From The Centre for Thrombosis and Vascular Research, School of
Pathology, The University of New South Wales, and Department of
Haematology, The Prince of Wales Hospital, Sydney,
New South Wales 2052, Australia
Platelet-derived growth factor (PDGF)
B-chain gene is differentially expressed in smooth muscle cells (SMCs)
derived from the rat aortic wall. SMCs cultured from two week-old rats
(pups) express high levels of PDGF-B mRNA, whereas cells isolated from three month-old rats (adults) express low levels of PDGF-B. Nuclear run-off experiments indicate that increased PDGF-B gene expression in
pups is mediated, at least in part, at the transcriptional level. We
used electrophoretic mobility shift assays and Western blot analysis to
demonstrate that levels of Sp1 and Sp3, two zinc finger transcription
factors which mediate basal expression of the PDGF-B gene, are elevated
in pup nuclei compared with adult nuclei. The immediate-early
transcription factor, Egr-1, which footprints the PDGF-B promoter, is
also constitutively expressed in these cells. Transient transfection
and binding studies show that these factors interact with a region in
the proximal PDGF-B promoter key for basal expression in pup cells.
Mutation of this proximal element in transfected pup cells attenuates
reporter gene expression to levels observed in adult cells. Conversely, overexpression of Sp1 in adult cells augments PDGF-B
promoter-dependent expression. Elevated PDGF-B expression
in cultured newborn rat SMCs may therefore require high constitutive
expression of a number of zinc finger transcription factors and their
specific interactions with the proximal PDGF-B promoter.
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INTRODUCTION |
Platelet-derived growth factor
(PDGF)1 consists of an
A-chain and a B-chain held together in heterodimeric or homodimeric
configuration by disulfide bonds (1). Multiple cell types, including
vascular smooth muscle cells (SMCs) (2), produce and respond to this potent mitogen and chemoattractant in culture. Seifert et
al. (3) observed over a decade ago that PDGF may be
developmentally regulated in these cells. SMCs cultured from the aortae
of 2-week-old rats (pups) (WKY12-22 cells) secreted almost 200-fold
greater levels of PDGF than cells isolated from 3-month-old rats
(adults) (WKY3M-22 cells). Subsequent Northern blot analysis determined that PDGF-B gene expression is at least 60-fold greater in cultured pup
SMCs than adult cells (4). Evidence that differential PDGF-B expression
is mediated at the level of transcription was obtained from nuclear
run-off analysis (5). Several other genes, such as tropoelastin and
1-procollagen (type I), were found to be overexpressed
in pup cells relative to adult cells by cDNA library screening
strategies. Cloning pup cells by dilution plating revealed that
subpopulations expressed high levels of elastin, CYPIA1 and osteopontin
mRNA (6). Cultured pup and adult SMCs also display marked
differences in morphology. The epithelioid and cobblestone shape of pup
cells contrasts with the spindle-like, elongated bipolar nature of
adult SMCs (4).
Basal and inducible expression of the PDGF-B gene is mediated by
nucleotide elements located approximately 40 bp upstream of the TATA
box. We and others showed that Sp1 binds to the 5'-CCACCC-3' motif and
drives basal expression in vascular endothelial (7) and osteosarcoma
cells (8). This is consistent with in vivo footprinting
studies which indicate that this region is occupied by nuclear factors
in intact cells (9). The related zinc finger transcription factor, Sp3,
also interacts with this region of the promoter and activates
transcription (8, 10). Egr-1, also known as TIS8, zif268, and
NGFI-A (11), plays a positive regulatory role in PDGF-B
promoter-dependent expression in cells exposed to phorbol
esters or subjected to mechanical injury (12, 13). Egr-1 binds to a
cryptic recognition element in the PDGF-B promoter and can displace Sp1
from overlapping binding sites (13). This region also mediates
inducible PDGF-B promoter-dependent gene expression in
endothelial cells exposed to thrombin (14); however, functional
trans-acting factors mediating this process have not yet
been identified.
We hypothesized that the restricted expression of PDGF-B in
subpopulations of cultured SMCs may be regulated by functional interactions of key zinc finger transcription factors with nucleotide elements in the proximal PDGF-B promoter. Here, we report that Sp1,
Sp3, and Egr-1 are constitutively elevated in pup SMCs compared with
adult cells. Promoter deletion and transient transfection analysis in
pup cells revealed that the region bound by these factors is critical
for basal expression directed by the PDGF-B promoter. Mutation of this
region attenuates expression to levels comparable with those in adult
cells. These findings provide mechanistic insights into the
differential expression of PDGF-B gene in SMCs.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Pup and adult SMC lines were cultured in
Waymouth's MB752/1 medium (Life Technologies, Inc.), supplemented with
10% fetal calf serum, 30 µg/ml L-glutamine, 10 units/ml
penicillin, and 10 µg/ml streptomycin at 37 °C and 5%
CO2. Cultures were passaged every 3-4 days in 75 cm2 flasks. Cells seeded for preparation of nuclear
extracts were grown to 80% confluence and washed twice in
phosphate-buffered saline (PBS), pH 7.4, then returned to the incubator
in 1% growth medium for 24 h prior to extraction.
Transient Cell Transfections--
SMCs were seeded in 100-mm
tissue culture plates 2 days prior to transfection. When approximately
60% confluent, the cells were transfected with 10 µg of the
indicated promoter-reporter plasmid along with 2 µg of pTKGH to
correct for transfection efficiency. In overexpression studies, SMCs
were cotransfected with 3 µg of either Sp1-pcDNA3 (CMV promoter
based), 5 µg of Egr-1-pcDNA3, or pcDNA3 alone. Transfections
were performed using Superfect (Qiagen). A precipitate was formed using
5 µl of Superfect reagent/µg of plasmid DNA and the transfection
mix made up to 300 µl with serum- and antibiotic-free Waymouth's
medium. After incubation at 22 °C for 10 min, 3 ml of complete
Waymouth's growth medium was added, and the entire mixture was then
added to cells previously washed once in PBS, pH 7.4. Following a 3-h
incubation at 37 °C in an atmosphere of 5% CO2, the
precipitate was gently aspirated, and the monolayers were washed once
with 4 ml of PBS, pH 7.4, prior to the addition of 10 ml of complete
Waymouth's growth medium. For overexpression experiments, SMCs were
refed with 2% Waymouth's medium. Two days post-transfection, the
conditioned medium was collected for determination of soluble growth
hormone by enzyme-linked immunosorbent assay (Bioclone Australia), and
cell lysates were prepared for assessment of chloramphenicol
acetyltransferase activity as described (7).
Preparation of Nuclear Extracts--
SMCs were washed twice with
PBS, pH 7.4, at 4 °C and removed from the culture dish by scraping.
The cells were pelleted by centrifugation at 1200 rpm for 10 min at
4 °C. The pellet was resuspended in cold PBS, pH 7.4, and the
suspension was transferred to Eppendorf tubes. Cells were repelleted in
a microcentrifuge by spinning at 14,000 rpm for 20 s at 4 °C.
The cells were lysed by resuspending the pellet in an ice-cold Buffer
A, which consisted of 10 mM HEPES, pH 8, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), 200 mM sucrose, 0.5% Nonidet P-40,
0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml
leupeptin, and 1 mg/ml aprotinin. After incubation on ice for 5 min,
the suspension was recentrifuged and the nuclei were lysed in ice-cold
Buffer C, which consisted of 20 mM HEPES, pH 8, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml aprotinin. Cellular debris was removed by centrifugation, and the
supernatant containing DNA-binding proteins was combined with an equal
volume of ice-cold Buffer D (20 mM HEPES, pH 8, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, and 1 mg/ml aprotinin). Extracts were immediately frozen on dry ice and then
stored at
80 °C until use.
Western Blot Analysis--
Electrophoresis, transfer, and
immunodetection were performed as described previously (13).
Oligonucleotide Synthesis, Purification, and
Radiolabeling--
Oligonucleotides were synthesized with trityl
groups off and purified by gel electrophoresis. Oligonucleotides were
radiolabeled with [
-32P]ATP (Bresatec) using
T4 polynucleotide kinase (New England Biolabs) and purified
by centrifugation in Chromaspin-10 columns
(CLONTECH).
Electrophoretic Mobility Shift Assay (EMSA)--
Binding
reactions for gel shift assays were performed in 20 µl of 10 mM Tris-HCl, 50 mM MgCl2, 1 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM PMSF, 1 µg of salmon sperm DNA (Sigma), 5% sucrose, 1 mg of poly(dI·dC)-poly(dI·dC) (Sigma), 32P-labeled
oligonucleotide probe (150,000 cpm), and 3 µg of nuclear extract
(determined by Bio-Rad Protein Assay). The reaction was incubated for
35 min at 22 °C. In supershift studies, 2 µl of the appropriate
affinity-purified antipeptide polyclonal antibody (Santa Cruz
Biotechnology) was incubated with the binding mix for 10 min before
addition of the probe. These antibodies do not cross-react with the
others' protein target. Bound complexes were separated from free probe
by loading samples onto a 6% non-denaturing polyacrylamide gel and
electrophoresing at 200 V for 2.5 h. The gels were vacuum dried at
80 °C and subjected to autoradiography overnight at
80 °C.
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RESULTS |
Certain Zinc Finger Transcription Factors Are More Abundant in
Cultured Pup Aortic SMCs than Adult SMCs--
Previous investigations
by our group (7, 13) and others (8, 14, 15) determined that PDGF-B gene
expression in cell types other than SMCs is under the transcriptional
control of the zinc finger nuclear factors, Sp1, Egr-1, and Sp3. Since the PDGF-B gene is expressed at higher levels in cultured pup rat
aortic SMCs than SMCs derived from adult rats, we hypothesized that
elevated PDGF-B gene expression may be due to elevated levels of Sp1,
Egr-1, and Sp3 in the former cell type. Using a 32P-labeled
double-stranded oligonucleotide bearing consensus sites for this
superfamily of regulatory proteins together with nuclear extracts from
cultured pup or adult aortic SMCs, we observed the formation of several
distinct nucleoprotein complexes, designated a-g
(Fig. 1). Differences were observed in
several of these complexes between the two cell subtypes. By
densitometric assessment, complexes a, b,
c, and d were approximately 12-, 11-, 4-, and
5-fold more abundant using extracts from pup cells as compared with
adult cells, respectively.

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Fig. 1.
Greater abundance of G+C-rich binding factors
in nuclei of pup aortic SMCs than adult cells. A
32P-labeled oligonucleotide bearing consensus elements for
several zinc finger transcription factors with preference for G+C-rich sequences (32P-Oligo GC); 5'-GGG GGG GGC GGG GGC
GGG GGC GGG GGA GG-3', sense strand) was incubated with nuclear
extracts from pup or adult SMCs. Binding reactions and gel
electrophoresis were performed as described under "Experimental
Procedures." Nucleoprotein complexes a-d are indicated by
arrows. Differences in band intensity were assessed by
scanning densitometry and normalized to complex f.
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Antibody inhibition experiments were performed to identify the protein
components of these complexes. Inclusion of polyclonal antibodies
directed against Sp1 in the binding reaction attenuated formation of
complex a (Fig.
2A), the most intense of these
complexes. Antibodies raised against Egr-1 eliminated complex
b (Fig. 2B), while antibodies recognizing Sp3
completely abolished the formation of complex c (Fig.
2C). Interestingly, the Sp1 antibody slightly attenuated the
intensity of complex c while the Sp3 antibody weakly inhibited the formation of complex a (Figs. 2, A
and C). This raises the possibility that Sp1 and Sp3 occupy
the promoter fragment simultaneously in these cells. Indeed, the
incomplete supershift obtained using the Sp1 antibody (Fig.
2A) has been observed previously by several groups
(16-19).

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Fig. 2.
Sp1, Egr-1, and Sp3 binding activity is
greater in nuclei of pup aortic SMCs than adult SMCs.
32P-oligo GC was incubated with nuclear extracts from
pup or adult SMCs, and nucleoprotein complexes were resolved by gel
electrophoresis. Polyclonal antibodies directed toward Sp1
(A), Egr-1 (B), or Sp3 (C) were
incubated with the extracts 10 min prior to the addition of
32P-oligo GC. Nucleoprotein complexes containing Sp1,
Egr-1, and Sp3 are indicated by arrows. D, Nuclear proteins
binding to the serum response element of the egr-1 promoter
are more abundant in adult SMCs than pup cells. Pup and adult SMCs were
incubated with 32P-Egr-1 prom (5'-AGG ATC CCC
CGC CGG AAC AAC CCT TAT TTG GGC AGC ACC-3', sense strand), and the
nucleoprotein complex was resolved by gel electrophoresis as described
under "Experimental Procedures". The nucleoprotein complex is
indicated by the arrow.
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Several criteria indicate that differences in Sp1, Egr-1, and Sp3
binding activity observed between pup and adult SMCs was not a
consequence of unequal protein content in each preparation of extract.
First, identical amounts of pup and adult nuclear extracts were used
for each EMSA. Second, the intensity of nucleoprotein complexes
e, f, and g was similar between pup
and adult samples. Third, the nucleoprotein complex obtained when a
32P-labeled oligonucleotide bearing a serum response
element in the egr-1 promoter was incubated with these
extracts was slightly more intense when adult samples were used (Fig.
2D).
We performed Western blot analysis to provide confirmatory evidence
that Sp1, Egr-1, and Sp3 are present in greater levels in nuclear
extracts from pup cells than adults since DNA binding activity and
levels of protein may not necessarily correlate. Findings from
immunoblot analysis indicate that Sp1, Egr-1, and Sp3 levels are indeed
greater in pup nuclei than adults (Fig. 3), consistent with observations from gel
and supershift studies (Fig. 2). The Coomassie Blue-stained gel
indicates equal protein loading (Fig. 3). Taken together, these results
demonstrate that Sp1, Egr-1, and Sp3 are constitutively expressed in
pup SMCs and that these levels exceed those in adult cells.

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Fig. 3.
Western blot analysis of nuclear extracts
from pup and adult SMCs. Nuclear extracts were resolved by
electrophoresis prior to transfer to PVDF membranes and exposure to
polyclonal antibodies directed toward Sp1, Egr-1, and Sp3.
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Pup Aortic SMCs Support High Basal Expression of a Reporter Gene
Driven by the Zinc Finger Transcription Factor Binding Site--
To
demonstrate a functional consequence of the disproportionate existence
of these transcription factors in the nuclei pup and adult SMCs, we
mobilized the gel shift oligonucleotide upstream of CAT cDNA in
pCATprom and performed transient transfection analysis using the
heterologous promoter-reporter construct in each cell type. Normalized
CAT activity was severalfold greater in pup SMCs than adult cells (Fig.
4). The higher levels of reporter gene expression in pup cells obtained using this construct are thus consistent with the greater abundance of factors binding the element in
this cell type (Fig. 1).

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Fig. 4.
Transient transfection analysis in pup SMCs
and adult cells. Pup and adult SMCs were transiently transfected
with a CAT reporter construct, pCATprom, bearing oligo GC upstream of the start site. CAT activity in the lysates was normalized to levels of
growth hormone secreted into the culture medium as described under
"Experimental Procedures."
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Nucleotide Elements in the Proximal PDGF-B Promoter Are Required
for High Basal PDGF-B Promoter-dependent Expression in Pup
Aortic SMCs--
Nucleotide regulatory elements mediating basal
expression of the PDGF-B gene have not yet been defined in vascular
SMCs. Transient transfection analysis was performed in pup cells with a
series of PDGF-B promoter fragments cloned upstream of a promoterless CAT cassette (7). CAT activity directed by construct d26, which extends
153 bp upstream of the TATA box, was comparable with expression generated from construct d77, which bears 82 bp of promoter sequence (Fig. 5). However, CAT expression
produced by construct d75, which bears 19 bp of promoter sequence, was
only 9% that of d77 (Fig. 5). These findings indicate that the region
between the 5' end points of the PDGF-B fragments in d77 and d75
contain nucleotide elements crucial for basal expression in pup
cells.

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Fig. 5.
Serial 5' deletion and transient transfection
analysis using PDGF-B promoter-CAT constructs. Pup SMCs were
transiently transfected with a series of PDGF-B promoter-CAT
constructs, and CAT activity in the lysates was determined as described
under "Experimental Procedures." CAT activity was normalized to
levels of growth hormone secreted into the culture medium.
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Sp1, Egr-1, and Sp3 Bind to the Proximal PDGF-B Promoter in
Cultured Pup Aortic SMCs--
We used EMSA and supershift studies to
determine whether Sp1, Egr-1, and Sp3 interact with the region in the
PDGF-B promoter responsible for high basal expression in pup SMCs. A
32P-oligonucleotide spanning the
30/
13 region of the
PDGF-B promoter in competition with its unlabeled cognate revealed the
formation of several specific nucleoprotein complexes (Fig.
6, complexes a-d, compare
lanes 2 and 4). Complexes a-c were
barely detectable when adult nuclei were used in the EMSA (lane
3). Complexes a-d were abrogated in the presence of a
molar excess of oligo GC (lane 6), whereas an irrelevant
oligonucleotide, E74, was without effect (lane 5). The oligo
GC result strongly suggested the involvement of zinc finger
transcription factors in these complexes. Antibody inhibition
experiments provided definitive evidence that Egr-1 (lane
7), Sp3 (lane 8), and Sp1 (lane 9) associate
with this region of the PDGF-B promoter preferentially in pup nuclei.
Interestingly, the partial supershift obtained using the Sp1 antibody
alone was completely abrogated by coincubation with the Sp3 antibody
(lane 10). These findings indicate that Sp1, Egr-1, and Sp3
bind to the proximal PDGF-B promoter in pup cells although, of all
these factors, Sp1 associates with this region of the promoter most abundantly.

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Fig. 6.
Sp1, Egr-1, and Sp3 bind to the proximal
PDGF-B promoter in a specific manner. Nuclear extracts from pup
rat aortic SMCs and adult SMCs were incubated with a
32P-labeled fragment of the proximal PDGF-B promoter
(32P-Oligo B) and adducts separated by gel
electrophoresis as described under "Experimental Procedures." For
competition or antibody inhibition studies, either a 100-fold molar
excess of unlabeled oligonucleotide or 2 µl of the appropriate
antibody was incubated with the binding mixture for 10 min prior to the
addition of the 32P-labeled probe. The specific
nucleoprotein complexes a-d are indicated by
arrows.
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The Proximal Region of the PDGF-B Promoter Required for Basal
Expression in Pup SMCs Supports Inducible Expression in Adult
Cells--
Transfection of construct d77 into pup SMCs resulted in
high levels of PDGF-B promoter-dependent gene expression
(Fig. 7A, column
1). In comparison, reporter gene expression driven by this construct was low in adult cells (column 3). Mutation of the
region bound by these factors in the PDGF-B promoter resulted in an
87% decrement in normalized CAT activity in pup cells (column
2). Interestingly, reporter gene expression directed by the mutant construct in pup cells was comparable with levels obtained using the
wild-type construct in adult cells (column 3). Mutation of the zinc finger binding site (
34/
18) only slightly attenuated promoter-dependent expression in adult cells (column
4). When wild type d77 was cotransfected with a cytomegaloviral
promoter-driven Sp1 expression construct in adult cells, reporter
expression increased to levels comparable with those in pup cells
(column 5). Overexpression of Egr-1, either alone or in
combination with Sp1, also resulted in inducible PDGF-B
promoter-dependent expression (Fig. 7B). These binding, mutational, and overexpression studies demonstrate that elevated PDGF-B expression in cultured pup SMCs is influenced by zinc
finger nuclear factors binding to regulatory elements in the proximal
region of the PDGF-B promoter.

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Fig. 7.
The proximal PDGF-B promoter supports
elevated basal expression in pup aortic SMCs but not adult cells.
A, SMCs were transiently transfected with construct d77 or
d77 bearing a mutation in the site bound by Sp1, Egr-1, and Sp3,
d77m (13). B, Cotransfection of d77 with
Sp1-pcDNA3 and Egr-1-pcDNA3. Transfections were performed as
described under "Experimental Procedures." CAT activity was normalized to levels of grown hormone secreted into the culture medium.
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DISCUSSION |
In this paper, we have investigated the transcriptional mechanisms
underlying the high constitutive expression of PDGF-B in SMCs isolated
from the aortae of pup rats. Curiously, SMCs cultured from adult rats
of the same strain express low levels of PDGF-B. EMSA and Western blot
analysis revealed that levels of Sp1 and two other zinc finger
transcription factors, Sp3 and Egr-1, are markedly elevated in the
nuclei of pup SMCs compared with adult cells. 5' deletion and transient
transfection analysis defined the proximal region of the PDGF-B
promoter as that which mediates high basal expression of the gene in
pup cells. EMSA using extracts from pup cells revealed that Sp1, Egr-1,
and Sp3 bind to this region of the promoter in a specific manner.
Mutation of the proximal element abolished basal PDGF-B
promoter-dependent expression in these cells. Conversely,
overexpression of Sp1 and/or Egr-1 in adult cells increased reporter
gene expression. These studies define a critical role for the proximal
region of the PDGF-B promoter in the elevated expression of the gene in
pup SMCs and identifies several nuclear proteins that bind to these
elements in a specific and functional manner.
While the basic structure of the rat aorta is already formed at birth,
the vessel wall continues to develop within the first three months
postpartum. SMCs proliferate and differentiate most actively in the
first few weeks and deposit large amounts of connective tissue matrix
components into the extracellular space while the artery undergoes
remodeling (20). The migratory and proliferative effects of PDGF-BB on
cultured aortic SMCs (2) and the chemotactic effects of PDGF-BB on SMCs
in rat (21) and porcine (22) models suggest that the PDGF B-chain may
play a developmental role. A thinner aortic wall, among other
abnormalities, in PDGF-B null mice is consistent with a role for PDGF-B
in blood vessel development (23). High constitutive PDGF-B expression
in pup SMCs that decline in older mice lends support to this notion
prima facie. However, Northern blot analysis indicates that
PDGF-B mRNA is present only in low amounts in aortic SMCs in pup
and adult rats (24). Placement of pup SMCs in culture results in a
dramatic change in PDGF-B phenotype, so much so that abundant levels of
PDGF-B mRNA are observed after six passages (4). Moreover, SMCs
isolated from the neointima of adult rats 2 weeks after balloon
catheter injury have an epitheloid morphology strongly resembling that
of pup SMCs (25). Steady-state PDGF-B mRNA levels in these pup-like neointimal cells also increase with time in culture (26). In situ hybridization using rat-specific cDNA probes and an
en face technique shed light on this apparent conundrum. It
appears that only a subpopulation of SMCs can actually express
PDGF-B in the aortic media. SMC cultures expressing high levels of
PDGF-B after successive passages may arise from clonal expansion of the
PDGF-B-expressing SMC subpopulation (27). Cloning pup SMCs by dilute
plating have generated isolates that either express or do not express
PDGF-B mRNA (6). Aortic SMCs are, therefore, a heterogeneous
population with diverse patterns of growth and gene expression (6).
Such gross differences in PDGF-B expression between cultured pup and adult SMCs provide a valuable resource to investigate transcriptional mechanisms that contribute to cell-specific gene expression. In addition, the similarity of the PDGF-B phenotype between cultured pup
SMCs and SMCs isolated from the balloon-injured rat adult artery wall
(26) implicates a role for zinc finger transcription factors in the
vascular response to injury.
Differences in nuclear transcription factor content have previously
been suggested as a regulatory mechanism underlying cell-specific gene
expression. For example, interleukin-2 expression in murine Th1 cells,
but not Th2 cells, may be due to the presence of NFAT in the former
cell type (28). Second, expression of Bruton's agammaglobulinemia
tyrosine kinase (Btk) gene in B-cells, but not K562 cells or Jurkat
T-cells, may be due to the presence of both Spi-1/PU.1 and Spi-B in
B-cells (29). Third, murine surfactant protein A (SP-A)
promoter-dependent expression in mouse lung epithelial (MLE-15) cells, but weakly, if at all, in HeLa, 3T3, or H441 cells, may
be due to the preferential expression of thyroid transcription factor-1
(TTF-1) in the former cell type (30). Finally, MyoD, the skeletal
muscle lineage control gene product that converts adult SMCs to
skeletal muscle myoblasts, is unable to alter the phenotype of pup SMCs
(31), suggesting that pup and adult SMCs differ in nuclear factors able
to interact with MyoD (6). The present study also extends recent
findings demonstrating no major differences between the content of
NF-
B and AP-1 in the nuclei of pup and adult cells (32). Our
findings also demonstrate that overexpression of Sp1 and Egr-1 could
augment PDGF-B promoter-dependent expression in rat SMCs
and that these effects were not additive under these conditions. Gel
shift studies indicate that in addition to Sp1 and Egr-1, Sp3 and other
factors, yet to be defined, bind specifically to the proximal PDGF-B
promoter (Fig. 6). While overexpression studies are helpful in
determining the functional importance of transcription factors,
simultaneous overexpression of multiple factors with a reporter may not
necessarily approximate the expression pattern of the authentic gene.
This process is likely to be influenced by the nature, concentration,
stoichiometry, and even the order of assembly of nuclear factors on the
promoter.
While the present findings indicate that the proximal region of the
PDGF-B promoter is required for high basal PDGF-B
promoter-dependent expression in pup cells and that this
region is bound by a large number of nuclear factors, elevated PDGF-B
expression may not be mediated by these trans-acting factors
alone. For example, basal and inducible PDGF-A transcription is, like
PDGF-B, dependent upon specific interactions of Sp1, Sp3, and Egr-1
with the proximal promoter (33). Unlike PDGF-B, however, steady-state
PDGF-A mRNA levels are reportedly similar in pup and adult SMCs but
do increase with time in culture (4, 6). This may result from
differences in the methylation state of the authentic promoter, or
combinatorial interactions between transcription factors over promoter
elements. Methylation of the herpes simplex virus thymidine kinase
promoter results in loss of DNase I hypersensitivity and inhibition of transcription (34). Prolactin promoter-dependent gene
expression in pituitary tumor cells is inversely correlated with CpG
methylation of the promoter and the ability of transcription factors to
bind (35). The G+C-rich nature of the PDGF-A promoter adds support to
this possibility (36). Second, transcription factors undergo ordered
cooperative interactions over promoter elements to form stereospecific
enhancer complexes that augment rates of transcription (37). Sp1 bends
DNA upon promoter binding (38) and interacts synergistically with YYI
(39), Ets-1 (40), HIV Tat (41), GATA-1 (42, 43), and p53 (44).
Differences in the nature or content of other regulatory factors may,
therefore, influence this highly selective process and contribute to
the preferential expression of PDGF-B in cultured pup SMCs.
We would like to thank Drs. U. M. Malayankar, C. M. Giachelli, and S. M. Schwartz (Department
of Pathology, University of Washington, Seattle) for their kind gift of
pup and adult rat aortic SMCs. We are grateful to Mr. Fernando S. Santiago for excellent technical assistance.