in vascular smooth muscle cells. Our
findings indicate that glucose induces PAI-1 gene expression through
two Sp1 sites located within 100 bases upstream of the transcription
start site of PAI-1 gene.
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EXPERIMENTAL PROCEDURES |
Materials--
Fetal bovine serum was obtained from Hyclone
Laboratory (Logan, UT). Tissue culture medium, transferrin, and
ascorbic acid were from Life Technologies, Inc. (Gaithersburg, MD).
Glucose, lactate, and insulin were from Sigma. Poly(dI-dC) was from
Pharmacia Biotech (Uppsala, Sweden). Restriction enzymes and calf
intestinal alkaline phosphatase were from Promega (Madison, WI). T4 DNA
ligase, T4 DNA kinase, and Klenow fragment of DNA polymerase I were
from New England Biolabs (Beverly, MA). Antibodies against Sp1 and Sp3
were from Santa Cruz Biotechnologies (Santa Cruz, CA) and monoclonal
antibody against Rb protein was from Pharmingen (San Diego, CA).
[
-32P]ATP (3000mCi/mmol), [
-32P]dUTP,
and [
-33P]dATP were from NEN Life Science Products
Inc. (Boston, MA).
Cell Culture--
Rat aortic smooth muscle (RASM) cells were
isolated as described previously (15). Cells were maintained in DMEM
with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin
(100 µg/ml), and 25 mM HEPES. For these studies, RASM
cells subcultured 10-16 times. The cells were identified as smooth
muscle cells by both appearance and positive fibrillar staining with a
smooth muscle specific
-actin antibody (Boehringer Mannheim,
Germany).
Plasmid Construction--
PAI-1-luciferase reporter vectors were
constructed by cloning various portions of the PAI-1 promoter into the
pGL2-Basic luciferase vector (Promega, Madison, WI). Progressive
deletions from the 5' end of the human PAI-1 gene promoter fragments
were constructed by using restriction endonuclease cleavage sites
present in the PAI-1 promoter or using polymerase chain reaction
methods to create fragments of PAI-1 promoter. All fragments had
identical 3' ends (i.e. the EcoRI site at
position +71) and different 5' ends. The following sites were used: for
pGLuc 3.4k, the XbaI site at position
3.4 kilobase pairs,
for pGLuc 1.5k, the KpnI site at
1.5 kilobase pairs, and
for pGLuc 884, the HindIII site at
884 bp. Specific oligonucleotide primers were used to synthesize smaller fragments using
the polymerase chain reaction. The DNA fragments
107 bp,
85 bp,
54 bp, and
42 bp were treated with Klenow fragment to generate
blunt ends, ligated to the pGL2 Basic luciferase reporter gene vector,
and designated as follows: pGLuc 107, pGLuc 85, pGLuc 54, and pGLuc 42. The luciferase reporter gene vector (
6.4 kilobase pairs) of the PAI-1
promoter was kindly provided by Dr. David J. Loskutoff (Scripps
Research Institute, La Jolla, CA).
Site-directed Mutagenesis--
Base substitutions were made by
oligonucleotide-mediated mutagenesis in three different regions
identified as Sp1A, Sp1B, P-box I, and P-box
II, corresponding to nucleotides
85 to
63,
53 to
30, and
71
to
47, respectively. The mutant oligonucleotides used as primers are
listed in Table I together with the
corresponding wild-type PAI-1 sequences. The mutations were either made
with the Altered Sites in vitro mutagenesis kit (Promega) or
carried out by the polymerase chain reaction method as described
previously (16). Mutation accuracy was checked by restriction enzyme
mapping and direct sequencing.
Transfection Assay--
For transient transfection experiments,
RASM cells were cultured in 35-mm 6-well plates at density of 1 × 105 cells/well. After cells reached 70-80% confluence,
they were transfected using the DEAE-dextran method (17), with 1 µg
of the PAI-1 pGL-2 construct and 0.8 µg of the plasmid pMVS-
-Gal, which contains the Lac Z gene under control of the Molony sarcoma virus
long terminal repeat. The control plasmid has been examined on several
occasions and has never demonstrated any response to glucose (data not
shown). After overnight incubation, the media was replaced with serum
starvation media (5.5 mM glucose, DMEM containing 5 × 10
7 M insulin, 5 µg/ml transferrin, and 0.2 mM ascorbic acid) for 24 h. This media has been
demonstrated to maintain to smooth muscle cells in a quiescent,
noncatabolic state for at least 72 h (18). The following
morning, the cells were then kept in the glucose-deficient DMEM
supplemented with either lactate (10 mM, final
concentration) or glucose (25 mM, final concentration).
After 48 h, the cells were harvested with reporter lysis buffer.
The luciferase assays were performed according to the protocol of
Promega. A 50-µl sample of the extract was used for determination of
-galactosidase activity by a colorimetric assay (19). The luciferase
activity was corrected for
-galactosidase activity and is presented
as corrected light units. To calculate relative induction, the
corrected light unit of glucose-treated cells was divided from the
corrected light unit of lactate-treated cells.
Northern Blot Analysis--
For the Northern blot analysis,
cells were grown in 100-mm tissue culture dishes. When the cells
reached 80% confluence, the cells were then kept in the DMEM
supplemented with different concentrations of glucose and glucosamine.
Total cellular RNA isolation and Northern blot analysis were carried
out as described (20).
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts from RASM cells prepared as described previously (17). EMSAs
were performed on 4% acrylamide gels with a buffer containing 50 mM Tris (pH 8.5), 0.38 M glycine, 2 mM EDTA, and 0.5 mM
-mercaptoethanol.
Reactions were carried out in a 20-µl volume containing 10 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 6% glycerol, 1 µg of
poly(dI-dC)·poly(dI-dC), and 3-6 µg of nuclear extract. The
mixture was incubated at room temperature for 10 min and then 10,000 cpm of 32P-labeled double-stranded oligonucleotides
(0.1-0.6 ng). Following an additional incubation for 10 min at room
temperature, the samples were electrophoresed at 200 V at 4 °C for
2 h. The gel was then dried and exposed to Kodak X-AR film. For
competition experiments, 0.2-0.6 µg of unlabeled double-stranded
oligonucleotides were added to the initial incubation mixture. For
supershift experiments, 2 µg of anti-Sp1 antibody (Santa Cruz
Biotechnologies, Santa Cruz, CA) was added to the reaction mixture and
incubated for 2 h at 4 °C prior to adding the labeled probes.
The following oligonucleotides covering sequences of 5'-flanking region
of the PAI-1 gene were synthesized in the Molecular Biology Core
facility of Vanderbilt University:
85/
63 sense, CAGTGAGTGGGT
GGGGCTGGAAC;
85/
63 antisense, GTTCCAGCCCCACCCACTCACTG;
85/
63
mutated Sp1A sense, CAGTGAGTGAATTCGGCTGGAAC;
85/
63
mutated Sp1A antisense, GTTCCAGCCGAATTCACTCACTG;
53/
30 sense, CATCTATTTCCTGC CCACATCTGGTA;
53/
30 antisense,
TACCAGATGTGGGCAGGAAATAGATG;
53/
30 mutated Sp1B
sense, CATCTATTTCTAGATCACATCTGG;
53/
30 mutated Sp1B
antisense, CCAGATGTGATCTAGAAATAGATG. Sp1 consensus oligonucleotides
were purchased from Promega, and were comprised of the following
nucleotide sequence: sense, ATTCGATCGGGGCGGGGCGAG; antisense,
CTCGCCCCGCCCCGATCGAAT.
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RESULTS |
Effects of Glucose on Levels of PAI-1 mRNA in Rat and Bovine
Aortic Smooth Muscle Cells--
To determine whether glucose regulates
PAI-1 gene expression, the relative expression of PAI-1 mRNA in
RASM cells was measured by Northern blotting. In these experiments,
confluent cultures of RASM cells were exposed to glucose over the
concentration range of 2.5-25 mM in serum-free DMEM (Fig.
1A) or DMEM with 10% serum (Fig. 1B) for 48 h. Glucose resulted in a
dose-dependent increase in the expression of PAI-1 gene in
RASM cells. No PAI-1 mRNA was detected in RASM cells treated with
2.5 mM glucose in the serum-free DMEM. On average, there
was a 4.2 ± 2.2-fold increase in PAI-1 message induced by glucose
(25 mM) compared with 5 mM glucose (p = 0.0013, n = 6). A
time-dependent effect of glucose on PAI-1 gene expression
in RASM cells was also demonstrated. The induction of PAI-1 mRNA
expression produced by glucose is maximal at 72 h in RASM cells
(Fig. 2). The specificity of this effect
of glucose was studied by performing comparable studies using
glucosamine, the hexosamine pathway metabolite of glucose. In the
presence of 5 mM glucose, incremental concentrations of
glucosamine failed to induce PAI-1 mRNA expression under serum-free
conditions (Fig. 1A). Similarly, in serum-replete DMEM,
increasing concentrations of glucosamine failed to induce PAI-1
mRNA expression in RASM cells (Fig. 1B).

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Fig. 1.
The effect of glucose and glucosamine on
PAI-1 expression in cultured RASM cells. A, 80% confluent
cultures of RASM cells were exposed to glucose over the
concentration range of 2.5-25 mM and glucosamine over
concentration range of 3.75-15 mM in the presence of 5 mM glucose in DMEM without serum for 48 h.
B, 80% confluent cultures of RASM cells were exposed to
glucose over the concentration range of 2.5-25 mM and
glucosamine over concentration range of 3.75-15 mM in the
presence of 5 mM glucose in DMEM with 10% serum for
48 h.
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Fig. 2.
Time-dependent effect of glucose
on PAI-1 expression in cultured RASM cells. Confluent RASM cells
were incubated in glucose-deficient DMEM supplemented with either 10 mM lactate or 25 mM glucose for 24-72 h.
kb, kilobase pair(s).
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Mapping of the Glucose-responsive Element in PAI-1
Promoter--
The regulatory elements responsible for the induction of
PAI-1 in RASM cells were localized with transient transfection assays. A schematic diagram of the PAI-1-1 5'-flanking region and the described regulatory elements is shown in Fig.
3. Two putative sequences (CACGTG)
identical to glucose response elements in the rat S14 gene (13) were
found at sites
683 bp and
562 bp in the PAI-1 promoter. To localize
the regulatory elements required for transcriptional activation of the
PAI-1 gene by glucose, progressive 5' promoter deletion constructs
containing different portions of the PAI-1 promoter and 72 nucleotides
of the PAI-1 5'-untranslated region were fused to a luciferase reporter
gene. The constructs, schematically depicted in Fig.
4A, were transiently
transfected into RASM cells and the luciferase activity was measured
48 h after lactate or glucose treatment of serum-starved RASM
cells. To normalize for transfection efficiency, each construct was
co-transfected with a constitutively expressed Molony sarcoma
virus-
-galactosidase reporter construct. All experiments were
repeated a minimum five times, and results are reported as mean ± S.E. In the presence of 25 mM glucose, there was a 3.7-fold
increase in luciferase activity in RASM cells transfected with pGLuc884
construct, which includes both copies of the putative glucose response
elements. To determine if the presence of the sequence motif CACGTG
confers glucose responsiveness to a heterologous promoter, multiple
copies of regions between
571 and
548 and between
698 and
664
of PAI-1 promoter were fused to the SV40 promoter-luciferase reporter gene vector as described above. These constructs were transfected into
RASM cells and were not inducible by glucose (data not shown). As seen
in Fig. 4B, constructs comprising 6.4k, 3.0k, 1.5k, 107, and
85 nucleotides of upstream sequences from the PAI-1 promoter all
exhibited glucose responsiveness that averaged between 3.2-4.5-fold compared with controls. However, a substantial reduction in glucose responsiveness was observed in further truncated constructs, with pGLuc
54 exhibiting a nearly 50% reduction in glucose responsiveness. A
further truncated construct, pGLuc 42, containing only PAI-1 TATA box,
was not induced by glucose in comparison with the luciferase reporter
construct pGL-2 Basic. These results indicate that the major sequence
determinants of glucose responsiveness in the PAI-1 promoter reside
between 85 and 42 nucleotides upstream from the transcription start
site.

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Fig. 3.
Schematic diagram of the upstream
5'-regulatory region of the PAI-1 gene showing the major described
enhancer elements. The TATA box and transcription start site are
shown. Putative carbohydrate response elements are indicated by
ChoRE.
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Fig. 4.
A, schematic diagram of PAI-1 promoter
deletion constructs used in transient transfection assays. Sequential
deletions from the 5' end were generated with the designation based on
the number of base pairs upstream from the PAI-1 transcriptional start
site as indicated. B, induction of luciferase activity with
glucose in RASM cells transfected with PAI-1 promoter/luciferase
reporter gene constructs. Bars represent the relative
induction of luciferase activity after treatment with 25 mM
glucose as compared with lactate-treated control cells. Results
represent mean ± S.E. of at least five independent
experiments.
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Mutagenesis of the Glucose-responsive Regions of PAI-1
Promoter--
To further define the sequence(s) involved in the
glucose responsiveness of PAI-1 promoter, site-directed mutagenesis was performed on the sequence between
85 and
42 in the pGLuc 107 and
pGLuc 85 constructs. There are four recognized regulatory elements in
the region, including two Sp1 sites, a P-box, and a D-box (Fig. 3). The
Sp1 site is the binding site of the ubiquitously expressed
transcription factor Sp1. The P box is similar in sequence to phorbol
ester response elements and cAMP response elements. The D-box, a
phorbol ester response elements-like sequence, has a weak affinity for
transcription factor AP-1 (21, 22). In these experiments, four single
mutants and different double mutants were constructed as shown in Fig.
5A. Mutations in P-box I or P-box II had no effect on the glucose responsiveness. Mutations in
either Sp1A or Sp1B resulted in an
approximately 50% decrease of glucose responsiveness compared with
wild type pGLuc 85 construct. Mutations in Sp1A + Sp1B resulted in a loss of responsiveness to glucose (Fig.
5B). Thus, both Sp1 sites appear to be essential for glucose
induction of PAI-1. These results support the involvement of the Sp1
transcriptional factor in the glucose activation of PAI-1 gene.

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Fig. 5.
A, schematic representation of the PAI-1
promoter mutation constructs used in transient transfection assays.
Mutations in a particular element are indicated by a X.
B, induction of luciferase activity with glucose in RASM
cells transfected with PAI-1 promoter/luciferase reporter gene
constructs. Bars represent the relative induction of
luciferase activity after treatment with 25 mM glucose as
compared with lactate-treated control cells. Results represent
mean ± S.E. of at least five independent experiments.
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Identification of the Nuclear Factor(s) Involved in the Glucose
Response--
To test if transcription factor Sp1 binds to the site
Sp1A and Sp1B in RASM cells, EMSAs were
performed with RASM nuclear extracts prepared from either
lactate-treated cells or glucose-treated cells and
32P-labeled oligonucleotides consisting of Sp1A
and Sp1B sequences spanning
85 to
63 and
53 to
30
of the PAI-1 promoter. As seen in Fig. 6,
nuclear extracts from lactate-treated cells formed one unique complex
with 32P-labeled
85/
63 probe (lane 1) and
four distinct complexes indicated by arrows on the
right of Fig. 6 with the 32P-labeled
53/
30
probe (lane 3). Extracts from glucose-treated cells formed
different protein-DNA complexes with distinctly differing mobility.
Three major complexes were formed with 32P-labeled
85/
63 probe (lane 2) and two major complexes were formed
with 32P-labeled
53/
30 probe (lane 4). The
DNA-protein complexes from glucose-treated cells migrated more rapidly
than those from lactate-treated cells.

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Fig. 6.
EMSA with RASM cell nuclear extracts.
The double-stranded oligonucleotides 85/ 63 and 53/ 30 of PAI-1
were end labeled and incubated with nuclear extracts from RASM cells
grown in lactate- (lanes 1 and 3) or
glucose-containing medium (lanes 2 and 4).
Solid arrows indicate distinct bands identified under
control (10 mM lactate) conditions and broken
arrows indicate distinct bands present with high glucose.
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To determine the specificity of the DNA-protein complexes formed with
the
85/
63 and
53/
30 probes, excess unlabeled consensus Sp1,
wild-type, and mutant oligonucleotides were included in the binding
reactions. The results of these competitive analyses on the formation
of DNA-nuclear protein complexes are shown in Figs. 7 and 8. As
seen in Fig. 7, one band from lactate-treated nuclear extract and three
bands from glucose-treated nuclear extract are specific since
competition with 100 molar excess unlabeled oligonucleotide (corresponding with sequence
85/
63) and consensus Sp1
oligonucleotides effectively prevented complex formation (lanes
2, 4 and 6, 8). The Sp1A site is critical
for this binding, as unlabeled oligonucleotides with a mutated
Sp1A site did not compete for the binding (lanes 3 and 7). In addition, excess unlabeled of wild-type
53/
30 oligonucleotide (containing Sp1B) partially
competed the bands (data not shown). As seen in Fig. 8, in either
lactate-treated (lanes 1-5) or glucose-treated (lanes
6-10) nuclear extracts, several proteins bind specifically to the
32P-labeled
53/
30 oligonucleotide containing
Sp1B site (lanes 1 and 6). The
specificity of these proteins is demonstrated by the fact that they
were effectively competed by excess unlabeled wild-type
53/
30
oligonucleotide (lanes 2 and 7). The unlabeled oligonucleotide with a mutated Sp1B did not compete for
binding (lanes 3 and 8). Interestingly, in the
lactate-treated nuclear extract, the unlabeled wild-type
85/
63
oligonucleotide containing the Sp1A site reduced the
apparent intensity of bands 1 and 3 (lane 4) while the
consensus Sp1 oligonucleotide eliminated only band 1 (lane
5). In the glucose-treated nuclear extract, unlabeled wild-type
85/
63 oligonucleotide effectively eliminated bands 1 and 2 (lane 9) and the unlabeled consensus Sp1 oligonucleotide eliminated band 1 (lane 10).

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Fig. 7.
EMSA with 32P-labeled 85/ 63
oligonucleotide and RASM cell nuclear extract. The double-stranded
oligonucleotides 85/ 63 of PAI-1 were end labeled and incubated with
nuclear extracts from RASM cells grown in lactate- (lanes
1-4) or glucose-containing medium (lanes 5-8).
Lanes 1 and 5 were positive controls (no
unlabeled competitor added). The unlabeled wide-type 85/ 63
oligonucleotide (lanes 2 and 6), or 85/ 63
oligonucleotide with mutant Sp1A site (lanes 3 and 7), or consensus Sp1 oligonucleotide (lanes 4 and 8) were added to the binding mixture in the mobility
shift assay. Arrows indicate the specific bands.
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Fig. 8.
EMSA with 32P-labeled 53/ 30
oligonucleotide and RASM cell nuclear extract. The double-stranded
oligonucleotides 53/ 30 of PAI-1 were end labeled and incubated with
nuclear extracts from RASM cells grown in lactate- (lanes
1-5) or glucose-containing medium (lanes 5-10).
Lanes 1 and 6 were positive controls (no
unlabeled competitor added). The unlabeled wide-type 53/ 30
oligonucleotide (lanes 2 and 7), or 53/ 30
oligonucleotide with mutant Sp1B site (lanes 3 and 7), or wild-type 85/ 63 oligonucleotide (lanes
4 and 9), or consensus Sp1 oligonucleotide (lanes
5 and 10) was added to the binding mixture in the
mobility shift assay. Arrows indicate the specific
bands.
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Supershift assays were performed using antibody against Sp1, which has
been reported to mediate the glucose response in the acetyl-CoA
carboxylase gene (14). In the lactate-treated nuclear extract, the
antibody against Sp1 yielded a novel band with reduced mobility using
the 32P-labeled
86/
63 probe (Fig.
9A, lane 3). The antibody
against Sp1 also supershifted band 1 formed with the
32P-labeled
53/
30 probe (Fig. 9B, lane 3).
In contrast, in the glucose-treated nuclear extract, the antibody
against Sp1 formed a larger, supershifted band with the
32P-labeled
86/
63 probe (Fig. 9A, lane 5)
and a very faint supershifted band with 32P-labeled
53/
30 probe. Therefore, it appears that Sp1 is present in the
complexes formed with the
85/
63 oligonucleotide under control and
hyperglycemic conditions. Sp1 also appears to be present in complexes
formed with the
53/
30 oligonucleotide under control conditions, but
its presence under hyperglycemic conditions is uncertain. In similar
experiments, a polyclonal antibody against Sp3 and a monoclonal
antibody against Rb protein failed to induce a similar supershift (data
not shown). These results indicate that Sp1 is one of the factors
binding to the Sp1A and Sp1B sites located
between nucleotides
85 and
30 of the PAI-1 promoter and suggest
that the transcriptional complex or accessibility to antibody differs
in the protein-DNA complexes bound to the two oligonucleotides.

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Fig. 9.
Supershift assays with Sp1 antibody.
A, the wild-type 85/ 63 oligonucleotide was end labeled and
added to the reaction mixture after the antibody reaction. The RASM
cell nuclear extract was first incubated with (lanes 3 and
5) and without (lanes 2 and 4)
anti-Sp1 antibody. Lane 1 is a negative control (no nuclear
extract added). B, the wild-type 53/ 30 oligonucleotide
was end labeled and added to the reaction mixture after the antibody
reaction. The RASM cell nuclear extract was first incubated with
(lanes 3 and 5) and without (lanes 2 and 4) anti-Sp1 antibody. Lane 1 is a negative
control (no nuclear extract added).
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DISCUSSION |
Previous experimental studies have indicated that increased
ambient glucose concentrations stimulate the expression of PAI-1 in
cultured endothelial cells (9-11). The present studies extend this
observation to include vascular smooth muscle cells. Increased glucose
appears to induce dose-dependent and protracted increases in PAI-1 mRNA levels in cultured RASM cells. This effect is not merely a response to altered media osmolarity, as control experiments using equivalent concentrations of mannitol failed to produce similar
effects (data not shown). Furthermore, glucosamine, a metabolite of
glucose and the product of hexosamine pathway, did not mimic the effect
of glucose in stimulating PAI-1 gene expression.
The recent description and identification of specific glucose-response
elements in the regulatory regions of several genes prompted a
systematic search for elements in the PAI-1 promoter that contribute to
this response (14, 23, 24). Although sequence analysis suggested that
candidate sequence motifs were present at
683 and
562 of the PAI-1
promoter, sequential deletion reporter constructs indicated that
glucose responsiveness does not reside in these regions. Instead,
glucose responsiveness was maintained in the reporter constructs down
to a length of
85. Beyond that point, glucose responsiveness was lost
in a stepwise manner following deletion to
54 and obliterated in
deletion constructs down to
45. In this sequence reside two distinct
Sp1 sites. Given previous reports that Sp1 sites mediate the glucose
response in the acetyl-CoA carboxylase gene (14, 25), these sites were examined for their functional role in glucose responsiveness by examining the effects of site-specific mutations to these regions. Indeed, mutations in either Sp1 site of PAI-1 promoter independently reduced glucose responsiveness by approximately 50%, while constructs engineered to have mutations in both sites lost their glucose responsiveness. The specificity of these mutations is supported by the
observation that mutation of the intervening P-box, located between the
two Sp1 sites, failed to diminish glucose responsiveness. These
findings confirm and extend previous observations that the glucose
responsive region of the transforming growth factor-
promoter maps
to a region including Sp1 sites (26, 27). Taken together, it appears
that Sp1 sites may provide a mechanism for glucose responsiveness in
vascular tissue.
The systematic analysis of the response of serially truncated or
mutated reporter constructs merely identifies potential
glucose-responsive regions of the PAI-1 promoter. Although Sp1 sites
are identified, this does not establish that the ubiquitous
transcription factor Sp1 is responsible for this effect. However, EMSA
studies confirmed that the electrophoretic mobility of oligonucleotides
comprised of these regions of interest was altered by increased glucose concentrations. Furthermore, immunological analyses of these complexes suggested that authentic Sp1 was present under control conditions and
remained associated to at least one of the labeled Sp1 sites in the
presence of increased glucose. Taken together, these findings suggest
the possibility that Sp1 is bound to the putative Sp1 sites under
normal conditions, but that transcription is suppressed by the presence
of a bound repressor. In the presence of increased glucose, the
repressor molecules are released, leaving Sp1 bound to the Sp1 sites,
and transcription ensues. Ample precedent exists to permit this
mechanism to be entertained as a potential explanation for the effects
of glucose described in this study and in fact, a similar mechanism has
recently been described through which Sp1 mediates induced expression
of the acetyl carboxylase gene (14). Alternatively, glucose has been
reported to induce gene expression by promoting the binding of
transcriptional activators (14, 24, 28). Although the present findings
are not entirely consistent with this possibility, it cannot be
completely excluded.
We have suggested that hyperglycemia promotes the displacement of
a transcriptional repressor present in the Sp1 transcriptional complex.
However, the identity of this repressor protein or proteins is unknown
at present. Studies with antibodies against the retinoblastoma gene
product and against the related protein Sp3 have failed to establish
the presence of these proteins in EMSA studies. Further studies are now
underway to identify potential negative and positive co-factors in the
Sp1 regulatory complex localized to the Sp1 sites in the PAI-1
promoter.
As atherosclerotic lesions progress in terms of severity (Stage IV and
beyond), there is increased deposition of fibrin, fibrinogen, and
collagen in areas of advanced atherosclerosis (29). Advanced lesions
are also characterized by smooth muscle cell invasion and
proliferation. The fibrinolytic system, i.e. plasmin, is
primarily responsible for the proteolytic degradation of fibrin and
fibrinogen. Furthermore, plasmin is indirectly responsible for the
dismantling of excess collagen by virtue of the role it plays in the
activation of matrix metalloproteinases (30). Plasmin also plays a role in regulating the smooth muscle cell content of atherosclerotic lesions
through its role in activating latent transforming growth factor-
(31), which has potent antimigratory and antiproliferative effects on
vascular smooth muscle cells. Thus, it is quite evident that plasmin
plays a multifaceted housekeeping role in the vessel wall, and that
fibrinolytic activity likely retards the progression of atherosclerotic
lesions. This view is supported by recent studies indicating that
plasminogen deficiency accelerated the development of atherosclerotic
lesions in ApoE knockout mice (32). Apart from its effects on
intraluminal fibrinolytic balance, PAI-1 is topologically distributed
to influence fibrinolytic function in the vessel wall. Several studies
have now confirmed the presence of increased amounts of PAI-1 in human
atherosclerotic plaques (7, 33, 34). The presence of excess PAI-1 in
atherosclerotic lesions reduces local plasmin generation (35). In such
a milieu, a relative paucity of plasmin production likely reduces
matrix remodeling capacity. Furthermore, since transforming growth
factor-
has potent antiproliferative effects on vascular smooth
muscle cells (36), a reduction in the plasmin-dependent
activation of this critical cytokine likely exerts a permissive effect
on the growth of an atherosclerotic lesion. Thus, increased local production of PAI-1 likely contributes to the progression of
atherosclerotic lesions, and PAI-1 expression appears to be regulated
by glucose in vascular tissues. This relationship may play an important
role in the pathogenesis of atherothrombotic disease in diabetes.
In conclusion, these studies indicate that hyperglycemia directly
stimulates PAI-1 expression in cultured VSMC. This response appears to
be localized to two independent Sp1 sites just upstream from the
transcription start site of PAI-1. The present findings suggest that
glucose induces this effect on PAI-1 transcription by dislodging an
unidentified transcriptional repressor from the Sp1 complex. The
ability of glucose to stimulate directly the expression of PAI-1 in
vascular tissue may have important clinical ramifications. Local
overexpression of PAI-1 in the blood vessel wall would be expected to
contribute to the development of atherosclerosis and fibrosis. The
development of diffuse and severe vascular disease in diabetes is an
all too common manifestation of the disease, and is responsible for a
great deal of the excess cardiovascular morbidity and mortality seen in
this disorder. Improved glucose control would be expected to result in
reduced vascular PAI-1 expression, which in turn may contribute to a
reduction in the development of vascular disease and its
complications.